Signaling mediated by c AMP and protein kinase

Signaling mediated by c. AMP and protein kinase A Hormone receptor G Adenylate cyclase G-protein Inctive Protein Kinase A c. AMP ATP Plasma membrane Active Protein Kinase A Nuclear membranes PO 4 Active p. KA 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

Adenylate cyclase ATP c. AMP (3’, 5’cyclic AMP) PPi pyrohosphatase 2 Pi c. AMP formation H 2 O phoshodiesterase AMP c. AMP 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 Ca. M kinases phosphorylate multiple targets including ser 133 of CREB Calmodulin activates Ca. M 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: CREB 1 stimulates transcription CREB 2 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 (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 c. AMP, 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 T 3 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 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 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 p 160 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 (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 c. AMP, 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 G 2 4 hours G 1 12 hours G 0 indefinite S (DNA synthesis) 6 hours

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

HER 2: Human Epidermal Growth Factor Receptor 2 Immunohistochemical analysis of paraffin-embedded human breast carcinoma, using HER 2/Erb. B 2 (29 D 8) Rabbit m. Ab HER 2 FISH

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 PO 4 Ras-GDP Plasma membrane One receptor’s tyrosine kinase activity phosphorylates the other receptor. Nuclear membranes Fos gene Other genes: SRF Serum regulatory factor

SH 2 domain PO 4 Grb 2 PO 4 Ras-GDP Plasma membrane Grb 2 binds to phosphorylated receptor (via SH 2 domain) Nuclear membranes PO 4 Fos gene Other genes: SRF Serum regulatory factor

1) GRB 2 changes conformation and binds SOS SH 2 domain PO 4 Grb 2 PO 4 SH 3 domain SOS GTP Plasma membrane Ras-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 Ras-GTP sos SOS is a GTP exchange protein. It removes the GDP and replaces it with GTP B. Inactivation of Ras-GTP H 2 0 Pi Ras-GDP Ras contains a GTPase activity (it continually hydrolyzes the associated GTP)

PO 4 Grb 2 PO 4 SOS 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 Raf (a kinase) Ras-GTP B. OH ATP ADP Pi Pi MAP kinase C. OH ATP ADP Substrate proteins Pi MAP kinase Pi Substrate proteins

PO 4 Grb 2 PO 4 SOS Plasma membrane Ras-GTP Raf (a kinase) ATP Transcription factors are activated by phosphorylation ADP MAP Kinases Nuclear membranes PO 4 Fos gene Other genes: SRF Serum regulatory factor

PO 4 Grb 2 PO 4 SOS Plasma membrane Ras-GTP Raf (a kinase) ATP Fos protein Fos m. RNA ADP MAP Kinases Nuclear membranes The Fos protein is synthesized PO 4 Pol II Other genes: Fos gene Fos pre m. RNA

PO 4 Grb 2 PO 4 SOS Plasma membrane Ras-GTP Raf (a kinase) ATP Fos protein Fos m. RNA ADP MAP Kinases Jun Nuclear membranes PO 4 Pol II Other genes: Fos gene Fos pre m. RNA Fos/Jun stimulates transcription of many genes

New proteins are formed PO 4 Grb 2 PO 4 SOS Plasma membrane Ras-GTP Raf (a kinase) ATP Fos protein Fos m. RNA ADP MAP Kinases Jun Nuclear membranes Some of the new proteins stimulate cell Other genes: division PO 4 Pol II Cell Division Fos gene Fos pre m. RNA

Deactivating the pathway PO 4 Grb 2 PO 4 SOS Plasma membrane Ras-GTP Raf (a kinase) ATP Fos protein Fos m. RNA ADP MAP Kinases Jun Nuclear membranes PO 4 Pol II Other genes: Cell Division Fos gene Fos pre m. RNA

Deactivating the pathway Plasma membrane SOS Grb 2 Ras-GTP Raf (a kinase) Fos protein Fos m. RNA H 2 O ATP Pi ADP MAP Kinases Jun Nuclear membranes PO 4 Pol II Other genes: Cell Division Fos gene Fos pre m. RNA

Deactivating the pathway SOS Grb 2 Plasma membrane Ras-GDP Raf (a kinase) Fos protein Pi inactivation H 2 O Fos m. RNA MAP Kinases Jun Nuclear membranes PO 4 Pol II Other genes: Cell Division Fos gene Fos pre m. RNA

Deactivating the pathway SOS Grb 2 Plasma membrane Ras-GDP Raf (a kinase) Fos protein Pi inactivation H 2 O Fos m. RNA MAP Kinases Jun Nuclear membranes HO Other genes: Cell Division Fos gene

Deactivating the pathway SOS Grb 2 Plasma membrane Ras-GDP Raf (a kinase) Pi inactivation H 2 O Fos m. RNA & protein degraded MAP Kinases Jun Nuclear membranes HO Other genes: Cell Division Fos gene

Deactivating the pathway SOS Grb 2 Plasma membrane Ras-GDP Raf (a kinase) Pi inactivation H 2 O Fos m. RNA & protein degraded MAP Kinases Nuclear membranes HO Other genes: Cell Division Fos gene

Deactivating the pathway SOS Grb 2 Plasma membrane Ras-GDP Raf (a kinase) Pi inactivation H 2 O Fos m. RNA & protein degraded MAP Kinases Nuclear membranes HO Other genes: Fos gene

Deactivating the pathway SOS Grb 2 Plasma membrane Ras-GDP Raf (a kinase) Pi inactivation H 2 O Fos m. RNA & protein degraded MAP Kinases Nuclear membranes HO Other genes: Fos gene

Problems that can lead to cancer PO 4 Grb 2 PO 4 SOS Plasma membrane Ras-GTP Raf (a kinase) ATP Fos protein Fos m. RNA ADP MAP Kinases Jun Nuclear membranes PO 4 Pol II Other genes: Cell Division Fos gene Fos pre m. RNA