Cell Signaling II: A circuitous pursuit

Transcription

1 Cell Signaling II: A circuitous pursuit Joe W. Ramos, Ph.D. joeramos@hawaii.edu www2.hawaii.edu/~joeramos From Genes and the Biology of Cancer, Varmus and Weinberg,

2 Epinephrine binds β adrenergic receptors on liver cells to stimulate increase in glucose levels in response to stress PKA=A Kinase=cAPK 2

3 Receptor tyrosine kinases and Ras RTK pathways are involved in regulation of: cell proliferation (EGF) and differentiation (FGF) promotion of cell survival (NGF) modulation of cellular metabolism (Insulin) RTKs transmit a hormone signal to Ras, a GTPase switch protein that passes on the signal on to downstream components 3

4 Six subfamilies of tyrosine kinase receptors Figure from Molecular Biology of the Cell, 4th edn. 4

5 Structure of Platelet derived growth factor (A) The dimeric structure of the protein, with the receptor-binding regions shaded in yellow. The dimer is held together by three disulfide bonds (not shown). (B) Because PDGF is a dimer with two receptor-binding sites, it can cross-link adjacent receptors to initiate the intracellular signaling process. Figure from Molecular Biology of the Cell, 4th edn. 5

6 Ligand binding leads to autophosphorylation of RTKs 6

7 Different Ligands induce RTK dimerization Figure from Molecular Biology of the Cell, 4th edn. 7

8 Activation of receptor tyrosine kinase: forms complex

9 Activation of Ras by an activated receptor kinase Ras is a small monomeric G-protein similar to the alpha subunit of the trimeric G-proteins. It s tethered to the PM. 9

10 10

11 Ras Ras is a small GTPase that controls both proliferation and differentiation pathways. A single amino acid mutation of Ras is found in more than 30% of all cancers. As many as 90% of certain human tumors like pancreatic carcinomas have mutant Ras! Activates Raf/MAP kinase, PI 3 Kinase, RalGDS 11

12 Ras Superfamily of small GTPases All are highly similar and are regulated by GTP binding- they have different effectors and locations in the cell 12

13 Ras cycles between active and inactive forms GEF=Guanine nucleotide Exchange Factor GAP=GTPase Activating Proteins The cytosol concentration of GTP is 10x that of GDP 13

14 Structure of Ras in GTP-bound form 14

15 Structures of Ras GDP-Sos complex and Ras GTP GDP GTP Ras-GDP SOS Ras-GTP 15

16 Mochizuki et al

17 Flourescence Resonance Energy Transfer (FRET) Inactive Ras Active Ras Single protein contains Ras, the Ras binding domain of Raf, and two fluorophores: Yellow fluorescent protein (YFP) and cyan fluorescent protein (CFP). Excite CFP=the energy emitted by CFP is partly captured by YFP which emits energy that peaks at 527 nm. Similar setup for Rap1, another small GTPase. N. Mochizuki, et al., Nature 411: (2001) 17

18 Fig.1: FRET works 18

19 EGF induced Ras activation The Movie Phase FRET CFP YFP Orange FRET = high Ras activity 19 N. Mochizuki, et al., Nature 411: (2001)

20 Ras and Rap1 activation in living cells:different location of active GTPases 20 N. Mochizuki, et al., Nature 411: (2001)

21 Fig 3. NGF Stimulation 21

22 An adapter protein and GEF link most activated RTKs to Ras How do Ras and G Proteins compare? 22

23 Modular binding Domains 23

24 Models of SH2 and SH3 domains bound to short target peptides SH2 SH3 SH2 domain in GRB2 adapter protein binds to a specific phosphotyrosine peptide (Src here) in an activated tyrosine kinase. RTKs Proline rich sequence in Sos, a GEF, binds to SH3 domains in GRB2. 24

25 The yeast two-hybrid system can be used to demonstrate interaction of components in the pathway 25

26 Generalized mass-spectrometry-based proteomics workflow Fractionation can include Immunoprecipitation, affinity resin, subcellular fractionation MS and MS/MS spectra are matched against protein sequence databases The outcome of the experiment is the identity of the peptides and therefore the proteins making up the purified protein population. Nature 422, (13 March 2003) 26

27 Specific Identification of Tyrosine Kinase Substrates Using [ 13 C]Tyrosine This is a MS-based method to identify proteins that are tyrosine phosphorylated in response to a stimulus JBC, vol. 279 no

28 Ras Effectors RBD=Ras Binding Domain RA=Ras Associated domain 28

29 Ras effectors involved in cancer 29

30 Ras transformation in tissue culture 30

31 Analysis of eye development in Drosophila has provided insight into RTK signaling pathways A single ommatidium diagrammed SEM of Drosophila eye: Note the individual ommatidia Wild-type sevenless mutant 31 Figure 20-24

32 Early cell-signaling events in R7 development Figure from Molecular Biology of the Cell, 3rd edn. 32

33 MAP kinase pathways Activated Ras induces a kinase signal cascade that culminates in activation of MAP kinase MAP kinase is a serine/threonine kinase that can translocate into the nucleus and phosphorylate many different proteins, including transcription factors that regulate gene expression 33

34 A Ras activated phosphorylation cascade Ras also activates PI3 kinase and RalGDS pathways. 34

35 Signals pass from activated Ras to a cascade of protein kinases Raf MEK MAP Kinase Raf Raf MAP Kinase MEK MEK MAP Kinase MAP Kinase 35

36 Raf Activation Cycle CR=Conserved Region1,2,3 CRD= Cysteine Rich Domain, Binds Ras/required for activation. Red serines =inhibitory phosph Green serines =activating phosph 36

37 craf regulation by PKA Dumaz and Marais, FEBS J, 272:3491 (2005) PKA prevents craf activation by preventing Ras displacement of

38 Cancer-associated B-Raf mutations Yellow=residues phosphorylated for kinase activity Cancer Mutated residues are in bold type; activating=green; inhibiting=red 38

39 Signals pass from activated Ras to a cascade of protein kinases Raf MEK MAP Kinase Raf Raf MAP Kinase MEK MEK MAP Kinase MAP Kinase 39

40 Phosphorylation of a tyrosine and a threonine activates MAP kinase Substrate sequence is S/ TP Note conformational change in activation loop containing TEY Melanie Cobb has helped define the mechanism of ERK function. 40

41 Selected signaling tools (+) (-) RTKs Dominant negative mutants (Cterm deletions, Kinase mutants Y-mutants) Can t hydrolyse GTP Activated Ras (G12V Mutant) H-Ras Dominant Negative Ras (T17N Mutant) Can t bind Raf Membrane localized Activated Raf (Raf-CAAX) Raf Dominant-negative Raf (RafN4 Mutant) Can t bind MEK Kinase active Activated MEK (222/226D Mutant) MEK Chemical Inhibitors (PD98059) And Dominant-negative mutants Block Raf activation of MEK And Can t bind ERK ERK Dominant-negative ERK (K17A Mutant) Kinase inactive 41

42 Drugs affect function Ozzie! 42

43 Drugs can be used to block or activate specific kinases AG494 (-) (EGFR) Forskolin (+) ZM (-) (c-raf) U0126 (-) (MEK1/2) KT5720 (-) KN62 (-) Chelerythrine (-) PMA(+) 43

44 A MAP Kinase Network: Rac Ligands/Stimuli A MAP kinase cascade Ras PAK *1 Ras MEKK *2 *3 *4 Raf Mos Tpl MEKK MUK SPRK MLK TAK ASK TAO MEKK Tpl MEK MEK1 MEK2 MEK4 MEK7 MEK3 MEK6 MEK5 MAPK ERK1 ERK2 JNK *5 *6 *7 SAPK p38 ERK5 Target Elk-1 Sap1 bhlh Elk-1 Jun *8 Elk-1 Sap1 MEF2C Sap1 MAPKAP RSK NFAT4 ATF2 MAPKAP RSK MEF2C *1: PAK1 PAK2 PAK3 *2: MEKK1 MEKK2 MEKK3 MEKK4 *3: TAO1 TAO2 *4: MEKK2 MEKK3 *5: JNK1 JNK2 JNK3 *6: SAPKα SAPKβ SAPKγ *7: p38α p38β p38γ p38δ *8: Jun-B Jun-D c-jun 44 Courtesy of Melanie Cobb

45 Much work on MAP kinases has been done in yeast Mating pathway in S. cerevisiae Two secreted peptide pheromones called a and α factors control mating between haploid yeast cells. The receptors for these factors are GPCR that activate MAP kinase pathways. Figure from Molecular Biology of the Cell, 3rd edn. 45

46 Multiple MAP kinase pathways are found in Yeast Figure

47 Scaffolds organize MAP kinase pathways:yeast Specificity of Ste11 signal from the scaffold involved 47

48 Scaffolds organize MAP kinase pathways: Vertebrates Ras Raf-1 MEK ERK Membrane Ras Raf MEK1ERK KSR KSR Creates the Scaffold for Ras Activation of ERK 48

49 ERK Regulation Image: Creative Diagnostics, Inc. 49

50 Overview of ERK in Growth and differentiation 50

51 ERK Substrates and Functions 51

52 TGFβ Signaling TGFβRI and TGFβRII are both serine/ threonine kinase transmem- brane receptors. Upon formation of the TGFβ ligated tetrameric receptor complex, TGFβRII phosphorylates the cytoplasmic tail of TGFβRI. This phosphorylation activates the kinase activity of the TGFβRI cytoplasmic domain resulting in recruitment of either Smad2 or Smad3, which are themselves phosphorylated by TGFβRI. Controls proliferation, differentiation, apoptosis and migration. Important in Cancer, autoimmune disorders, infectious diseases and fibrosis among others. 52

53 TNFα receptors signal to NFkB Ubiquitylation is a common signal transduction mechanism (see regulation of cyclins for another example) IKKK=IkB Kinase kinase NFkB activates transcription of several anti-apoptotic proteins including IAPs and Bcl-2. 53

54 Interaction and regulation of signaling pathways The effects of activation of GPCRs and RTKs is more complicated than a simple step-by-step cascade Interaction of different signaling pathways permits fine-tuning of cellular activities 54

55 Four parallel intracellular pathways and their connections 55

56 Two simple mechanisms of signal integration 56

57 Questions 57