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Plasma membrane

Signaling mediated by cAMP and protein kinase A. Hormone. Hormone receptor. Plasma membrane. ATP. G. Adenylate cyclase. G-protein. cAMP. Active Protein Kinase A. Inctive Protein Kinase A. Nuclear membranes. Active pKA enters the

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Plasma membrane

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  1. Signaling mediated by cAMP and protein kinase A Hormone Hormone receptor Plasma membrane ATP G Adenylate cyclase G-protein cAMP Active Protein Kinase A Inctive Protein Kinase A Nuclear membranes Active pKA enters the nucleus and phosphorylates CREB on Serine 133 PO4 PO4 Core promoter Phosphorylation of CREB: - stimulates interactions with several core promoter proteins - induces binding of HAT and acetylation of histones

  2. Adenylate cyclase ATP cAMP (3’,5’cyclic AMP) PPi H2O H2O phoshodiesterase pyrohosphatase AMP 2Pi cAMP formation cAMP hydrolysis

  3. O ║ Substrate─O ─P ─O─ │ OH ATP ADP Substrate-OH Kinase ATP ADP O ║ Protein ─O ─P ─O─ │ OH Protein-OH Protein Kinase Several specific Proteins Including CREB Several specific proteins including CREB O ║ Serine ─O ─P ─O─ │ OH ATP ADP Serine-OH Protein Kinase A

  4. CREB and Memory

  5. Signaling by calcium ions Ca++ enters cell Ca++ activates calmodulin CaM kinases phosphorylate multiple targets including ser 133 of CREB Calmodulin activates CaM kinases

  6. CREB and Memory

  7. A. Amplification: C/EBP is another transcription factor. It activates many genes. (CCAAT box enhancer binding protien) CREB andMemory( in Aplysia, a slug) B. Stimulates long term memory Inhibits long term memory Two forms of CREB: CREB1 stimulates transcription CREB2 represses transcription Bailey, Kandel & Kausik (2004) Neuron 44:49-57

  8. A model promoter HAT Core Promoter Binds general transcriptional machinery Histone Acetyl Transferase CREB CREB Fos Jun NR=“nuclear receptor” pol II SP-1 D B NR NR H F TGACTCA GACGTC TATAAA HRE GGGCGG E HRE (Hormone Response Element) Regulation by hormones such as estrogen which enter the cell SP-1 Site Provides basal unregulated transcriptional activity. Many genes have multiple SP-1 sites AP-1 (Fos-Jun binding site) Regulation by growth factors, stress, and various transmembrane signals CRE (Cyclic AMP Response Element) Regulation by cAMP, and by Ca+. Interacts with core promoter (through CREB Binding Protein) and modifies chromatin structure (through HAT). Positions of these elements are relatively unimportant

  9. Nuclear receptors: An important class of transcription factors Nuclear receptors include: Steroid hormone receptors: Glucocorticoid receptor Estrogen receptors α and β Androgen receptor Progesterone receptor Mineralocorticoid receptor Vitamin D receptor Thyroid hormone T3 receptor Fatty acid receptors PPARα, PPARδ and PPARγ Bile salt receptor Oxysterol receptor Many others

  10. Example of Nuclear receptor functionStimulation of transcription by glucocorticoids Glucocorticoid receptor + glucocorticoid Glucocorticoid receptor Binds to specific DNA sequences and stimulates transcription of glucocorticoid-responsive genes

  11. DNA binding domain of the glucocorticoid receptor (a zinc finger protein)

  12. Activation of steroid receptors The cooactivator functions to recruit other factors that stimulate transcripton such as CBP Example: estrogen The “LLXXL” domain (Leu Leu X X Leu) of the coactivator. Tamoxifen inhibits the movement of helix 12 Binding of estrogen moves helix 12, allowing binding of the coactivator’s LLXXL helix

  13. Phosphorylation of CREB and the CREB binding protein (CBP) HAT coactivator p160 p160 NR NR HRE

  14. SERMS Selective estrogen receptor modifiers SERM Bone Breast Cardio- Uterus vascular 17-Estradiol + + + + ICI 182,780 (antiestrogen) - - - - Tamoxifen + - + + Raloxifene + - + - + Agonist - Antagonist

  15. A model promoter HAT Core Promoter Binds general transcriptional machinery Histone Acetyl Transferase CREB CREB Fos Jun pol II SP-1 D B NR NR H F TGACTCA GACGTC TATAAA HRE GGGCGG E HRE (Hormone Response Element) Regulation by hormones such as estrogen which enter the cell SP-1 Site Provides basal unregulated transcriptional activity. Many genes have multiple SP-1 sites AP-1 (Fos-Jun binding site) Regulation by growth factors, stress, and various transmembrane signals CRE (Cyclic AMP Response Element) Regulation by cAMP, and by Ca+. Interacts with core promoter (through CREB Binding Protein) and modifies chromatin structure (through HAT). Positions of these elements are relatively unimportant

  16. The Cell Cycle M (Mitosis) 2 hours G2 4 hours G1 12 hours  G0 indefinite S (DNA synthesis) 6 hours

  17. Growth factor Receptor Growth factor Receptor Plasma membrane Ras-GDP Nuclear membranes Fos gene Other genes: SRF Serum regulatory factor

  18. HER2: Human Epidermal Growth Factor Receptor 2 Immunohistochemical analysis of paraffin-embedded human breast carcinoma, using HER2/ErbB2 (29D8) Rabbit mAb HER2 FISH

  19. Growth factor Growth factor binds Growth factor Receptor Growth factor Receptor Plasma membrane Ras-GDP Growth factor receptors form dimers Nuclear membranes TFIID Fos gene Other genes: SRF Serum regulatory factor

  20. Growth factor Receptor Growth factor Receptor Plasma membrane Ras-GDP PO4 PO4 One receptor’s tyrosine kinase activity phosphorylates the other receptor. Nuclear membranes Fos gene Other genes: SRF Serum regulatory factor

  21. SH2 domain Plasma membrane Ras-GDP Grb2 PO4 PO4 Grb2 binds to phosphorylated receptor (via SH2 domain) Nuclear membranes PO4 Fos gene Other genes: SRF Serum regulatory factor

  22. 1) GRB2 changes conformation and binds SOS SH2 domain Plasma membrane Grb2 Ras-GTP PO4 SOS PO4 SH3 domain GTP GDP 2) SOS exchanges GDP on Ras for GTP Nuclear membranes Fos gene Other genes: SRF Serum regulatory factor

  23. A. Activation of Ras GTP GDP Ras-GDP Ras-GTP sos SOS is a GTP exchange protein. It removes the GDP and replaces it with GTP B. Inactivation of Ras H20 Pi Ras-GTP Ras-GDP Ras contains a GTPase activity (it continually hydrolyzes the associated GTP)

  24. Plasma membrane Grb2 Ras-GTP PO4 SOS PO4 Raf (a kinase) ATP ADP A Series of kinases are activated by phopshorylation MAP Kinases Nuclear membranes Fos gene Other genes: SRF Serum regulatory factor

  25. Activating a chain of MAP kinases (mitogen activated protien kinases) A. ATP ADP OH Pi MAP kinase kinase MAP kinase kinase Raf (a kinase) Ras-GTP B. ATP ADP OH Pi MAP kinase Pi MAP kinase MAP kinase kinase C. ATP ADP OH Pi Substrate proteins Substrate proteins Pi MAP kinase

  26. Plasma membrane Grb2 Ras-GTP PO4 SOS PO4 Raf (a kinase) ATP ADP Transcription factors are activated by phosphorylation MAP Kinases Nuclear membranes PO4 Fos gene Other genes: SRF Serum regulatory factor

  27. Plasma membrane Grb2 Ras-GTP PO4 SOS PO4 Raf (a kinase) ATP ADP Fos protein Fos mRNA MAP Kinases Nuclear membranes PO4 The Fos protein is synthesized Pol II Fos gene Other genes: Fos pre mRNA

  28. Plasma membrane Grb2 Ras-GTP PO4 SOS PO4 Raf (a kinase) ATP ADP Fos protein Fos mRNA MAP Kinases Jun Nuclear membranes PO4 Pol II Fos gene Other genes: Fos pre mRNA Fos/Jun stimulates transcription of many genes

  29. New proteins are formed Plasma membrane Grb2 Ras-GTP PO4 SOS PO4 Raf (a kinase) ATP ADP Fos protein Fos mRNA MAP Kinases Jun Nuclear membranes Some of the new proteins stimulate cell division PO4 Pol II Fos gene Other genes: Cell Division Fos pre mRNA

  30. Deactivating the pathway Plasma membrane Grb2 Ras-GTP PO4 SOS PO4 Raf (a kinase) ATP ADP Fos protein Fos mRNA MAP Kinases Jun Nuclear membranes PO4 Pol II Fos gene Other genes: Cell Division Fos pre mRNA

  31. Deactivating the pathway Plasma membrane Grb2 Grb2 Ras-GTP SOS Raf (a kinase) ATP ADP H2O Pi Fos protein Fos mRNA MAP Kinases Jun Nuclear membranes PO4 Pol II Fos gene Other genes: Cell Division Fos pre mRNA

  32. Deactivating the pathway Plasma membrane Grb2 Grb2 Ras-GDP SOS Raf (a kinase) Pi H2O inactivation Fos protein Fos mRNA MAP Kinases Jun Nuclear membranes PO4 Pol II Fos gene Other genes: Cell Division Fos pre mRNA

  33. Deactivating the pathway Plasma membrane Grb2 Grb2 Ras-GDP SOS Raf (a kinase) Pi H2O inactivation Fos protein Fos mRNA MAP Kinases Jun Nuclear membranes HO Fos gene Other genes: Cell Division

  34. Deactivating the pathway Plasma membrane Grb2 Grb2 Ras-GDP SOS Raf (a kinase) Pi H2O inactivation Fos mRNA & protein degraded MAP Kinases Jun Nuclear membranes HO Fos gene Other genes: Cell Division

  35. Deactivating the pathway Plasma membrane Grb2 Grb2 Ras-GDP SOS Raf (a kinase) Pi H2O inactivation Fos mRNA & protein degraded MAP Kinases Nuclear membranes HO Fos gene Other genes: Cell Division

  36. Deactivating the pathway Plasma membrane Grb2 Grb2 Ras-GDP SOS Raf (a kinase) Pi H2O inactivation Fos mRNA & protein degraded MAP Kinases Nuclear membranes HO Fos gene Other genes:

  37. Deactivating the pathway Plasma membrane Grb2 Grb2 Ras-GDP SOS Raf (a kinase) Pi H2O inactivation Fos mRNA & protein degraded MAP Kinases Nuclear membranes HO Fos gene Other genes:

  38. Problems that can lead to cancer Plasma membrane Grb2 Ras-GTP PO4 SOS PO4 Raf (a kinase) ATP ADP Fos protein Fos mRNA MAP Kinases Jun Nuclear membranes PO4 Pol II Fos gene Other genes: Cell Division Fos pre mRNA

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