The interaction between mixed-lineage kinase 3 and the tumor suppressor protein merlin

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1 The University of Toledo The University of Toledo Digital Repository Theses and Dissertations 2007 The interaction between mixed-lineage kinase 3 and the tumor suppressor protein merlin Amanda M. Stewart The University of Toledo Follow this and additional works at: Recommended Citation Stewart, Amanda M., "The interaction between mixed-lineage kinase 3 and the tumor suppressor protein merlin" (2007). Theses and Dissertations This Honors Thesis 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.

2 The Interaction Between Mixed-Lineage Kinase 3 and the Tumor Suppressor Protein Merlin Amanda M Stewart As partial fulfillment of the requirements for the Bachelor of Sciences Degree with Departmental Honors Advisor: Dr. Deborah N. Chadee Department Chair: Dr. Douglas Leaman Director of the Honors Program: Dr. Thomas E. Barden The University of Toledo May 2007

3 ii Abstract Neurofibromatosis type 2 (NF2) is an autosomal dominant disorder of the nervous system which affects 1 in 30,000 individuals. The NF2 gene encodes a 595 amino acid tumor suppressor protein called Merlin that is a member of the ezrin, radixin, moesin (ERM) family of proteins. Patients with Neurofibromatosis Type 2 exhibit loss of function mutations in the NF2 gene and develop schwannomas and meningiomas. Mixed Lineage Kinase 3 (MLK3) is a mitogen activated protein kinase kinase kinase (MAP3K) that activates multiple MAPK signaling pathways. Stable overexpression of wildtype MLK3 in NIH3T3 cells is transforming and we previously observed that depletion of MLK3 inhibits proliferation of different types of normal and tumor cells. We also found that overexpression of Merlin inhibited MLK3 kinase activity and blocked the interaction between MLK3 and B-Raf. The goal of this research is to identify the region of Merlin that is required for the interaction with MLK3 and the inhibition of MLK3 kinase activity. We purified GST fusion proteins of Merlin expressed in bacteria consisting of the full length (aa 1-590), the N-terminus (aa 1-332) and two additional deletion mutants lacking the N-terminus (aa and aa ). Full-length histidine-tagged MLK3 (His-MLK3) was also expressed and purified from bacteria and in vitro binding assays were performed with His-MLK3 and the different deletion mutants of GST-Merlin. We observed that His-MLK3 protein bound to full length Merlin and the Merlin deletion mutants lacking the N-terminus (aa and aa ). However, the Merlin deletion mutant containing the alpha helical region and the C-terminus (aa 1-332) did not interact with His-MLK3. We conclude that the region of Merlin that is required for the interaction with MLK3 lies within aa

4 iii Acknowledgements I would like to thank Dr. Chadee for her help in coordinating and funding this project, and for her help in understanding the intricate details of molecular biology. I would like to thank Nidhi Modi as well for her guidance and patience during my initial research and for working with me on the MMP Gelatinase Assay. Thanks also to Cathy Zhan for the use of her FLAG WB and her advice and guidance, and to Eric Cole and Amanda Korchnack for guidance in my early research. I am grateful also to the UT Honors and Biology Departments for their funding and support of this project, which has enhanced my understanding of biology and of research in general, and has been a wonderful culminating experience of my time in the Biological Sciences at UT.

5 iv List of Abbreviations MAPK... Mitogen Activated Protein Kinase MAP3K or MKKK..... MAP Kinase Kinase Kinase MAP2K... MAP Kinase Kinase GFs.. Growth Factors SAPK/JNK.. Stress Activated Protein Kinase/c-Jun N-Terminal Kinase ASK 1.Apoptosis Inducing Kinase 1 MLKs. Mixed Lineage Kinases DLKs.. Duel-Leucine-Zipper-Bearing Kinases ZAK.. Zipper-Sterile-α-motif kinase SH3. SRC-Homology 3 CRIB. Cdc42/Rac Interactive Binding Domain Slpr... Slipper TNF Tumor Necrosis Factor NGF.. Neuronal Growth Factor SCG.. Superior Cervical Ganglion JIP1. JNK Interacting Protein 1 NF-κB. Nuclear Factor κb MMP... Matrix Metalloproteinase NF2 Neurofibromatosis Type 2 Merlin..Moesin-ezrin-radixin-like-protein NIH National Institute of Heath ERM.. Ezrin, Radixin and Moesin

6 v FERM. Four-point-one, Ezrin, Radixin and Moesin PTPH... Protein Tyrosinase Phosphatases NBL4... Novel Band 4.1-Like 4 DAL Differentially Expressed in Adenocarcinomas of the Lung PTB Phosphotyrosinase Binding DRM... Detergent Resistent Membrane PAKs... p21-activated Kinases MYPT-1-PP1δ myosin phosphatase GH... Growth Hormone Magicin Merlin and Grb2 Interacting Cytoskeleton Protein Hpo. Hippo RalGDS.. Ral guanidine nucleotide dissociation stimulator Mst2.. Mammalian Sterile-20-link Kinase 2

7 vi Table of Contents Title Page i Abstract... ii Acknowledgements.. iii List of Abbreviations.... iv Table of Contents.. vi List of Figures.. vii Introduction 1 Materials and Methods. 33 Results.. 38 Discussion 48 References

8 vii List of Figures Figure 1: The Phosphorylation Cascade of the MAPK pathways Figure 2: The Three Major MAPK pathways Figure 3: Silencing MLK3 Inhibits Matrixmetalloproteinase 2 (MMP-2) activity in SKOV3 cells. Figure 4: Expression of the GST-Merlin Full Length Truncation Mutants Figure 5: Interaction Between His-MLK3 and GST-Truncation Mutants of Merlin Figure 6: The Effect of GST-Merlin Mutants on MLK3 Activity Figure 7: Anti-FLAG blot of MLK3 Kinase Activity Experiment

9 1 Introduction The human body is made up of trillions of cells on average, one billion cells per gram of tissue. These cells communicate using proteins. Every cell then relies on a complicated network of proteins to deliver these messages from the outside of the cell to the nucleus, where changes in gene expression can be made. This project involves the mitogen activated protein kinase (MAPK) pathway, which is critically important in relaying signals to generate intracellular responses in all eukaryotic cells (1). The MAPK pathway is also called the extracellular signal related kinase (ERK) pathway, and is often referred to as MAPK/ERK (1). MAPK pathways are organized into a three tiered system, which they use to relay messages from the outside of the cell to the nucleus. A MAP Kinase Kinase Kinase (MAP3K or MKKK) phosphorylates a MAP Kinase Kinase (MAP2K) at its serine and threonine residues, activating it. The active MAP Kinase Kinase then phosphorylates and activates MAP tyrosine and threonine residues (1). The MAPK can then phosphorylate cytosplasmic substrates such as phospholipases, proteins that associate with the cytoskeleton and protein kinases, or transcription factors, which can then bind to DNA and activate transcription (1). MAP3Ks are often regulated by small GTPases in the Ras superfamily, such as Raf kinases, and by phosphorylation, oligomerization and scaffolding (1). Regulation by scaffolding can allow the cell to distinguish between the many functions of one protein (1). Although the exact number is not clearly known, there could be as many as 13 MAPK genes, a fact that highlights the complexity and importance of these pathways. The MAPK pathways help regulate a cell s response to

10 2 growth factors (GFs) and stress, controlling whether or not a cell grows, divides or undergoes apoptosis (1). Figure 1. The Phosphorylation Cascade of the MAPK Pathways. A MAP kinase kinase kinase, such as Raf in the MAPK/ERK pathway, passes on the extracellular signal by phosphorylating a MAP kinase kinase, such as MEK in the MAPK/ERK pathway. The MAP kinase kinase then phosphorylates a MAP kinase, which leads to its activation and translocation to the nucleus, where it phosphorylates transcription factors that regulate gene transcription. Three major MAPK pathways have been characterized. The MAPK/ERK pathway responds primarily to mitogens, which are substances that stimulate mitosis,

11 3 such as epidermal growth factor (EGF). EGF binds to the EGF receptor, which autophosphorylates (1). Grb2 is an adaptor protein that binds to the EGF receptor and to SOS, a guanine nucleotide exchange factor. This stimulates Ras, a GTPase, to exchange its GDP for GTP. Active Ras then activates the MAPK Raf. The pathway then proceeds through the typical MAPK phosphorylation steps (1). The two other pathways are the stress activated protein kinase/c-jun N-terminal kinase (SAPK/JNK) and p38 pathway, which are also organized in a similar three level phosphorylation cascade that is characteristic of the MAPK pathways. The SAPK/JNK and p38 pathways respond primarily to stress, such as UV light, inhibition of protein glycosylation, heat shock, and inflammatory cytokines, rather than mitogens. In the JNK pathway, JNKs activate c-jun, which controls transcription of cytokines and other genes (1). JNK can also be activated by apoptosis inducing kinase 1 (ASK 1), MEK kinases and others. Deregulation of these MAPK pathways is important in tumor development, inflammation and differentiation (1).

12 4 Figure 2. The Three Major MAPK Pathways. The MAPK/ERK pathway responds mostly to mitogens, such as epidermal growth factor (EGF), while the SAPK and p38 pathways tend to respond to stress stimuli, which can include UV light or oxidation. All three pathways affect the ability of a cell to divide, differentiate and undergo apoptosis. MLK3 Structure and Regulation by Rho GTPases Mixed-Lineage Kinases (MLKs) are a group of proteins within the serine/threonine MAP3Ks and have been shown to play a role in activating all three major MAPK pathways. There are seven different MLK proteins that have been characterized. They share eleven conserved subdomains, of which I-VII are similar to serine/threonine kinases, such as MEK Kinases and Raf. Subdomains VIII-XI are similar

13 5 to tyrosine kinases (1). The name Mixed-Lineage is derived from this seeming split of functions. However, it s clear that they are true serine/threonin kinases, as only this function has been observed (1). Three mammalian subfamilies of MLKs are MLKs (Mixed Lineage Kinases), DLKs (Duel-leucine-zipper-bearing kinases) and ZAK (Zipper-sterile-α-motif kinase). The proteins are divided based on their sequence similarities in their catalytic domains and on their domain arrangements (1). The MLK subgroup contains MLKs 1-4 and begins with an N-terminal SRC-Homology 3 (SH3) domain, which is about 50 amino acids long and recognizes and binds sequences rich in proline. The SH3 domain is followed by a kinase domain, then a region that contains a leucine zipper, and finally a Cdc42/Rac interactive binding (CRIB) motif. CRIB motifs allow interactions between Rac and Cdc42 that are bound to GTP and signaling molecules (1). For MLKs 1-4, 75% of the catalytic domain is conserved. Their C-terminals are different, but all have large amount of proline, for which the function is not yet known. MLK3 is one of the best understood MLKs. It s also called the SH3-domaincontaining-proline-rich-kinase and protein-tyrosine-kinase 1 (1). Multiple names for the MLKs are common because many were identified by different research groups around the same time. MLK3 has an N-terminus that is rich in glycine and proline, which is different from the other MLKs. The homologue of MLK3 in Drosophila is called Slipper (Slpr). It contains the same domains as the MLK subfamily of MLKs and has been shown to play a role in the JNK pathway, as the MLKs do (1). C. Elegans has proteins that appear to have similar characteristics, such as the SH3 domain and a catalytic domain that is close to the MLK family, but no clear, useful link has been established (1).

14 6 All of the MLKs contain a leucine zipper. Leucine zippers usually contain a leucine residue every seven amino acids, which allow it to form coiled coils via dimerization or oligomerization. These coils are stabilized at the interfaces of opposing helices by hydrophobic, non-aromatic interactions by residues such as methionine (1). It is proposed that these leucine zipper regions help regulate selectivity, because there is 75% sequence conservation among the MLK subfamily of MLKs, but only 35% similarity with the DLK subfamily (1). Dimerization using the leucine zipper also plays an important role in activating MLK3. It has been demonstrated when a proline residue is added, which destabilizes dimerization, MLK3 cannot auto-phosphorylate and thus, cannot activate the JNK pathway. Therefore, the leucine zipper is vital for phosphorylation and substrate interaction (2). MLK3 can also be regulated by its SH3 domain. The SH3 domain is known to help localize MLKs to proline residues on other proteins for signal regulation and localization, but is has also been shown that the MLK3 SH3 domain can have an autoinibitory function on its kinase activity (1). It was shown that the SH3 domain binds to a proline sequence on the protein that lies between the leucine zipper and the CRIB motif (2). This was demonstrated by disrupting the domain with an alanine at residue 52, which increased MLK3 kinase activity a first for a Ser/Thr kinase. In addition to regulation by its own domains, MLK3 regulation can also be mediated by Rho GTPases. Rho GTPases are proteins that help regulate growth, cellular transport, cytoskeleton motility and signaling pathways, including the MAPK pathways (1). In particular, Rac and Cdc42 are known to play an important role in activating MLK3. MLK3 has been shown to bind to activated Cdc42 and Rac, and, when bound to

15 7 Cdc42, MLK3 activity and JNK activation both increase (1). It has been demonstrated that Cdc42 could still induce auto-phosphorylation when the leucine zipper was disrupted (though phosphorylation of T258 could not occur, which is consistent with the dimerization requirement for activation), so Cdc42 must act on a separate region (1). One proposed mechanism for this activation is that GTPases interrupt the autoinibition of the SH3 domain, allowing dimerization, auto-phosphorylation, and localization to the membrane, all of which can increase MLK3 catalytic activity (2). It was also demonstrated that Cdc42 could use prenylation to translocate MLK3 to the proper place on cellular membranes (2). MLK3 in the JNK, p38 and MAPK/ERK Pathways MLK3 has been shown to activate the JNK, p38 and MAPK/ERK pathways, helping to coordinate signaling through complicated protein networks. MLK3 was originally identified primarily as a JNK activator, and secondarily as a p38 activator, both related to stress stimuli. However, JNKs are occasionally activated by mitogens and JNK can function with ERK to promote c-fos transcription by phosophorylation of Elk1 (3). Although they are considered downstream, MLK3 can be activated by proteins involved with the MAPK pathways that respond to stress, such as JNK Interacting Protein 1 (JIP1), JNK or p38. MLK3 contains an activation loop in its C terminal residues 277 to 281, which is within its catalytic site. The sequence for these residues is TTXXS, with X standing for any amino acid. This sequence is conserved throughout mammalian MLKs, and is an important region for regulation by phosphorylation (1). When Cdc42 is bound to this site, threonine 277 and serine 281 are more easily phosphorylated, (2, 3).

16 8 JNK or p38 can phosphorylate MLK3 at this site, and recent studies have also shown that without JNK, in vivo phosphorylation of MLK3 does not occur. In addition, if JNK is completely blocked, all MLK3 remains in its inactive form (2). In general, when phosphorylation levels of MLK3 change, total levels of MLK3 remain constant, though distribution does change dramatically (2). In fact, inhibition of JNK for long periods of time may even lead to degradation of MLK3 (2). However, some studies have shown that inhibition of p38 and ERK did not affect levels of MLK3 or active MLK3. This implicates JNK as a specific inhibitor of MLK3 which acts as a negative feedback control mechanism, since MLK3 is an upstream activator of JNK, which can then regulate MLK3 (1). This may be especially important because when MLK3 is phosphorylated by JNK, it can lead to intensified JNK signaling, which can cause apoptosis (2). In addition, in the absence of tropic factor or neuronal growth factor (NGF), MLK3 activity can increase, which increases JNK activation and can result in apoptosis, as studied in the superior cervical ganglion (SCG). When NGF is lost, this is the response that appears to dominate. Even inactive MLK3 appears capable of blocking apoptosis in SCGs (1). JIP1 is a scaffolding protein that participates in the JNK pathway. It has been demonstrated that JIP1 binds to the MLKs and plays a role in the MLK3 activation of the p38 pathway (1). MLK3 can also be inhibited by CEP-1347, which is an ATP competitor (1, 4). In the MAPK/ERK pathway, MLK3 forms complexes with Rafs, such as MLK3- B-Raf-Raf-1 and Raf-1-Mst-2 (3). B-Raf was first identified as an oncogene in Ewing Sarcoma, while Raf-1 was discovered as an oncogene of a murine retrovirus. Raf-1 is a protein that is downstream of Raf and stimulates ERK activation. Raf-1 is activated by

17 9 phosphorylation, and B-Raf also requires several phosphorylation steps. It appears that MLK3 is integral in activating B-Raf, but not Raf-1 (3). Regardless, however, both Raf-1 and B-Raf are needed to phosphorylate MEK and transmit the mitogen signal (3). B-Raf can transactivate Raf-1, but Raf-1 cannot do the same to B-Raf. In addition, many B-Raf mutations result in cancer or serious diseases (3). Neither B-Raf nor Raf-1 can function alone without B-Raf, ERK activation fails to occur, and without Raf-1, there is a hightened sensitivity to apoptogenic signals. The Rafs also require signaling complexes involving chaperonins Hsp 90 and Cdc37, which MLK3 also requires (3). These complexes often provide scaffolding that enhances activation, which is true for a protein called kinase suppressor of Ras-1 (Ksr1) and connector-enhancer of Ksr (CNK), which is known to bind to MLK3 (3). Different scaffolds can form depending on the stimulation conditions, which can help control specificity. It seems that MLK3 s role in ERK activation is mostly noncatalytic, instead promoting and maintaining the interaction between B-Raf and Raf-1 that allows the signal transmittance to occur (3, 4). MLK3 has been identified as critical in this interaction, however, because when MLK3 is silenced, there is no interaction between B- Raf and Raf-1 and no ERK signaling. When MLK3 is reintroduced, signaling returns (4). A proposed mechanism is that MLK3 activates B-Raf and Raf-1 by binding GTP-Ras, which results in Raf phosphorylation. How the phosphorylation might occur, however, is still unclear (4). MLK3 appears to affect cell cycle entry, and when MLK3 is silenced, the cell does not progress through the G1/S checkpoint (3).

18 10 MLK3 in Cancer One important line of study involves investigating the role of MLK3 in cancer. Cancer progresses through five stages at a cellular level: from a normal cell, through neoplastic transformation, tumorigenesis, invasion, and metastasis. In cellular transformation, complex genetic alterations take place that give the cell an advantage in growth, such as anchorage independent growth and lack of dependence on GFs. It has been demonstrated that increased levels of active MLK3 resulted in increased transformation in NIH3T3 fibroblasts (1). Inactive MLK3 did not have this affect, however, indicating that MLK3 s role in cancer does involve its catalytic activity (1). It has been shown that MLK3 affects the proliferation of SKOV3 cells, an ovarian cancer cell line (5). This is evidence that MLK3 plays an important role in cancer. Matrix metalloproteinases (MMPs) often play an important part in cancer cell invasiveness because they degrade the extracellular matrix, making metastasis possible. Neurofibromatosis Type 2 One negative regulator of MLK3 is the tumor suppressor protein called merlin (moesin-ezrin-radixin-like-protein, also called schwannomin). Neurofibromatosis Type 2 (NF2) is an autosomal dominant predisposition gene located on chromosome 22 that encodes for the 67kDa protein merlin. Merlin inhibits the JNK and SAPK pathway signaling and cell proliferation. Loss of function mutations in NF2 lead to NF2 disease, which affects the central and peripheral nervous systems (6). It presents with schwannomas and meningiomas, both of which are benign tumors of the nervous system (7). The incidence rate is reported to be 1 in 30,000, and although many of these may be

19 11 carriers, it has also been reported that nearly 100% of all carriers die from multiple tumors (8, 9). Schwannomas are encapsulated tumors that form from Schwann cells, and are the most common tumor type in NF2 (7). Spinal schwannomas present with a variety of symptoms depending on their location, and can include sensory abnormalities, pain that radiates or remains localized, bladder changes, and muscle weakness. The most common symptoms are hearing loss, tinnitus, weakness, seizures and paralysis because tumors often reside on the vestibular nerve (7). Ependymomas also make up a small percentage of tumors that can occur in NF2 as a result of loss of merlin or other 4.1 family proteins (7). Out of all brain tumors, 20-30% are meningomas. Some meningiomas occur with intradural or subarachnoidal spread. In NF2, tumors occur frequently and in large numbers (7). NF2 can also present with eye conditions such as cataracts, and skin tumors (8). Clinical diagnosis of NF2 can be quite difficult, and different criteria for diagnosis have developed. Bilateral vestibular schwannomas are almost always present in NF2 patients, and a first degree relative with NF2 is often a strong indicator in diagnosis (7). The criteria established by the National Institute of Health (NIH) requires bilateral vestibular schwannomas or family history plus other symptoms, which excludes many patients with NF2-like symptoms (8). The Manchester criteria is most lenient and includes all patients with bilateral vestibular schwannomas regardless of family history or other symptoms, and also uses tumors of other nerves for diagnosis of those without bilateral vestibular schwannomas (8).

20 12 A complication of diagnosis is that NF2 can have childhood or adult onset. Childhood onset NF2 usually does not include 8 th cranial nerve or vestibular symptoms, while adult onset does (8). Genetic testing is available but only detects about 60% of mutations when no family history is present (7). Mosaicism also occurs in some cases, and can be very difficult to identify with a genetic test (7). MRI screening can also detect tumors in suspected cases or with family history (7). Oddly, some patients have no merlin loss, but still exhibit multiple meningiomas, which also makes diagnosis difficult (7). In addition, a related disease called schwannomatosis is often confused with mosaic NF2. Schwannomatosis involves multiple schwannomas, but usually not bilateral vestibular nerve tumors. Schwannomatosis tends to be sporadic, as NF2 is, and is usually more painful but less deadly. Schwannomatosis patients can also exhibit NF2 mutations in their tumors, which blurs the lines between mosaic NF2 and schwannomatosis (7). Treatment of NF2 is also difficult because of the great variation between patients, and even within the same patient (8). This high degree of variability may stem from random events, but it is more likely that the mechanisms of this disease are not yet well enough understood to predict the progression of the disease. Fifty percent of NF2 cases have a family history component, but even in these cases, the rates and progression of the disease can be quite different from family member to family member (7, 8). In general, the tumor reoccurrence rate is so high that treating NF2 is a lifetime battle (7). Deciding how to treat tumors is difficult for several reasons. First, the tumors do not respond well to chemotherapy, and radiation is only recommended for especially aggressive tumors, if the patient is a poor candidate for surgery, or for elderly patients (8). This is because radiation in NF2 patients can lead to more tumors by increasing the

21 13 incidence of mutation of the second allele (7). Instead, surgery is often recommended, and can prevent hearing loss if tumors are removed in time (7). In addition, some tumors grow so slowly that it may be possible not to treat them if they are carefully monitored (7). Despite these sometimes slow growth rates, many patients with NF2 have a shortened life span due to swallowing problems caused by the tumors. In addition, it s difficult to predict growth rates, even based on mutation type, because there are so many factors (10). Fatality risk correlates strongly with age at diagnosis, which seems to be the most important factor, as well as number of intracranial meningiomas, type of mutation, and type of treatment center (8). Because treatment is long term and requires doctors in many different specialties, such as neurologists, neurosurgeons, otolaryngologists, geneticists and audiologists, specialty centers are often best suited for treating NF2 (7, 8). Those treated at specialty centers rather than elsewhere tend to have a lower mortality rate as well (8). NF2 Mutations For NF2 to develop, mutations in both NF2 alleles must occur. The mutation of one allele is inherited, causing a dominant predisposition to acquiring NF2. If the second allele becomes mutated, the gene will be dysfunctional and NF2 will develop (7, 11). This is common in tumor suppressor genes and is typically referred to as the two-hit mechanism (11). Inactivation of NF2 in mice tissues cause schwannomas or meningiomas based on the location of the inactivation (12). The NF2 mutation rate is thought to be 6.5 x 10-6, with 25-30% of those mutations occurring as mosaic mutations

22 14 (10). If p53, another tumor suppressor gene, is also mutated, tumors occur much more frequently (12). The type of mutation that occurs greatly affects the severity and progression of the disease, with missense, large deletions, and mosaicism being the mildest (10). Although mosaic mutations tend to be milder than constitutional mutations in NF2, the mutation can be passed off to offspring as a widespread mutation. In addition, mosaicism can be more difficult to detect and diagnose, especially somatic mosaicism, which occurs at a level difficult to detect with DNA screening (7, 8). Splice mutations, which 8 defines as single base pair alterations that occur within 5 GT or 3 AG conserved regions, deletions in coding sequences or in these conserved regions, or mutations of a conserved base or bases near the slice site, can also be very damaging, which 5 more so than 3 (10). From studies been done with the information in the International NF2 Mutation Database, it has been determined that nonsense and frameshift mutations, which make up about half of all constitutional NF2 mutations, correlate with earlier onset and higher tumor frequency (10). It must be recognized, however, that because of their severity, these mutations are the easiest to recognize, which may contribute their high reported incidence rate (10). In general, nonsense mutations occur more frequently in constitutional mutations than frameshift mutations do, but this ratio is reversed in somatic mutations. In addition, the ratio of frameshift to nonsense mutations increases with age, probably because DNA repair becomes less efficient with age. It has been observed that end joining accrues more errors as age increases, which can lead to an increase in mutations (10).

23 15 NF2 mutations occur most frequently at CGA codons, because CpG nucleotides are particularly susceptible to mutation. This is mostly likely because the methyl cystosine is easily deaminated to thymine (10). When this occurs, CGA is converted to a stop codon, TGA, which results in a truncated merlin protein. The susceptibility of the CGA condons are increased when they are next to a 5 pyrimidine, which relates to many CGA sequences found in NF2 (10). In fact, 54% of constitutional mutations in NF2 were C to T conversions in CGA codons. Mutations were also very common in the six base pair CT repeat that occurs in codons 12-14, probably via replication slippage, which results in an insertion or deletion mutation (10). Interestingly, when mutations in the CGA codons are excluded, exon 15 has one of the highest rates of NF2 constitutional splice site mutations, but none are known to exist in exon 16 or exon 17. Exon 16 exists only in the form of merlin that does not act as a tumor suppressor protein, so it makes sense that no mutations would be observed in NF2 patients in this exon (10). That no disease-causing mutations are found in Exon 17, however, is more interesting, as it encodes Phe 592, a hydrophobic residue that forms a H bond with Asn 226 to help form the closed conformation of active merlin. However, it is proposed that merlin contains sufficient structural flexibility to accommodate these mutations without symptoms (10). It is also possible that embryos with this mutation do not live long enough for the mutation to appear in studies. Oddly, very few mutations in exons 14 and 15 can be found in sporadic meningiomas (10). It has been proposed that these exons are important in suppressing schwannoma formation, but do not play a strong role in preventing meningiomas. Because of the large number of protein interactions each protein participates in, this

24 16 theory is reasonable (10). The average frequency of mutations in the N-terminal is slightly higher than in the helix region and C-terminal domain, but this is likely because CGA distribution heavily favors the C-terminus (10). There are very few mutations observed in exon 9, and it has been proposed by Baser that this is because embryos with these mutations do not survive (10). It is also reasonable to theorize that mutations of residues in contact with the solvent are less likely to produce disease than those that serve to pack the subunits of the protein or form interfaces, salt bridges or hydrogen bonds. In other words, those residues that are integral to folding and function are those most likely to cause NF2, and so are more likely to be seen in NF2 patients, unless they are so damaging that the embryos do not survive (10). These patterns are based on available genetic data, but more studies with more patients will help provide stronger conclusions. Although transformation and malignancy in schwannomas is quite rare, there is a tentative correlation between NF2 mutations and cancers that are capable of invasion and metastasis. Mutations of NF2 have been found in breast and colon cancer tissue, but there is not yet evidence that it is a primary cause (11). When mutation of the second NF2 allele occurs in mice with a genetic predisposition to NF2 highly metastic tumors are formed during development. Oddly, no meningiomas or schwannomas were found in some of these mice (8, 11). In others, osseous metastases, osteosarcomas and other aggressive malignancies were found in addition to schwannomas and cateracts (13, 16). In addition, non-metastatic schwannomas can actually move into the nerve, and nonmetastatic meningiomas can travel along leptomeninges (8). In NF2 patients, mesotheliomas commonly occur with NF2 mutations (15). Mesotheliomas are malignant tumors of the plural membrane which often occur in

25 17 conjunction with NF2 mutations that result in truncated merlin (11). These tumors are highly invasive, unlike schwannomas and meningiomas. Nonsense mutations appeared to be particularly common in NF2 patients who exhibited both classical NF2 symptoms and mesothelioma (10). It has been proposed that nerve cells are particularly affected in NF2 because they proliferate mainly in adolescence, while cells that are constantly renewed, like lung cells, require more drastic changes. This help may explain why mesotheliomas are not seen in every NF2 case (13). There is not yet any direct evidence for a causeeffect relationship between NF2 inactivation and mesothelioma, but it is clear that a correlation exists (11). NF2 is also very important in embryogenesis and tissue differentiation. Mice cannot survive if NF2 is completely inactivated during their initial development. These mice are shown to have completely disorganized ectoderms (12). In Drosophila, the overexpression of a murine mutant led to cellular transformation (11). Merlin also interacts closely with Rac pathways, and Rac has been implicated in cellular transformation (14). Merlin performs growth suppression functions in cells other than meningiomas and schwanommas and appears to have a metastasis suppression function as well, which could mean a close link to cancer (11, 15). Structure and Conformational Regulation of Merlin The NF2 gene encompasses about 100 kb on the 22 nd chromosome (6). It was isolated using genetic linkage analysis and tumor deletion mapping (11). The NF2 product, merlin, is similar to a large group of proteins called ERM for Ezrin, Radixin and

26 18 Moesin, and belongs to the Protein 4.1 superfamily of proteins (12). All Protein 4.1 proteins have a FERM (Four-point-one, Ezrin, Radixin and Moesin) domain and most have a spectrin/actin domain and are involved in cytoskeleton organization as well (6, 12). In the name, 4.1 refers to a gel position from the originally identified protein. Now the superfamily has more than 40 members which are divided into five subgroups: Protein 4.1 subgroup, ERMs, talin-related, protein tyrosinase phosphotases (PTPH) and novel band 4.1-like 4 (NBL4), the last two of which lack actin binding sites. These tend to be similar structurally and sometimes in the way they are expressed (12). Merlin is thought to be the only protein in the 4.1 superfamily to be involved in a hereditary tumor syndrome, even though Protein 4.1B and DAL-1 (DAL stands for Differentially expressed in adenocarcinomas of the lung) are known to be tumor suppressors (11, 12). In support of this, there is no alteration of ERM protein levels in schwannomas or meningiomas (8). Merlin is thought to have three domains, a FERM domain from residues 1-302, an α-helix domain from residues and the remainder of the C-terminus from or 595 (12). It exists in two isoforms, (I) and (II), which differ in the splicing of the 16 th and 17 th exon. Isoform I consists of exons 1-15 and 17, while isoform II consists of exons 1-15 and 16 (11). Exon 16 contains a stop codon which prevents exon 17 from being translated (12). The FERM domain is highly conserved in ERM proteins, which are known to anchor membrane proteins to the cytoskeleton. The FERM domain eases interactions with membrane binding partners. Merlin showns 63% homology with the N-terminus of ERMs (13). Merlin s FERM domain has been crystallized, and three subdomains have

27 19 been observed, termed A, B, and C (12). Subdomain A is similar to ubiquitin, while subdomain B resembles acyl-co A binding protein. Subdomain C seems to be related to signaling domains in phosphotyrosinase binding (PTB) and others (11). These three subdomains create a globular, clover-like structure using salt bridges at key residues, such as Asp70 in subdomain A to Arg291 and Lys 289 in subdomain C (6, 12). Subdomain B of the FERM domain also contains a region called the Blue Box, which is a 7 amino acid stretch that is crucial for merlin function. Substitutions in this region in Drosophila have resulted in increased cell proliferation that was ameliorated by the addition of functional merlin (11). Interestingly, the Blue Box is conserved in the Drosophila version of merlin, but not in the ERM proteins (12). It appears that the FERM domain interacts with merlin s C-terminus using the Blue Box region, a fact important for both the regulation of merlin and understanding how mutations may change proteinprotein interactions (11). In ERM proteins, the C-terminus contains a region that binds to actin, which allows them to regulate cytoskeleton organization. In merlin, this region is not conserved, meaning merlin has no known way to bind actin directly (11, 12). Instead, the C-terminus is only 28% homogolous to ERMs and, in merlin, appears to be involved primarily in regulating its activity. ERM proteins do contain what is called the ERM-tail, which interacts with the FERM domain (10). This consists of a β-sheet at the beginning, followed by four major helices lettered A through D. αa interacts with the FERM subdomain B, while αd fits between the two β-sheets of subdomain C (10). In addition, Arg588 forms a salt bridge with Glu260 and hydrophobic Van der Waals interacts with Ile254. Similarly, Phe592 forms a hydrogen bond with Asn226 (10). Also, the C-terminus

28 20 Ser518 gets phosphorylated, which prevents merlin from forming a closed, active conformation (11). In fact, merlin mutants that lack the C-terminus can no longer perform their growth suppressive function (11). The potential interaction between the head and tail of merlin is very important for regulation, as it is in ERM proteins. It has been observed that changes in merlin folding correspond to changes in tumor suppressor activity (6). N-terminal folding must occur first, and then the N-terminus and C-terminus interact (6). Both ERMs and merlin are regulated by their open or closed conformation. When FERM domain residues bind with tail residues , a closed, circular conformation occurs. Interestingly, a closed conformation in merlin corresponds to the active form, but a closed conformation of ERM proteins renders them dormant (11). The closed form can only occur with Isoform I because the last five amino acids allow the C to N terminal binding to occur (7). Another difference is that ERMs have aromatic residues in the C subdomain of the FERM which push away the loop from subdomain A, while merlin has salt bridges that connect subdomain A to subdomain C (12). Merlin can heterodimerize with ERM proteins using residues in its C- terminus to bind to ezrin s N-terminus. This allows ezrin to negatively regulate merlin, because ezrin has a higher affinity to merlin than merlin s own C-tail, and when heterodimerization takes place, merlin cannot form its closed, active conformation (12). When merlin is phosphorylated, this heterodimerization forms even easier because merlin is stuck in the open, inactive position. The differences between ezrin and merlin and information about how they interact may provide insights about the mechansism for their antagonistic functions: merlin acts

29 21 as a tumor suppressor, while ERMs tend to increase cell proliferation and growth (16). It is also known that ERMs and Merlin bind to identical binding sites on the plasma membrane, which is consistent with their antagonistic relationship (9). Merlin s tumor suppressor function has been demonstrated by decreasing levels of merlin in human cells, which leads to an increase in cell spreading and proliferation (7). This affect can be corrected by the re-addition of merlin (7). In addition, schwannomas can be forced to undergo apoptosis and the proliferation of meningomas can be inhibited in vitro by Isoform I (1-595) of merlin (11). Merlin Expression and Localization For any protein, information on where the protein is expressed and localized can give clues to its structure and function. For merlin, there is conflicting data on whether it is expressed ubiquitously across all types of tissues or not, but it is certainly prominent in tissues of the retinas, lenses, testis, ovaries, adrenal glands and nerves (7, 12). If it is in all tissues, it is thought that the lack of merlin in other tissue types is compensated for by other proteins in NF2 patients but not in tissues like the nerves and eyes, which leads to the characteristic schwannomas, meniginiomas and cataracts (7). It has been demonstrated, however, that high levels of merlin are seen in a wide variety of tissues in embryos, so it is clear that it plays an important role in development (8). Within a cell, merlin is primarily localized to the cell membrane, especially to regions that participate in cell to cell contact or cell to matrix contact (7, 8). These areas are often rich in actin, and may include membrane ruffles, microvilli and lamellipodia (14). Depending on the cell cycle, merlin may also move to the nucleus (7). Merlin is not

30 22 soluble in triton, a nonionic detergent, and this is classically taken as evidence that merlin is attached to the cytoskeleton (16). However, a recent theory is that a lack of solubility may be because a protein is attached to a part of the membrane called a Detergent Resistent Membrane, or DRM. DRMs are small pieces of the membrane made up of glycosphingolipids and cholesterol molecules, which do not dissolve easily (16). There is already accumulating evidence that proteins associated with DRMs are not soluble in detergents, and many of these are signaling proteins such as integrins and Rac, or membrane trafficking proteins; some have even been shown to bind with merlin as well (16). In the case of signaling proteins, a theory is that these DRMs form a docking station for signals, increasing their effectiveness. There are two types: lipid rafts, and caveolae, which differ based on structure and variety of function. Lipid rafts tend to be associate with signaling molecules primarily and have short lives, while caveolae perform a number of different functions and are more stable (16). Kissil et al demonstrated that merlin was involved in these lipid rafts, though ezrin was not. Using localization visualization and immunoblotting, they showed that merlin s insolubility stems from both association with actin and association with DRMs (16). When merlin becomes growth suppressive, they showed, there is a loss of actin interaction that occurs within lipid rafts (16). They found that all merlin is localized to DRMs, whether active or inactive. Bouyancy, however, was shown to change based on whether merlin was active or inactive, indicating, perhaps, that is associates with different proteins based on its conformation (16). Specifically, merlin may dissociate from actin when it is acting as a growth suppressor, moving from a heavier raft to a

31 23 lighter one. This idea was supported by evidence obtained from disrupting the actin network (16). Kissil et al proposes that this protein translocation can regulate signaling in a very specific manner, because it can cause a protein to initiate or break contact with another protein (16). Since lipid rafts are already thought to influence cell growth, it is not difficult to imagine that merlin can disrupt these signals when necessary (16). In addition NF2 patients have mutated merlin that is more soluble in triton, suggesting that merlin may not associate properly with a DRM in NF2 (16). Regulation of Merlin by Phosphorylation: Rac, Cdc42 and PAKs Merlin is known to interact with many different signaling partners, which include proteins involved in cell proliferation, cell motility, and cell signaling. It has been proposed that receptor tyrosine kinase (RTK) inactivates merlin by phosyphorylation (8). Merlin can be also be activated by paxillin, which has a cytoplasmic tail that can bind to merlin s residues and helps it to move to the cytoskeleton, where it binds proteins on the cell s surface, such as BII Spectrin and CD44 (8, 12). CD44 is a glycoprotein that spans the membrane and functions as a hyaluronic acid receptor, which also binds to several nucleotide exchange factors and can activate Rac 1 (12, 14). Merlin interacts with the cytoplasmic tail of CD44 using residues 1-50, and this interaction gives merlin sensitivity to extracellular stimuli because CD44 mediates signaling from the outside of the cell to the inside (12). It has been proposed that the damage in NF2 is caused by the loss of merlin s ability to respond to the extracellular growth suppressive cues, and indeed, in a merlin mutant without residues 1-50, increased tumor growth was observed in mice (6, 8). These growth suppressive clues

32 24 can include cell density, serum deprivation and other measures of growth and nutrient availability. In many cell types, the ratio of active merlin to inactive merlin increases significantly with an increase in cell density, which supports its tumor suppressor function (14, 16). When conditions do not encourage growth, the levels of phosphorylated merlin decrease (14). It appears that merlin works as a growth suppressor only when large numbers of cells are present (11). It has been proposed, then, that the complex formed by merlin and CD44 forms a molecular switch which controls whether growth can occur conditions are favorable, merlin is phosphorylated and unbound to CD44 or growth in suppressed conditions are unfavorable, merlin is dephosphorylated and bound to CD44 (6). When conditions for growth are unfavorable, ERMs are also dephosphorylated, putting them in a closed, inactive state. This emphasizes how merlin and ERMs work together in the cell (12). When conditions are favorable for growth, the activity of GTPases increase, causing them to phosphorylate ERMs, which unfold and bind to CD44, becoming active. Small GTPases Rac and Cdc42 can promote phosphorylation of merlin on Ser518, inactivating merlin and increasing cellular proliferation. Rac and Cdc42 mostly involved in the organization of the cytoskeleton and in changing gene expression, which corresponds well to merlin s role in cell motility (14). In many cells, when merlin is lost, Rac is activated at the membrane. This is a non-localized, long term effect that can lead to non-polar cells, a phenomenon that may help explain why Schwann cells fail to myelinate their axons (7). It has been shown that p21-activated kinases (PAKs), Rac/Cdc42 kinase effectors, phosphorylate merlin, which acts as an inhibitor by preventing the closed,

33 25 active conformation. This encourages an increase in cellular proliferation and can also promote a loss of adhesion (6, 11). PAKs decreases the phosphorylation of merlin by three or four fold even if they are deactivated, meaning that they function in a dominantnegative manner (14). The model currently proposed suggests that normal levels of merlin down regulate Rac/Cdc42 which allows phosphorylation of merlin, causing growth (14). MYPT-1-PP1δ (myosin phosphotase) dephosphorylates merlin, achieving a balance based on cellular density and other conditional signals (6). As a feedback mechanism, merlin can also inhibit PAK1 by binding within merlin residues This binding prevents Rac from binding and activating PAK1. When merlin is lost, Rac activity increases, which means merlin can negatively regulate signaling and cell transformation that is mediated by Rac (6, 11). Since JNK, p38 and NF-κB are downstream of Rac, merlin levels can affect these as well (11). Merlin also participates in complexes with B-1 integrin, which may help coordinate motility and proliferation (11). Proper regulation and activation of merlin as described above is important to maintain the proper balance of cell motility and growth suppressive functions. Schwannomas often exhibit cellular motility defects, such as aberrant cell ruffling, which can be corrected by restoring merlin levels (11, 16). Merlin controls cell adhesion and motility by controlling actin, so in cells with little or no merlin, adhesion to the matrix or other cells is decreased, which helps explain why schwannomas do not wrap around the nerve as Schwann cells normally do (7, 11). Schwannomas can be rescued and normal spreading, organization and attachement result from overexpression of merlin in these cells. However, this does mean reduced motility for these cells as well, which may

34 26 interfere with normal processes (11). In truncation mutants with no FERM domain, cell adhesion was completely lost, even after wild type merlin was added. Merlin loss correlates with invasiveness and metastasis in mouse models, indicating the importance of normal, functional motility (11). Merlin and the MAPK/ERK pathway As more studies are done, evidence is accumulating that merlin is involved in pathways that are downstream of growth factors, or GFs (12). One of these pathways is the MAPK/ERK pathway. When the MAPK/ERK pathway is stimulated by serum or growth hormone (GH), growth is increased, but this effect can be inhibited by the addition of merlin (17). Similarly, the phosphorylation of the MAPK/ERK pathway is blocked by the expression of merlin. This inhibits the transcription of SRE, which in turn decreases c-fos expression, as measurd by a luciferase reporter (17). Cells with reduced levels of merlin fail to check growth in response to cell-to-cell contact, do not have functional adherens junctions, and exhibit high levels of ERK and JNK (9). Because of this correlation, drugs that inhibit the MAPK/ERK pathway are being successfully tested on NF2 disease models (7). Integrins, proteins upstream of Ras and Raf in the MAPK/ERK pathway, form coreceptors for merlin with CD44. In Drosophila models, cells with no merlin demonstrate an increased amount of EGF receptors (9). Activation of the receptors recruits adaptor protein Grb2 to the membrane, which draws SOS to itself to form a complex. This complex can then receive and transmit growth signals to Ras, which is the beginning of the MAPK/ERK phosphorylation cascade (17). It has been demonstrated that merlin does

35 27 not interact with or affect the Grb2-SOS complex in vitro or in vivo, though the complex does contain ERM proteins which were shown necessary for Rac activation (9). Merlin did appear to disrupt Rac interactions with the Grb2-SOS complex, however (9). Because these studies also supported that phosphorylation of ERK and JNK were inhibited by merlin, it was concluded that merlin must affect the MAPK/ERK pathway downstream of the Grb2-SOS complex (9). Magicin (Merlin and Grb2 Interacting Cytoskeleton Protein) is a ubiquitously expressed protein that binds to the C-terminus of either isoform of merlin, from residue 340 to 590 or 595 (6). Magicin is thought to play a role in Ras signaling, and upregulation of magicin has been observed in endothelial cells. A pulldown experiment has shown that magicin forms a complex with Grb2 and Merlin, which may help regulate Ras, Rho and Rac at a high level in the pathway (6, 17). In addition, magicin overexpression activates ERK, JNK and p38, and has an increased expression in breast, colon and prostate cancers (6). It has been clearly demonstrated that Merlin does affect Ras, though the binding may be indirect and mediated by a guanine-nucleotide exchange factor (9). Overexpression of Ras results in anchorage-independent growth, which is a characteristic of cellular transformation, but this effect can be amerliorated by overexpression of merlin (9, 11, 13). Non-functional merlin mutants do are not able to affect the cells transformation, indicating that merlin activity helps rescue the cells (11). Merlin blocks activation of Ras and Rac and reduces cell colonies, even when a dominant-active form of Ras or Rac is introduced. However, a dominant-active form of MEK could overcome merlin s growth suppressive effects, indicating that merlin s interaction with the pathway

36 28 takes place above MEK (9). It can be concluded, then, that merlin negatively regulates Ras and Rac based on the cell s environment (9, 11). Ras activates the transcription factor AP-1, which binds to the promoter region of genes responsible for cell proliferation, metastasis, and growth (13). Increasing AP-1 activity increases cyclin D1 expression, which helps control cell cycle progression. High levels of cyclin D1 lead to increased growth (11, 13). It has been proposed that merlin interacts with cyclin D1, perhaps through PAK, which would potentially provide a Rasindependent mechanism for control of cyclin D1 (11). Ras is also responsible for promoting phosphorylation of a protein named Rb, as well as increasing levels of active E2F-1, which is also responsible for transcriptional activation of several key growth and proliferation proteins (13). In this way, merlin affects the levels of proteins that are activated by Ras AP-1, cyclin D1 and E2F-1, and may also interact with cyclin D1 to suppress growth (11, 13). Immunoprecipitation and western blotting have demonstrated that merlin inhibits the growth hormone (GH) stimulated interaction of Ras and Raf-1 in both wild type and dnf2 deficient mouse embryonic fibroblast cells (17). It is thought to use an indirect mechanism, however, because merlin was not in the Ras-Raf-1 complex. Raf-1 can be activated by localizing to the membrane, an action that is mediated by Ras, and also by phosphorylation on multiple residues (17). Merlin can decrease the phosphorylation of these residues, which indicates that merlin can inhibit the MAPK/ERK pathway via Raf-1 as well (17). The ability of merlin to regulate the MAPK/ERK pathway through Raf has also been observed in osteosarcoma cells (6).

37 29 In addition, merlin also inhibits RalGDS (Ral guanidine nucleotide dissociation stimulator), a protein that operates downstream of Ras in response to growth stimuli (6, 17). Merlin interacts directly with RalGDS using residues and (6). Thus, merlin inhibits RalGDS directly and indirectly, through suppression of Ras signaling. As demonstrated by the multiple proteins that merlin interacts with, merlin can affect the MAPK/ERK pathway on many different levels and through direct and indirect mechanisms. Tumor suppression may require a combination of some or all of these pathways, and likely involves at least Ras and Rac inhibition (9). Merlin and MLK3 Studies in Drosophila have indicated that merlin has significant apoptotic and antiproliferic effects. The Hippo (Hpo) pathway in Drosophila is often used as a model for the MAPK pathways in humans because the protein hpo helps control the normal cell cycle, and decreased hpo results in increased proliferation and lack of normal apoptosis (3). The Drosophila analog of merlin (also called merlin) regulates hpo. In mammals, Mst2 (mammalian sterile-20-line kinase 2) is a homologue of hpo, and Raf is a likely negative regulator of Mst2. MLK3 is known to regulate this interaction (3). If there is mechanism conservation between Drosophila and mammals in this particular interaction, it could mean that the MLK3-Raf regulation of Mst2 also involves merlin (3). Already it is known that merlin can disrupt the B-Raf, Raf-1 complex, so it would not be surprising if merlin and MLK3 interact to antagonistically regulate the B-Raf, Raf-1 complex (3). In fact, recent studies have shown that merlin does disturb complexs of MLK3/B- Raf and MLK3/Raf-1, though merlin s affect on the MLK3/B-Raf/Raf-1 complex has not

38 30 yet been observed (4). In addition, merlin has been shown to inhibit JNK activation that occurs through MLK3, possibly by decreasing the effect of extracellular signals that JNK would otherwise receive (4). Although not inducible by external stimuli, normal levels of MLK3 in cells may help regulate ERK signaling (4). MLK3 and merlin have also been co-precipitated from HEK293 cells, and it was determined that levels of this complex were not changed by serum deprivation (4). It has also been shown that proliferation of NF2 cells requires MLK3, and merlin expression inhibits MLK3 kinase activity (3, 4). Another piece of evidence supporting a potential connection between merlin and MLK3 lies in their localization on the cell membrane. As described previously, merlin is thought to reside in lipid rafts (16). Other experiments have shown that MLK3 can be triton insoluble, and it is proposed that inactive MLK3 may be found in caveloae or lipid rafts, perhaps where merlin resides (2). Thus, there is a tentative connection between MLK3 and merlin that, once explored, could have important implications for NF2, neurodegenerative diseases or even cancer, especially as both merlin and MLK3 have been potentially linked to cancer. Therefore, the purpose of this study is to explore the relationship between merlin and MLK3 by determining which portion of merlin binds to MLK3 and whether that portion of merlin is sufficient to inhibit MLK3 kinase activity.

39 31 Materials and Methods Gelatinase Assay SKOV3 cells, a line of ovarian cancer cells, were cultured at 37 C and 5% CO 2 in DMEM, with 10% fetal bovine serum, treptomycin and penicillin. SKOV3 cells were transfected with no sirna, murine MLK3 sirna and human MLK3 sirna. For transfection, 5 µl Lipofectamine 2000 (Invitrogen) was incubated with the sirna for 20 minutes to make tranfection complexes. The transfection complexes were then incubated with the cells for four hours. After this time, fresh media with fetal bovine serum was added. The media was saved for the gelatin gel, and then 6x sample buffer was added to cells. The cells were scraped into tubes and the sample was homogenized to get the cell extracts for the SDS PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) gel. The media from these cultures were ran on an SDS PAGE gelatin gel (10 ml of 30% acrylamide, 7.5 ml of 4xTris at ph 8.8, 9.48 ml water, 160 µl APS, 16 µl Temed, 3 ml of 10 mg/ml geletin) at 65 V for about 18 hours. The gel was incubated in washing buffer for three hours (2.5% triton in water), digestion buffer for one week (10 mm CaCl, 20 mm Tris acetic acid, ph 7), coomassie blue stain (40% methanol, 10% acetic acid, 0.4 g coomasie blue), and destain (70% methanol in water). A picture was then taken of the gel. The cell extracts were also run in duplicate on an SDS PAGE gel (15 ml of 30% low bis acrylamide, 7.5 ml of 4xTris ph 8.8, 7.5 ml water, 120 µl APS and 12 µl Temed) at 65 V for about 18 hours and it was subjected to Western Blotting. A protein marker (EZ Run Pre-Stained Protein Ladder, Fisher) was run as well in order to visualize

40 32 the sizes of the resulting bands. The proteins were transferred from the gel onto a polyviniylidene diflouride membrane (Millipore) membrane using 500 mamps for about 3 hours. The membrane was cut so that each half contained one of each sample and the cut membranes were stained with coomassie blue stain for 5 minutes, then destained. They were rinsed with PBS (phosphate buffered saline) and then put into blocking solution (5% dried, non-fat milk in PBS) for 3x15 minutes. One cut membrane was then incubated in ERK2 antibody, and the other in MLK3 antibody at 1:1000 dilutions in antibody buffer (PBS, 0.05% Tween-20 and 5% dried, non-fat milk) for 2 hours with constant rotation. They were washed with washing buffer (PBS and 0.05% Tween-20) for 3x15 minutes then blotted with a secondary antibody, GAR (goat anti-rabbit) conjugated with horseradish peroxidase, overnight at a 1:5000 dilution in antibody buffer with constant rotation. The membrane was removed from the secondary antibody buffer, washed in washing buffer 3x15 minutes and developed using Immobilion chemiluminescence-enhancing solutions (Millipore). Protein Expression and Purification His-tagged full length MLK3 and GST fusions of merlin aa 1-332, , , and were expressed in BL21 One Shot Cells (Invitrogen). IPTG was added to induce expression. The cells were lysed in lysis buffer (50 mm Tris, ph 7.8, 60 mm NaCl, 2mM DTT, 1 mm EDTA, 4mM benzamide, 0.05% w/v Triton X-100, 1 mm PMSF). Lysozyme and DNAse was added, and after centrifuging, the supernatant was saved and GSH beads (for the GST-Merlin fusion proteins) or Nickel Agarose beads (for the His-tagged MLK3) were added. The proteins were eluted by washing in lysis buffer

41 33 and adding free gluthathione (adjusted to ph 7.8) for the GST fusion proteins or imidazole for the His-tagged proteins. They were then dialyzed overnight with 25 mm Tris, ph 7.4, 5 mm EGTA, 2 mm DDT, 0.1% w/v Trition X-100, and 50% v/v glycerol. The resulting purified protein was stored at -20 C until run on a gel. Western Blot Analysis of GST-Merlin Fusions and Binding Assay The purified full length and truncation mutants of merlin and His-MLK3 were incubated together in vitro and pulled down with glutathione agarose. For the incubation, Tubes 1-3 consisted of 10 µl His-tagged full length MLK3; 15 µl merlin fragment (Tube 1), 15 µl merlin fragment (Tube 2) or 15 µl merlin fragment (Tube 3); plus 20 µl of GST beads, 0.5 µl PMSF, and 500 µl lysis buffer. Tube 4 consisted of 10 µl His-tagged full length MLK3, 40 µl merlin 1-590, 20 µl GST beads, 0.5 µl PMSF and 500 µl lysis buffer. Tube 5 was a control and consisted of 10 µl Histagged full length MLK3, 20 µl GSH beads, 0.5 µl PMSF and 500 µl lysis buffer. These were incubated for three hours, centrifuged, washed once in lysis buffer and twice in wash buffer, boiled to denature the proteins and ran in duplicate on an SDS gel at 50 V overnight. The membrane was cut in half in order to visualize both the GST-fusion proteins and the results of the binding assay by western blotting. For one half, the primary antibody was GST antibody at a 1:1000 dilution in antibody buffer and the secondary antibody, GAR (goat anti-rabbit) conjugated with horseradish peroxidase, at a 1:5000 dilution in antibody buffer with constant rotation overnight. For the second half of the membrane, the primary antibody was anti-mlk3, at a 1:1000 dilution in antibody buffer

42 34 and the secondary antibody, GAR at a 1:5000 dilution in antibody buffer with constant rotation overnight. The membranes were removed from the secondary antibody buffer, washed in washing buffer 3x15 minutes and developed using Immobilion enhancedchemiluminescence solutions (Millipore). Merlin Truncation Mutant Transfection and Western Blot Analysis HEK 293 cells (Human Embryonic Kidney cells) were cultured at 37 C and 5% CO 2 in DMEM, with 10% fetal bovine serum, treptomycin and penicillin. Transfection complexes were made in four tubes. Tube 1-3 contained 5 µg of mammalian constructs expressing HA-MLK3 and either FLAG-merlin 1-595, FLAG-merlin or FLAGmerlin 1-332, 15 µl Lipofectamine 2000 (Invitrogen) and 1 ml serum-free medium. Tube 4 contained no DNA, but had 15 µl Lipofectamine 2000 (Invitrogen) and 1 ml serumfree medium. These were incubated for 20 minutes before being placed on the cells. After six hours, 5 ml of 20% FBS was added to each dish. Cell extracts were made by adding 600 µl of 6x sample buffer to each dish. The extracts were left on ice for five minutes before being scraped and the sample homogenized with a syringe. The samples were then boiled to denature the proteins and frozen at -20 C until they were run on a gel. The samples were then run on an SDS PAGE gel in duplicate and were subjected to western blotting as described above. Three primary antibodies were used on three sections of the membrane. The first was probed with phosphomlk3 (Thr 277/Ser 281, Cell Signaling Technology) as a primary antibody, the second was probbed with anti- MLK3 (Santa Cruz Technology), and the third was probed with HA-FLAG (Stratagene). All primary antibodies were at 1:1000 dilutions. The secondary antibody for pmlk3 and

43 35 MLK3 was GAR, while the secondary antibody for HA-FLAG was goat anti-mouse (GAM). Both were used at 1:5000 dilutions in antibody buffer with constant rotation. For the pmlk3 blotting, 5% BSA was added to the primary antibody buffer instead of the dried milk. The membranes were removed from the secondary antibody, washed in washing buffer 3x15 minutes and developed using Immobilion enhancedchemiluminescence solutions (Millipore).

44 36 Results Effect of Silencing MLK3 on Metalloproteinase 2 (MMP-2) in SKOV3 cells Matrix Metalloproteinases are enzymes that are known to degrade the extracellular matrix, which allows a cell to take on increased invasiveness and possibly metastasize. It has been shown that MLK3 affects SKOV3 cell invasiveness, but the mechanism for this was not clear. This experiment was intended to determine whether MLK3 s affect on SKOV3 cell invasiveness was mediated by MMP-2. MMPs are secreted from cells into their medium, so in a gelatinase assay, cell media are taken from cultures and ran on an SDS PAGE gelatin gel. This will separate them according to their size. When separated on the gel, the active MMPs will digest the gelatin that is in the gel just as they would do to the extracellular matrix in a cellular environment. When the gelatin gel is stained with coomassie blue, uniform binding occurs everywhere except where there is MMP activity. In these areas, a white band appears because there is no gelatin to which the coomassie blue can bind. In the results presented here, the uniform binding on the gel can be seen with white bands for MMP-2 activity. It was determined that the bands shown are MMP-2 because of its size compared to the standard. The negative control and mouse sirna control, which were transfected with no sirna or murine MLK3 sirna show similar levels of MMP-2. When human MLK3 sirna is transfected into the SKOV3 cells, however, the activity of MMP-2 clearly decreases. The immunoblot shows that the MLK3 silencing was effective and the equal levels of ERK indicate equal levels of expression and loading of cells.

45 37 Figure 3: Silencing MLK3 Inhibits Matrixmetalloproteinase 2 (MMP-2) Activity in SKOV3 Cells. SKOV3 cells were transfected with no sirna, murine MLK3 sirna and human MLK3 sirna and the cell medias were subjected to a gelatinase assay. Cell extracts were separated by SDS PAGE and Western blotting was performed with anti- MLK3 and anti-erk2 antibodies. Western Blot of Expression of GST-Merlin Truncation Proteins It has been shown previously that merlin and MLK3 associate together and can be coimmunoprecipitated (4). In addition, merlin has been implicated in the regulation of the MAPK pathways and shown to reside in lipid rafts where MLK3 may also be found (2, 3, 4, 16).

46 38 One of the project goals for this experiment was to determine which region of merlin bound to MLK3 and to demonstrate using purified proteins isolated from bacteria that this binding was indeed a direct association rather than one mediated by another protein. To explore this, purified His-tagged MLK3 and GST-fused merlin fragments of 1-332, and were incubated together with GSH beads, as was a sample with His-tagged MLK3 and GST-fused full length merlin, which served as a positive control. A negative control consisting of His-MLK3 and GSH beads with no merlin was also incubated and ran alongside the other samples on an SDS-PAGE gel. In order to visualize whether or not the GST-merlin truncation and full length proteins had been successfully transfected and expressed, the binding assay samples were blotted with anti- GST. Here, the samples for the binding assay were separated on an SDS gel and Western blotted using anti-gst antibody. This serves as a control by ensuring that the results from the binding assay are due to relatively equal levels of GST. The negative control had MLK3 but no merlin fragments, so no strong band is evident, as is expected because it would not have been pulled down by GSH beads. The sample in lane 2 is 1-332, and it shows a large amount of protein. Merlin shows a smaller amount and merlin shows a moderate amount. The full length band should appear around 92 kda, but it did not show up well. This is common in many publications. Instead, a band appears at a smaller size similar to the merlin fragments, which may because of degradation. Other blots ran in the lab have showed the full length at the appropriate size. Despite not seeing the full length, we can see that all of the merlin mutants did successfully express.

47 39 GST fusion with full length merlin GST fusion with merlin truncation mutants Figure 4: Expression of the GST-Merlin Full Length and Truncation Mutants. Histagged MLK3 was incubated with GST-merlin 1-332, , or truncation mutants and GSH beads. A negative control consisted of His-tagged MLK3 with GSH beads but no merlin was included. The incubation lasted three hours, after which the samples were heated and then the proteins separated by SDS-PAGE. Anti-GST was used as a primary antibody and GAR-HRP as secondary antibody.

48 40 Western Blot Analysis of a Binding Assay Between His-MLK3 and GST-Merlin Full Length and Truncation Mutants As described previously, a goal of this project was to determine what region of merlin bound to MLK3 directly. MLK3 has been implicated in cancer and is known to stimulate the three major MAPK pathways: ERK, SAPK and p38. MLK3 is known to associate with merlin, and there are several theories as to how this might come about, including through interactions that take place on lipid rafts and through other complexes, such as those involving Raf (2, 3, 4, 16). To establish evidence for a direct binding between one terminus of merlin and MLK3, rather than only an indirect association, a binding assay was set up in order to describe the interactions between GST-fusion merlin truncation mutants and His-tagged MLK3, using GST-fusion full length merlin and His-tagged MLK3 as a positive control incubation and His-tagged MLK3 with no addition of GST-merlin as a negative control. GSH beads would bind specifically with the GST-fusion proteins so that fragments of merlin that bound to MLK3 could be specifically identified. These samples were incubated for three hours and then ran on an SDS-PAGE gel, and immunoblotted using anti-mlk3 antibody. This gave bands only where MLK3 could be seen. Because the experiment used GSH beads to pull down the samples, only those samples that had specific MLK3-merlin interactions should be evident on the western blot (Figure 5). The negative control shows minimal amount of binding, which, because there is no GST-merlin in the sample to bind to the GSH beads, indicates non-specific binding. The merlin mutant aa shows a small amount of binding, similar to the negative

49 41 control. The aa mutant of merlin shows no clear band, but aa shows excellent binding, as does the full length form of GST-merlin. This means that the N- terminus is binding more strongly than the C-terminus. His-MLK3 Figure 5: Interaction Between His-MLK3 and GST-Truncation Mutants of Merlin. His-tagged MLK3 was incubated with full length merlin, merlin 1-332, merlin and merlin GST pulldowns were performed and the samples were separated by SDS PAGE and Western blotting was performed with anti-mlk3 antibody.

50 42 Effect of Merlin Mutant on MLK3 Kinase Activity Merlin s C-terminus has been shown to be different than the closely related ERM proteins, and is important in folding, regulation by dimerization, and regulation by phosphorylation. Magician, a protein that may be important in the Ras pathway, is also thought to bind with merlin s C-terminus (6, 12). Because it was determined that the C-terminus of merlin was binding directly to MLK3, an experiment was next performed in order to determine if the aa region of Merlin was sufficient to inhibit MLK3 kinase activity. Full length merlin exists in two isoforms: Isoform I consists of exons 1-15 and 17, or amino acids 1-595, while Isoform II consists of exons 1-16, or amino acids In previous experiments, Isoform I was used, but Isoform II has been shown to have more growth suppressive activity, so it was used for this experiment in order to get the best idea of how it affects MLK3 kinase activity. Mammalian constructs expressing HA-MLK3 and full length, aa or aa FLAG-Merlin were transfected into HEK293 cells. Cell extracts were subjected to Western blotting with anti-phospho MLK3 (Thr 277/Ser 281) antibody (Figure 6). In the untransfected control cells, active MLK3 was detected. In the cells expressing the merlin mutant, MLK3 kinase activity appears at the same level as the control. Cells expressing the merlin mutant however, show reduced MLK3 kinase activity, as seen by the decreased levels of pmlk3. The cells expressing full length merlin also show have reduced MLK3 kinase activity, as is expected. The levels of total MLK3 in each cell extract is comparable, which serves as a control for loading.

51 43 MLK3 + Merlin mutants AB: pmlk3 MLK3 + Merlin mutants AB: MLK3 pmlk3 Total MLK3 Figure 6: The Effect of GST-Merlin Mutants on MLK3 Activity. Mammalian HA- MLK3 was transfected into HEK293 cells with full length, aa or aa FLAG-tagged merlin. Cell extracts were prepared and separated on an SDS-gel. Immunoblotting of the cell extracts was performed with anti-phospho MLK3 (Thr 277/Ser 281) antibody and anti-mlk3 antibody. The membrane was also probed with anti-flag antibody in order to visualize the merlin -truncation mutants (Figure 7). From this blot, it can be seen that the mutant was expressed. The mutant expression was much lower but was evident in a longer exposure. Despite these low levels, the mutant still inhibits MLK3 kinase activity, as seen in Figure 6.

52 44 Shorter Exposure Longer Exposure Figure 7: Anti-FLAG blot of MLK3 Kinase Activity Experiment. Mammalian HA- MLK3 was transfected into HEK293 cells with full length, aa or aa FLAG-tagged merlin. Cell extracts were prepared and separated on an SDS-gel. Immunoblotting of the cell extracts was performed with anti-flag antibody to visualize each of the merlin truncation mutants.