The physiological function of beclin : a novel BCL-2 interacting protein in protein trafficking

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1 The University of Toledo The University of Toledo Digital Repository Theses and Dissertations 2005 The physiological function of beclin : a novel BCL-2 interacting protein in protein trafficking Xuehuo Zeng Medical College of Ohio Follow this and additional works at: Recommended Citation Zeng, Xuehuo, "The physiological function of beclin : a novel BCL-2 interacting protein in protein trafficking" (2005). Theses and Dissertations This Dissertation is brought to you for free and open access by The University of Toledo Digital Repository. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of The University of Toledo Digital Repository. For more information, please see the repository's About page.

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3 The Physiological Function of Beclin, a Novel Bcl-2 Interacting Protein, in Protein Trafficking Xuehuo Zeng Medical College of Ohio 2005

4 DEDICATION This dissertation is dedicated to my father Hanting Zeng, my mother Juhua Ye, my wife Guirong Wang, and my son Jacob Zeng for their encouragement and love. ii

5 ACKNOWLEDGEMENTS First of all, I would like to thank my major advisor, Dr. William Maltese, for his expert guidance, support and encouragement. Without his assistance and direction this dissertation would not be possible. I would also like to thank my committee member Drs. James Trempe, Han-Fei Ding, Manohar Ratnam, and Linda Dokas for their time and wonderful advice. I would like to thank current and former members in our lab. Dr. Jean Overmeyer taught me a lot of techniques and provided the control sirna vector. Jane Ding helped me with the FPLC size exclusion chromatography experiments. Erin Johnson helped me with the HRP uptake assay. Brandy Alexander, Gwen Soule, Aparna Kaul and Sue Wilson all helped me a lot over the past years. I would like to thank Dr. Tamotsu Yoshimori for providing the antibody against LC3. iii

6 TABLE OF CONTENTS Dedication..ii Acknowledgements...iii Table of Contents...iv Introduction...1 Literature...7 Materials and Methods.43 Results..54 Discussion...96 Conclusions.104 Summary.105 References Abstract iv

7 INTRODUCTION Beclin was isolated in a search for Bcl-2 interacting proteins using a yeast twohybrid system in which human Bcl-2, lacking the C-terminal signal anchor sequence, was used as a bait and an adult mouse brain cdna library served as a target (Liang et al., 1998). The human beclin gene maps to a region on chromosome 17q21 that is commonly deleted in breast, ovarian, and prostate cancers (Aita et al., 1999). It encodes a 450 aa, 60- kda coiled-coil protein. Beclin shares no significant structural homology with any other known mammalian proteins, except for a limited homology with myosin-like proteins within its coiled-coil domain (Aita et al., 1999). Expression of beclin mrna is widespread in both mouse and human adult tissues such as spleen, thymus, kidney, heart and liver. It is expressed at highest levels in mouse and human skeletal muscle (Liang et al., 1998). Heterozygous disruption of beclin in mice increases the frequency of spontaneous malignancies (Qu et al., 2003; Yue et al., 2003). This indicates that Beclin is a haploinsufficient tumor suppressor. Some evidence suggests that Beclin may be involved in apoptosis. The role of Bcl-2 family proteins in the regulation of mitochondria-dependent apoptosis has been clearly established (Green and Reed, 1998). Apoptosis is a morphological phenomenon which is characterized by chromatin condensation and nuclear fragmentation, plasma membrane blebbing, and cell shrinkage. Eventually, the cells break into small membranesurrounded fragments (apoptotic bodies), which are cleared by phagocytosis without inciting an inflammatory response (Kerr et al., 1972). Apoptotic cell death ensures the elimination of cells that become irrelevant or potentially damaging for the organism (Danial and Korsmeyer, 2004). Bcl-2 family proteins include pro- and anti-apoptotic 1

8 molecules sharing homology in any of the four Bcl-2 homology (BH) domains (BH1 to 4) (Adams and Cory, 1998). As mentioned above, Beclin was originally isolated as a novel Bcl-2 interacting protein by yeast two-hybrid screening (Liang et al., 1998). Further studies using fluorescence resonance energy transfer (FRET) microscopy from Liang et al. (1998) have shown that Beclin associates with Bcl-2 when the proteins are co-expressed in African green monkey kidney COS-7 cells (Liang et al., 1998). Overexpression of Beclin reduces Sindbis virus replication, decreases Sindbis virusinduced apoptotic cell death in mouse brains and protects against fatal Sindbis virus encephalitis (Liang et al., 1998). Therefore, one possibility for the function of Beclin is that it plays a role in regulating apoptosis through its interaction with Bcl-2. In addition to the above possible role of Beclin in apoptosis (type I cell death), Beclin also has been demonstrated to function in macroautophagy (type II cell death). In macroautophagy, cytoplasmic constituents and organelles such as mitochondria are enwrapped by a membrane sac, the isolation membrane. Closure of the isolation membrane results in the formation of double membrane structures called autophagosomes. Fusion of the autophagosomes and late endosomes or lysosomes generates autolysosomes (Meijer and Codogno, 2004). Viruses and bacteria can be trapped and degraded in autophagosomes (Rich et al., 2003; Nakagawa et al., 2004). Therefore, the prevention of cell death by Sindbis virus infection may also result from promoting macroautophagy and thus reducing Sindbis virus replication in neurons. There is considerable evidence indicating that Beclin is essential for macroautophagy. In yeast, Atg6/Vps30, the yeast homologue of Beclin, is required for nitrogen deprivation-induced macroautophagy. Atg6/Vps30 deletion mutant yeast cells 2

9 show a defect in macroautophagy (Kametaka et al., 1998). Gene transfer of beclin cdna into the Atg6/Vps30 deletion mutant complements the macroautophagy defect in those cells. Furthermore, overexpression of Beclin promotes macroautophagy in response to nutrient deprivation in human breast carcinoma MCF-7 cells (Liang et al., 1999). More recently, some evidence supports the essential role of Beclin in macroautophagy. Reduction of Beclin expression in mouse L929 fibroblastic cells by RNAi decreases the macroautophagic cell death triggered by zvad [benzyloxycarbonyl-valyl-alanyl- Aspartic-acid (O-methyl)-fluoromethylketone], a caspase inhibitor (Yu et al., 2004). Silencing of Beclin inhibits etoposide-induced macroautophagic cell death in Bax/Bak -/- knockout mouse embryonic fibroblast cells (Shimizu et al., 2004). Despite the accumulating evidence that Beclin is involved in the regulation of macroautophagy, the underlying molecular mechanisms are still not very clear. In Saccharomyces cerevisiae (S. cerevisiae), Atg6/Vps30, the yeast homologue of Beclin, functions in macroautophagy as a subunit of Vps34 phosphatidylinositol 3 -kinase (PtdIns 3-kinase) complex (Kihara et al., 2001b). As in yeast, Beclin, the mammalian homologue of Atg6/Vps30, also has been shown to form a complex with mammalian Vps34 (mvps34) in human Hela cells (Kihara et al., 2001a). mvps34 has been reported to be required for macroautophagy under starvation conditions in human colon HT-29 cells (Petiot et al., 2000) and in mouse myotube C2C12 cells (Tassa et al., 2003). Inhibition of the PtdIns 3-kinase activity by 3-methyladenine (3-MA) prevents the increased macroautophagic response to nutrient deprivation in human breast carcinoma MCF-7 cells overexpressing Beclin (Liang et al., 1999). This indicates that the role of Beclin in macroautophagy may depend on its interaction with mvps34 PtdIns 3-kinase. 3

10 PtdIns (3)P serves as membrane lipid anchor for recruitment of core proteins involved in the vesicle docking and fusion machinery (Wurmser et al., 1999). These proteins typically contain a specific PtdIns(3)P binding domain termed the FYVE finger (Stenmark et al., 2002; Fruman et al., 1999) or PX domains (Song et al., 2001; Cheever et al., 2001). The role of these PtdIns(3)P binding proteins in vesicular transport has been reviewed (Deneka and Van der Sluijs, 2002) and will be discussed in more detail in the LITerature section. In addition to its role in macroautophagy, mvps34 also functions in normal lysosomal enzyme sorting and protein trafficking in the endocytic pathway. PtdIns 3- kinase inhibitors, wortmannin and LY294002, inhibit the processing and delivery of newly synthesized cathepsin D, a lysosomal enzyme, to the lysosomes (Davidson, 1995; Brown et al., 1995). Overexpression of a kinase-deficient mvps34 (dominant-negative) inhibits cathepsin D maturation (Row et al., 2001). These data demonstrate that mvps34 is essential for protein trafficking from the trans-golgi network (TGN) to the lysosomes. A number of studies also support a role for mvps34 in protein trafficking in the endocytic pathway. Wortmannin, which inhibits mammalian PtdIns 3-kinase, blocks the sorting of endocytosed platelet-derived growth factor receptors (PDGFR) to the lysosomes (Shpetner et al., 1996). Microinjection of cells with an inhibitory neutralizing antibody against mvps34 causes mis-localization of the early endosome antigen (EEA1) (Siddhanta et al., 1998) and generation of enlarged multivesicular bodies (MVB)/late endosomes (Futter et al., 2001), and blocks the transit of internalized PDGFR to a perinuclear compartment (Siddhanta et al., 1998). 4

11 In summary, two major classes of interacting proteins for Beclin have been identified: Bcl-2 and mvps34. This raises two possible mechanisms whereby Beclin may affect cell survival and leads to my first hypothesis that mvps34 is the primary target for Beclin. To test this hypothesis, I conducted a series of co-immunoprecipitation studies. My results show that Beclin interacts with Bcl-2 or Bcl-X L when the proteins are coexpressed in cultured cells. However, under conditions that work well with the overexpressed proteins, no endogenous Beclin was found to co-precipitate with Bcl-2 or Bcl- X L. In contrast, endogenous Beclin can easily co-immunoprecipitate with endogenous mvps34. Furthermore, upon gel filtration analysis, Beclin and mvps34 co-elute together in cytosol fractions from control cells, whereas the size of the cytosolic mvps34 complex is reduced from 500 kda to 200 kda when Beclin expression is ablated in the Beclin knockdown cells. These observations demonstrate that mvps34 rather than Bcl-2 is the primary target for endogenous Beclin. Since Beclin forms a complex with mvps34, it is very important to determine if Beclin affects the function of this PtdIns 3-kinase in lysosomal enzyme sorting and protein trafficking in the endocytic pathway. My second hypothesis is that Beclin functions selectively in macroautophagy. To address this issue, I generated Beclin knockdown (KD) U251 human glioblastoma cells using RNAi-mediated gene silencing. My results show that macroautophagic response to nutrient deprivation or C2-ceramide treatment is impaired in Beclin KD U251 cells. However, the trafficking of procathepsin D from the TGN to the lysosomes is normal. Suppression of Beclin expression in U251 cells produced no effects on cell growth, endocytic uptake of HRP, or internalization/degradation of EGF receptor. These data clearly suggest that Beclin 5

12 specifically engages mvps34 PtdIns 3-kinase to the macroautophagic pathway, but is not required for the functions of mvps34 in lysosomal enzyme sorting and protein trafficking in the endocytic pathway. 6

13 LITERATURE Biological Properties of Beclin The beclin gene comprises 12 exons extending over a 12 kb region of the genome. Exons 6-8 contain the predicted coiled-coil domain. The complete cdna of human beclin consists of 2098 bp, including a 120-bp 5 UTR, 1353-bp coding region, and 625- bp 3 UTR. The 2.2-kb Beclin mrna is detectable by Northern blot analysis in many tissues such as spleen, thymus, kidney, heart, lung and liver and present at highest levels in human skeletal muscle. In some tissues, additional 1.7- and 1.4-kb transcripts were observed, suggesting the presence of alternatively spliced transcripts (Liang et al., 1998). Functions of these splice variants are still unknown. PROSITE analysis of human Beclin identified several potential glycosylation, phosphorylation, and myristoylation sites but no other functional sequence motifs (Liang et al., 1998). Human beclin shares 93% homology at the nucleotide level and 98% homology at the amino acid level with the mouse beclin sequence. Human Beclin is also homologous with the Caenorhabditis elegans T19E7.3 gene product (GenBank accession no: U42843) and the S. cerevisiae gene product Lph7p (GenBank accession no: U43503) (38 and 37% identical over 145 and 137 residues, respectively), indicating a high degree of evolutionary conservation (Liang et al., 1998). Yeast Homologue of Beclin: Atg6/Vps30 Human Beclin shares 24.4% amino-acid identity (and 39.1% conservation) with yeast Atg6/Vps30 (Liang et al., 1999). VPS30 encodes a hydrophilic protein containing 556 amino acids which has a predicted molecular mass of 65 kd. Like the mammalian 7

14 Beclin, the central portion of Vps30 (residues 186 to 322) is predicted to form coiled-coil structures (Lupas et al., 1991). Vps30 is partly associated with membrane fractions, while most (~80%) was fractionated in the cytosolic pool (Kihara et al., 2001b). Macroautophagy in Yeast Atg6/Vps30 plays an essential role in macroautophagy in yeast. In response to nutrient starvation, a double membrane structure encloses a portion of the cytosol and/or organelles and autophagosomes are formed. The outer membrane of the autophagosome then fuses with the vacuolar membranes. The inner membrane structures of the autophagasomes are released into the vacuoles as the autophagic bodies (Baba et al., 1994). Vacuolar hydrolases then disintegrate the membranes of autophagic bodies and its cytosolic contents for reuse (Huang and Klionsky, 2002). Incubated in a nitrogendepleted medium, wild-type yeast cells were shown to accumulate autophagic bodies, the final membrane structure of macroautophagy. In contrast, no autophagic bodies in the vacuole were observed in Atg6/Vps30 deletion mutant cells, suggesting that Atg6/Vps30 is essential for macroautophagy in yeast. (Kametaka et al., 1998) Vacuolar Protein Sorting in Yeast In the yeast vacuolar hydrolase sorting pathway, newly synthesized vacuolar proteins traverse the Golgi complex. Vps10 functions as a receptor to vacuolar hydrolases in the late-golgi (Graham and Emr, 1991) and transport them to the prevacuolar endosomes. In the endosomes, the hydrolases dissociate from Vps10 and are sorted to the vacuoles. One of the marker proteins typically used to study this pathway is carboxypeptidase Y (CPY). Carboxypeptidase Y is a glycoprotein initially synthesized as an inactive preproenzyme, which undergoes signal sequence cleavage upon translocation 8

15 across the endoplasmic reticulum (ER) (Blachly-Dyson and Stevens, 1987). The movement of the proenzyme from the ER to the Golgi apparatus can be monitored by a change in the apparent molecular mass of the protein from the 67 kd form in the ER to the 69 kd form in the Golgi compartment (Stevens et al., 1982). A post-sorting proteolytic activation of procpy to mature CPY (61 kd) takes place at the vacuole (Stevens et al., 1982). Atg6/Vps30 has been shown to be involved in the vacuolar protein sorting pathway. In Atg6/Vps30 deletion mutant yeast cells, Vps10 was mislocalized to the vacuoles (Seaman et al., 1997), resulting in missorting of the Golgi form of precursor CPY to the extracellular medium. This indicates that Atg6/Vps30 is responsible for correct vacuolar hydrolase sorting in yeast. It was proposed that Vps30 is required for the retrieval process of Vps10, the CPY receptor, from the endosome back to late-golgi (Seaman et al., 1997). In the absence of Vps30, Vps10 is not recycled back to the late- Golgi and fails to transport the Golgi-form of CPY to the prevacuolar endosome. Therefore, CPY cannot mature in the vacuoles and is secreted into the extracellular medium. Vps34, an interacting partner for Atg6/Vps30 as discussed below, also is required for protein delivery to the vacuole in the yeast S. cerevisiae. In wild-type yeast cells, >95% of the newly synthesized CPY was present as a 61 kd mature form. In contrast, strains which were deleted for the Vps34 gene or contained Vps34 point mutations in the lipid kinase domain, lacked PtdIns 3-kinase activity and exhibited severe defects in the localization of CPY. Less than 5% of the CPY was present as a mature form. The majority of the CPY was present as the Golgi-modified 69 kda precursor form and 90% 9

16 of this precursor CPY was secreted to the extracellular medium (Herman and Emr, 1990; Schu et al., 1993). This information demonstrates that Vps34 is required for the vacuolar protein sorting pathway in yeast. Consistent with the above findings, it was further reported that Atg6/Vps30 functions in macroautophagy and vacuolar protein sorting as a subunit of two distinct large PtdIns 3-kinase complexes: Complex I and II (Fig. 1). Each complex contains a specific component Atg14 (complex I) or Vps38 (complex II) together with three common proteins-vps34, Vps15, and Vps30 (Kihara et al., 2001b). Complex I functions in macroautophagy and complex II functions in CPY sorting. In complex I, Atg14 serves as an adaptor for Atg6/Vps30-Vps34 interactions (Kim et al., 2002) while Vps38 links Atg6/Vps30 to Vps34 in complex II. Vps15-Vps34 is the core of these two complexes. Vps15 is a serine/threonine kinase that interacts with Vps34 (Stack et al., 1993). Vps15 protein kinase activity is required for the Vps15-Vps34 interaction and the PtdIns 3- kinase activity of Vps34 (Stack et al., 1995). Proteins with FYVE or PX domains specifically interact with PtdIns(3)P, the product of Vps34 PtdIns 3-kinase. In S. cerevisiae, PX domain containing proteins, Atg20 and Atg24, specifically bind PtdIns(3)P and interact with an macroautophagyspecific protein, Atg17, which associates with Atg1 kinase. In Atg14 deletion mutant cells, the punctate perivacuolar localization of Atg20 and Atg24 is lost, suggesting that the Atg6/Vps30-Atg14 dependent Vps34 PtdIns 3-kinase complex functions in producing PtdIns(3)P and recruiting PX domain containing proteins, Atg20 and Atg24, to the preautophagosome structures (Fig. 2) (Nice et al., 2002). 10

17 Fig. 1. Model for Two Distinct Vps34 PtdIns 3-kinase Complexes in S. cerevisiae (Adapted from Kihara et al., 2001b) Vps30 P Apg14 Complex I P Vps34 P Vps15 PtdIns PtdIns(3)P membrane Macroautophagy Vps30 P Vps38 Complex II P Vps34 P Vps15 PtdIns PtdIns(3)P membrane CPY sorting Vps30 is the Beclin homologue. Vps15 is anchored to the pre-autophagosomal membrane by myristic attached to the N-terminus of Vps15. Apg14 and Vps38 act as connectors between Vps30 and Vps15-Vps34. Phosphorylation of Vps34 by Vps15 is required for the interaction between Vps34 and Vps15. Complex I and complex II function in macroautophagy and CPY sorting, respectively. 11

18 Fig. 2. Model for Recruitment of PX domain-containing Atg20 and Atg24 Complex by PtdIns(3)P during the Induction of Macroautophagy. (Modified from Klionsky, 2005) Macroautophagy P P Complex I Vps30 Apg14 Vps34 P Vac8 P P Atg13 Atg1 Atg17 Atg11 Atg24 Atg20 P P P Vps15 PtdIns PtdIns(3)P PtdIns(3)P membrane In yeast, Vps34 Ptdins 3-kinase complex I, consisting of Vps15, Vps34, Atg6/Vps30 and Atg14 is required for macroautophagic activity and may function at the preautophagosomal membrane. Vps34 PtdIns 3-kinase phosphorylates PtdIns and produces PtdIns(3)P, which serves as membrane lipid anchor for recruitment of PX domaincontaining Atg20 and Atg24. Atg20 and Atg24 are in complex with Atg1 and several other proteins. Atg1 is a protein kinase required for autophagosome formation. Atg13 and Atg17 appear to modulate the kinase activity of Atg1. Vac8 is implicated in homotypic vacuole fusion and Atg11 is thought to play a role in cargo selection during the process of autophagosome biogenesis. As part of the unified macroautophagy nomenclature agreed upon the yeast research community, the former names for Atg20 and Atg24 are Cvt20 and Cvt13, respectively. (Klionsky et al., 2003) 12

19 The roles of Atg6/Vps30 in macroautophagy and CPY sorting in yeast indicate that Beclin may also function in macroautophagy and lysosomal enzyme sorting (mammalian equivalent to CPY sorting in yeast) in mammalian cells. Macroautophagy in Mammalian Cells Eukaryotic cells have two major protein degradation systems: the ubiquitinproteasome pathway and the lysosomal system. Most intracellular short-lived proteins and misfolded proteins are selectively degraded by the ubiquitin-proteasome pathway (Hershko and Ciechanover, 1998). The lysosomal system is responsible for degradation of most long-lived proteins. The mechanisms to transport cytoplasmic components to the lysosomes are called autophagy in general. There are three types of autophagy: chaperone-mediated autophagy, microautophagy and macroautophagy (Seglen and Bohley, 1992; Dunn, 1994; Blonmaart et al, 1997b). In chaperone-mediated autophagy, cytoplasmic proteins containing KFERQ-like motifs are recognized by a cytoplasmic chaperone, heat shock cognate protein of 73 kda (HSC73). The resulting complex then binds to a lysosomal receptor, LAMP2a (lysosome-associated membrane protein type 2a) and the target proteins are unfolded and delivered into the lumen for degradation (Cuervo and Dice, 1996). In microautophagy, part of the cytoplasm is engulfed by the lysosomal membrane itself. This process is involved in the uptake of macromolecules and organelles such as peroxisomes (Ogier-Denis and Codogno, 2003). Macroautophagy is believed to be responsible for the majority of the intracellular protein degradation. In macroautophagy, cytoplasmic constituents including organelles such as mitochondria, are enwrapped by a membrane sac called isolation membrane. Closure of the isolation membrane results in formation of autophagosomes, a double membrane structures. 13

20 Autophagosomes then fuse with endosome and become amphisomes (Nara et al., 2002). Finally, autolysosomes are generated by the fusion of the outer membranes of the autophagosomes and lysosomes. Lysosomal hydrolases degrade the cytoplasm-derived contents of the autophagosome together with its inner membranes (Fig. 3) (Meijer and Codogno, 2004). For the purpose of discussion, macroautophagy can be divided into three stages: induction, formation of autophagosome, and maturation of autophagosome and its fusion with lysosome (Kirkegaard et al., 2004). Induction of Macroautophagy The induction of macroautophagy can be triggered by a variety of extracellular signals including nutrient starvation, C2-ceramide and rapamycin. Nutrient Deprivation Nonspecific macroautophagy is inhibited under nutrient-rich conditions and is induced by nutrient deprivation. In mammalian cells, phosphorylation of ribosomal protein S6 strongly correlates with inhibition of of macroautophagy (Blommaart et al., 1995). The activity of p70s6 kinase is regulated by mammalian Target of Rapamycin (mtor) kinase (Thomas and Hall, 1997). Inhibition of phosphorylation resulting from inactivation of mtor kinase induces macroautophagy. The mtor kinase exerts a negative regulatory effect on macroautophagy when cells are growing under nutrient-rich conditions. mtor is a sensor for amino acids and energy (Rohde et al., 2001; Dennis et al., 2001). While the mechanism by which mtor senses amino-acid levels is still not clear, two models have been proposed. First, mtor may sense the charging of aminoacylated trna, since amino-acid alcohols, which are competitive inhibitors of 14

21 Fig. 3. Macroautophagy Pathway C2-ceramide Nutrient deprivation fusion with late endosome or lysosome Activation of autophagy derived from ER isolation membrane autophagosome autolysosome Upon nutrient deprivation or C2-ceramide treatment, macroautophagy will be activated. Initially, double membrane structures, derived from endoplasmic reticulum (ER), enclose part of the cytoplasm which may contain nonfunctional proteins and damaged organelles. Autophagosomes then are formed. The autophagosomes fuse with late endosomes or lysosomes and develop into autolysosomes in which the cytoplasmic content is degraded by acid lysosomal hydrolases together with the autophagosome inner membrane. 15

22 aminoacyl-trna synthetases, inhibit mtor regulation of p70s6 kinase (Iiboshi et al., 1999). Second, it also has been suggested that mtor may be regulated directly or indirectly by intracellular amino acids or by their metabolites (Beugnet et al., 2003). After nutrient deprivation, the Tor kinase is inactivated and the negative regulation is relieved leading to induction of macroautophagy (Klionsky and Emr, 2000). C2-ceramide Ceramide can be produced de novo or by the hydrolysis of sphingomyelin. Ceramide synthase also produces ceramide de novo through N-acylation of sphinganine and the addition of a double bond. Ceramide also can be produced when neutral or acidic sphingomyelinases are activated to cleave the bond between ceramide and the phosphoric acid of sphingomyelin (Senchenkov, 2001). Ceramide-mediated cell signaling has been shown to contribute to cell proliferation, differentiation and apoptosis (Hannun, 1994; Kolesnick and Kronke, 1998; Obeid et al., 1993). Recent reports have shown that C2- ceramide can induce macroautophagic cell death in human glioma U373 and T98G cells (Daido et al., 2004) and human colon cancer HT-29 cells (Scarlatti et al., 2004). One possible mechanism is that C2-ceramide inhibits protein kinase B (PKB)/Akt activation and thus relieves the inhibitory effect of the class I PtdIns 3-kinase pathway on macroautophagy (Scarlatti et al., 2004). In mammalian cells, class I PtdIns 3-kinase is stimulated in response to the binding of a ligand to a receptor such as the insulin receptor. PtdIns(3,4)P2 and PtdIns(3,4,5)P3 generated by the activated class I PtdIns 3-kinase at the plasma membrane allow the binding and activation of 3-phosphoinositide-dependent protein kinase 1 (PDK1) and PKB/Akt (Brazil and Hemmings, 2001; Katso et al., 2001). Akt inhibits the GTPase-activating tuberous sclerosis complex (TSC1-TSC2) (Tee et al., 16

23 2002), resulting in the stabilization of RhebGTP, which activates mtor, resulting in the inhibition of macroautophagy (Garami et al., 2003; Tabancay et al., 2003). Rapamycin Rapamycin is a lipophilic macrolide. The intracellular rapamycin receptor in all eukaryotic cells is a small and ubiquitous protein termed FKBP12 (12 kda FK506 Binding Protein). Disruption of FKB1, the gene for FKBP, resulted in the failure of rapamycin to induce macroautophagy, suggesting that the action of rapamycin in inducing macroautophagy is mediated by FKBP (Noda et al., 1998). The rapamycin- FKBP12 gain of function complex interacts specifically with the evolutionarily conserved Tor protein, a negative regulator for macroautophagy. Rapamycin was reported to inhibit the stimulation of mtor kinase activity in response to insulin treatment (Scott et al., 1998). Rapamycin addition to yeast cultures or mammalian cells in culture induces macroautophagy, even in a nutrient-rich medium (Noda et al., 1998; Blommaart, 1995). Tor directly or indirectly causes hyperphosphorylation of Atg13 (Funakoshi et al., 1997; Scott et al., 2000). This form of Atg13 has a lower affinity for the kinase with which it interacts, Atg1, and the reduced interaction inhibits macroautophagy (Kamada et al., 2000). Inhibition of Tor through treatment with rapamycin results in partial dephosphorylation of Atg13 and allows macroautophagic induction (Noda and Ohsumi, 1998). Formation of Autophagosomes The formation of autophagosomes is dependent upon two conjugation systems summarized in Fig. 4: Atg5-Atg12 conjugation and LC3 conjugation systems. Atg5- Atg12 conjugation system is essential for the elongation of the isolation membrane. At 17

24 Fig. 4. Two Conjugation Systems Essential for Formation of Autophagosomes during Macroautophagy A Atg12 Atg7 Atg12 Atg10 Atg12 Atg5 Atg12 Atg5 Atg5 Atg12 Atg16 Atg16 Atg16 Atg16 Atg12 Atg5 Atg5 Atg12 Elongation of isolation membrane B LC3 LC3-I Atg7 LC3-I LC3-II * PE Formation of autophagosomes Atg4 Atg3 (A) Atg5-Atg12 conjugation system. The C-terminal glycine of Atg12 is activated by transient covalent linkage first to Atg7 and then to Atg10, before becoming covalently attached to Lys130 of Atg5. Atg5-Atg12 conjugate binds Atg16 noncovalently and the self-interaction of Atg16 allows multimerization of the complex. (B) LC3 conjugation system. The C-terminal amino acids of LC3 are cleaved by the cysteine protease Atg4 to reveal a glycine residue. Cleaved LC3 is then activated by Atg7, transferred to Atg3, and finally conjugated to phosphatidylethanolamine (PE). * indicates the crosstalk between the two conjugation systems. Overexpression of Atg3, the activating enzyme for LC3-PE conjugation, facilitates the conjugation of Atg12 to Atg5. LC3-PE conjugation is blocked in the absence of Atg5-Atg12 conjugate. 18

25 the initial step of this conjugation reaction, the C-terminal glycine residue of mammalian Atg12 is activated by Atg7 and forms an Atg12-Atg7 thioester intermediate (Tanida et al., 2001). Atg12 is then transferred to mammalian Atg10, resulting in formation of an Atg12-Atg10 thioester intermediate (Mizushima et al., 2002a). Atg10 may serve as an Atg5 recognition molecule and catalyze the final conjugation of Atg12-Atg5 (Mizushima et al., 2002a). Atg16 then associates with Atg12-conjugated Atg5 and mediates the formation of the Atg12-Atg5.Atg16 multimeric complex (Kuma et al., 2002; Mizushima et al., 1999). Most Atg12-Atg5.Atg16 complex resides in the cytosol while a small fraction localizes on the isolation membrane throughout its elongation process. Atg12- Atg5.Atg16 at first associates evenly with the membranes of small vesicles. As the isolation membrane elongates, Atg12-Atg5.Atg16 shows asymmetric localization, with most of them associating with the outer side of the isolation membrane. Right before or after the completion of autophagosome formation, Atg12-Atg5.Atg16 dissociates from the membrane (Mizushima et al., 2001). The second conjugation system is the LC3 (microtubule-associated protein light chain 3) system. This conjugation system is required for the formation of autophagosome. LC3 is the mammalian orthologue of Atg8. Immediately after synthesis, the C-terminal region of LC3 is cleaved (Kabeya et al., 2000). The proteolytic modification is catalyzed by Atg4. This processed form of LC3 has a glycine residue at the C-terminus and resides in the cytosol. It is called LC3-I. Activated by Atg7, LC3-I is then transferred to Atg3 (Tanida et al., 2001, 2002). In the final step, LC3-I is covalently conjugated to phosphatidylethanolamine (PE) through an amide bond between the C-terminal glycine of LC3-I and the amino group of PE to become LC3-II. The presence of this LC3-II-PE 19

26 conjugate in both sides of the isolation membrance is essential for autophagosome completion. LC3-II targets to the isolation membrane throughout the course of membrane elongation and remains on the autophagosome membrane after the completion of autophagosome formation (Kabeya et al., 2000). Immunoblotting of LC3 usually gives two bands: LC3-I (16 kda) and LC3-II (14 kda). The amount of LC3-II or the LC3II/LC3-I ratio is correlated with the number of autophagosomes and the immunoblot analysis of LC3 is one of the most reliable methods to predict macroautophagic activity in mammalian cells (Kabeya et al., 2000) Maturation of Autophagosome and its Fusion with Lysosome Prior to its fusion with lysosomes, autophagosomes mature by fusing with endosomes or endosome-derived vesicles (Tooze et al., 1990; Liou et al., 1997). These structures are called intermediate autophagic vacuoles (Avi/d). The significance of endosome fusion is still unclear. One possible explanation is that fusion with endosomes provides nascent autophagosomes with some machinery required for the fusion with lysosomes. The fusion of autophagosomes with lysosomes is impaired when Rab7 expression is ablated in Rab7 knockdown cells (Jager et al., 2004), indicating that Rab7, a small GTP binding protein, plays an essential role in the maturation of autophagosomes. After the fusion of autophagosomes with lysosomes, the inner memberane of the autophagosomes and the protein and organelle contents are degraded by lysosomal hydrolases (Meijer and Codogno, 2004). The maturation of autophagosomes to autolysosomes is accompanied by a loss of LC3-II-PE, which is present on the membranes of autophagosomes (Mizushima, 2004). 20

27 Functions of Macroautophagy Role of Macroautophagy in Cell Homeostasis and Physiological Processes It is generally accepted that macroautophagy is constitutive in all cell types containing a lysosomal compartment and carries out the housekeeping function involved in cytoplasmic homeostasis. It controls the turnover of peroxisomes, mitochondria and long-lived proteins (Lenk et al., 1992; Luiken et al., 1992; Xue et al., 2001). The role of macroautophagy in adaptation to starvation has been widely investigated because this stress response is physiologically important in the liver and in cultured cells (Blommaart et al., 1997a; Mortimore, 1989). Mammalian cells use macroautophagy during periods of starvation to degrade nonessential cellular components and produce amino acids and other elements needed for vital biosynthetic pathways. Macroautophagy also is involved in certain tissue-specific functions. Macroautophagy was shown to be associated with the intracellular biogenesis of surfactant in pneumocytes II (Hariri et al., 2000). During erythroid maturation, after expulsion of the nucleus, macroautophagy is responsible for eliminating organelles such as mitochondria and ribosomes (Takano-Ohmuro et al, 2000; Holm et al., 2002). Macroautophagy in Pathology Macroautophagy is also implicated in some human diseases such as pathogen infection, myopathy, neurodegenerative disorders and cancer. One role of macroautohagy in cellular defense is to remove invading pathogens. Although bacterial pathogens that invade cells through endocytosis are usually delivered to lysosomes and degraded there (by a process termed phagocytosis). Some of them escape this defense mechanism by blocking or altering the maturation of the sequestering 21

28 vesicles. However, the bacteria may then be trapped by autophagosomes and further degraded after their fusion with lysosomes (Rich et al., 2003; Nakagawa et al., 2004). The sequestration of bacteria into autophagosomes is enhanced by macroautophagy induction through serum withdrawal, whereas the macroautophagy inhibitor 3-MA block uptake of the bacteria (Kirkegaard et al., 2004). These data suggest that macroautophagy can protect against bacterial infection. Macroautophagy may also play a role in the clearance of some viruses. The appearance of autophagosome-like structures occurs after viral infection (Suhy et al., 2000). Macroautophagy is induced through the activation of the double-stranded RNA-activated protein kinase R (PKR) following infection by herpes simplex virus (HSV) (Talloczy et al., 2002). The PKR is a eukaryotic translation initiation factor-2α (eif2α) kinase. Phosphorylation of eif2α by PKR results in translational arrest, which contributes to the induction of macroautophagy and results in the inhibition of viral replication (Kirkegaard et al., 2004). Several hereditary muscular disorders are associated with the accumulation of autophagic or lysosomal vacuoles. These pathologies are characterized by the accumulation of multiple vacuoles from lysosomal and autophagic origin. Based on morphological, biochemical and genetic criteria, vacuolar myopathy has been classified into three groups: rimmed vacuolar myopathy, Danon disease and X-linked vacuolar myopathy (Nishino, 2003). The rimmed vacuolar myopathy is characterized by the presence of apparent small holes in muscle fiber lined by rims, which are red granules observed by modified Gomori trichrome staining. Danon disease is caused by mutation in the LAMP-2 gene. Muscle biopsies of Danon disease patients show an accumulation of small autophagic vacuoles (Eskelinen et al., 2002). X-linked myopathy is similar to 22

29 Danon disease and is characterized by progressive muscle atrophy, involving cardiac and respiratory muscle degeneration (Kalimo et al, 1988; Chabrol et al., 2001). Muscle biopsy shows similar features to those observed in Danon disease, but no LAMP-2 mutations have been observed in patients of X-linked myopathy. The accumulation of autophagic vesicles also has been observed in many neurodegenerative disorders such as Parkinson s and Huntington s disease. Parkinson s disease is characterized by the accumulation of aggregates called Lewy bodies in neurons. Mutations in α-synuclein, a major protein in Lewy bodies, cause early onset Parkinson s disease. Similar to the situation with mutant α-synuclein, the expression of mutant huntingtin also induces the accumulation of autophagic vesicles (Kegel et al., 2000). Treatment of those mutant huntingtin and α-synuclein expressed cells with rapamycin promotes autophagic degradation of mutant huntingtin and α-synuclein and prevents the accumulation of aggregates. This suggests a protective role for macroautophagy in Parkinson s and Huntington s disease (Ravikumar et al., 2002). In contrast to the protective function of macroautophagy, some forms of neuronal cell death may involve uncontrolled macroautophagy. It has been shown that death of Purkinje cells in heterozygous Lurcher mice is associated with the presence of an excess of autophagosomes (Selimi et al, 2003). Furthermore, Lurcher cell death is caused by the expression of a constitutively activated form of the glutamate receptor GRID2, which interacts with a protein complex that contains Beclin (Yue et al., 2002). These observations indicate a functional role for macroautophagy in neuronal cell death. The characterization of the tumor suppressor activity of the macroautophagy gene beclin suggests the role of macroautophagy in cancer development. Overexpression of 23

30 Beclin in MCF-7 cells promotes macroautophagy in response to nutrient deprivation and inhibits tumorigenesis, which suggests that macroautophagic activity is associated with inhibition of cellular proliferation (Liang et al., 1999). In addition, Beclin heterozygous mice suffer from a high incidence of spontaneous tumors (Qu et a., 2003; Yue et al., 2003). Macroautophagy may thus function as a tumor suppressor mechanism. Some anticancer drugs also have been shown to act through macroautophagy. C2-ceramide inhibits protein kinase B activation and relieves the inhibitory effect of the class I Ptdins 3-kinase pathway on macroautophagy (Scarlatti et al., 2004). Rapamycin inhibits the stimulation of mammalian Tor (mtor) kinase activity and induces macroautophagy (Scott, 1998). Role of Beclin in Macroautophagy in Mammalian Cells Accumulating evidence suggests that Beclin functions in macroautophagy. Beclin can complement macroautophagy defect in Atg6/Vps30-disrupted yeast and overexpression of Beclin promotes starvation-induced macroautophagy in human breast carcinoma MCF-7 cells. Beclin expression is upregulated during macroautophagy induced by C2-ceramide in human colon cancer HT-29 cells. More recently, Yu et al. (2004) have shown that suppression of Beclin expression in mouse L929 fibroblastic cells by RNAi strongly inhibits macroautophagic cell death induced by the caspase inhibitor zvad (Yu et al., 2004). It also was reported that Beclin is required for the macroautophagic cell death induced by etoposide (an inhibitor of topoisomerase and a common apoptotic inducer) in embryonic fibroblast cells from Bax/Bak double knockout mice (Shimizu et al., 2004). These studies strongly indicate that Beclin plays an essential role in macroautophagy in mammalian cells. 24

31 Despite the apparent importance of Beclin in the regulation of macroautophagy, the underlying molecular mechanisms are still not very clear. As in yeast, Beclin, the mammalian homologue of Atg6/Vps30, also has been shown to form a complex with mammalian Vps34 (mvps34) (Kihara et al., 2001b). The mvps34 has been reported to be required for macroautophagy under starvation conditions in human colon HT-29 cells (Petiot et al., 2000) and in mouse myotube C2C12 cells (Tassa et al., 2003). Inhibition of the PtdIns 3-kinase activity by 3-MA prevents the increase in macroautophagic response to nutrient deprivation in human breast carcinoma MCF-7 cells overexpressing Beclin (Liang et al., 1999). This indicates that the role of Beclin in macroautophagy may depend on mvps34 PtdIns 3-kinase and its product, PtdIns(3)P. Beclin also was reported to contain a leucine-rich nuclear export signal (NES) that is essential for its role in macroautophagy. Leucine-rich short amino acid sequences responsible for efficient nuclear export have been identified in a number of different cellular and viral proteins (Nigg, 1997; Gorlich, 1998). Functional NESs normally have consensus sequence (Lx (2 3) Lx (2 3) LxL). The functional NES forms a complex with the nuclear export receptor, chromosome maintenance region 1 (CRM1), and thus mediates nuclear protein export (Fornerod et al., 1997). Leptomycin B, a fungicide, can target CRM1 and inhibit the formation of this complex and then block nuclear export of NEScontaining proteins (Stade et al., 1997; Wolff et al., 1997). Inhibition of the CRM1 nuclear export pathway by leptomycin B or mutation of the conserved leucine residues (amino acids ) within the Beclin NES blocks the nuclear export of Beclin, thereby interefering with the function of Beclin in promotion of nutrient-deprivation induced macroautophagy in MCF-7 cells (Liang et al., 2001). This result suggests that 25

32 cycling of Beclin through nuclear compartments is important for the function of Beclin in macroautophagy. Beclin macroautophagy gene is important for cell growth control and tumorigenesis. Heterozygous disruption of beclin in mice (beclin +/- ) resulted in significant increase of spontaneous tumor formation in comparison to wild-type mice. About 59% beclin +/- mice developed tumors, whereas only 14% wild-type mice developed tumors. beclin +/- mice not only had a higher cancer rate, but also a different spectrum of tumor types. The tumors formed in beclin +/- mice include lung adenocarcinoma, heptacellular carcinomas, B cell lymphomas and lymphoblast cell lymphoma, while the tumors present in wild-type mice were all lymphomas (Yue et al., 2003). Heterozygous disruption of beclin in mice also accelerates the progression of hepatitis B virus (HBV) -induced liver cell neoplasia. The severity of preneoplastic changes, characterized by the extent of liver with small-cell dysplasia an important histopathologic predictor of malignant transformation was significantly increased in the beclin +/ compared with wild-type mice. Thus, beclin heterozygous deletion both increases susceptibility to spontaneous malignancies and accelerates HBV-induced hepatocellular carcinogenesis (Qu et al., 2003). Biological Functions of Vps34 in Mammalian Cells Three Classes of PtdIns 3-kinase Unlike in Saccharomyces cerevisiae, which has a single PtdIns 3-kinase gene, there are three classes of PtdIns 3-kinase in mammalian cells. Class I enzymes are composed of catalytic p110 subunits and p85 adaptors (Fruman et al., 1998). The SH2 (Src homology domain 2) motif, which is contained in p85 adaptors, binds to 26

33 phosphorylated tyrosine residues, thereby linking the catalytic subunit p110 to receptor tyrosine kinase signaling pathway (Class I A ) (Backer et al., 1992). The class I B enzymes (PI 3Kγ) are activated by βγ subunits from heterotrimeric G-proteins (Stephens et al., 1997). Class I PtdIns 3-kinases phosphorylate PtdIns, PtdIns(4)P and PtdIns(4,5)P2. However, PtdIns(4,5)P2 is likely to be the favored substrate in vivo. The class II PtdIns 3-kinases are large monomeric enzymes of approximately kda (Virbasius, 1996). These enzymes phosphorylate PtdIns and PtdIns(4)P in vitro, but not PtdIns (4,5)P2. They are activated in insulin-stimulated cells, but the mechanism is not yet known (Brown et al., 1999). Class III PtdIns 3-kinases (mvps34) are homologous to the archetypal Vps34 characterized in Saccharomyces cerevisiae. The mvps34 only produces PtdIns(3)P (Schu et al., 1993). In yeast, PtdIns(3)P is found on vacuoles and endosomal membranes (Gillooly, 2000). In mammalian cells, it is found in membranes of early endosomes and the internal vesicles of multivesicular endosomes (Gillooly, 2000). The localization of PtdIns(3)P to endosomes and yeast vacuoles suggests that it functions by recruiting effector proteins to these membranes. PtdIns(3)P can be recognized specifically by two distinct protein domains: FYVE domain and Phox homology (PX) domain. These domains are found in many proteins that function in protein trafficking. Hence, production of PtdIns(3)P by mvps34, possibly regulated by interaction with Beclin, might greatly influence the membrane recruitment of these proteins. 27

34 FYVE Domain The FYVE domain was named after the first letter of the first four proteins in which it was found (Fab1p, YOTB, Vac1p and EEA1)(Stenmark et al., 1996). The FYVE domains contain two β-hairpins plus a small C-terminal α-helix that are held together by two Zn 2+ -binding clusters in a Zinc finger-like structure (Misra S. et al., 1999). The characteristic cluster of basic residues is located on the β-strand and the following β-turn. Together with the conserved arginine residue, this domain forms a basic pocket for interaction with the negatively charged 1,3-bisphosphomyo-inositol head group of PtdIns(3)P. The shallow groove that forms the PtdIns(3)P binding site is too small for PtdIns(4,5)P2 or PtdIns(3,4,5)P3 and is incompatible with other PtdIns such as PtdIns(4)P, thereby accounting for the binding specificity of the motif (Misra et al., 1999). In addition, hydrophobic residues preceding the first β-strand are predicted to form a hydrophobic loop for interaction with the membrane (Kutateladze, 2001). The FYVE domain proteins are involved in endocytic membrane trafficking. EEA1, a large coiled-coil protein with a C-terminal FYVE domain, is the best studied FYVE domain protein. Endosomal targeting of EEA1 requires both PtdIns(3)P binding by the FYVE domain and binding to Rab5:GTP (Simonsen et al., 1998). EEA1 functions in endosome fusion. Depletion of EEA1 inhibits and excess EEA1 promotes this process in vitro (Christoforidis, 1999a). Rabenosyn-5, another FYVE domain protein, is the mammalian homologue of Vac1p. It contains an internal FYVE domain and also has been shown to play a role in endosome fusion (Nielsen et al., 2000). As with EEA1, Rabenosyn-5 is an effector of Rab5:GTP and localized to early endosome in a PtdIns(3)P-dependent manner. The exact mechanism for how EEA1 and Rabenosyn-5 28

35 regulate endosome membrane traffic still remains to be elucidated. One possibility is that these proteins interact with syntaxin-6 and syntaxin-13, which are thought to mediate endocytic membrane fusion through the engagement in heteromeric complexes with other SNARE proteins (Simonsen, 1999). Another FYVE domain containing protein, PIKfyve, acts on PtdIns(3)P through the interaction of its FYVE domain with PtdIns(3)P to generate PtdIns(3,5)P2 that is required for multivesicular bodies inward vesiculation and/or storage of internal vesicles (Ikonomov et al., 2003). Hepatocyte growth factorregulated tyrosine kinase substrate (Hrs) is an early endosomal protein (Komada et al., 1997). The FYVE domain of Hrs also binds specifically to PtdIns(3)P in vitro and can replace the FYVE domain of EEA1 with respect to the endosomal targeting of EEA1(Gaullier et al., 1998). In addition, a point mutation in the FYVE domain of Hrs, which reduces the binding affinity with PtdIns(3)P, displaced Hrs out of the early endosomal membranes (Raiborg et al., 2001a). This indicates that the early endosomal localization of Hrs is mediated through its FYVE domain by binding to PtdIns(3)P. Overexpression of Hrs inhibited trafficking of epidermal growth factor receptor (EGFR) from early endosomes, resulting in an accumulation of EGFR on early endosomes in both EGF-stimulated and unstimulated cells (Morino et al., 2004). Hrs contains a ubiquitininteracting motif and the C-terminus of Hrs binds to clathrin (Raiborg et al., 2001b), suggesting that Hrs could recruit ubiquitinated receptors into clathrin-coated subdomains of early endosomes. This could serve to sort receptors that are destined for late endosomes and lysosomes. Besides their functions in endocytic membrane trafficking, one FYVE domain containing protein also has been found to be associated with macroautophagy. Alfy 29

36 (autophagy-linked FYVE protein), a novel FYVE domain containing protein, binds to and partially colocalizes with PtdIns(3)P. Upon amino acid starvation, Alfy accumulates on cytoplasmic structures and colocalizes with macroautophagic marker proteins Atg5 and LC3. Inhibition of proteasomal protein degradation results in a strong increase in the number of Alfy-positive cytoplasmic structures and these structures were often detected inside autophagosomes by electromicroscopy. These observations suggest that Alfy may target cytosolic protein aggregates for autophagic degradation (Simonsen et al., 2004). PX Domain The PX domain is approximately 120 residues long. It contains a number of basic residues and a proline-rich stretch (Ponting, 1996). Nuclear Magnetic Resonance (NMR) spectroscopy studies of PX domains from p47 phox and Van7p suggested that the overall structure of the domain is a three stranded β-sheet followed by three α-helices (Cheever et al., 2001; Hiroaki et al., 2001). The majority of PX domains studied so far show binding selectivity for PtdIns(3)P. The PtdIns(3)P headgroup binds in a pocket between the β-sheet and the helical subdomain (Bravo et al., 2001). The ability of the PX domain to bind PtdIns(3)P results in the association of the host protein with components of the endocytic pathway. In this respect, PX domains resemble FYVE domains, although they are structurally distinct (Bravo et al., 2001; Dumas et al., 2001). Most PX domain-containing proteins are involved in vesicular trafficking, protein sorting or lipid modification. Several PX domain-containing proteins such as MVP1p, Grd19p and vps5p/grd2p in yeast have been characterized to function in protein trafficking. MVP1 is involved in sorting proteins in the late Golgi for delivery to the vacuole. Grd19p consists essentially of a PX domain and is suggested to function as a 30

37 component of the retrieval machinery by direct interaction with certain late Golgi proteins. Vps5p/Grd2p was identified as a protein responsible for normal traffic to the vacuole. A defective Vps5p results in the secretion of CPY with Vps10p being misdelivered to and rapidly degraded in the vacuole. Mammalian sorting nexins (SNX) are a family of related proteins implicated in the endocytic pathway that also contain PX domains (Teasdale et al., 2001). SNX1 is found to be present in endosomal structures which also contain the EGF receptor following ligand-induced internalization (Kurten et al., 1996). Overexpression of SNX1 dramatically decreases the half-life of the EGF receptor, suggesting that it may be involved in trafficking from endosomes to the lysosomes (Zhong et al., 2002). SNX3 is similarly enriched in the early endosome. Micro-injection of SNX3 antibodies affects the transport of transferrin receptor from the early endosome to the recycling endosome (Xu et al., 2001). The PX domain is required for its membrane association and for association with the PDGF receptors (Phillips et al., 2001). Taken together, the above data clearly document the role of FYVE finger or PX domain proteins in the protein trafficking pathways. Through its product PtdIns(3)P, which recruits or activates FYVE finger or PX domain containing proteins, mvps34, an interacting partner for Beclin, could be involved in three pathways: (i) lysosomal enzyme sorting, (ii) endocytic trafficking and sorting of cell surface receptors, and (iii) macroautophagy. Since Beclin physically interacts with mvps34, it is important to determine how this interaction may affect the physiological function of mvps34. 31

38 mvps34 Functions in Lysosomal Enzyme Sorting in Mammalian Cells In mammalian cells, there is an analogous pathway to the yeast vacuolar protein sorting pathway. New synthesized lysosomal hydrolases are modified in the Golgi by the attachment of a mannose 6-phosphate on their N-linked oligosaccharide side chains. In the TGN, this tag is recognized by a set of two mannose 6-phosphate receptors (MPRS) that mediate sorting of the hydrolases dissociated from the MPRs due to the acidic environment in the endosome. Finally, the hydrolases are delivered to the lysosomes while MPRs are recycled back to the TGN for further rounds of sorting (Von Figura and Hasilik, 1986; Kornfeld and Mellman, 1989). Cathepsin D can be used as a marker protein for the lysosomal enzyme sorting in mammalian cells (Fig. 5). Cathepsin D is synthesized as a precursor of 53 kda (TGN form). After transport from TGN to the late endosomes, it is processed to an intermediate of 47 kda (late endosome form). This protein matures to a heterodimeric form consisting of a 31 kda heavy and a 14 kda light chain in the lysosomes (lysosome form) (Gieselmann et al., 1983). A fraction of the 53 kda precursor form is secreted into the medium of cultured cells (Hasilik et al., 1980). There is much evidence to show that mvps34 is essential for this pathway. In Normal Rat Kidney (NRK) fibroblastic cells, PtdIns 3-kinase inhibitor wortmannin inhibits the normal processing and delivery of newly synthesized cathepsin D and diverts it to the constitutive secretory pathway. At concentrations which dramatically reduced cellular levels of PtdIns(3)P, the product of mvps34, wortmannin inhibits the processing and delivery of newly synthesized cathepsin D to lysosomes in NRK cells. As a result, newly synthesized procathepsin D is secreted into the extracellular medium (Brown et al., 32

39 Fig. 5. Cathepsin D Maturation Pathway A TGN procathepsin D (52 kd) late endosome intermediate cathepsin D (47 kd) lysosome mature cathepsin D (31 kd) B 50 kda pro intermediate 36 kda mature (A) Cathepsin D is initially synthesized as a ~52-kD unprocessed protein. After delivery from the trans-golgi network to the late endosome compartment, cathepsin D is processed to yield a ~47 kd form. After reaching the lysosomes, cathepsin D is further processed into ~31 kda and ~14 kda forms. (B). A typical example of the three forms of cathepsin D is observed when whole cell lysate from U251 human glioblastoma cells was subjected to SDS-PAGE and immunoblot analysis with an antibody against cathepsin D. 33

40 1995). Overexpression of a kinase-deficient mvps34 in Hela cells inhibited cathespin D maturation (Row et al., 2001). Involvement of Vps34 in Endocytic Protein Trafficking There is considerable evidence to support a requirement for Vps34 in the endocytosis pathway (Fig. 6). All eukaryotic cells internalize cell surface proteins and material from their environment by endocytosis. Typically, receptors and bound ligand are targeted to early endosomes. Early endosomes have a slightly acidic ph (5.9-6) and this can release the receptor from ligand. The receptor may be recycled to the surface by vesicles that bud from the endosome and then target the plasma membrane. After these recycling vesicles fuse with the plasma membrane, the receptor is returned to the cell surface. After ligands dissociate from their receptors at the mildly acidic internal ph of the early endosome, they are delivered to degradative late endocytic compartments. Also, clathrin-coated vesicles from the TGN carry degradative enzymes to the late endosome and fuse with these structures, releasing their contents. Late endosomes include multivesicular bodies (MVB) and contain whorls or vesicles of membranes inside. Finally, ligands travel to lysosomes to be degraded (Mukherjee et al., 1999). In the yeast Saecharomyces cerevisiae, Vps34 is responsible for efficient delivery of endocytic cargo to the vacuole. Wurmser et al. (1998) monitored the endocytosis of FM4-64 in Vps34 PtdIns 3-kinase inactivated cells. FM4-64 is a fluorescent lipophilic molecule which intercalates into the plasma membrane when added exogenously to yeast cells. The endocytic progress of FM4-64 to the vacuole can be monitored by fluorescence microscopy. The cells were pulsed with FM4-64 and the trafficking of FM4-64 to the vacuole was monitored at various chase times in FM4-64 free media. They found that 34

41 Fig. 6. Receptor-mediated Endocytosis Pathway Endocytosis pathway Receptor-mediated EGF Mitogenic signaling Phosphorylation Early Endosome Inward Vesiculation Multivesicular Endosome (Late Endosome) Lysosome Receptor Degraded The binding of a growth factor to its specific receptor such as epidermal growth factor receptor (EGFR) on the plasma membrane causes the initiation of the signal transduction cascade which results in cell proliferation. This mitogenic signaling can be attenuated by endocytosis of the ligand-receptor complexes from the cell surface. The ligand bound receptors are internalized and transported to early endosomes. They are further sorted in the MVB/late endosomes and delivered to the lysosomes to be degraded. 35

42 FM4-64 accumulated adjacent to vacuoles and poor vacuole staining was observed even after a 50 min chase in Vps34 PtdIns 3-kinase inactivated cells. However, in wild-type cells, the bulk of FM4-64 reached the vacuole after 10 min of chase (Wurmser et al, 1998). This indicates that loss of Vps34 function results in a block in endocytic transport from prevacuolar compartments to the vacuole, which is characterized by the accumulation of FM4-64 in prevacuolar endocytic compartments. Similar effect was observed in another yeast Candida albicans null mutant cells (Bruckmann et al., 2001; Gunther et al., 2005). In mammalian cells, substantial evidence also documents the role of mvps34 in the endocytic protein trafficking. Addition of the PtdIns 3-kinase inhibitor, wortmannin, blocks the sorting of endocytosed platelet-derived growth factor (PDGF) receptors to the lysosomes (Shpeter et al., 1996). Experiments using antibodies against mvps34, which significantly reduced its PtdIns 3-kinase activity, blocked the transit of internalized PDGF receptor to a perinuclear compartment (Siddhanta et al., 1998). These data indicate that mvps34 plays an essential role in endocytic trafficking. The mvps34 may function in both early endosomes and late endosomes. Microinjections with inhibitory antimvps34 cause mis-localization of the early endosome antigen (EEA1) (Siddhanta et al., 1998) and the generation of enlarged MVBs/late endosomes (Futter et al., 2001). As in yeast, the production of PtdIns(3)P by mvps34 PtdIns 3-kinase is believed to be responsible for the role of mvps34 in endocytic protein trafficking. The mvps34 PI 3- kinase may be recruited onto the endosomal membrane by Rab5 GTPase (Murray et al., 2002). PtdIns(3)P in turn acts as a second messenger through recruitment of FYVE containing effector proteins. One such effector, EEA1, is believed to mediate membrane 36

43 heterotypic fusions involving endosomes (Rubino et al., 2000; Mills et al 1998; Stenmark et al., 1999) and to act as a tethering factor (Pfeffer, 1999) mediating endosomeendosome docking (Christoforidis et al., 1999b). A second effector, Rabenosyn-5, which also has been shown to localize to the early endosome in a manner dependent on its FYVE domain and PtdIns(3)P, plays a role in endosome fusion (Nielsen et al., 2000; Patki et al., 1998) Functions of mvps34 in Macroautophagy In addition to the above functions in lysosomal enzyme sorting and endocytic protein trafficking, accumulating evidence suggests that mvps34 is involved in macroautophagy. As mentioned earlier, Vps34 has been shown to be required for formation of autophagosomes and macroautophagic activity in yeast S. cerevisiae (Kihara et al., 2001a). This function is conserved in mammalian cells. In isolated rat hepatocytes, the PtdIns 3-kinase inhibitors, wortmannin and LY294002, inhibit macroautophagy, indicating that PtdIns 3-kinase is required for macroautophagy. Wortmannin, LY and 3-Methyladenine, which inhibited PtdIns 3-kinase activity, disrupted macroautophagic sequestration of electroinjected cytosolic [ 14 C] sucrose and abrogated autophagosome formation observed by electron microscopy in isolated rat hepatocytes (Blommaart et al., 1997b). It also has been shown that wortmannin and LY impaired macroautophagic sequestration of the cytosolic enzyme lactate dehydrogenase (LDH) in human colon HT-29 cells (Petiot et al., 2000). These data suggest that PtdIns 3-kinase is required for macroautophagy in mammalian cells. It was further reported that class I PtdIns 3-kinase inhibits macroautophagic pathway while class III PtdIns 3-kinase (mvps34) stimulates this pathway. Introduction of anti-sense oligonucleotides targeting 37

44 class III PtdIns 3-kinase (mvps34) into HT-29 cells strongly inhibited class III PtdIns 3- kinase activity and interrupted macroautophagic activity induced by nutrient deprivation. Overexpression of p150, the mvps34 adaptor, stimulated both the synthesis of PtdIns(3)P and macroautophagic sequestration(petiot et al., 2000). Consistent with this finding, mvps34 was also found to be implicated in macroautophagy induced by amino acid starvation in mouse myotube C2C12 cells. Delivery of inhibitory anti-vps34 into C2C12 myotubes dramatically inhibited mvps34 PtdIns 3-kinase activity and reduced rates of macroautophagic degradation (Tassa et al., 2003). All this information demonstrates that mvps34 is required for the macroautophagic pathway, and may explain why Beclin plays a key role in macroautophagy. Involvement of Beclin in Apoptosis? After mouse Beclin was isolated as a Bcl-2 interacting protein by yeast twohybrid screening, additional yeast two-hybrid studies confirmed that the region of human Beclin that corresponds to the mouse gene product isolated in the yeast two-hybrid screen (aa 1 to 236) also interacts with Bcl-2. However, full length-beclin does not interact with Bcl-2 in the yeast two-hybrid system. Besides the above yeast two-hybrid system, fluorescence resonance energy transfer (FRET) analysis also has been used to confirm the interaction of Beclin and Bcl-2 after they were co-expressed in African green monkey kidney (COS-7) cells (Liang et al., 1998). Fluorescence resonance energy transfer is a fluorescence technique that can be used as a spectroscopic ruler to study and quantify the interactions of cellular components with each other (Clegg, 1992). In FRET, a fluorophore (donor) in an excited state may transfer its excitation energy to a neighboring chromophore (acceptor) nonradiatively through dipole-dipole interactions. The efficiency 38

45 of this process varies as the inverse of the sixth power of the distance separating the donor and acceptor chromophores and, in practice, requires the distance between the donor and acceptor fluorophores to be short (usually less than 50 Å). The dependence of the energy transfer efficiency on the donor-acceptor separation provides the basis for the utility of this phenomenon in the study of cell component interactions (Selvin, 1995). In addition to the above interaction of Beclin and Bcl-2, there is some evidence showing that Beclin may be involved in apoptosis. It was reported that overexpression of Beclin can reduce Sindbis virus replication, decrease Sindis virus-induced apoptotic death in mouse brains and protect against fatal Sindbis virus encephalitis. However, it is not known whether Beclin can prevent neural cell death as a consequence of reducing Sindbis virus replication through macroautophagy or whether Beclin exerts direct antiapoptotic effects (Liang et al., 1998). On the contrary, one study also shows that Beclin is not essential for apoptotic cell death induced by ultraviolet (UV) irradiation or serum deprivation. In response to UV light, embryonic stem (ES) cells undergo classic apoptosis in which p53 and Bcl-2 family proteins are involved (Chao et al., 2000; Xu et al., 2002). In contrast, upon serum withdrawal a second form of apoptotic cell death is induced in ES cells which involves apoptosis-inducing factor (AIF) and is caspase-independent (Joza et al., 2001). There were no differences in cell death between wild-type ES cells and beclin -/- ES cell lines in response to either UV irradiation or serum deprivation (Yue et al., 2003). These data suggest that the interaction between Beclin and Bcl-2 and the role of Beclin in apoptosis need to be further investigated. 39

46 Apoptosis is a morphological phenomenon with characteristics including chromatin condensation and nuclear fragmentation, plasma membrane blebbing, and cell shrinkage. Eventually, the cells break into small membrane-surrounded fragments (apoptotic bodies), which are cleared by phagocytosis without inciting an inflammatory response (Kerr et al., 1972). The mitochondria-dependent pathway for apoptosis is governed by Bcl-2 family proteins. Bcl-2 family proteins can be divided into 3 groups (Fig. 7): 1. Anti-apoptotic members such as Bcl-2 and Bcl-XL, which share Bcl-2 homology (BH) domain 1 through Pro-apoptotic members such as Bax and Bak. They share sequence homology in BH1, BH2, and BH3 but not in BH4. 3. BH3-only proteins such as Bad and Bid (Reed., 2000; Tsujimoto et al., 2000). It was proposed that Bax exists in three conformations: cytosolic (state 1), loosely attached to the outer mitochondrial membrane (state 2), and integral to the mitochondrial membrane (state 3) (Harris and Johnson., 2001; Mihhailov et al., 2001). Stimulated by some apoptotic signals such as staurosporine or γ irradiation (Hsu et al., 1997; Wolter et al., 1997), Bax can be translocated from the cytosol to the mitochondria and undergo conformational changes to form oligomers and channels that permit the release of cytochrome c or apoptosis inducing factors (AIF), and then activate a downstream signaling pathway (Mikhailov et al., 2001; Lee et al., 2001; Bedner et al., 2000). Bcl-2/Bcl-X L can form heterodimers with Bax on the mitochondrial membrane and prevent Bax oligomer/channel formation, thereby inhibiting apoptosis (Murphy et al., 2000; Putcha et al., 1999; Gottlieb., 2000; Mihhailov et al., 2001). 40

47 Fig. 7. Three Groups of Bcl-2 Family Proteins 1. ANTI-APOPTOTIC BCL-2 BCL-X L BH 4 BH 3 BH 1 BH 2 TM BH 4 BH 3 BH 1 BH 2 TM 2. PRO-APOPTOTIC BAX BAK 3. BH 3-ONLY BAD BID BH 3 BH 1 BH 2 TM BH 3 BH 1 BH 2 TM BCL-X S BH 4 BH 3 TM BH 3 BH 3 Named after the founding member of the family, which was isolated as a gene involved in B-cell lymphoma (hence the name bcl), the Bcl-2 family is composed of over a dozen proteins, which have been classified into three functional groups. Members of the first group, such as Bcl-2 and Bcl-X L, are characterized by four conserved Bcl-2 homology (BH) domains (BH1-BH4). The key feature of group I members is that they all possess anti-apoptotic activity, and protect cells from death. In contrast, group II consist of Bcl-2 family members with pro-apoptotic activity. Members of this group, which includes Bax, Bak and Bcl-X S, share sequence homology in BH1, BH2, and BH3 but not in BH4. Group III consists of BH3-only proteins such as Bad and Bid. 41

48 Summary Beclin was originally isolated in a yeast two-hybrid screen for proteins that associate with Bcl-2, a key regulator of programmed cell death (apoptosis). After it was discovered, very limited data demonstrate the interaction between Beclin and Bcl-2 except for the FRET studies showing that Beclin and Bcl-2 are in close proximity (< 50 Å) when the proteins are co-expressed in cultured cells. Interestingly, mvps34 PtdIns 3- kinase also has been identified to be an interacting partner for Beclin. Hence, which protein is the major target for Beclin in mammalian cells needs to be determined. mvps34 is the class III PtdIns 3-kinase. It uses PtdIns as a substrate to produce PtdIns(3)P, which serves as membrane lipid anchor for recruitment or activation of FYVE finger or PX domain proteins. These FYVE finger or PX domain proteins play essential roles in lysosomal enzyme sorting, endocytic protein trafficking and macroautophagy. Since Beclin interacts with mvps34, it is very important to determine if this interaction affects the functions of mvps34 in those pathways. Beclin has been shown to play an essential role in macroautophagy and thus function as a tumor suppressor. However, the anti-tumor activity of Beclin also may be related to its interaction with the anti-apoptotic protein Bcl-2 or to its regulation of the function of mvps34 in normal vesicular trafficking pathways. For example, it is possible that loss of Beclin expression might potentiate growth factor signaling pathways by disrupting mvps34-dependent steps required for post-endocytic sorting. All the above interesting issues will be addressed in this dissertation. 42

49 MATERIALS AND METHODS Generation of Construct for Expression of Flag-tagged Beclin The cdna encoding Beclin was obtained by PCR amplification using Pfu polymerase (Stratagene, La Jolla, CA) from a cdna template, reverse transcribed from human embryonic kidney (HEK) 293 cell mrna. The PCR product was cloned into pcmv5 and modified by addition of a 5 sequence encoding the Flag epitope (DYKDDDDK). Transient Transfection Cells were seeded in 60-mm dishes on the day before transfection. Transient transfection using Lipofectamine Plus reagent was performed according to the manufacturer s instructions (Invitrogen, Carlsbad, CA). Briefly, to prepare DNA-Plus- Lipofectamine complex, 2 µg plasmid DNA was mixed with 8 µl of Plus Reagent and then 12 µl of Lipofectamine Reagent. The DNA-Plus-Lipofectamine complex was added to cells containing fresh serum-free Dulbecco s modified Eagle medium (DMEM) and incubated for 3 h. The medium then was replaced with fresh DMEM with 10% (v/v) Fetal Bovine Serum (FBS). For transfection of cells cultured in 100-mm dishes, 4 µg DNA was mixed with 20 µl of Plus Reagent and 30 µl of Lipofectamine Reagent. Stable Transfection of Flag-Beclin into MCF-7 Tet-off Cells MCF-7 Tet-off cells were purchased from Clontech (Palo Alto, CA) and were maintained in DMEM, supplemented with 10% FBS and 100 µg/ml G418. Flag-Beclin was subcloned into a pbi-egfp expression vector (Clontech, Palo Alto, CA) that allows simultaneous expression of EGFP and the gene of interest. MCF-7 Tet-off cells were transfected with the Flag-Beclin/ pbi-egfp plasmids using Lipofectamine Plus as described above. To facilitate clonal selection, cells were co-transfected with a ptk-hyg 43

50 vector (Clontech, Palo Alto, CA). Transfected clones resistant to 200 µg/ml hygromycin B (Clontech, Palo Alto, CA) were selected and tested for expression of EGFP by fluorescence microscopy and expression of Flag-Beclin by immunoblot analysis with mouse monoclonal anti-flag (Sigma, St. Louis, MO) in the presence or absence of 1 µg/ml doxycycline hydrochloride (Dox, Sigma, St. Louis, MO). In the presence of Dox (+Dox), transcription of Flag-Beclin or EGFP is not induced; in the absence of Dox (- Dox), transcription of Flag-Beclin or EGFP is induced. Positive clones were maintained continuously under +Dox conditions until experiments were initiated. To turn on the expression of Flag-Beclin, the cells were washed with Hanks balanced salt solution (HBSS) to remove residual Dox and incubated in the absence of Dox for 4 d. Subcellular Fractionation Cells were trypsinized, washed in HBSS, and allowed to swell for 10 min in hypotonic buffer (10 mm HEPES, ph 7.5, 1.5 mm MgCl 2, 10 mm KCl, 1 mm dithiothreitol and protease inhibitors). Cells were disrupted with 20 strokes of a Teflon homogenizer and sucrose was added to a final concentration of 0.25 M. The cell lysate then was centrifuged at 100,000 g for 1 h at 4 C, resulting in soluble (S100) and particulate (P100) fractions. An equal percentage by volume from each fraction was subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot analysis to detect Beclin and mvps34. To generate the cytosol and membrane fractions for immunoprecipitation of Beclin or Flag-Beclin complexes, cells were harvested, washed in HBSS and suspended in hypotonic buffer (50 mm Tris, ph 7.4, 50 mm NaCl) and allowed to swell for 10 min on ice. The cells then were lysed by 15 passages through a 27 gauge needle and 2 salt 44

51 solution was added to obtain final concentration of 50 mm Tris, ph 7.4, 150 mm NaCl, 1 mm EDTA and protease inhibitor cocktail (Roche Molecular Biochemicals, Indianapolis, IN). The cell lysate was centrifuged for 5 min at 500 g (4ºC) to remove unbroken cells and the supernatant was centrifuged for 1 h at 100, 000 g (4ºC) to obtain soluble (S100) and particulate (P100) fractions. The particulate fractions were extracted with 1% Triton X-100 in 50 mm Tris, ph 7.4, 150 mm NaCl, 1 mm EDTA and protease inhibitor cocktail and then centrifuged for 1 h at 100, 000 g (4ºC). The soluble fraction (S100) and membrane extract were subjected to immunoprecipitation as described below. Immunoprecipitation of Flag-Beclin Protein Complexes Using Anti-Flag M2 Affinity Gel Pull-down Assay Anti-Flag M2 affinity gel (hereafter referred to simply as mouse Flag-beads) (Sigma, St. Louis, MO) is a purified murine IgG1 monoclonal antibody covalently attached to agarose by hydrazide linkage. It is useful for purification or immunoprecipitation of Flag fusion proteins. HEK293 cells were purchased from American Type Culture Collection (Manassas, VA) and were maintained at 37 C in a 5% CO 2 / 95% air atmosphere in DMEM containing 10% FBS. To immunoprecipate Flag-Beclin from HEK293 cells expressing Flag-Beclin and Bcl-2 family proteins, HEK293 cells were seeded in 100-mm dishes in DMEM with 10% FBS on the day before transfection. Plasmids encoding Flag-Beclin and Bcl-2 family proteins then were co-transfected into those cells using Lipofectamine Plus reagent. Twenty-four hours after the transfection, the cells were washed three times with HBSS, scraped from the dish and homogenized in IP buffer: 50 mm Tris-HCl, ph 7.4, 150 mm NaCl, 1 mm EDTA, 1% Triton X-100, and protease inhibitor cocktail. The lysate was 45

52 centrifuged at 10,000 x g for 15 min at 4 C and the supernatant solution was incubated with mouse Flag-beads (pre-washed with 0.2 M glycine-ph 3.0 twice, and then washed three times with IP buffer) for 30 mins at 4 C. The beads then were collected by centrifugation for 20 sec at 10,000 g and the supernatants were removed by aspiration. The pellets were washed three times with IP buffer. After aspirating the final wash supernatants, bound proteins were eluted from the beads in 100 µl 0.2 M glycine-hcl (ph 3.0). The eluate was neutralized with 10 µl 2 M Tris-HCl (ph 8.0), and proteins were solubilized with 25 µl 5 SDS sample buffer. Aliquots of whole cell lysate (1/10) before addition of mouse Flag beads or immunoprecipitated proteins were subjected to SDS- PAGE and immunoblot analysis as described previously (Wilson et al., 1996). Primary antibodies used for immunoblot analysis included rabbit polyclonal anti-flag (Sigma), rabbit polyclonal against Bcl-2 and rabbit polyclonal against Bcl-X L/S (Santa Cruz Biotechnology, Santa Cruz, CA). Plasmids used to express Bcl-2 family proteins included Bcl-2/pUSE plasmid (Upstate Biotechnology, Charlottesville, VA), Bcl- X L /porf and Bcl-X S /porf (InvivoGen, San Diego, CA). To immunoprecipitate Flag-Beclin from MCF-7 Tet-off cells stably expressing Flag-Beclin, doxycycline was removed from the culture medium to turn on the expression of Flag-Beclin. Then the cells were harvested and fractionated into cytosol and membrane as described above. The membrane fraction was extracted with 1% Trition X-100 in 50 mm Tris, ph 7.4, 150 mm NaCl, 1 mm EDTA and protease inhibitor cocktail. The soluble fraction and membrane extract then were subjected to mouse Flag bead pull-down assay as described above. Aliquots of the soluble and membrane fractions (1/10) before addition of mouse Flag beads or immunoprecipitated proteins 46

53 were subjected to SDS-PAGE and immunoblot analysis to detect Flag-Beclin and endogenous Bcl-2. Immunoprecipitation of Endogenous Beclin Protein Complexes MCF-7 and U251 cells were obtained from the National Cancer Institute Frederick Cancer DCT Tumor Repository (Frederick, MD) and were maintained at 37 ºC in a 5% CO 2 / 95% air atmosphere in DMEM supplemented with 10% FBS. MCF-7 or U251 cells were grown to 80% confluence in 100-mm dishes in DMEM with 10% FBS. The cells were washed three times with HBSS, scraped from the dish and homogenized in IP buffer. The lysate was centrifuged at 10,000 x g for 15 min at 4 C and the supernatant solution was incubated with goat polyclonal IgG against Beclin (2 h at 4 C), followed by 1 h incubation with protein A sepharose beads. The beads were washed three times with IP buffer, twice with phosphate-buffered saline (PBS), and then the immune complexes were eluted from the beads and subjected to SDS-PAGE and immunoblot analysis. Primary antibodies used for immunoblot analysis included mouse monoclonal against Beclin (BD Biosciences, San Diego, CA), rabbit polyclonal against mvps34 (Zymed Laboratories, South San Francisco, CA), mouse monoclonal against Bcl-2, and rabbit polyclonal against Bcl-XL (Santa Cruz Biotechnology, Santa Cruz, CA). Size Exclusion Chromatography Washed cells pooled from six 100-mm cultures were suspended in 50 mm Tris- HCl (ph 7.4) and allowed to swell for 10 min on ice. The cells then were lysed by nine passages through a 27 gauge needle and 10 salt solution was added to obtain final concentrations of 50 mm NaCl, 50 mm NaF and 1 mm EDTA. The cell lysate was 47

54 centrifuged for 1 h at 100, 000 g (4 ºC) and the supernatant solution was subjected to FPLC size exclusion chromatography on a Superose-12 HR 10/30 column (Pharmacia, Picataway, NJ). The elution positions of the Beclin and mvps34 were determined by immunoblot analysis of the fractions, using a Kodak 440CF Image Station to quantify the signals. Marker proteins were: thyroglobulin (670 kda), apoferritin (443 kda), β-amylase (200 kda) and bovine serum albumin (66 kda) (Sigma, St. Louis, MO). sirna-mediated Silencing of Beclin To screen sirna constructs that efficiently knock down Beclin expression, I designed five sirna oligonucleotides. Those sirna oligonucleotides were subcloned into psilencer 1.0-U6 (Ambion, Austin, TX) and co-transfected with Flag-Beclin into 293T cells (American Type Culture Collection, Manassas, VA) by Lipofectamine Plus as describe above. The cells then were subjected to SDS-PAGE and immunoblot analysis with a mouse monoclonal antibody against Flag (Sigma) to determine the expression of Flag-Beclin. From those five sirna constructs, I obtained one sirna (20/21) which completely suppressed the expression of Flag-Beclin. This efficient sirna then was used to knock down endogenous Beclin. Initially I used the effective sirna (20/21) to knock down endogenous Beclin in 293T cells by sequential transfection. 293T cells were initially transfected with Beclin sirna in psilencer using Lipofectamine Plus. On the next day the cells were transfected a second time with the same sirna using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer s instructions. Twenty-four hours after the second transfection, the cells were harvested to check the expression of endogenous Beclin. 48

55 To obtain stable and better suppression of endogenous Beclin, I subcloned the effective Beclin sirna into psuper.retro.puro vector (OligoEngine, Seattle, WA), a retroviral sirna expression vector. The oligonucleotide sequence (20/21) used for successful sirna interference with Beclin expression corresponded to nucleotides ( 5 -GGCAAGAUUGAAGACACAG-3 ) downstream of the transcription start site of beclin (GeneBank accession number: AF077301), followed by a 9-nucleotide noncomplementary spacer (TATCTTGAC) and the reverse complement of the initial 19- nucleotide sequence. A control vector was constructed with a similar insert where the 19- nucleotide sequence had no homology to any known human gene sequence. Retrovirus was produced in 293 GPG packaging cells (Ory et al., 1996) maintained in DMEM + 10% heat-inactivated FBS with 1 µg/ml puromycin, 300 µg/ml G418, and 2 µg/ml doxycycline. For transfection, the 293 GPG cells were seeded at cells/dish on 100-mm dishes in DMEM containing 10% heat inactivated FBS. Twenty-four hours later 293 GPG cells were transfected with the psuper.retro.puro constructs using Lipofectamine-Plus reagent (Invitrogen, Carlsbad, CA). Forty-eight and 72 h after transfection, the virus-enriched medium was collected and passed through a 0.22 µm filter. Infections of the U251 cells were performed on two sequential days in the presence of 4.0 µg/ml hexadimethrine bromide (Sigma, St. Louis, MO). Twenty-four hours after the second infection the cells were trypsinized and re-plated in selection medium containing 1 µg/ml puromycin. After a selection period of 6 d, the surviving cells were pooled and used for studies described in the following sections. 49

56 Immunofluorescence Microscopy Organelle morphology was assessed in control and Beclin KD cells grown on laminin-coated glass coverslips for 24 h. For detection of EEA1, cells were fixed with 3% paraformaldehyde and permeabilized with 0.05% saponin in PBS. For LAMP1 or GM130, cells were fixed with ice cold methanol for 10 min. All cells were blocked with 10% goat serum in PBS for 30 min and the following monoclonal antibodies were applied for 1 h in PBS with 10% goat serum: anti-lamp1 (University of Iowa Developmental Studies Hybridoma Bank, Iowa City, IA), anti-gm130 and anti-eea1 (BD Biosciences, San Diego, CA). Cells then were washed three times with 10% goat serum in PBS and incubated for 1 h with Alexa Fluor-568 goat anti-mouse IgG (Molecular Probes, Eugene, OR). Photomicrographs were taken with a Nikon Eclipse 800 fluorescence microscope equipped with a digital camera. Images were acquired and processed using ImagePro software (Media Cybernetics, Silver Spring, MD). Induction of Macroautophagy Macroautophagy was induced by nutrient starvation or exposure to C2-ceramide (N-Acetyl Derythro-sphingosine; Calbiochem, La Jolla, CA). For starvation, cells were washed with HBSS three times and then incubated in HBSS for 4 h. For C2-ceramide treatment, cells were incubated with 10 µm or 20 µm C2-ceramide in DMEM+0.1% FBS for 24h. Ceramide was dissolved in dimethlysulfoxide (DMSO), and control cultures contained equal amounts of vehicle. The ratio of endogenous LC3 in the unmodified form (LC3-I) and the phosphatidylethanolamine-conjugated form (LC3-II) was determined by immunoblot analysis of whole-cell lysate, using a rabbit polyclonal antibody against LC3 (Kabeya et al., 2000) kindly provided by Dr. Tamotsu Yoshimori. 50

57 Detection and Quantification of Acidic Vesicular Organelles with Acridine Orange (AO) Vital staining of cells with acridine orange (Molecular Probes, Eugene, OR) was performed essentially as described (Paglin et al., 2001). Cells were grown on laminincoated coverslips (for fluorescence microscopy) or in 96-well plates (for quantification of red fluorescence) and treated with C2-ceramide or vehicle (DMSO) for the indicated time. Acridine orange was added for 15 min at a final concentration of 1 µg/ml, and the cells then were washed three times with PBS. Unfixed cells were examined immediately by fluorescence microscopy using a Nikon Eclipse 800 microscope with the red filter set (G-2E/C; excitation , emission ). Red fluorescence was quantified with a microplate fluorimeter (Molecular Devices, Gemini EM) with excitation and emission wavelengths set at 488 nm and 655 nm, respectively. To normalize the measurements to the number of cells present in each well, a solution of ethidium bromide was added to a final concentration of 0.2 µm and the fluorescence emitted from the DNA complexes was measured at 530 nm (excitation), 590 nm (emission). The AO red fluorescence was expressed as a ratio to the ethidium bromide (EB) fluorescence. Cathepsin D Processing Steady-state levels of intracellular cathepsin D were measured in whole-cell lysates by SDS-PAGE and immunoblot analysis, using goat anti-cathepsin D from Santa Cruz Biotechnology (Santa Cruz, CA). To measure the kinetics of cathepsin D processing, U251 cells were pulse-labeled for 30 min in methionine-free DMEM containing 10% FBS and 100 µci/ml [ 35 S]methionine (Easy Tag express labeling mix; 1175 Ci/mmol, Perkin Elmer, Boston, MA), then chased for 4 h in DMEM containing 51

58 10% FBS, 200 µm methionine and 200 µm cysteine. Cells were washed three times with PBS three times, harvested using a cell scraper, homogenized and solubilized in 50 mm Tris-HCl, ph 7.4, 150 mm NaCl, 1% Nonidet P40, 0.5% sodium deoxycholate, 0.1% SDS, 5 mm EDTA. Insoluble material was removed by centrifugation at 100,000 x g for 45 min at 4 C and the lysates were precleared with protein A Sepharose. Samples then were incubated for 2 h with a polyclonal antibody against cathepsin D (Biodesign International, Saco, Maine). Immune complexes then were collected on protein A sepharose and subjected to SDS-PAGE and fluorography as described previously (Wilson et al., 1996). Endocytosis of Horseradish Peroxidase (HRP) Cells grown to approximately 80% confluence were washed with DMEM and then incubated at 37 C with HRP in (2 mg/ml) in DMEM containing 1% BSA (bovine serum albumin) for the time periods indicated in the figure. Cells were placed on ice, washed three times with ice-cold PBS containing 1% BSA and one time with PBS. Cells then were scraped into PBS and collected by centrifugation at 390 g for 4 min at 4 C. Cell pellets were washed once with PBS and lysed in PBS containing 0.5% Triton X-100. Lysates were cleared by centrifugation for at 10,000 g for 10 min at 4 C, and equal aliquots were removed for peroxidase assay, using the One-Step Turbo TMB enzymelinked immunosorbent assay kit (Pierce Chemical, Rockford, IL). After addition of sulfuric acid stop solution, absorbance at 450 nm was measured and the enzyme activity was normalized to total protein, determined using a colorimetric assay (Bio-Rad, Hercules, CA). 52

59 Measurement of EGF Receptor Degradation Parallel cultures of control or Beclin KD cells were seeded at 200,000 cells/dish on laminin-coated cover slips in 60-mm dishes and grown for 48 h. The cells then were washed with PBS and maintained in serum-free DMEM overnight to allow the EGFR to accumulate on the cell surface. The EGFR internalization was stimulated by incubating the cells 200 ng/ml EGF (Upstate Biotechnology, Charlottesville, VA) in HBSS containing 20 mm HEPES and 0.2% BSA for 30 min or 70 min at room temperature. Immunofluorescence localization of the EGFR was performed using anti-egfr monoclonal antibody (Upstate Biotechnology), as described earlier for other organelle markers. In a separate study the cells were harvested in SDS sample buffer at 30 min or 70 min after stimulation with EGF, and aliquots containing equal amounts of total cell protein were subjected to SDS-PAGE and immunoblot analysis for EGFR. 53

60 RESULTS Beclin Interacts with Bcl-2 and Bcl-X L, but not Bcl-X S when the Proteins are Coexpressed Beclin has been proposed to interact with Bcl-2 and mvps34 in mammalian cells. Therefore, I first performed extensive studies to determine which protein is the physiological interacting partner for Beclin. Beclin has been reported to interact with Bcl-2 in a yeast two-hybrid system and FRET analysis also has shown that the two proteins, when co-expressed in COS-7 cells, are in close proximity (<50Å) (Liang et al., 1998). However, no one has demonstrated the interaction between Beclin and Bcl-2 using co-immunoprecipitation techniques. I conducted a series of such studies to observe the association of Beclin and Bcl-2 family proteins in transiently transfected HEK293 cells. As described earlier, Bcl-2 family proteins can be divided into three groups based on their structural homology and functions: anti-apoptotic proteins containing BH1 to BH4 domains such as Bcl-2 and Bcl- X L ; pro-apoptotic proteins containing BH1 to BH3 domains such as Bax and Bcl-X S ; BH3 only proteins such as Bad. Using mouse Flag-bead pull-down assay, I tested if Beclin interacts with Bcl-2 family proteins when they are co-expressed in HEK293 cells. Plasmids encoding Flag-Beclin and Bcl-2 family proteins were co-transfected into HEK293 cells and Flag-Beclin was immunoprecipitated from whole cell lysate using mouse Flag beads. Associated proteins were probed by immunoblot analysis with antibodies against Bcl-2 (Fig. 8), Bcl-X L (Fig. 9), Bcl-X S (Fig. 10). These results for the first time show that Flag-Beclin can associate with Bcl-2 and Bcl-X L but not Bcl-X S when these proteins are co-expressed in HEK293 cells. 54

61 Fig. 8. Bcl-2 Co-immunoprecipitates with Flag-Beclin when Both Proteins are Coexpressed in HEK293 cells Flag-Beclin Bcl Mouse Flag bead pull-down Cell lysate kda Flag-Beclin 45 kda 30 kda Bcl-2 Plasmids encoding Flag-Beclin and Bcl-2 were co-transfected into HEK293 cells by Lipofectamine Plus. Twenty four hours after the transfection, Flag-Beclin was immunoprecipitated with mouse Flag beads. Aliquots of whole cell lysate (1/10) (right panel) before addition of mouse Flag beads or immunoprecipitated proteins (left panel) were subjected to SDS-PAGE and immunoblot analysis with rabbit polyclonal anti-flag and rabbit polyclonal anti-bcl-2. 55

62 Fig. 9. Bcl-X L Co-immunoprecipitates with Flag-Beclin when Both Proteins are Coexpressed in HEK293 Cells Flag-Beclin Bcl-X L Mouse Flag bead pull-down Cell lysate kda 44 kda Flag-Beclin 30 kda Bcl-X L 20 kda Bcl-X L was transiently co-expressed with Flag-Beclin in HEK293 cells. Flag-Beclin complexes were collected from cell lysates using mouse Flag beads as described in Fig. 8 and the Methods. Aliquots of whole cell lysate (1/10) (right panel) before addition of mouse Flag beads or immunoprecipitated proteins (left panel) were subjected to SDS- PAGE and immunoblot analysis with rabbit polyclonal anti-flag and rabbit polyclonal anti-bcl-x L. 56

63 Fig. 10. Bcl-X S does not Co-immunoprecipitate with Flag-Beclin when Both Proteins are Co-expressed in HEK293 Cells Flag-Beclin Bcl-X S 66 kda 45 kda Mouse Flag bead pull-down Flag-Beclin Cell lysate kda Bcl-X S Bcl-X S was transiently co-expressed with Flag-Beclin in HEK293 cells. Flag-Beclin complexes were collected from cell lysates using mouse Flag beads as described in Fig. 8 and Methods. Aliquots of whole cell lysate (1/10) (right panel) before addition of mouse Flag beads or immunoprecipitated proteins (left panel) were subjected to SDS-PAGE and immunoblot analysis with rabbit polyclonal anti-flag and rabbit polyclonal anti-bcl-x S. 57

64 Stable Transfection of Flag-Beclin into MCF-7 Tet-off Cells The physical interaction of Flag-Beclin with overexpressed Bcl-2 in HEK293 cells prompted us to determine if Beclin interacts with endogenous Bcl-2. To achieve this goal, MCF-7 Tet-off cells were stably transfected with Flag-Beclin to obtain a cell line in which a higher percentage of cells express Flag-Beclin. Here MCF-7 cells were used because they express a very high level of endogenous Bcl-2. In the MCF-7 Tet-off system, Beclin expression is turned on when tetracycline (Tc) or doxycycline (Dox; a Tc derivative) is added. There are two plasmids involved in this system (Fig. 11A). One is the ptet-off regulatory plasmid which encodes a fusion protein TetR/VP16, a tetracycline-controlled transcriptional activator (tta). The other is the response plasmid which expresses Beclin under control of the tetracycline-response element (TRE), located just upstream of the minimal CMV promoter (P mincmv ). P mincmv lacks the strong enhancer elements normally associated with the CMV immediate early promoter. In the presence of doxycycline, tta cannot bind the TRE on the response plasmid and the transcription of Beclin is inactivated. After doxycycline removal, tta will be able to bind the TRE and turn on transcription. Flag-Beclin was subcloned into pbi-egfp response plasmid, in which Beclin and enhanced green fluorescence protein (EGFP) are both expressed from a bi-directional tetracycline-responsive promoter. The EGFP is useful for screening positive colonies. Flag-Beclin in pbi-egfp and ptk-hyg were co-transfected into MCF-7 Tet-off cells, and stable clones were selected by growth with hygromycin. After removal of doxcycline from the culture medium, hygromycin resistant colonies were screened by examination under fluorescence microscopy to check green fluorescence and by immunoblot analysis with anti-flag. Based on these two screening 58

65 Fig. 11. Selection of Stable MCF-7 Cells with Inducible Expression of Flag-Beclin A Transcription activator PCMV tetr VP16 ptet-off regulatory plasmid TetR VP16 +Tc (or Dox ) -Tc ( or Dox) tta TetR VP16 Transcription TRE PminCMV Flag-beclin pbi-egfp response plasmid B Beclin clone 6 Doxycycline - Beclin clone kda 66 kda Flag-Beclin 45 kda (A) Summary of the MCF-7 Tet-off system. tta binds to TRE in the silent promoter and activates transcription in the absence of Dox. (B) Expression of Flag-Beclin in two stable Tet-off MCF-7 clones. The stable MCF-7 cell lines were maintained in 100 ng/ml doxycycline. Four days after doxycycline removal, the cells were harvested to check Flag-Beclin expression. (+), maintained with doxcycline; (-), doxcycline removed for 4 days 59

66 methods, two clones (clone 6 and 16) (Fig. 11B) were obtained in which 100% of the cells express EGFP, indicating that they are homogenous clones. Overexpressed Flag-Beclin does not Associate with Endogenous Bcl-2 in MCF-7 Cells The availability of cell lines where Flag-Beclin could be induced in the entire cell population allowed us to examine the potential association of Flag-Beclin with endogenous proteins. The expression of Flag-Beclin was induced by removal of Dox in clone 16 and cells were fractionated into the cytosol and membrane fractions. Flag-Beclin then was immunoprecipitated either from the cytosol fraction or from a 1% Triton X-100 solubilized membrane fraction. Immunoprecipitated proteins eluted from Flag beads were analyzed by immunoblot analysis with anti-bcl-2. Most of Flag-Beclin was found in the cytosol fraction while endogenous Bcl-2 was detected exclusively in the particulate fraction. Furthermore, no endogenous Bcl-2 from the membrane fraction was coprecipitated with Flag-Beclin (Fig. 12). Endogenous Beclin does not Form a Complex with Endogenous Bcl-2 or Bcl-X L In case the Flag tag may change the physiological conformation of Flag-Beclin and interfere with the interaction of Flag-Beclin and endogenous Bcl-2. Endogenous Beclin was immunoprecipitated from MCF-7 whole cell lysate and the associated proteins were probed by immunoblot analysis with antibodies against Bcl-2. Consistent with the immunoprecipitation results from MCF-7 cells expressing Flag-Beclin, 60

67 Fig. 12. Endogenous Bcl-2 does not Co-immunoprecipitate with Flag-Beclin in MCF-7 Cells Mouse Flag bead Pull-down Cell lysate S100 P100 S100 P kda Flag-Beclin 50 kda 36 kda IgG light chain Bcl-2 22 kda Four days after Dox removal from the culture medium, MCF-7 Tet-off cells expressing Flag-Beclin were harvested and fractionated into soluble (S100) and particulate (P100) fractions. The particulate fraction was solubilized with 1% Triton X % of each fraction was saved for immunoblot analysis. The remainder of each fraction was used for mouse Flag bead pull-down assay. Immunoprecipitated proteins were subjected to SDS- PAGE and immunoblot analysis with rabbit polyclonal anti-flag and rabbit polyclonal anti-bcl-2 to detect endogenous Bcl-2. 61

68 no endogenous Bcl-2 was found to be co-precipitated with endogenous Beclin in MCF-7 cells (Fig. 13). Similarly, no endogenous Bcl-2 was detected to be associated with endogenous Beclin in NIH3T3 cells (Fig. 14) and in U251 glioblastoma cells (Fig. 15). Endogenous Bcl-X L also failed to co-precipitate with endogenous Beclin in the glioblastoma cells (Fig. 15). These results suggest that Bcl-2 and Bcl-X L are not the primary targets for Beclin in mammalian cells, except under artificial conditions where Beclin and Bcl-2 or Bcl-X L are overexpressed. Beclin Forms a Complex with mvps34 in Mammalian Cells In addition to its potential interaction with Bcl-2, Beclin has been reported to associate with the mvps34 PtdIns 3-kinase in Hela cells (Kihara et al., 2001b). To determine if mvps34 is an interacting partner for Beclin in MCF-7, I performed coimmunoprecipitation studies similar to those described for Bcl-2. The expression of Flag- Beclin was induced by removal of Dox in the MCF-7 Tet-off cells (clone 16) and cells were fractionated into the cytosol and membrane fractions. Flag-Beclin then was immunoprecipitated either from the cytosol fraction or from a 1% Triton X-100 solubilized membrane fraction. Immunoprecipitated proteins eluted from Flag beads were analyzed by immunoblot analysis with anti-mvps34. Unlike the situation with endogenous Bcl-2, endogenous mvps34 was found to associate with Flag-Beclin in MCF-7 cells (Fig. 16). To further explore if mvps34 interacts with endogenous Beclin, the latter was immunoprecipitated from whole-cell lysate and the associated proteins were probed by immunoblot analysis with a rabbit polyclonal antibody against mvps34. In contrast to 62

69 Fig. 13. Endogenous Bcl-2 does not Co-immunoprecipitate with Endogenous Beclin in MCF-7 Cells Anti- Beclin IP Control IgG Cell lysate 64 kda Beclin 50 kda 36 kda Bcl-2 22 kda Cells lysates were prepared from parallel cultures as described in the Methods and 10% of each lysate was saved for immunoblot analysis. The remainder of each lysate was immunoprecipitated with either IgG against Beclin or a control IgG against an unrelated protein (SPARC, Santa Cruz Biotechnology). Equal aliquots of the immune complexes eluted from protein A sepharose beads were probed by immunoblot analysis using the antibodies against Beclin and Bcl-2. 63

70 Fig. 14. Endogenous Bcl-2 does not Co-immunoprecipitate with Endogenous Beclin in NIH3T3 Cells IP Anti-Beclin Control IgG Cell lysate 66 kda Beclin 45 kda 30 kda Bcl-2 20 kda Immunoprecipitation and immunoblot analysis was performed as described in Methods and Fig

71 Fig. 15. Endogenous Bcl-2 or Bcl-X L does not Co-immunoprecipitate with Endogenous Beclin in U251 Cells Anti- Beclin IP Control IgG Cell lysate 64 kda Beclin 50 kda 36 kda Bcl-2 22 kda 36 kda Bcl-x L 22 kda Immunoprecipitation and immunoblot analysis was performed as described in Methods and Fig. 13. Equal aliquots of the immune complexes eluted from protein A sepharose beads were probed by immunoblot analysis using the antibodies against Beclin, Bcl-2 and Bcl-X L. 65

72 Fig. 16. Endogenous mvps34 Co-immunoprecipitated with Flag-Beclin from both Cytosol and Membrane Fractions in MCF-7 Cells Mouse Flag bead Pull-down Cell lysate S100 P100 S100 P kda Flag-Beclin 50 kda 98 kda mvps34 Four days after doxycycline removal to turn on the expression of Flag-Beclin, MCF-7 Tet-off cells (clone 16) were fractionated into soluble and particulate fractions as described in Methods. The particulate fractions were solubilized with 1% TX-100. Both soluble fraction and membrane extract were subjected to mouse Flag-bead pull-down assay. Immunoprecipitated proteins were analyzed by SDS-PAGE and immunoblotting to detect Flag-Beclin and mvps34. 66

73 endogenous Bcl-2, endogenous mvps34 was co-precipitated with endogenous Beclin in MCF-7 (Fig. 17) or U251 cells (Fig. 18). Beclin and mvps34 exhibited similar subcellular distributions when U251 cells were fractionated into soluble and particulate components. Approximately 60% of the total Beclin and mvps34 was found in the soluble fraction (Fig. 19A). Furthermore, mvps34 was found to associate with Beclin in both soluble and particulate fractions in U251 cells (Fig. 19B). The above results clearly demonstrate that mvps34 rather than Bcl-2 is the primary target for endogenous Beclin in mammalian cells. sirna-mediated Suppression of Beclin in Mammalian Cells Since mvps34 is the major target for Beclin as determined above, next I started to test my second hypothesis that this interaction specifically affects the physiological function of mvps34 in macroautophagy. To achieve this goal, sirna-mediated suppression of Beclin expression was performed. Small interfering RNAs (sirnas) are nucleotides double-stranded RNA molecules that can target complementary mrnas sequences for degradation via a cellular process termed RNA interference (Elbashir, 2001). To specifically suppress Beclin expression in mammalian cells, five Beclin sirna target sites (Table I) that spanned the full length of Beclin mrna were selected. Two complementary oligonucleotides for each sirna were synthesized and annealed. The oligonucleotides encode a hairpin structure with a 19-nucleotide stem derived from the Beclin mrna target sites. Initially, I subcloned the Beclin sirna inserts into the sirna expression 67

74 Fig. 17. Endogenous mvps34 Co-immunoprecipitated with Endogenous Beclin in MCF-7 Cells Anti- Beclin IP Control IgG Cell lysate 64 kda Beclin 50 kda 97 kda mvps34 Immunoprecipitation was performed as described in the Methods and Fig. 13. The same samples used for Bcl-2 analysis (Fig. 13) also were checked for presence of mvps34. Equal aliquots of the immune complexes eluted from protein A sepharose beads were probed by immunoblot analysis using the antibodies against Beclin and mvps34. 68

75 Fig. 18. Endogenous mvps34 Co-immunoprecipitated with Endogenous Beclin in U251 Cells IP Anti- Beclin Control IgG Cell lysate 64 kda Beclin 50 kda 148 kda 97 kda mvps34 Immunoprecipitation was performed as described in Methods and Fig. 13. The same samples used for Bcl-2 analysis (Fig. 15) also were checked for presence of mvps34. Equal aliquots of the immune complexes eluted from protein A sepharose beads were probed by immunoblot analysis using the antibodies against Beclin and mvps34. The band appearing just below Beclin in the upper panel is a non-specific cross-reacting protein. 69

76 Fig. 19. Endogenous Beclin Associates with Endogenous mvps34 in both Cytosol and Membrane Fractions in U251 Cells A S100 P100 a 97 kda mvps34 64 kda a Beclin B S100 IP P100 IP 64 kda S100 Cell lysate Anti- Beclin Control IgG Cell lysate P100 Anti- Beclin Control IgG Beclin 50 kda IgG heavy chain 97 kda mvps34 (A) Subcellular distribution of mvps34 and Beclin in U251. Cells were fractionated as described in Methods. Equal percentages of the soluble and particulate fractions (13% by volume) were subjected to SDS-PAGE and immunoblot analysis to determine the distribution of endogenous Beclin and mvps34 between soluble (S100) and particulate (P100) fractions. The percent of each protein in the soluble fraction was 59% for Beclin and 64% for mvps34. (B) Endogenous mvps34 co-immunoprecipitates with endogenous Beclin from both cytosol and membrane fractions in U251 cells. Cells were fractionated and the resulting soluble fraction and membrane extracts were used for immunoprecipitation of Beclin as described in Methods. 70

77 Table. I. Five Beclin sirna Target Sequences Beclin sirna constructs Target sequence in beclin mrna (downstream of the transcription start site) 12/13 5 -(756) CCAGATGCGTTATGCCCAG (774)-3 14/15 5 -(1041) GGAGCTGCCGTTATACTGT (1059)-3 16/17 5 -(277) GTGCCAACAGCTTCACTCT (295)-3 18/19 5 -(555) GGAGCTGGCACTAGAGGAG (573)-3 20/21 5 -(1201) GGCAAGATTGAAGACACAG (1219)-3 Five sirna target sites were chosen by scanning beclin mrna sequence for AA dinucleotides, recording the 19 nucleotides immediately downstream of the AA, and then comparing the sirna target sequences with National Center for Biotechnology Information (NCBI) GenBank database to eliminate any sequences with significant homology to other genes. 71

78 vector psilencer 1.0-U6 (Fig. 20A). To screen sirnas which efficiently suppress Beclin expression, those sirna constructs were co-transfected with Flag-Beclin into 293T cells. Suppression of Flag-Beclin expression was analyzed by SDS-PAGE and immunoblot analysis with anti-flag monoclonal antibody. I obtained one Beclin sirna construct (20/21) which completely suppressed the expression of Flag-Beclin (Fig. 20B). This efficient Beclin sirna construct was used to knock down endogenous Beclin in 293T cells by sequential transfection. In this sequential transfection procedure, 293T cells were initially transfected with Beclin sirna in psilencer using Lipofectamine Plus. On the next day the cells were transfected a second time with the same sirna using Lipofectamine Through this sequential transfection, I reduced the expression of endogenous Beclin by 75% (Fig. 21); however, this reduction in Beclin expression is transient. To stably suppress Beclin expression, I subcloned the efficient Beclin sirna (20/21) into psuper.retro.puro (Fig. 22A), a retroviral sirna expression vector that drives the expression of RNAi sequence and confers puromycin resistance on infected cells. This RNAi system has been used to obtain persistent suppression of gene expression, allowing the analysis of loss-of-function phenotypes that develop over longer periods of time (Brummelkamp et al., 2002). Augmentation of Beclin expression in MCF-7 cells promotes macroautophagy in response to nutrient deprivation and inhibits tumorigenesis (Liang et al., 1999). Therefore, to further explore the physiological role of Beclin in MCF-7 cells, I first attempted to knock down endogenous Beclin in this cell line using the psuper RNAi system. As shown in Fig. 22B, Beclin expression was reduced by 75% in puromycin resistant cells that received the Beclin knockdown 72

79 Fig. 20. Screening sirna Constructs that Efficiently Suppress Beclin Expression A B 97 kda 14/15 16/17 18/19 20/21 * Empty vector 64 kda Flag- Beclin 50 kda (A) sirna expression plasmid psilencer 1.0-U6. This vector features a mouse U6 RNA polymerase III promoter. It can only be used for transient transfection. (B) Suppression of Flag-Beclin expression. Flag-Beclin and Beclin sirna constructs were co-transfected into 293T cells by lipofectamine plus. 24 h after the transfection, the cells were harvested and equal amounts of protein from each sample were subjected to SDS-PAGE and immunoblot analysis with a mouse monoclonal antibody against Flag epitope. * indicates the Beclin sirna construct that completely suppressed the expression of Flag-Beclin. 73

80 Fig. 21. sirna-mediated Suppression of Endogenous Beclin Expression in 293T by Sequential Transfection Control Beclin KD 66 kda Beclin 45 kda To obtain significant suppression of Beclin expression in 293T cells, cells were tranfected with Beclin sirna construct (20/21) first by Lipofectamine Plus and then by Lipofectamine Twenty four hours after the second transfection, the cells were harvested. Equal amounts of protein from control and Beclin knockdown (KD) cells were subjected to SDS-PAGE and immunoblot analysis with a monoclonal anti-beclin IgG as described in Methods. The band appearing just below Beclin is a non-specific crossreacting protein. 74

81 Fig. 22. sirna-mediated Suppression of Beclin Expression in MCF-7 Breast Carcinoma and U251 Glioma Cells by Retroviral Infection B A Target sequence sense strand Predicted transcript against Beclin U C A U GGCAAGATTGAAGACACAG A UU CCGTTCTAACTTCTGTGTC A transcription GGCAAGATTGAAGACACAG TTCAAGAGA CTGTGTCTTCAATCTTGCC TTTTT G A G Target sequence anti-sense strand Bgl II Hind III H1-RNA promoter LTR Puro LTR Ampicillin 64 k Control psuper.retro.puro Beclin KD Beclin C 66 kda Control Beclin KD Beclin 45 kda (A) psuper.retro.puro is a retroviral sirna expression vector containing a puromycin resistance gene. It uses the polymerase-iii H1-RNA gene promoter and has a welldefined start of transcription and a termination signal consisting of five thymidines in a row. Beclin sirna insert (20/21) was subcloned into this vector at Bgl II and Hind III. MCF-7 (B) or U251 (C) cells infected with retroviral vectors and the cells surviving after 6 days in medium containing 1 µg/ml puromycin were pooled to generate stable cell lines. Equal amounts of protein from control and Beclin KD cells were subjected to SDS- PAGE and immunoblot analysis with a monoclonal anti-beclin IgG as described in Methods. Beclin expression was reduced by 75% in MCF-7 cells and by 90-95% in U251 cells. 75

82 vector, compared with cells that were infected with the control vector. In order to obtain better reduction of Beclin expression, we tested a few other cell lines infected with a GFP reporter construct and found that the human U251 glioma cell line showed the highest initial infection efficiency. Thus, I also used the psuper RNAi system to reduce Beclin expression in U251 cells. As shown by the immunoblots in (Fig. 22C), expression of Beclin was almost undetectable in Beclin knockdown cells, compared with cells that were infected with the control vector. Expression of unrelated proteins such as lactate dehydrogenase and LAMP1 was not reduced (data not shown), indicating that the loss of Beclin expression was not due to a general effect of the sirna on protein synthesis in the KD cells. Suppression of Beclin Expression Causes a Reduction in the Size of the Cytosolic mvps34 Complex Before the cells with stable suppression of Beclin expression were used to test if the interaction of Beclin with mvps34 affects the physiological functions of mvps34 in protein trafficking, I first determined if the mvps34 PtdIns 3-kinase complex was affected in the Beclin KD cells. When the soluble fractions from control MCF-7 cells were subjected to size exclusion chromatography, Beclin and mvps34 eluted together in a broad peak in the range of kda (Fig. 23A). Because Beclin expression was only reduced by 75% in MCF-7 cells, there were two peaks for cytosolic mvps34 elution profile from Beclin KD MCF-7 cells (Fig. 23B). One peak suggestive of a kda complex overlaps with the mvps34 elution peak in the control MCF-7 cells, indicating 76

83 66 K 670 K 66 K Fig. 23. Suppression of Beclin Expression Causes a Reduction in the Size of the Cytosolic mvps34 Complex in MCF-7 Cells A Arbitrary Units kda 500 K 200 K Beclin mvps Fraction Number kda 670 K 500 K 200 K B mvps34 (Arbitrary Units) mvps34 in Control cells mvps34 in Beclin KD cells Fraction Number (A) Endogenous Beclin co-elutes with mvps34 in the soluble fraction from control MCF- 7 cells. Control MCF-7 cells were fractionated into soluble and particulate components as described in Methods. The soluble fraction then was subjected to FPLC size exclusion chromatography on a Superose 6 column. The elution positions of the Beclin and mvps34 were determined by immunoblot analysis of the fractions, using a Kodak 440CF Image Station to quantify the signals. (B) Superose-6 elution profile for mvps34 in the soluble fraction from Beclin KD MCF-7 cells, overlaid with the mvps34 profile from control cells. 77

84 that it represents the mvps34 complex with Beclin. The size of the other peak was reduced to kda, suggesting that it represents the mvps34 complex without Beclin. Since I could not obtain better reduction of Beclin expression in MCF-7 cells, I extended this study in U251 cells in which I could suppress the Beclin expression by 90-95%. Consistent with the observation in MCF-7 cells, cytosolic mvps34 co-elutes with Beclin in a peak of kda complex in control cells (Fig. 24A). The molecular mass of the cytosolic mvps34 complex was reduced to approximately kda (Fig. 24B) when Beclin expression was ablated in the KD cells. These results suggest that suppression of Beclin expression interferes with the mvps34 PtdIns 3-kinase complex. In agreement with earlier immunoprecipitation results, these findings also indicate that Beclin is a component of the mvps34 PtdIns 3-kinase complex in MCF-7 and U251 cells. Suppression of Beclin Expression Impairs Macroautophagy Induced by Various Stimuli Next I used the U251 cells with stable suppression of Beclin expression to determine if Beclin interaction with mvps34 is required for the functions of mvps34 in macroautophagy, lysosomal enzyme sorting and endocytic protein trafficking. To assess the consequences of Beclin knockdown for the induction and progression of macroautohagy in U251 cells, I subjected the control and Beclin KD cells to two established pro-autophagic stimuli: nutrient deprivation (Klionsky et al., 2000) or treatment with C2-ceramide (Daido et al., 2004; Scarlatti et al., 2004). MAP-LC3 processing was used as a marker to monitor autophagosome biogenesis. LC3 is the mammalian homologue of the yeast macroautophagy protein, Atg8. As described earlier, 78

85 66 K 66 K Fig. 24. Suppression of Beclin Expression Causes a Reduction in the Size of the Cytosolic mvps34 Complex in U251 Cells A Arbitrary Units Arbitrary Units kda 670 K 500 K 443 K Fraction Number 200 K mvps34 Beclin B Vps34 (Arbitrary Units mvps34 (Arbitrary Units) Units) kda mvps34 in Control cells 670 K 500 K 443 K Fraction Number 200 K mvps34 in Beclin KD cells (A) Superose-12 elution profile for Beclin and mvps34 in the soluble fraction from control U251 cells. (B) Superose-12 elution profile for mvps34 in the soluble fraction from Beclin KD U251 cells, overlaid with the mvps34 profile from control cells. 79

86 immediately after synthesis, the C-terminal region of LC3 is cleaved (Kabeya et al., 2000) and becomes LC3-I. The latter resides in the cytosol. After macroautophagy is induced, LC3-I is conjugated to phosphatidylethanolamine (PE) and becomes LC3-II. The latter associates with the isolation membrane throughout the course of the membrane elongation and remains on the autophagosome membrane after the completion of autophagosome formation (Kabeya et al., 2000). Immunoblot analysis of whole-cell lysate against LC3 usually reveals two bands: LC3-I (16 kda) and LC3-II (14 kda). It is well established that the processing of LC3-I to LC3-II is closely correlated with the numbers of autophagosomes, and the determination of the ratio of LC3-II to LC3-I is one of the most reliable methods to quantify macroautophagic activity of mammalian cells (Mizushima, 2004). As measured by this LC3 processing assay, reduction of Beclin expression dramatically inhibits macroautophagy in U251 cells. Nutrient deprivation induced a three-fold increase in LC3-II/LC3-I in the control cells, but only a slight induction was observed in the Beclin KD U251 cells (Fig. 25). C2-ceramide treatment caused an even greater (seven-fold) increase in LC3-II/LC3-I in the control cells, with a markedly attenuated response again seen in the Beclin KD U251 cells (Fig. 26). The maturation of autophagosomes to autolysosomes is accompanied by a loss of LC3-II and an increase in the acidity of the lumen (Mizushima, 2004). Therefore, to measure the relative number of autolysosomes in control versus Beclin KD U251 cells, I used an assay that measures supravital staining of acidic compartments with the lysosomotropic agent, acridine orange (AO). Acridine orange is a weak base that moves freely across biological membrane when uncharged. Its protonated form becomes trapped 80

87 Fig. 25. Suppression of Beclin Expression Impairs the Autophagy-associated Posttranslational Processing of Endogenous LC3-I to LC3-II Induced by Nutrient Deprivation Control Beclin KD unstarved HBSS unstarved HBSS 2222k kda 1616k kda LC3-I LC3-II Ratio of LC3-II to LC3-I unstarved HBSS unstarved HBSS Control Beclin KD Cells were incubated in DMEM+ 10% FBS (unstarved) or HBSS (starved) for 4 h. The cells then were subjected to SDS-PAGE and immunoblot analysis to detect the endogenous cytosolic LC3-I and the PE-conjugated LC3-II. The ratios of LC3-II to LC3- I represented in the bar graphs were determined from scans of the blots performed with a Kodak 440CF Image Station. 81

88 Fig. 26. Suppression of Beclin Expression Impairs the Autophagy-associated Posttranslational Processing of Endogenous LC3-I to LC3-II Induced by C2- ceramide p Control Beclin KD C2-ceramide kda 22k 16 kda 16k LC3-I LC3-II Ratio of LC3-II to LC3-I untreated C2-Cer Control untreated C2-Cer Beclin KD Cells were treated with vehicle (DMSO) or 10 µm C2-ceramide (+ ceramide) for 24 h. The cells were subjected to SDS-PAGE and immunoblot analysis to detect the endogenous cytosolic LC3-I and the PE-conjugated LC3-II. The ratios of LC3-II to LC3- I represented in the bar graphs were determined from scans of the blots performed with a Kodak 440CF Image Station. 82

89 and accumulates in acidic compartments, where it forms aggregates that fluoresce bright red (Arvan et al., 1984; Mains et al., 1988). The intensity of the red fluorescence is correlated with the degree of acidity or the volume of the cellular acidic compartments. A substantial increase in AO-positive acidic vesicular organelles (AVOs) has been observed in conjunction with the induction of macroautophagy in malignant glioma cells (Kanzawa et al., 2003; Daido et al., 2004; Takeuchi et al., 2004). Consistent with the LC3 processing results, a general increase in the intensity of AVOs could be observed in C2- ceramide treated control cells, but not in the parallel C2-ceramide treated Beclin KD cells (Fig. 27A). To measure the C2-ceramide induced increase in the amount of AO sequestered in acidic compartments. Cells were stained with AO and the red fluorescence emitted from AO was quantified by a fluorometer and normalized to DNA (ethidium bromide fluorescence). In line with the results observed under fluorescence microscopy, Beclin knockdown in U251 cells dramatically reduced C2-ceramide induced increase in the amount of AO sequestered into acidic compartments, compared with that in control cells (Fig. 27B). Suppression of Beclin Expression does not Block Protein Trafficking from the TGN to the Lysosomes The transport pathway of hydrolase to the vacuole/lysosome are similar in both yeast and mammals. In yeast, Atg6/Vps30, the homologue of Beclin, functions as a subunit of a Vps34 PtdIns 3-kinase complex in sorting soluble proteinases such as carboxypeptidase Y (CPY), to the vacuole (Kametaka et al., 1998). As reviewed earlier, treatment of mammalian cells with the PtdIns 3-kinase inhibitor, wortmannin, results in a 83

90 Fig. 27. Accumulation of Acidic Vesicular Organelles (AVOs) Induced by Ceramide Treatment is Impaired in Beclin KD Cells A Untreated Control Beclin KD + C2-ceramide Control Beclin KD B Fluorescence (AO/EB) C2-Ceramide (µm) (A) Control and Beclin KD cells were maintained for 24 h in medium with or without 10 µm C2-ceramide, as indicated. Cells then were incubated with acridine orange (AO) and examined by fluorescence microscopy, using a filter combination to detect red fluorescence. All digital micrographs were taken at the same exposure setting. The scale bar represents 50 µm. (B) Control and Beclin KD cells were treated with 0, 10 or 20 µm C2-ceramide for 24 h and incubated with AO. The relative amount of AO trapped in vesicular compartments (red fluorescence; excitation at 488 nm, emission at 655nm) was measured and normalized to cellular DNA detected with ethidium bromide (EB). The data represent the mean ± S.E. of three determinations from parallel cultures. 84

91 block in trafficking of procathepsin D from the TGN to the late endosomes and lysosomes (Brown et al., 1995; Davidson et al., 1995). Furthermore, overexpression of a kinase-deficient mvps34 dominant-negative inhibits the cathepsin D maturation (Row et al., 2001). Based on the above information, it is important to determine if Beclin interaction with mvps34 may be required for the ability of mvps34 to function in targeting of lysosomal enzymes to the lysosomes. To address this issue, I used cathepsin D maturation as a marker for this pathway. Three major forms of cathepsin D can be distinguished during maturation of this lysosomal hydrolase. Cathepsin D is synthesized as a precursor of 53 kda (TGN form). After transport from TGN to the late endosomes, it is processed to an intermediate of 47 kda (late endosome form). This protein matures to a heterodimeric form consisting of a 31 kda heavy and a 14 kda light chain in the lysosomes (lysosome form) (Gieselmann et al., 1983). A fraction of the 53 kda precursor form is secreted into the medium of cultured cells (Hasilik and Neufeld, 1980). I used these three different forms of cathepsin D as hallmarks of the lysosomal enzyme sorting pathway to determine if Beclin is essential for the transport from TGN to the late endosomes and lysosomes. As shown by the immunoblots in Fig. 28A. The steady-state levels of the 53 kda procathepsin D, the 47 kda intermediate and 31 kda mature cathepsin D were similar in the control and Beclin KD U251 cells. However, when control cells were treated with ammonium chloride, as a positive control, to raise the ph of the endosomal and lysosomal compartments, a substantial decrease in the level of mature cathepsin D and a great increase in the levels of precursor and intermediate cathepsin D were observed. 85

92 Fig. 28. Suppression of Beclin Expression does not Impede Lysosomal Enzyme Sorting as Measured by Cathepsin D Processing in U251 Cells A 50 kda p Control Beclin KD NH 4 Cl pro intermediate 36 kda mature B Cell medium Cell medium Cell medium 50 kda pro intermediate 36 kda mature Chase time 0 h 4 h Control 4 h 0 h 4 h 4 h 4 h 4 h Beclin KD NH 4 Cl (A) Immunoblot analysis was performed on endogenous cathepsin D in whole cell lysates from control and Beclin KD cells. To demonstrate inhibition of cathepsin D processing, a separate control culture was incubated with 10 mm NH 4 Cl for 24 h prior to harvest. Equal amounts of protein were loaded in each lane. The part of the blot above the dashed line was exposed seven times longer than the lower portion, to permit detection of the precursor forms of cathepsin D. (B) Cells were labeled with 100 µci/ml 35 S-methionine, then harvested immediately or chased in medium with unlabeled methionine for 4 h. A separate control culture was incubated with 15 mm NH 4 Cl during the 4-h chase. Cathepsin D was immunoprecipitated from both cell lysates and culture medium and subjected to SDS-PAGE and fluorography. The forms of cathepsin D are labeled as follows: pro, proenzyme; intermediate, endosomal intermediate; mature, mature lysosomal enzyme. 86

93 To obtain a more direct assessment of the processing of newly synthesized procathepsin D, pulse-chase and immunoprecipitation of cathepsin D was carried out. Control and Beclin KD U251 cells were metabolically labeled with 35 S-methionine for 30 min, chased for 4h, then harvested and lysed in RIPA buffer. A rabbit polyclonal cathepsin D antibody which recognizes all three forms of cathepsin D was used for immunoprecipitation. Consistent with the above cathepsin D immunoblot results, all of the radiolabeled protein was in the 53 kda precursor form in both control and Beclin KD U251 cells when 35 S-methionine labeled cathepsin D was immunoprecipitated immediately after 30 min pulse. After a 4 h chase, the mature 31 kda cathepsin D was the predominant form detected in both control and Beclin KD cells, with no residual 53 kda procathepsin D and only a small amount of the 47 kda intermediate. In contrast, control cells treated with ammonium chloride, which serves as a positive control, produced no mature cathepsin D during the 4 h chase. Immunoprecipitation of 35 S-methionine-labeled cathepsin D from the culture medium showed a similar amount of secreted procathepsin D in control and Beclin KD U251 cells. By comparison, a large amount of newly synthesized procathepsin D was secreted into the culture medium in control cells treated with ammonium chloride during the 4 h chase (Fig. 28B). These results clearly demonstrate that Beclin is not required for mvps34 PtdIns 3-kinase dependent lysosomal enzyme sorting in mammalian cells. 87

94 Suppression of Beclin Expression does not Interfere with Endocytic Trafficking Beclin KD U251 cells have normal staining pattern for early endosomes, lysosomes and Golgi membranes Two major hypotheses have been proposed for the organization of the endocytic pathway. The maturation model suggests that the endocytic organelles form de novo by the involution of plasma membrane-derived coated vesicles, which gradually acquire new properties during their intracellular transport (Murphy, 1991). The vesicle shuttle model, on the other hand, suggests that transport between pre-existing endocytic organelles is mediated by intermediate transport vesicles (Dell Angelica et al., 2000). However, both models deal with the same major stations along the pathway: the early endosomes containing specific marker proteins EEA1 (Mu et al., 1995) and Rab5 (Gorvel et al., 1991), the late endosomes and the lysosomes. The latter two stations share the established marker protein lysosome associated membrane protein (LAMP1). It has been shown that treatment of cultured cells with the PtdIns 3-kinase inhibitor, wortmannin, results in marked swelling and vacuolation of late endosome compartments (Reaves et al., 1996; Brown et al., 1995). Similar vacuolation of late endosome compartments has been observed in cells microinjected with an antibody against mvps34 (Tuma et al., 2001) and in our mvps34 knockdown U251 cells (unpublished). Although Beclin associates with mvps34, suppression of Beclin expression had no detectable effect on the immunofluorescence distribution of established marker proteins for early endosomes (EEA1), late endosomes/lysosomes (LAMP1) or Golgi membranes (GM130) (Fig. 29). 88

95 Fig. 29. Morphology of Endosomes, Lysosomes and Golgi Membranes is Similar in Beclin KD Cells Compared with Controls Control Beclin KD EEA1 (Early endosomes) LAMP 1 (Late endosomes and lysosomes) GM130 (Golgi membranes) Control or Beclin KD cells were seeded in parallel dishes at the same density and examined by immunofluorescence microscopy after 2 d, using the primary antibodies indicated at the left of the figure. The scale bar represents 20 µm. 89

96 Suppression of Beclin Expression does not Block Endocytosis of a Fluid-phase Marker, Horseradish Peroxidase (HRP) Horseradish Peroxidase is a marker for fluid-phase endocytosis. Horseradish Peroxidase is constitutively internalized via clathrin-coated and non clathrin-coated invagination of the plasma membrane, reaching endosomes within approximately 5 min (Casey et al., 1986; Cupers et al., 1994). From this compartment, about 20-40% is returned to the medium via endosome recycling (Adams et al., 1982; Cupers et al., 1994) and the remainder is transferred to and sequestered in lysosomes. The latter are fully accessed after about min and are responsible for HRP degradation (Secinman et al., 1974; Storrie et al., 1984). Treatment with wortmannin, an inhibitor of PtdIns 3-kinase, has been shown to dramatically interfere with fluid-phase endocytosis (Li et al., 1995; Clague et al., 1995; Araki et al., 1996). PIKfyve, which acts on PtdIns(3)P (the product of mvps34) to generate PtdIns(3,5)P2, is essential for the late stage of fluid-phase endocytosis of HRP (Ikonomov et al., 2003). These observations prompted us to determine if Beclin, as an interacting partner for mvps34 PtdIns 3-kinase, plays a role in fluid-phase endocytosis of HRP. As shown in Fig. 30A, the rate of uptake of HRP in the Beclin KD cells was similar to that observed in the control cells. Similar results were obtained from different batch of Beclin KD U251 cells (Fig. 30B). These results indicate that Beclin is not essential for fluid-phase endocytosis. 90

97 Fig. 30. Suppression of Beclin Expression does not Interfere with Endocytosis of a Fluid Phase Marker, Horseradish Peroxidase (HRP) (A) HRP Uptake (Units/µg protein) Time (min) Control Beclin KD (B) HRP Uptake (Units/µg protein) Time (min) Beclin KD Control (A) Control (closed square) and Beclin KD cells (open circle) were seeded at the same density, grown for 2 d and then incubated for the indicated periods of time with 2 mg/ml HRP in DMEM + 1% BSA. Washed cells were lysed and HRP activity was determined as described in Methods. Each point represents the mean ± S.E. from three determinations on parallel cultures. (B) Similar results were obtained when the experiment was repeated with different batches of control and Beclin KD cells derived from separate retroviral infections and puromycin selections. 91

98 Suppression of Beclin Expression does not Prevent Post-endocytic Degradation of the Epidermal Growth Factor Receptors (EGFR) Next I examined if Beclin affects receptor-mediated endocytic protein trafficking. The mechanisms of EGFR signaling and down-regulation are probably the best understood among growth factor receptors in mammalian cells (Schlessingel. 2002). Upon EGF binding, EGFR becomes activated and triggers dimerization and trans- or autophosphorylation of several tyrosine residues within the C-terminal region of the receptor (Yarden et al., 2001). This is followed by activation of downstream mitogenic signalling pathways (Van der Geer et al., 1994). This mitogenic signaling is attenuated by internalization and degradation of the EGF-EGFR complex. Internalized receptors are initially delivered to early endosome and then to late endosomes/multivesicular bodies (Waterman and Yarden, 2001). In these structures, EGFRs undergo sorting and are either recycled back to the plasma membrane or directed to the lysosome for degradation (Waterman and Yarden, 2001). Microinjections with inhibitory anti-mvps34 cause mislocalization of the early endosome antigen (EEA1) (Siddhanta et al., 1998) and the generation of enlarged MVBs/late endosomes (Futter et al., 2001), indicating that mvps34 plays an essential role in endocytic trafficking. To test if Beclin, an interacting partner for mvps34, is required for endocytic protein trafficking, I followed the fate of activated EGFR. In the absence of EGF, localization of EGFR by immunofluorescence showed that most of the receptors were present in the peripheral cell membrane. Within 30 min after addition of EGF, most of the receptors were found in small internal vesicles diffusely arranged throughout the cytoplasm, typical of early endosomes and MVBs. By 70 min, most of the EGFR-positive 92

99 structures were clustered in the juxtanuclear region in a pattern typical of late endosomes or lysosomes. At all of these stages, there were no consistent differences in EGFR localization between the control and Beclin KD cells (Fig. 31A). To complement the above morphological studies, I examined the degradation efficiency of endogenous EGFR (Fig. 31B). Control and Beclin KD U251 cells were serum-deprived and after stimulation with EGF for indicated times, the total EGFR levels were determined by immunoblot analysis with anti-egfr monoclonal antibody. In both control and Beclin KD cells, I observed a similar time-dependent decrease in the immunoreactive EGFR after addition of EGF, consistent with ligand-stimulated receptor degradation. These results indicate that the presence or absence of Beclin is irrelevant for mvps34 to function in endocytic protein trafficking. Suppression of Beclin Expression in U251 Cells does not Affect Cell Growth Rate Disruption of endocytic trafficking of growth factor receptors enhances growth factor dependent cell proliferation by preventing degradation (Veiira et al., 1996; Wells et al., 1990). To indirectly test if Beclin is required for endocytic protein trafficking, I compared the rate of cell proliferation for control and Beclin KD cells. Consistent with the normal morphology and endocytic trafficking of HRP or EGFR, the suppression of Beclin expression had no detectable effect on proliferation of U251 cells (Fig. 32). 93

100 30 min 70 min No EGF Fig. 31. Suppression of Beclin Expression does not Disrupt EGF-stimulated Endocytosis or Post-endocytic Degradation of the EGF Receptor A Control Beclin KD B (B) EGFR Control Beclin KD Min after EGF EGFR (% of time 0) Control Beclin KD (A) Control or Beclin KD cells were fixed and processed for immunofluorescence detection of EGFR after overnight growth in serum-free medium (No EGF), and after 30 min or 70 min incubation with 200 ng/ml EGF. Scale bar = 10 µm. (B) Cells were harvested at the indicated times after addition of EGF and subjected to immunoblot analysis for total EGFR (upper panel). The bar graph (lower panel) shows the data derived from Kodak Imager scans performed on blots from three cultures harvested at each time point. 94