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Kinases and Phosphatases in cell signaling Giulio Taglialatela, Ph.D. John Sealy Professor and Vice Chair Dept. of Neurology Director, Mitchell Center for Neurodegenerative Diseases The University of Texas Medical Branch at Galveston

phosphatases and kinase in cell signalling

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Page 1: phosphatases and kinase in cell signalling

Kinases and Phosphatases in cell signaling

Giulio Taglialatela, Ph.D.John Sealy Professor and Vice Chair

Dept. of NeurologyDirector, Mitchell Center for Neurodegenerative Diseases

The University of Texas Medical Branch at Galveston

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Signals are changes in homeostasis

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To be recognized as a signal, an event must be:

1. Sudden.

Page 4: phosphatases and kinase in cell signalling

To be recognized as a signal, an event must be:

1. Sudden.

2. Short-lived.

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0

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TIME

INT

EN

SIT

YGood signals

0

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TIME

INT

EN

SIT

Y

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0

5

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TIME

INT

EN

SIT

YGood signals

0

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TIME

INT

EN

SIT

Y

0

5

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TIME

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SIT

Y

0

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TIME

INT

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SIT

Y

Bad signals

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The balanced activity between kinases and phosphatases provides sudden and (often) short lived modifications of proteins, resulting in effective signaling events

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Kinase signal cascade

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Kinases

Phosphorylases

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The human kinome

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Kinases share a highly conserved catalytic core

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Three classes of protein kinases

1. Serine/threonine kinases

2. Tyrosine kinases3. Mixed kinases

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Three classes of protein kinases

1. Serine/threonine kinases

2. Tyrosine kinases3. Mixed kinases

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Serine/threonine protein kinases

1. DAG/Ca2+ dependent (PKC)

2. cAMP-dependent (PKA)

3. Ca2+/Calmodulin-dependent (CaMKII)

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1. DAG/Ca2+ dependent (PKC)

2. cAMP-dependent (PKA)

Serine/threonine protein kinases

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G-PROTEIN-LINKED RECEPTORS

Binding domain

Docking domain

CYTOSOL G-PROTEIN (inactive)

subunit

subunit subunit

GDP

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G-PROTEIN-LINKED RECEPTORS

CYTOSOL G-PROTEIN (inactive)

Signaling molecule

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G-PROTEIN-LINKED RECEPTORS

CYTOSOL G-PROTEIN (inactive)

Signaling molecule

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G-PROTEIN-LINKED RECEPTORS

CYTOSOL G-PROTEIN (inactive)

Signaling molecule

GTP

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G-PROTEIN-LINKED RECEPTORS

CYTOSOL G-PROTEIN (active)

Signaling molecule

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G-PROTEIN-LINKED RECEPTORS

CYTOSOL G-PROTEIN (active)

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G-PROTEIN-LINKED RECEPTORS

CYTOSOL G-PROTEIN (active)

Pi

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G-PROTEIN-LINKED RECEPTORS

CYTOSOL G-PROTEIN (inactive)

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G-PROTEIN-LINKED RECEPTORS

CYTOSOL G-PROTEIN (inactive)

Signaling molecule

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G-PROTEIN-LINKED RECEPTORS

CYTOSOL G-PROTEIN (inactive)

Signaling molecule

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G-PROTEIN-LINKED RECEPTORS

CYTOSOL G-PROTEIN (inactive)

Signaling molecule

GTP

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G-PROTEIN-LINKED RECEPTORS

CYTOSOL G-PROTEIN (active)

Signaling molecule

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G-PROTEIN-LINKED RECEPTORS

CYTOSOL G-PROTEIN (active)

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G-PROTEIN-LINKED RECEPTORS

CYTOSOL G-PROTEIN (active)

Pi

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G-PROTEIN-LINKED RECEPTORS

CYTOSOL G-PROTEIN (inactive)

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G-PROTEIN-LINKED RECEPTORS

CYTOSOL

G-PROTEIN (active)

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G-PROTEIN-LINKED RECEPTORS

CYTOSOL

G-PROTEIN (active)

Adenylate cyclase

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G-PROTEIN-LINKED RECEPTORS

CYTOSOL

G-PROTEIN (active)

Adenylate cyclase

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G-PROTEIN-LINKED RECEPTORS

CYTOSOL

G-PROTEIN (active)

Adenylate cyclase

ATP cAMP

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G-PROTEIN-LINKED RECEPTORS

CYTOSOL

G-PROTEIN (active)

Adenylate cyclase

ATP cAMP

A-kinase

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G-PROTEIN-LINKED RECEPTORS

CYTOSOL

G-PROTEIN (active)

Adenylate cyclase

ATP cAMP

A-kinase

Nucleus

CREB

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G-PROTEIN-LINKED RECEPTORS

CYTOSOL

G-PROTEIN (active)

Adenylate cyclase

ATP cAMP

A-kinase

Nucleus

TranscriptionCREB

CBP

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G-PROTEIN-LINKED RECEPTORS

CYTOSOL

G-PROTEIN (active)

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G-PROTEIN-LINKED RECEPTORS

CYTOSOL

G-PROTEIN (active)

Phospholipase C (PLC)

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G-PROTEIN-LINKED RECEPTORS

CYTOSOL

G-PROTEIN (active)

Phospholipase C (PLC)

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G-PROTEIN-LINKED RECEPTORS

CYTOSOL

G-PROTEIN (active)

Phospholipase C (PLC)

PIP2

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G-PROTEIN-LINKED RECEPTORS

CYTOSOL

G-PROTEIN (active)

Phospholipase C (PLC)

PIP2

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G-PROTEIN-LINKED RECEPTORS

CYTOSOL

G-PROTEIN (active)

Phospholipase C (PLC)

DAG

IP3

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G-PROTEIN-LINKED RECEPTORS

CYTOSOL

G-PROTEIN (active)

Phospholipase C (PLC)

C-Kinase

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G-PROTEIN-LINKED RECEPTORS

CYTOSOL

G-PROTEIN (active)

Phospholipase C (PLC)

C-Kinase

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G-PROTEIN-LINKED RECEPTORS

CYTOSOL

G-PROTEIN (active)

Phospholipase C (PLC)

C-Kinase

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G-PROTEIN-LINKED RECEPTORS

CYTOSOL

G-PROTEIN (active)

Phospholipase C (PLC)

C-Kinase

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Serine/threonine protein kinases

3. Ca2+/Calmodulin-dependent (CaMKII)

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Ca2+-sensitive proteins:

A story of co-operation

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Calmodulin

A ubiquitous mediator of Ca2+ signals

Target protein

Page 51: phosphatases and kinase in cell signalling

Calmodulin

A ubiquitous mediator of Ca2+ signals

Target protein

Increased affinity

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Calmodulin

A ubiquitous mediator of Ca2+ signals

Target protein

Page 53: phosphatases and kinase in cell signalling

Calmodulin

A ubiquitous mediator of Ca2+ signals

Target protein

Page 54: phosphatases and kinase in cell signalling

Calmodulin

A ubiquitous mediator of Ca2+ signals

Active target protein

P

Page 55: phosphatases and kinase in cell signalling

Calmodulin

A ubiquitous mediator of Ca2+ signals

Active target protein

PP

Secondary cytosolic effects

Page 56: phosphatases and kinase in cell signalling

Intracellular Ca2+ waves and oscillations

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The ability of intracellular Ca2+ stores to release Ca2+ is affected by the relative concentration of free cytosolic Ca2+:

It is increased by slightly elevated Ca2+ and decreased by very high Ca2+ concentrations.

0

2

4

6

8

10

12

Cytpsolic Ca2+ concentration

ER

Ca2

+ r

elea

se

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Intracellular Ca2+ increases are characterized by:

- frequency

- amplitude (intensity)

Consequently, cells have developed systems that will respond to either intensity or frequency (or both) of Ca2+ increases.

Page 85: phosphatases and kinase in cell signalling

CaM Kinase II, a direct substrate for calmodulin, is a Ca2+ spike frequency detector

In order for CaM Kinase to be fully active, each catalytic subunit must be autophosphorylated in a calmudulin-dependent fashion. Autophosphorylation of each subsequent subunit follows a co-operative kinetic.

Page 86: phosphatases and kinase in cell signalling

CaM Kinase II, a direct substrate for calmodulin, is a Ca2+ spike frequency detector

In order for CaM Kinase to be fully active, each catalytic subunit must be autophosphorylated in a calmudulin-dependent fashion. Autophosphorylation of each subsequent subunit follows a co-operative kinetic.

calmudulin

Page 87: phosphatases and kinase in cell signalling

CaM Kinase II, a direct substrate for calmodulin, is a Ca2+ spike frequency detector

In order for CaM Kinase to be fully active, each catalytic subunit must be autophosphorylated in a calmudulin-dependent fashion. Autophosphorylation of each subsequent subunit follows a co-operative kinetic.

Page 88: phosphatases and kinase in cell signalling

CaM Kinase II, a direct substrate for calmodulin, is a Ca2+ spike frequency detector

In order for CaM Kinase to be fully active, each catalytic subunit must be autophosphorylated in a calmudulin-dependent fashion. Autophosphorylation of each subsequent subunit follows a co-operative kinetic.

Page 89: phosphatases and kinase in cell signalling

CaM Kinase II, a direct substrate for calmodulin, is a Ca2+ spike frequency detector

In order for CaM Kinase to be fully active, each catalytic subunit must be autophosphorylated in a calmudulin-dependent fashion. Autophosphorylation of each subsequent subunit follows a co-operative kinetic.

Fully active CaM kinase

Page 90: phosphatases and kinase in cell signalling

time

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time

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time

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time

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time

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time

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time

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time

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time

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time

Fully active CaM Kinase

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CaM Kinase II, a direct substrate for calmodulin, is a Ca2+ spike frequency detector

Page 102: phosphatases and kinase in cell signalling

Three classes of protein kinases

1. Serine/threonine kinases

2. Tyrosine kinases3. Mixed kinases

Page 103: phosphatases and kinase in cell signalling

The receptor tyrosine kinases

Page 104: phosphatases and kinase in cell signalling

Binding domain

tyrosine kinase domain

Ligands that bind to specific RTK:

FGF, NGF, IGF, BDNF, ……, XXXF

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Binding domain

tyrosine kinase domain

P P

P P

Page 106: phosphatases and kinase in cell signalling

P P

P P

P P

P P

Receptor autophosporylation

Page 107: phosphatases and kinase in cell signalling

P

The phophotyrosine domain acts as a docking site for proteins possessing a particular domain called SH2

SH2

Page 108: phosphatases and kinase in cell signalling

P SH2

The SH2-containing protein is then activated by either conformational change or by phosphorylation promoted by the receptor itself.

P PLC

PI3-K

GAP (GEF)

IRS

Page 109: phosphatases and kinase in cell signalling

P SH2

PPLC PIP2/IP3/PKC

PI3-K IP2/PIP3/PKB

GAP (GEF) ras/MAPK

IRS

Multiple pathway activation

Page 110: phosphatases and kinase in cell signalling

Inactive Ras

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P P

Active/Inactive Ras

GTP/GDP

GAP

GNRPSH2SH3

SH3

Sem-5+

_

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Receptors associated with cytoplasmic tyrosine kinases

JAK JAK

cytosol

nucleus

Enzyme-linked receptors

Page 113: phosphatases and kinase in cell signalling

Receptors associated with cytoplasmic tyrosine kinases

JAK JAKP P

P P

STAT STATP P

cytosol

nucleus

SOCS

Enzyme-linked receptors

Page 114: phosphatases and kinase in cell signalling

Receptor Serine/Threonine Kinases

cytosol

nucleus

RIIRI

Enzyme-linked receptors

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Receptor Serine/Threonine Kinases

cytosol

nucleus

RIIRI

P

P SMAD

co-SMAD

Enzyme-linked receptors

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Three classes of protein kinases

1. Serine/threonine kinases

2. Tyrosine kinases3. Mixed kinases

Page 117: phosphatases and kinase in cell signalling

GTP

MAP KINASE

MAPK KINASE

MAPKK KINASE

P-SER

P-SER

P-SER

P-THR

P-THRP-TYR

P-TYR

Active Ras

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GTP

MAP KINASE

MAPK KINASE

MAPKK KINASE

P-SER

P-SER

P-SER

P-THR

P-THR

PKC

P-TYR

P-TYR

Active Ras

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MAP KINASEP-SER

P-THR

Direct: CREB. Indirect: JNK/c-jun

DNA

P-TYR

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MAP KINASEP-SER

P-THR

Direct: CREB. Indirect: JNK/c-jun

DNA

RNA Pol II

P-TYR

Page 122: phosphatases and kinase in cell signalling

To be recognized as a signal, an event must be:

1. Sudden.

2. Short-lived.

Page 123: phosphatases and kinase in cell signalling

Who cleans up the mess?

Page 124: phosphatases and kinase in cell signalling

Protein Phosphatases

Page 125: phosphatases and kinase in cell signalling

Protein Phosphatases (PPT)

Ser/Thr PPT Tyr PPT Mix PPT•MAPK PPT’s•CDC14’s•PTEN’s•CDC25’s

•RTP’s•nonRTP’s•LMPT•Eya1-4

•PP1•PP2A•PP2B (calcineurin)•PP2C

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The phosphatase reaction is conserved among all phosphatases and does not require energy

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Ser/Thr Protein Phosphatases

Ca/CaM-indep.

•PP2C•PP2B (calcineurin)

•PP1•PP2A

Ca/CaM-dep. Mg/Mn-dep.

Multimeric proteins formed by a catalytic subunit, a modulatory subunit and, in certain instances (PP2A), a scaffolding subunit

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Both calcineurin and CaMKII are essential to modulate the function of the synapse in

response to stimulatory Ca++ entry

Both calcineurin and CaMKII are activated by binding to Ca++/calmodulin

Page 130: phosphatases and kinase in cell signalling

Na+

Na+

Na+Na+ Ca2+

Ca2+

Ca2+

Na+

Na+

CaM

Ca2+

CaN

CaN

CaMKIICaMKII

Ca2+

pCREBpCREB

Ca2+

Ca2+

Ca2+

Ca2+

PP1

Page 131: phosphatases and kinase in cell signalling

Giulio [email protected]