MCB Cell Signaling Lecture 2

Transcription

1 MCB Cell Signaling Lecture 2 Ken Blumer Dept. of Cell Biology & hysiology 506 McDonnell Sciences kblumer@wustl.edu

2 Main concepts Lecture 2 Small GTases (Ras etc)- Differ from big (trimeric) G proteins Lots of them; do many things, often in cascades Mapping functions/pathways (D-neg & CA epistasis) GF receptors & Y kinases Drive prolif, differentiation, development, cancer Discovery involved accidents GFRs dimerize and trans phosphorylate Trigger diverse signaling via allostery, recruitment etc Yp produces protein binding sites Modular domains in signaling proteins (SH2, SH3, H etc) utting cascades together (MAKs, I3K, LCgamma etc) Sometimes switch-like behavior Shutting things off Lipid phosphatases (TEN) Yp phosphatases

3 Signal Transduction by G proteins Discovery and Structure of Heterotrimeric G proteins Signaling pathways of G proteins Receptors that activate G proteins Small G proteins-discovery and structure Activation and inactivation mechanisms Alliance for Cell Signaling (AfCS)

4 Ras genes first identified in 60 s as transforming genes of rat sarcoma viruses. Weinberg, Varmus, Bishop and others in the early 80 s showed that many cancer cells have mutated versions of ras. Discovery of Small G proteins Signaling GTases are Allosteric Switches Ras = classical monomeric GTase Activated form of ras found in 90% of pancreatic carcinomas, 50% of colon adenocarcinomas, and 20% of malignant melanomas. γ -phosphate Swi1 Ras-GT vs. Ras-GD Swi2 Binding γ-phosphate changes the conformations of two small surface elements, called switch 1 and 2

5 Gαt-GT vs. Ras-GT Swi3 Swi1 Swi2 Gα α-helical domain Ras

6 Ras relatives: Rho/Rac/Cdc42 In early 90 s, Alan Hall discovered that newly characterized Ras homologs (Rho, Rac, Cdc42) induced cytoskeletal changes. Actin Stress fibers Focal adhesions Lamellipodia Filopodia Hall, Science 1998

7 Ras superfamily of small G proteins Takai, et al. hysiological Reviews, 2001

8 GTases: How to use reverse genetics to identify their roles in cell regulation Depends on understanding how the machines work Epistasis question: Where in a pathway does a specific protein convey its particular message? A B C D E M N Q Response Idea: 1. Inhibit activity of the protein of interest 2. Increase activity of the protein of interest How to do this? Drugs, genetic diseases, mouse KOs, and...

9 Reverse genetics: express one or two mutant versions of the protein of interest Depends on understanding how the machines work 1. Inhibit activity of the endogenous protein with a dominant-negative interfering mutant of that protein The mutant titrates (binds up) a limiting component to block the normal protein s signal 2. Increase activity of the protein with a dominant-positive or constitutively active interfering mutant of the protein The mutant exerts the same effect as the normal protein would, if it were activated in the cell

10 Reverse genetics: small GTases as examples Depends on understanding how the machines work Dominant-negative mutation GD GEF Dominant-positive mutation Binds GEF but cannot replace GD by GT; so GEF not available for activating normal protein GEF GD GT Cannot hydrolyze GT, so remains always active i GA The mutant titrates (binds up) a limiting component to block the normal protein s signal The mutant exerts the same effect as the normal protein would, if it were activated

11 Reverse genetics: advantages/pitfalls of using dominant-interfering mutants ro: Quick-and-dirty; no biochem Many different families of signaling proteins amenable... once we understand how one of them works Examples: RTKs? Other kinases? Adaptors? Con: Dominant-negatives Over-expression can titrate too many proteins (or the wrong proteins Dominant positives Not always precise mimics of the normal protein (e.g., may be in the wrong place)) Can induce adaptation, turn-off mechanisms Therefore... Hard to apply to complex networks Still need biochemistry

12 Hierachy of small G protein activation Use of constitutively active or dominant negative mutant small G proteins revealed that ras and cdc42 can activate rac. Rac, in addition to inducing lamellipodia, also activates Rho. Ras Takai, et al. hysiological Reviews, 2001

13 Rho/Rac/Cdc42 signaling in actin assembly Takai, et al. hysiological Reviews, 2001

14 Signal Transduction by G proteins Discovery and Structure of Heterotrimeric G proteins Signaling pathways of G proteins Receptors that activate G proteins Small G proteins-discovery and structure Activation and inactivation mechanisms

15 Small G protein turn on mechanisms First mammalian GEF, Dbl, isolated in 1985 as an oncogene in NIH 3T3 focus forming assay. It had an 180 amino acid domain with homology to yeast CDC25. This domain, named DH (Dbl homology) is necessary for GEF activity. In 1991, Dbl shown to catalyze nucleotide exchange on Cdc42. Dbl= Diffuse B-cell lymphoma Schmidt & Hall, Genes & Dev. (2002)

16 Ras Turn Off: Identification of RasGA McCormick injected Xenopus oocytes with oncogenic ras (V12) versus wt ras (G12) and monitored germinal vesicle breakdown (GVB) (top panel) Then loaded ras with α- 32 GT, injected into oocytes, did immppt at increasing times and determined if GT or GD was bound (bottom panel) Rate of GT hydrolysis is 300-fold faster in oocytes than in vitro! urified the factor that promoted GTase activity, cloned and named it GA (or ras-ga). Another ras-ga later identified is NF1 (the gene mutated in neurofibromatosis, i.e., Elephant Man Syndrome). % GVB % Ras-GT V12 (oncogenic) Ras G12 (WT) Ras [ras] (ng) V12 G12 Time (min)

17 RhoGAs outnumber the small G proteins Rho/Rac/Cdc42 by nearly 5- fold. Why so much redundancy? Luo group did RNAi against 17 of the 20 RhoGAs in fly. Six caused lethality when expressed ubiquitously. Tissue specific expression of RNAi revealed unique phenotypes. 190RhoGA implicated in axon withdrawal. Increasing amounts of RNAi caused more axon withdrawal (panels C-G). Many RhoGAs Why so many RhoGAs? Billuart, et al. Cell (2001)

18 Rho/Rac/CDC42 activation of downstream effectors Rho Effectors: I 3-Kinase, LD, Rho Kinase, Rhophilin, and others. Rac-interacts via a CRIB domain in downstream effectors. CRIB (Cdc42/ Rac interacting binding) Effectors: NADH oxidase, AK, I 3-Kinase, MLK2,3, OSH, DGK Cdc42 Effectors: I ε-kinase, AK, WAS, S6-Kinase, MLK2,3, Borg

19 Conclusion: GTases are switches Schmidt & Hall, Genes & Dev. (2002)

20 Growth Factors and Receptor Tyrosine Kinases RTK s--how do they work? EGFR signaling and ras MA kinase cascades I3K, KB, LCγ Ts (rotein Tyrosine hosphatases)

21 Growth factor signaling: Learning from mistakes Neurotrophic growth factor (NGF) isolated from mouse submaxillary glands Side effects of impure NGF preps remature eyelid opening, tooth eruption ure Tooth-lid factor = EGF Regulates growth/differentiation of many target cells Levi-Montalcini Cohen no EGF EGF 1 µ g/kg 1986 Nobel prize

22 Mistake #2: Tony Hunter & hosphotyrosine Kinases phosphorylate tyrosine (Y*) residues of target proteins Y~ = target for distinctive protein binding pockets, with surrounding sequences lending specificity ALWAYS activate by promoting proximity of proteins A and B A (sometimes by allostery also) X B YK A X B Y~ provides long-lasting but erasable memory, which is terminated by DE-phosphorylation *Y = one-letter code for tyrosine; S = ser, T = thr, etc. A X In its new proximity to A, B s activity (= X) can now: hosphorylate or de-phosphorylate another protein Make or degrade a 2nd messenger Attract additional signaling molecules

23 hospho-tyrosine signals regulate growth & differentiation RTKs = Receptor Tyrosine Kinases Extracellular region variable, with many different motifs Usually cross membrane only once Intracellular region contains conserved catalytic domains ALSO: YK-linked receptors for: Antigens (receptors on B and T cells Growth hormone Interleukin-4 Erythropoietin, many others Alberts, 15-47

24 How RTKs (& YK-linked Rs) work 1. Ligand promotes formation of RTK dimers, by different mechanisms: Ligand itself is a dimer (DGF) One ligand binds both monomers (GH) 2. Dimerization allows trans-phosphorylation of catalytic domains, which induces activation of catalytic (Y-kinase) activity 3. Activated YK domains phosphorylate each other and proteins nearby, sometimes on multiple tyrosines 4. Y~ residues recruit other signaling proteins, generate multiple signals EGF receptor as a model 1st RTK to be characterized v-erbb oncogene = truncated EGFR

25 Evidence for EGFR dimerization Yarden & Schlessinger Rate of phosphorylation = k[egfr] 2, even in micelles! Therefore: 2 EGFRs required for phosphorylation Later confirmed by Chemical cross-linking FRET Dominant-negative mutants (e.g., kinase-dead EGFR) IMORTANT Dimerization/proximity = alternative to allostery (Shown by swapping EC/IC domains of EGFR, DGFR)

26 How do we know that the EGFR autophosphorylates in trans? Experiment: test WT and short EGFRs, each with or without a kin - mutation wt + wt kin short kin - + short kin Kinase phosphorylation Does this result rule out phosphorylation in cis as well? If not, how can you find out? S: What do trans and cis mean?

27 How can we know that the EGFR does not autophosphorylate in cis? Need an EGFR that cannot homodimerize EGFR family is huge, with many RTK members and many EGF-like ligands Such receptors often form obligatory heterodimers with a similar but different partner If A can dimerize only with A, then we can inactivate the kinase domain of A and ask whether A phosphorylates itself Answer: NO QED

28 How does dimerization activate RTKs? GFRs (like many kinases) have sites in their T loops at which phosphorylation activates Dimerization induces T-loop phosphorylation in trans T-loop Cat. loop hosphorylation of Y (one or more) in T-loop causes it to move out of the way of the active site. Y1162 occupies the active site Substrate Y sits in active site Y1162 flips out roximity by itself is usually enough to promote T-loop phosphorylation, but there is good evidence for allostery too Once activated, each monomer can phosphorylate nearby Y residues in the other, as well as in other proteins

29 Therapeutic relevance of receptor dimerization in macular degeneration and cancer

30 Growth Factors and Receptor Tyrosine Kinases RTK s--how do they work? EGFR signaling and ras MA Kinase Cascades I3K, KB, LCγ Ts (rotein Tyrosine hosphatases)

31 Signals generated by the EGFR Individual Y~ residues recruit specific proteins, generate different signals SOS, a Ras GEF LC-γ I3-kinase The activated dimer phosphorylates itself... T-loop only Docks via intermediate adapters to activate Ras Ras activates multiple targets (MAK) Multiple sites Docking of Y-kinases allows Tyr-phos n of LC-γ, which activates it Adapters again Docking allosterically activates I3K Each signal, in turn, activates a different set of pathways, which cooperate to produce the overall response.

32 Adapters connect A with B, B with C... to create complex, localized assemblies of signaling proteins Adapter 2 Each adapter has at least 2 interaction domains, and may have other functions as well A B C Types of adapter interactions Adapter 1 Y~ binding domains allow regulatable adapter functions SH2 Tyrosine phosphates Also TB SH3 DZ leckstrin homol. (H) Many others Tyrosine phosphates olyproline-containing sequences Specific 4-residue sequences at C-termini hosphoinositides

33 SH2 & SH3 domains--src homology domains SH domains are protein domains initially discovered in Src, a transforming tyrosine kinase found in Rous sarcoma virus. Sequences of many signaling proteins that interact with RTKs revealed multiple homologous domains to Src region 2 and region 3. SH2: rotein motif of ~100 amino acids, binds to phosphotyrosine peptide sequences. (87 SH2 in the human genome) SH3: ~60 amino acid domain, binds to R-X-X--X-X- peptide sequences. (143 SH3 in the human genome) Lodish, How would you determine the specificity of an individual SH2 domain for a phosphopeptide?

34 EGF activates the MAK pathway in multiple steps, with multiple mechanisms EGF EGFR Mechanism roximity Allostery EGFR~ Extracellular GF Grb2 Small GTase Covalent modification RTK SOS Ser kinase hospho-rtk Ras Tyr/thr kinase Adapter Raf Ser kinase Ras-GEF Mek Transcription factor ERKs C-Jun

35 Fly eye consists of ~800 ommatidia, an individual lens structure consisting of 22 cells (8 photoreceptor cells, R1-R8) Eye development is highly ordered process. RTK signaling is essential. Mutation in sevenless results in loss of R7. Additional mutations in pathway identified sos (son-of-sevenless), boss (bride of sevenless), Drk (downstream of receptor kinase) Fly genetics to the rescue Alberts, 15-53

36 EGFR Activation of Ras: roximity & Allostery RTK = EGFR The layers.. Ras GD Rat Sarcoma Small GTase, attached to M by prenyl group SH2 Grb2 SH3 SH3 GF receptor binding 2 Adapter, found in screen for binders to EGFR~ SOS Son of Sevenless GEF, converts Ras-GD to Ras-GT Found in Drosophila, homol. To S.c. Cdc25

37 EGFR Activation of Ras: roximity & Allostery Even before EGF arrives..... Ras GD SOS is ready to go : already (mostly) associated with Grb2 in cytoplasm, in the resting state SH2 Grb2 SH3 SH3 SOS

38 EGFR Activation of Ras: roximity & Allostery Then... Covalent modification.. Ras GD EGF-bound dimers trigger phosphorylation, in trans SH2 Grb2 SH3 SOS SH3

39 EGFR Activation of Ras: roximity & Allostery Then... roximity.. SH2 Grb2 SH3 SOS Ras GD SH3 Grb2 s SH2 domain binds Y~ on EGFR, bringing SOS to the plasma membrane

40 EGFR Activation of Ras: roximity & Allostery Then... Allostery.. SH2 Grb2 SH3 SOS Ras GD SH3 GD SOS now binds Ras-GD, causing GD to dissociate, and...

41 EGFR Activation of Ras: roximity & Allostery Then... Allostery continues.. SH2 Grb2 SH3 SOS Ras GT SH3 GT GT enters empty pocket on Ras, which dissociates from SOS and converts into its active conformation

42 EGFR Activation of Ras: roximity & Allostery Finally... roximity again!.. SH2 Grb2 SH3 SOS Ras GT Raf SH3 GT Ras-GT brings Raf to the M for activation, and the MAK cascade is initiated Raf MAK Cascade

43 How does Ras activate Raf? roximity vs. allostery? Allostery: Ras recruits Raf to the M and activates it directly Ras GT Ras GT Raf* Raf (Cytoplasmic) MAK Cascade roximity: Ras recruits Raf to the M, where it is activated by X Ras GT Ras GT Raf* X Raf (Cytoplasmic) MAK Cascade

44 How can we tell the difference? Does Raf signal (without Ras) when recruited to the M? Experiment Attach a CAAX* box to Raf s C- terminus Express Raf-CAAx in cells, measure activity of MEK, an enzyme downstream in the MAK pathway EXV Raf Raf+RasG12 RafCAAX RafCAAX+Ras17N RafCAAX+RasG12V Stokoe et al. (1994) Science Relative MEK activity *CAAX (A = aliphatic; C = cysteine) is a site for prenylation; prenylated proteins concentrate at the M Answer: proximity + Ras does localize Raf but does not activate it (other proteins do)

45 Growth Factors and Receptor Tyrosine Kinases RTK s--how do they work? EGFR signaling and ras MA Kinase Cascades I3K, KB, LCγ Ts (rotein Tyrosine hosphatases)

46 Diversity of MA Kinase Cascades generic identified by MAK type Johnson & Lapadat (2002) Science 298: 1911 Borrowed from Chan, STKE

47 The best understood MAK cascade MAK = Mitogen-activated protein kinase. Raf-1 A-raf B-raf MAKKK MEK1 MEK2 MAKK. hos n of T-loop Ser residues ERK1 ERK2 MAK. hos n of T-loop Thr and Tyr C-Jun hos n of Ser/Thr Altered gene expression

48 MAK cassettes mediate many different responses Vertebrates Frog oocyte S. cerevisiae Mitogens rogesterone Mating pheromone MAKKK MAKK MAK MAKKK MAKK MAK MAKKK MAKK MAK roliferation G2-M transition Cell cycle arrest, mating Different biology, similar cassettes: why 3 kinases? Additional sites for regulation Combinatorial diversity Magnitude amplification Switch-like responses

49 Switch-like behavior* Responses are not always graded Frog oocyte rogesterone Instead Response1.0 MAKKK MAKK MAK Stimulus (multiples of EC50) G2-M transition Amplified sensitivity: reduces low stimulus; reversible Bistable responses: off or on, often via positive feedback & used for irreversible responses (e.g., cell cycle) Other examples? *JE Ferrell, Tr Bioch Sci 22:288, 1997

50 All or nothing response in Xenopus oocytes rogesterone, or fertilization, induces germinal vesicle breakdown of Xenopus oocytes--a process mediated by the MAK cascade. Question: At a concentration of progesterone that halfmaximally activates MAK (0.01 um, panel A), are all the oocytes activated halfway (panel B), or are half of the oocytes activated fully (panel C)? Since Xenopus oocytes are HUGE, one can look at MAK on a cell by cell basis. Answer: All or nothing. Ferrell, et al., Science (1998)

51 Of course, life is not so simple... BONUS slide Does this work in mammalian cells? Blenis and co-workers used FACS and immunohistochemistry (anti-d ERK Ab) to look at EGF activation of ERK in Swiss 3T3 fibroblasts MacKeigan MCB 2005

52 Scaffolds for MA Kinase signaling Deletion analysis of the binding of JI-1 to JNK1, MKK7, MLK3, and DLK. JI-1 was expressed in cells as a GST fusion protein together with HK1 or epitope-tagged JNK1, MKK7, MLK3, and DLK (15, 16). The presence of these kinases in glutathione-agarose precipitates was examined by protein immunoblot analysis. HK=hematopoeitic progenitor kinase DLK=dual lineage kinase (member of the MLK family) Whitmarsh et. al. (1998) Science 281: 1671

53 Various scaffolds couple inputs to JNK activation Dhanasekaran (2007) Oncogene

54 Distinct scaffolds in ERK-signaling pathways Dhanasekaran (2007) Oncogene

55 Growth Factors and Receptor Tyrosine Kinases RTK s--how do they work? EGFR signaling and ras MA Kinase Cascades I3K, KB, LCγ Ts (rotein Tyrosine hosphatases

56 EGFR Activation of I3K combines roximity & Allostery.. SH2 I2 I3 SH2 p85 p110 Activated by EGFR/p85 Can also be activated by Rac or Ras! Recruitment from cytoplasm to M, via SH2 domains SH2 SH2 p85 p110 How do we know proximity is not enough? 1. p85 mutants that activate without binding to RTKs 2. Tethering to membrane does not activate

57 I3-K pathway and Cancer Syndromes GF RTK Cancer Syndromes I3 I3-K p Lipid Kinase GI, Brain, Ovarian TEN Lipid Tase Cowden s, Multiple Akt1/2 Ser/Thr Kinase ancreas (Tuberous Sclerosis Complex) Hamartin TSC1 Tuberin TSC2 Ras GA TSC (Ras-homology enriched in brain) (Target of rapamycin) RheB mtor Small GTase Kinase S6K 4EB-1 Kinase Inhibitor of eif4e rotein synthesis Kovich & Cohen (2004) Dematology Online Journal 10: 3. erelman (2004) Dematology Online Journal 10: 17. Cell growth/size/survival

58 I3 targets include many GEFs, many tyrosine kinases, and others, including... KB (aka Akt) = ser/thr kinase that promotes cell survival I3 (= membrane lipid) H K KB... is inactive in cytoplasm... contains a H (pleckstrin homology) domain & a kinase domain

59 Multi-step activation of KB: proximity I3 H roximity to M alone does not activate the kinase K H K H domain recognizes 3 - phosphate of I3, bringing kinase domain to the M

60 Multi-step activation of KB: covalent modification I3 H K DK1* H K Inactive KB Active (phos d) KB *DK1 is also recruited to the membrane via a I3-binding H domain Overall, two proximity steps plus (at least) one phosphorylation step

61 EGFR Activation of LCγ combines THREE inputs.. I3 I2 LCγ (Inactive, in cytoplasm) 1. ROXIMITY: Recruitment from cytoplasm to M, via SH2 domains SH2 SH2 H Catalytic

62 EGFR Activation of LCγ combines THREE inputs 3. ROXIMITY: Binds to I3 via H domain. 2. COVALENT: Activated by EGFR phosph n. SH2 SH2 H Catalytic I2 DAG Ins3

63 Summary: Many RTK effectors require two or more simultaneous inputs for activation I3K: recruitment via SH2, allosteric regulation by EGFR,p85 KB: recruitment, phos n by non-egfr-kinase(s) LCγ: recruitment, phos n, retention at M by binding I3 Why multiple inputs to each effector?

64 RTKs activate a complex network of interacting response pathways (and this is the simple version!) STAT LCγ I3K Active RTK SOS I3K STAT~ DAG Ins3 Rac Ras Cdc42 DK1 S6K KC CaMK ROS T MAK JNK KB GSK3 Targets Targets Targets Targets Targets Apoptosis Targets Targets Targets Targets Targets Targets Nuclear Transcription Factors

65 Growth Factors and Receptor Tyrosine Kinases RTK s--how do they work? EGFR signaling and ras MA Kinase Cascades I3K, KB, LCγ Ts (rotein Tyrosine hosphatases)

66 But how do you shut these things off? Family of rotein hosphatases Tonks & Neel, Curr Op Cell Bio (2001)

67 How Do Ts dephosphorylate specific targets? Intracellular targeting: zip code model Extra domains on Ts confer localization and protein-protein interactions Initially thought that catalytic domains possessed little specificity for RTKs. However, co-crystal structures and biochemistry reveal that some Ts catalytic domains exhibit exquisite sensitivity T-1B critical residues interact with Insulin Receptor T-loop residues Salmeen, et al Mol Cell (2000)

68 TEN opposes I3K by removing I3-phosphate TEN discovered as a tumor suppressor gene. Mutated in brain, breast and prostate cancers. Has homology to dual specificity phosphates, but shows little activity toward phosphoproteins. Was discovered to remove phosphates from Is; thereby providing likely mechanism for tumor suppression. Cantley & Neel, NAS (1999)

69 Gleevec--proof that you can target kinases for drug therapy Goldman & Melo, NEJM, Oct 9, 2003

70 Gleevec--proof that you can target kinases for drug therapy