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Signaling mediated by cAMP and protein kinase A Hormone Hormone receptor G Adenylate cyclase G-protein Inctive Protein Kinase A cAMP ATP Plasma membrane Active Protein Kinase A Nuclear membranes PO4 PO4 Active pKA enters the nucleus and phosphorylates CREB on Serine 133 Core promoter Phosphorylation of CREB: - stimulates interactions with several core promoter proteins - induces binding of HAT and acetylation of histones NH2 N O HO P OH O O P OH NH2 N N N O O P O N O N N O O OH N O P OH OH Adenylate cyclase ATP HO O OH cAMP (3’,5’cyclic AMP) PPi H2O pyrohosphatase 2Pi cAMP formation H2O phoshodiesterase AMP cAMP hydrolysis ATP ADP Substrate-OH Kinase ATP ADP Protein-OH Protein Kinase Several specific Proteins ATP ADP Serine-OH Including CREB Protein Kinase A O ║ Substrate─O ─ P ─ O─ │ OH O ║ Protein ─ O ─ P ─ O─ │ OH Several specific proteins O ║ Serine ─ O ─ P ─ O─ │ OH including CREB CREB and Memory Signaling by calcium ions Ca++ enters cell Ca++ activates calmodulin CaM kinases phosphorylate multiple targets including ser 133 of CREB Calmodulin activates CaM kinases CREB and Memory CREB and Memory ( in Aplysia, a slug) B. A. Amplification: C/EBP is another transcription factor. It activates many genes. (CCAAT box enhancer binding protien) 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 A model promoter HAT Fos Jun CREB Histone Acetyl Transferase CREB NR=“nuclear receptor” NR NR SP-1 TGACTCA HRE HRE (Hormone Response Element) Regulation by hormones such as estrogen which enter the cell Core Promoter Binds general transcriptional machinery AP-1 (Fos-Jun binding site) Regulation by growth factors, stress, and various transmembrane signals GACGTC 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 GGGCGG TATAAA SP-1 Site Provides basal unregulated transcriptional activity. Many genes have multiple SP-1 sites 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 Example of Nuclear receptor function Stimulation of transcription by glucocorticoids Glucocorticoid Glucocorticoid receptor receptor + glucocorticoid Binds to specific DNA sequences and stimulates transcription of glucocorticoid-responsive genes DNA binding domain of the glucocorticoid receptor (a zinc finger protein) 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. Binding of estrogen moves helix 12, allowing binding of the coactivator’s LLXXL helix Tamoxifen inhibits the movement of helix 12 Phosphorylation of CREB and the CREB binding protein (CBP) HAT coactivator p160 p160 NR NR HRE SERMS Selective estrogen receptor modifiers SERM 17-Estradiol ICI 182,780 (antiestrogen) Tamoxifen Raloxifene + Agonist - Antagonist Bone Breast Cardiovascular + + + + + + + Uterus + + - A model promoter HAT Fos Jun CREB Histone Acetyl Transferase CREB Core Promoter Binds general transcriptional machinery SP-1 NR NR TGACTCA HRE HRE (Hormone Response Element) Regulation by hormones such as estrogen which enter the cell AP-1 (Fos-Jun binding site) Regulation by growth factors, stress, and various transmembrane signals GACGTC 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 GGGCGG TATAAA SP-1 Site Provides basal unregulated transcriptional activity. Many genes have multiple SP-1 sites The Cell Cycle M (Mitosis) 2 hours G2 4 hours G1 12 hours G0 indefinite S (DNA synthesis) 6 hours Growth factor Receptor Growth factor Receptor Ras-GDP Plasma membrane Nuclear membranes Fos gene Other genes: SRF Serum regulatory factor HER2: Human Epidermal Growth Factor Receptor 2 Immunohistochemical analysis of paraffin-embedded human breast carcinoma, using HER2/ErbB2 (29D8) Rabbit mAb HER2 FISH Growth factor Growth factor Receptor Growth factor binds Growth factor Receptor Ras-GDP Plasma membrane Growth factor receptors form dimers Nuclear membranes TFIID Fos gene Other genes: SRF Serum regulatory factor Growth factor Receptor PO4 Growth factor Receptor PO4 Ras-GDP Plasma membrane One receptor’s tyrosine kinase activity phosphorylates the other receptor. Nuclear membranes Fos gene Other genes: SRF Serum regulatory factor SH2 domain Grb2 PO4 PO4 Ras-GDP Plasma membrane Grb2 binds to phosphorylated receptor (via SH2 domain) Nuclear membranes PO4 Fos gene Other genes: SRF Serum regulatory factor 1) GRB2 changes conformation and binds SOS SH2 domain Plasma membrane Grb2 PO4 PO4 SH3 domain Ras-GTP GTP GDP 2) SOS exchanges GDP on Ras for GTP Nuclear membranes Fos gene Other genes: SRF Serum regulatory factor A. Activation of Ras GTP Ras-GDP GDP Ras-GTP sos SOS is a GTP exchange protein. It removes the GDP and replaces it with GTP B. Inactivation of Ras Ras-GTP H20 Pi Ras-GDP Ras contains a GTPase activity (it continually hydrolyzes the associated GTP) Grb2 PO4 PO4 Plasma membrane Ras-GTP Raf (a kinase) ATP A Series of kinases are activated by phopshorylation ADP MAP Kinases Nuclear membranes Fos gene Other genes: SRF Serum regulatory factor Activating a chain of MAP kinases (mitogen activated protien kinases) A. OH ATP ADP Pi MAP kinase kinase MAP kinase kinase Raf (a kinase) Ras-GTP B. OH ATP ADP Pi Pi MAP kinase MAP kinase MAP kinase kinase C. OH ATP ADP Substrate proteins Pi MAP kinase Pi Substrate proteins Grb2 PO4 PO4 Plasma membrane Ras-GTP Raf (a kinase) ATP Transcription factors are activated by phosphorylation ADP MAP Kinases Nuclear membranes PO4 Fos gene Other genes: SRF Serum regulatory factor Grb2 PO4 PO4 Plasma membrane Ras-GTP Raf (a kinase) ATP Fos protein Fos mRNA ADP MAP Kinases Nuclear membranes The Fos protein is synthesized PO4 Pol II Other genes: Fos gene Fos pre mRNA Plasma membrane Grb2 PO4 PO4 Ras-GTP Raf (a kinase) ATP Fos protein Fos mRNA ADP MAP Kinases Jun Nuclear membranes PO4 Pol II Other genes: Fos gene Fos pre mRNA Fos/Jun stimulates transcription of many genes New proteins are formed Plasma membrane Grb2 PO4 PO4 Ras-GTP Raf (a kinase) ATP Fos protein Fos mRNA ADP MAP Kinases Jun Nuclear membranes Some of the new proteins stimulate cell Other genes: division PO4 Pol II Cell Division Fos gene Fos pre mRNA Deactivating the pathway Plasma membrane Grb2 PO4 PO4 Ras-GTP Raf (a kinase) ATP Fos protein Fos mRNA ADP MAP Kinases Jun Nuclear membranes PO4 Pol II Other genes: Cell Division Fos gene Fos pre mRNA Deactivating the pathway Fos protein Plasma membrane SOS Grb2 Grb2 Ras-GTP Raf (a kinase) H2O ATP Pi Fos mRNA ADP MAP Kinases Jun Nuclear membranes PO4 Pol II Other genes: Cell Division Fos gene Fos pre mRNA Deactivating the pathway SOS Grb2 Grb2 Plasma membrane Ras-GDP Raf (a kinase) Fos protein Pi inactivation H2O Fos mRNA MAP Kinases Jun Nuclear membranes PO4 Pol II Other genes: Cell Division Fos gene Fos pre mRNA Deactivating the pathway SOS Grb2 Grb2 Plasma membrane Ras-GDP Raf (a kinase) Fos protein Pi inactivation H2O Fos mRNA MAP Kinases Jun Nuclear membranes HO Other genes: Cell Division Fos gene Deactivating the pathway SOS Grb2 Grb2 Plasma membrane Ras-GDP Raf (a kinase) Pi inactivation H2O Fos mRNA & protein degraded MAP Kinases Jun Nuclear membranes HO Other genes: Cell Division Fos gene Deactivating the pathway SOS Grb2 Grb2 Plasma membrane Ras-GDP Raf (a kinase) Pi inactivation H2O Fos mRNA & protein degraded MAP Kinases Nuclear membranes HO Other genes: Cell Division Fos gene Deactivating the pathway SOS Grb2 Grb2 Plasma membrane Ras-GDP Raf (a kinase) Pi inactivation H2O Fos mRNA & protein degraded MAP Kinases Nuclear membranes HO Other genes: Fos gene Deactivating the pathway SOS Grb2 Grb2 Plasma membrane Ras-GDP Raf (a kinase) Pi inactivation H2O Fos mRNA & protein degraded MAP Kinases Nuclear membranes HO Other genes: Fos gene Problems that can lead to cancer Plasma membrane Grb2 PO4 PO4 Ras-GTP Raf (a kinase) ATP Fos protein Fos mRNA ADP MAP Kinases Jun Nuclear membranes PO4 Pol II Other genes: Cell Division Fos gene Fos pre mRNA
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