Plasma membrane

Nuclear membranes

Hormone receptor

Adenylate cyclase

ATP

Active Protein Kinase A

PO4

G

cAMP Inctive Protein Kinase A

PO4

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

ATP cAMP(3’,5’cyclic AMP)

PPi

2Pi

H2Opyrohosphatase

Adenylate cyclase

AMP

phoshodiesterase

H2O

cAMP formation cAMP hydrolysis

NO

O

OHO

PO

OH

N

NN

NH2

NO

O

OHOH

P

O

OH

P

O

O

OH

OH

OH

O

O

P N

NN

NH2

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 ADPSeveral 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 transcriptionCREB2 represses transcription

A.

B.

A model promoter

TGACTCA GACGTC GGGCGG

SP-1

CREBFos Jun CREB

AP-1 (Fos-Jun binding site)Regulation bygrowth factors,stress, and varioustransmembranesignals

CRE (Cyclic AMPResponse Element)Regulation by cAMP,and by Ca+. Interacts withcore promoter (through CREB Binding Protein)and modifies chromatin structure (through HAT).

SP-1 SiteProvides basalunregulatedtranscriptionalactivity. Manygenes have multiple SP-1 sites

Core PromoterBinds generaltranscriptionalmachinery

TATAAA

Positions of these elementsare relatively unimportant

HAT Histone Acetyl Transferase

HRENR NR

HRE (HormoneResponse Element)Regulation by hormonessuch as estrogen which enter the cell

NR=“nuclear receptor”

Nuclear receptors: An important class of transcription factors

Nuclear receptors include:Steroid hormone receptors:

Glucocorticoid receptorEstrogen receptors α and βAndrogen receptorProgesterone receptorMineralocorticoid receptor

Vitamin D receptorThyroid hormone T3 receptorFatty acid receptors

PPARα, PPARδ and PPARγBile salt receptorOxysterol receptorMany others

Glucocorticoid

Glucocorticoid receptor

receptor +

glucocorticoid

Example of Nuclear receptor functionStimulation 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 recruitother factors that

stimulate transcripton such as CBP

Example: estrogen

HAT

Phosphorylation of CREB and the CREB binding protein (CBP)

HRE

NR NR

p160 p160coactivator

SERMS Selective estrogen receptor modifiers

SERM Bone Breast Cardio- Uterus vascular17-Estradiol + + + +ICI 182,780 (antiestrogen) - - - -Tamoxifen + - + +Raloxifene + - + -

+ Agonist- Antagonist

A model promoter

TGACTCA GACGTC GGGCGG

SP-1

CREBFos Jun CREB

AP-1 (Fos-Jun binding site)Regulation bygrowth factors,stress, and varioustransmembranesignals

CRE (Cyclic AMPResponse Element)Regulation by cAMP,and by Ca+. Interacts withcore promoter (through CREB Binding Protein)and modifies chromatin structure (through HAT).

SP-1 SiteProvides basalunregulatedtranscriptionalactivity. Manygenes have multiple SP-1 sites

Core PromoterBinds generaltranscriptionalmachinery

TATAAA

Positions of these elementsare relatively unimportant

HAT Histone Acetyl Transferase

HRENR NR

HRE (HormoneResponse Element)Regulation by hormonessuch as estrogen which enter the cell

The Cell Cycle

M (Mitosis) 2 hours

S (DNA synthesis) 6 hours

G112 hours

G24 hours

G0indefinite

Plasma membrane

Nuclear membranes

Growth factorReceptor

SRFSerum regulatory factor

Growth factor Receptor

Fos geneOther 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 factorReceptor

TFIID

SRFSerum regulatory factor

Growth factor Receptor

Fos geneOther genes:

Growth factor

Growth factor receptors form dimers

Growth factor binds

Ras-GDP

Plasma membrane

Nuclear membranes

Growth factorReceptor

SRFSerum regulatory factor

Growth factor Receptor

Fos geneOther genes:

PO4PO4

One receptor’s tyrosine kinase activity phosphorylatesthe other receptor.

Ras-GDP

Plasma membrane

Nuclear membranes

Grb2

SRFSerum regulatory factor

PO4

Fos geneOther genes:

PO4PO4

SH2 domain

Grb2 binds to phosphorylated receptor (via SH2 domain)

Ras-GDP

Plasma membrane

Nuclear membranes

Grb2

SRFSerum regulatory factor

Fos geneOther genes:

PO4PO4

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-GTPRas-GDP

GTP GDP

sos

SOS is a GTP exchange protein. It removes the GDP and replaces it with GTP

Ras-GTP Ras-GDP

H20 Pi

A. Activation of Ras

B. Inactivation of Ras

Ras contains a GTPase activity(it continually hydrolyzes the associated GTP)

Plasma membrane

Nuclear membranes

Grb2

SRFSerum regulatory factor

Fos geneOther genes:

PO4PO4

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 kinase

MAP kinase MAP kinase

MAP kinase kinase

ATP ADP

Ras-GTP

Raf (a kinase)

MAP kinase

Substrate proteins

ATP ADP

Pi

Pi

Pi

Pi

PiSubstrate proteins

OH

OH

OH

A.

B.

C.

Transcription factors are activated by phosphorylation

Plasma membraneGrb2

SRFSerum regulatory factor

PO4

Fos geneOther genes:

PO4PO4

SOS Ras-GTP

Raf (a kinase)

ATP

ADP

MAP Kinases

Nuclear membranes

Plasma membraneGrb2

PO4

Fos geneOther genes:

PO4PO4

SOS Ras-GTP

Raf (a kinase)

ATP

ADP

MAP Kinases

Nuclear membranes

Fos pre mRNA

Fos protein Fos mRNA

Pol IIThe Fos protein is synthesized

Plasma membraneGrb2

PO4

Fos geneOther genes:

PO4PO4

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 membraneGrb2

PO4

Fos geneOther genes:

PO4PO4

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 membraneGrb2

PO4

Fos geneOther genes:

PO4PO4

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 membraneGrb2

PO4

Fos geneOther genes:SO

S 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

H2O Pi

Plasma membraneGrb2

PO4

Fos geneOther genes:

SOS Ras-GDP

Raf (a kinase)

Pi

H2O

MAP Kinases

Nuclear membranes

Fos pre mRNA

Fos protein Fos mRNA

Pol II

Jun

Cell Division

Deactivating the pathway

Grb2

inactivation

Plasma membraneGrb2

HO

Fos geneOther genes:

SOS Ras-GDP

Raf (a kinase)

Pi

H2O

MAP Kinases

Nuclear membranes

Fos protein Fos mRNA

Jun

Cell Division

Deactivating the pathway

Grb2

inactivation

Plasma membraneGrb2

HO

Fos geneOther genes:

SOS Ras-GDP

Raf (a kinase)

Pi

H2O

MAP Kinases

Nuclear membranes

Jun

Cell Division

Deactivating the pathway

Grb2

inactivationFos mRNA & protein degraded

Plasma membraneGrb2

HO

Fos geneOther genes:

SOS Ras-GDP

Raf (a kinase)

Pi

H2O

MAP Kinases

Nuclear membranes

Cell Division

Deactivating the pathway

Grb2

inactivation

Fos mRNA & protein degraded

Plasma membraneGrb2

HO

Fos geneOther genes:

SOS Ras-GDP

Raf (a kinase)

Pi

H2O

MAP Kinases

Nuclear membranes

Deactivating the pathway

Grb2

inactivationFos mRNA & protein degraded

Plasma membraneGrb2

HO

Fos geneOther genes:

SOS Ras-GDP

Raf (a kinase)

Pi

H2O

MAP Kinases

Nuclear membranes

Deactivating the pathway

Grb2

inactivationFos mRNA & protein degraded

Plasma membraneGrb2

PO4

Fos geneOther genes:

PO4PO4

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