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Hanahan and Weinberg (2000) Cell 100: 57.

Signal Transduction Cross-Talk Plays a major Role in Biology. Hanahan and Weinberg (2000) Cell 100: 57. Sixteen (or more) Different Classes of Receptor Signaling Pathways. from Pollard & Earnshaw '02. Overview of MAP kinase pathways. Growth factors and caclium. Cytokines Cellular stress.

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Hanahan and Weinberg (2000) Cell 100: 57.

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  1. Signal Transduction Cross-Talk Plays a major Role in Biology Hanahan and Weinberg (2000) Cell 100: 57.

  2. Sixteen (or more) Different Classes of Receptor Signaling Pathways from Pollard & Earnshaw '02

  3. Overview of MAP kinase pathways Growth factors and caclium Cytokines Cellular stress Stimulus Map KKK Raf MEKKs Upstream Kinases Map KK MEK MKKs MAP Kinase Map Kinase ERK JNK/p38 Proliferation Differentiation Cell survival Inflammation Cell death Response

  4. Map Kinase phospho-relay systems Johnson & Lapadat Science 2003

  5. The JNKS are Stress-Activated Protein Kinases • JNKs bind to and phosphorylate the DNA binding protein c-Jun and increase its transcriptional activity. • c-Jun is a component of the AP-1 transcription complex, which is an important regulator of gene expression. • AP-1 contributes to the control of many cytokine genes and is activated in response to environmental stress, radiation, and growth factors — all stimuli that activate JNKs. • JNKs are important in controlling programmed cell death or apoptosis.

  6. The Small G proteins 1. The small G proteins are about half the size of Gs or Gi. 2. They are active when GTP is bound. 3. They have intrinsic GTPase activity that inactivates them. 4. They serve as general coupling factors in a variety of signaling systems and provide points for signal transduction cross-talk.

  7. G protein Modulators 1.GEFs:Guanylyl Nucleotide Exchange Factors GEFs activate G proteins by stimulating the exchange of GTP for GDP. GEFs can be activated by second messengers. For example there are calcium and cAMP activated GEFs that stimulate Ras. 2. GAPs:GTPase Activating Proteins GAPs stimulate the GTPase activity of G coupling proteins and inhibit G-protein activity. 3. GDIs:GDP Dissociation Inhibitors GDIs block dissociation of GDP from G proteins, thereby inhibiting G protein activity. 4.SCOP: SCN Circadian oscillatory Protein A bifunctional protein that inhibits K-Ras and also has protein phosphatase activity.

  8. Ras GDP/GTP cycle GTP GDP GAP Pi GTPase GAPS = RGS = Regulators of G-protein Signaling

  9. Classic “Map Kinase” Pathway RTK RTK Shc Grb2 Hormone RTK = receptor tyrosine kinase Shc = adapter protein tyr P by growth factors contains PTB, SH2 domains and tyr P sites Grb2 = adapter protein with 1 SH2 & 2 SH3 domains SOS = son of sevenless = GEF for Ras SOS Ras = small G-protein, part of p21 Ras family including H-Ras, K-Ras, N-Ras, & R-Ras Ras GAP = GTPase activating protein GEF = Guanine Nucleotide Exchange Factor Raf cRAF1 = a tyrosine kinase MEKs = Map Kinase Kinases, unusual in that it will phosphorylate both thr and tyr (at least 7) MEK = MAPK/ERK Kinase MEK1/2 ERKs = Extracellular signal Regulated protein Kinase; MAP kinases (mitogen activated kinases) phosphorylated on TEY motif (others include JNKs, SAPKs, & p38 kinase) ERK1/2 Mitogenesis, Differentiation, Proliferation, development, neuronal survival, Memory formation Transcription Factors Cytoskeletal proteins Rsk , MSK1, etc

  10. Nine classes of receptor tyrosine kinases How does receptor occupancy yield activation? from Pollard & Earnshaw '02

  11. Dimerization/ Autophosphorylation Model First Second Schlessinger JBC 273:11987

  12. Schematic of the PDGF receptor Q: Why autophosphorylation? Src PI3K P120 RasGap SHP2 PLCg-1 Nature Reviews Molec Cell Biol 3; 177-186

  13. Diagram of SH2 domain Protein having specific P-tyr sequence Site for binding AA side chain Site for binding P-tyr Protein having SH2 binding site

  14. Cutaway view of an SH2 domain bound to the phosphopeptide, (PNY(P)EEI) The space filling model of the phosphopeptide shows the - backbone in yellow, - side chains in green, and - phosphate in white. Like a two pronged plug the P-Tyr and isoleucine fit into a two-pronged socket on the surface of the SH2 domain (red) Specificity in the sequence surrounding the P-Tyr from Waksman et al Cell 72:779

  15. Erk/MAP Kinase is Regulated by cAMP Cyclic AMP inhibits Erk/MAP kinase in most non-neuronal cells and is generally anti-proliferative. 2. Cyclic AMP stimulates Erk/MAP kinase in neurons and plays a major role in neuroplasticity. This is an example illustrating that signal transduction mechanisms are cell specific.

  16. Model of Rap1 activation by cAMP as regulator of ERKs in a cell type-specific manner Fig. 2. (a) Rap1 activation by cAMP inhibits ERKs. Hormonal stimulation of a Gas/cAMP/PKA module leads to Rap1 activation (GTP loading). Many cells express Raf-1 as the major Raf isoform. In these cells, GTP-loaded Rap1 blocks Ras activation of Raf-1, thereby inhibiting growth factor activation of ERKs and cell proliferation. (b) Some cells express B-Raf as well as Raf-1. In these cells, GTP-loaded Rap1 can activate B-Raf and the mitogen-activated protein (MAP) kinase cascade and hormonal stimulation of cAMP/PKA/Rap1 in these cells activates ERKs. Rap1 might also antagonize Ras activation of Raf-1, as in (a). Rap1 activation of B-Raf often predominates over the inhibition of Raf-1, resulting in a net effect of ERK activation. TRENDS in Cell Biology Vol.12 No.6 June 2002

  17. Protein kinase A antagonizes PDGF-induced MAP kinase signaling in human arterial smooth muscle 100 - 80 - 60 - 40 - 20 - 0 Fig. 2 Forskolin inhibits PDGF-BB-induced activation of MAPKK and MAPK and activates PKA in a dose dependent manner. Human arterial SMCs were incubated with forskolin or vehicle for 30 min. Cells then were stimulated with 0. 3 nM PDGF for 5 min and cell extracts assayed for: MAPK (), MAPKK (o), and PKA( ) activities. PKA % Activity MAP kinase So! Which step in MapK pathway is inhibited? MAP kinase kinase 0 -7 -6 -5 -4 Forskolin Graves et al. PNAS 90: 10300 Nov “93

  18. Likely mechanisms of cAMP/PKA inhibition of ERK activation PKA cAMP can activate Rap1 to antagonize Ras signaling to Raf-1. cAMP activation of PKA activates Rap1 via an Src-dependent pathway (1). PKA might also inhibit Raf-1 by direct phosphorylation at serines 43, 259 and 621. phosphorylation of serine 43 can inhibit the ability of Raf-1 to bind to GTP-loaded Ras (2). cAMP and PKA might interfere with the activation of Raf-1 by activating the serine/threonine kinase Akt, which can also inhibit Raf-1 by direct phosphorylation on serine 259 (3). PKA phosphorylation at serine 621 can inhibit isolated kinase domains, but might potentiate the activity of full-length Raf-1 through 14-3-3 binding (4). TRENDS in Cell Biology Vol.12 No.6 June 2002

  19. Graves and Krebs worked upstream from Raf-1 and MEK MEK overcomes cAMP inhibition of MAP kinase Raf-1 overcomes cAMP inhibition of MEK (B) Extracts from control, PDGF treated, control, forskolin treated and PDGF/forskolin treated cells were incubated with DE-53 ion exchange resin to prepare a partially purified fraction of MAPKK. The DE-52 samples were incubated with activated Raf-1 and Mg2+/ATP at 30o for 30 min, after which 5 ul was removed for MAPKK assay. The rightmost represents activated Raf-1 in the absence of cell extract. (A) Extracts of cells were incubated with purified MAPKK, and MAPK activity was measured in SDS/polyacrylamide gels containing MBP. The intensities of the bands representing the MAPks, (Erk1 and Erk2) were estimated by scanning the exposed film with a densitometer. Activated recombinant Erk2 was used as the standard. Q: how does this order the step for cAMP action? Graves et al. PNAS 90: 10300 Nov “93

  20. CT blocks proliferation cAMP does NOT block activation of Ras by EGFR cAMP does NOT block EGF auto-phosphorylation cAMP DOES block Activation of Raf by Ras D Cook and McCormick worked downstream from receptor CT blocks Erk-1 activity Grb-2/Shc binding Cook & McCormick Science 262: Nov ‘93

  21. Model of Rap1 activation by cAMP as negative regulator of ERKs Rap1 is a small GTP-binding protein having the same sequence at its effector domain as Ras. It therefore can inhibit Ras function (a) Rap1 activation by cAMP inhibits ERKs. Hormonal stimulation of a Gas/cAMP/PKA module leads to Rap1 activation (GTP loading). Many cells express Raf-1 as the major Raf isoform. In these cells, GTP-loaded Rap1 blocks Ras activation of Raf-1, thereby inhibiting growth factor activation of ERKs and cell proliferation. So, this is likely one mechanism by which an inhibitory effect can occur! Q: how is Rap1 regulated by cAMP/PKA? Direct PO4? TRENDS in Cell Biology Vol.12 June 2002

  22. Probably not; Rap 1 Stimulation by cAMP Requires SRC Family Kinases Whoops, yet another player - src

  23. PKA phosphorylates and activates src and active src activates Rap1 A B P- src will activate RAP1 Iso/cAMP --> PO4 src Cbl = a src kinase Note use of S/D and S/A mutants and FLAG tags What is purpose of S/D Src??? From Stork et al, JBC Nov 02

  24. Mechanism for Src Stimulation of Rap1 Activity From Stork et al, JBC Nov 02

  25. Likely mechanisms of cAMP/PKA inhibition of ERK activation cAMP can activate Rap1 to antagonize Ras signaling to Raf-1. cAMP activation of PKA activates Rap1 via an Src-dependent pathway (1). PKA might also inhibit Raf-1 by direct phosphorylation at serines 43, 259 and 621. PKA phosphorylation of serine 43 can inhibit the ability of Raf-1 to bind to GTP-loaded Ras (2). cAMP and PKA might interfere with the activation of Raf-1 by activating the serine/threonine kinase Akt, which can also inhibit Raf-1 by direct phosphorylation on serine 259 (3). PKA phosphorylation at serine 621 can inhibit isolated kinase domains, but might potentiate the activity of full-length Raf-1 through 14-3-3 binding (4). TRENDS in Cell Biology Vol.12 No.6 June 2002

  26. Then what about B-Raf and cAMP Stimulation of MAPK? PC-12 cells vs 3T3 cells

  27. cAMP-dependent activation of MAPK is potentiated by B-Raf In PC12 cells cAMP stimulates MAPK In 3T3 cells cAMP inhibits until B-Raf added THM: Cell type specificity Vossler et al Cell 89:73 ‘97

  28. What difference does it make whether or not a cell has Rap-1/B-Raf? Effect of EGF vs NGF on Duration of Action

  29. Model of Rap1 activation by cAMP as regulator of ERKs in a cell type-specific manner Fig. 2. (a) Rap1 activation by cAMP inhibits ERKs. Hormonal stimulation of a Gas/cAMP/PKA module leads to Rap1 activation (GTP loading). Many cells express Raf-1 as the major Raf isoform. In these cells, GTP-loaded Rap1 blocks Ras activation of Raf-1, thereby inhibiting growth factor activation of ERKs and cell proliferation. (b) Some cells express B-Raf as well as Raf-1. In these cells, GTP-loaded Rap1 can activate B-Raf and the mitogen-activated protein (MAP) kinase cascade and hormonal stimulation of cAMP/PKA/Rap1 in these cells activates ERKs. Rap1 might also antagonize Ras activation of Raf-1, as in (a). Rap1 activation of B-Raf often predominates over the inhibition of Raf-1, resulting in a net effect of ERK activation. TRENDS in Cell Biology Vol.12 No.6 June 2002

  30. Is this the whole story and what about activation of ERK by cAMP dependent GEFs that occurs in some cells ?

  31. Gefs: Direct binding of cAMP to Epac Active Epacs bind cAMP cAMP binding Kd ~ 1 uM Does binding do anything? deRooij & Bos Nature 396, 474 - 477 (1998)

  32. Time course of Rap1A activation by cAMP-GEF (EPAC) From Kawasaki et al Science 282 Dec ‘98

  33. Regulation of Raf/Map Kinases by PKA and GEFs X? X ERKactivity p90Rsk p90Rsk cAMP THM: There are many different mechanisms for regulation of Map Kinase pathways. Different cells and different parts of cell may utilize different mechanisms.

  34. Ca2+ Adenylyl Cyclase 1,8 CaM Ca2+ cAMP GEF Ras B-Raf Rap1 PKA MEK MAPK MAPK p90rsk2 MSK1 p p CBP CREB Transcription Neuroplasticity CRE

  35. High Frequency Stimulation of CREB Phosphorylation in Hippocampal Slices Is mediated Through Erk/MAPK P-CREB was monitored using a phospho-peptide specific antibody that recognizes P-CREB PD 98059 is a MEK inhibitor. KCl depolarizes and increases intracellular free calcium. Impey et al (1998) Neuron 21: 869

  36. Calcium and cAMP Synergistically Stimulate of CREB- Mediated Transcription in Neurons dep KCl depolarization increases intracellular free calcium Forskolin increases cAMP CRE-mediated transcription was monitored using cultured neurons from a CRE-lacZ reporter mouse strain Impey et al (1998) Neuron 21: 869

  37. Ca2+ Adenylyl Cyclase 1,8 CaM Ca2+ cAMP GEF Ras B-Raf Rap1 PKA MEK MAPK MAPK p90rsk2 MSK1 p p CBP CREB Transcription Neuroplasticity CRE

  38. PKA is Required for the Nuclear Translocation of Erk Rp is an Inhibitor of PKA Impey et al (1998) Neuron 21: 869

  39. Calcium Stimulates Erk/MAP Kinase in Neurons 1. Calcium activates adenylyl cyclases in neurons. 2. Calcium stimulates Ras through GEF’s. 3. Calcium stimulates the degradation of SCOP, a negative regulator of Ras.

  40. Mechanisms for Regulation of Adenylyl Cyclase 1. Calcium stimulation-mediated through calmodulin Examples: AC1 and AC8 2. Calcium inhibition of type 3 adenylyl cyclase Mediated through CaM Kinase II phosphorylation of AC3 3. Stimulation by Gs-coupled receptors 4. Inhibition by Gi-coupled receptors 5. Stimulation or inhibition by the beta/gamma complex of G-coupling proteins Examples: AC2 and AC4 are stimulated by beta/gamma. 6. Stimulation by protein kinase C 7. Inhibition by PKA Example: AC5

  41. Some Physiological Roles of the MAP Kinases • Ras/ Erk1,2 MAP Kinase and proliferation 2. Erk1,2 /MAP kinase and neuronal survival 3. Role of the Stress Activated JNK and p38 Kinases in neuronal apoptosis 4. Role of Erk1,2 /MAPK in Circadian Rhythm in the SCN 5. Cell cycle progression and Erk 5 6. Neuronal Cell Fate determination and Erk5 7. Memory Formation Erk1,2, Erk5 required for remote memory

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