Plasma membrane Nuclear membranes Hormone receptor Adenylate cyclase ATP Active Protein Kinase A PO 4 G cAMP Inctive Protein Kinase A PO 4 Active pKA enters the nucleus and phosphorylates CREB on Serine 133 Hormone G-protein Core promoter Phosphorylation of CREB: - stimulates interactions with several core promoter proteins - induces binding of HAT and acetylation of histones Signaling mediated by cAMP and protein kinase A

ATPcAMP (3’,5’cyclic AMP) PP i 2P i H2OH2O pyrohosphatase Adenylate cyclase AMP phoshodiesterase H2OH2O cAMP formation cAMP hydrolysis

Substrate-OH O ║ Substrate─O ─ P ─ O ─ │ OH Kinase ATP ADP Protein-OH O ║ Protein ─ O ─ P ─ O ─ │ OH Protein Kinase ATP ADP Serine-OH O ║ Serine ─ O ─ P ─ O ─ │ OH Protein Kinase A ATP ADP Several specific Proteins Including CREB Several specific proteins 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) Bailey, Kandel & Kausik (2004) Neuron 44:49-57 Stimulates long term memory Inhibits long term memory Amplification: C/EBP is another transcription factor. It activates many genes. (CCAAT box enhancer binding protien) Two forms of CREB: CREB1 stimulates transcription CREB2 represses transcription A. B.

A model promoter TGACTCA GACGTC GGGCGG SP-1 CREB Fos Jun CREB 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). SP-1 Site Provides basal unregulated transcriptional activity. Many genes have multiple SP-1 sites Core Promoter Binds general transcriptional machinery TATAAA Positions of these elements are relatively unimportant HAT Histone Acetyl Transferase HRE NR HRE (Hormone Response Element) Regulation by hormones such as estrogen which enter the cell NR=“nuclear receptor”

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

Glucocorticoid Glucocorticoid receptor receptor + glucocorticoid Example of Nuclear receptor function Stimulation of transcription by glucocorticoids 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 “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 The cooactivator functions to recruit other factors that stimulate transcripton such as CBP Example: estrogen

HAT Phosphorylation of CREB and the CREB binding protein (CBP) HRE NR p160 c oactivator

SERMS Selective estrogen receptor modifiers SERMBoneBreast Cardio- Uterus vascular 17-Estradiol ICI 182,780 (antiestrogen) Tamoxifen Raloxifene Agonist - Antagonist

A model promoter TGACTCA GACGTC GGGCGG SP-1 CREB Fos Jun CREB 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). SP-1 Site Provides basal unregulated transcriptional activity. Many genes have multiple SP-1 sites Core Promoter Binds general transcriptional machinery TATAAA Positions of these elements are relatively unimportant HAT Histone Acetyl Transferase HRE NR HRE (Hormone Response Element) Regulation by hormones such as estrogen which enter the cell

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

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

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

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

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

Plasma membrane Nuclear membranes Grb2 SRF Serum regulatory factor PO 4 Fos gene Other genes: PO 4 SH2 domain Grb2 binds to phosphorylated receptor (via SH2 domain) Ras-GDP

Plasma membrane Nuclear membranes Grb2 SRF Serum regulatory factor Fos gene Other genes: PO 4 SH2 domain 2) SOS exchanges GDP on Ras for GTP SH3 domain SOS Ras-GTP GTP GDP 1) GRB2 changes conformation and binds SOS

Ras-GTP Ras-GDP GTP GDP sos SOS is a GTP exchange protein. It removes the GDP and replaces it with GTP Ras-GTPRas-GDP H 2 0 P i A. Activation of Ras B. Inactivation of Ras Ras contains a GTPase activity (it continually hydrolyzes the associated GTP)

Plasma membrane Nuclear membranes Grb2 SRF Serum regulatory factor Fos gene Other genes: PO 4 SOS Ras-GTP Raf (a kinase) ATP ADP MAP Kinases A Series of kinases are activated by phopshorylation

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

Transcription factors are activated by phosphorylation Plasma membrane Grb2 SRF Serum regulatory factor PO 4 Fos gene Other genes: PO 4 SOS Ras-GTP Raf (a kinase) ATP ADP MAP Kinases Nuclear membranes

Plasma membrane Grb2 PO 4 Fos gene Other genes: PO 4 SOS Ras-GTP Raf (a kinase) ATP ADP MAP Kinases Nuclear membranes Fos pre mRNA Fos protein Fos mRNA Pol II The Fos protein is synthesized

Plasma membrane Grb2 PO 4 Fos gene Other genes: PO 4 SOS Ras-GTP Raf (a kinase) ATP ADP MAP Kinases Nuclear membranes Fos pre mRNA Fos protein Fos mRNA Pol II Jun Fos/Jun stimulates transcription of many genes

Plasma membrane Grb2 PO 4 Fos gene Other genes: PO 4 SOS Ras-GTP Raf (a kinase) ATP ADP MAP Kinases Nuclear membranes Fos pre mRNA Fos protein Fos mRNA Pol II Jun New proteins are formed Cell Division Some of the new proteins stimulate cell division

Plasma membrane Grb2 PO 4 Fos gene Other genes: PO 4 SOS Ras-GTP Raf (a kinase) ATP ADP MAP Kinases Nuclear membranes Fos pre mRNA Fos protein Fos mRNA Pol II Jun Cell Division Deactivating the pathway

Plasma membrane Grb2 PO 4 Fos gene Other genes: SOS Ras-GTP Raf (a kinase) ATP ADP MAP Kinases Nuclear membranes Fos pre mRNA Fos protein Fos mRNA Pol II Jun Cell Division Deactivating the pathway Grb2 H 2 O P i

Plasma membrane Grb2 PO 4 Fos gene Other genes: SOS Ras-GDP Raf (a kinase) PiH2OPiH2O MAP Kinases Nuclear membranes Fos pre mRNA Fos protein Fos mRNA Pol II Jun Cell Division Deactivating the pathway Grb2 inactivation

Plasma membrane Grb2 HO Fos gene Other genes: SOS Ras-GDP Raf (a kinase) PiH2OPiH2O MAP Kinases Nuclear membranes Fos protein Fos mRNA Jun Cell Division Deactivating the pathway Grb2 inactivation

Plasma membrane Grb2 HO Fos gene Other genes: SOS Ras-GDP Raf (a kinase) PiH2OPiH2O MAP Kinases Nuclear membranes Jun Cell Division Deactivating the pathway Grb2 inactivation Fos mRNA & protein degraded

Plasma membrane Grb2 HO Fos gene Other genes: SOS Ras-GDP Raf (a kinase) PiH2OPiH2O MAP Kinases Nuclear membranes Cell Division Deactivating the pathway Grb2 inactivation Fos mRNA & protein degraded

Plasma membrane Grb2 HO Fos gene Other genes: SOS Ras-GDP Raf (a kinase) PiH2OPiH2O MAP Kinases Nuclear membranes Deactivating the pathway Grb2 inactivation Fos mRNA & protein degraded

Plasma membrane Grb2 HO Fos gene Other genes: SOS Ras-GDP Raf (a kinase) PiH2OPiH2O MAP Kinases Nuclear membranes Deactivating the pathway Grb2 inactivation Fos mRNA & protein degraded

Plasma membrane Grb2 PO 4 Fos gene Other genes: PO 4 SOS Ras-GTP Raf (a kinase) ATP ADP MAP Kinases Nuclear membranes Fos pre mRNA Fos protein Fos mRNA Pol II Jun Cell Division Problems that can lead to cancer