1

Generic Signal Transduction Pathways

ProliferationSurvival

Differentiation

Apoptosis

Cytoskeleton

LIGANDS

RECEPTORS

ADAPTORS

EFFECTORS

OUTPUTS

Downward, J. 2001

Nature N&V

Ln

Rn

Gn

CHANNELS

AC cAMP cAKinase

PDE cGMP cGKinase

CHANNELS

PLA

PLC

Arachidonic

acid

Prostaglandins

Eicosanoids

PIP2

IP3

Ca++ CaMKinase

DAG

Kinase C

PI derivatives

Kinase B/Akt

PI3K

2

RECEPTORS TRANSDUCERS EFFECTORS

(proximal)

“SECOND

MESSENGERS”

SERPENTINE

(7TM)

HETEROTRIMERIC

G PROTEINS

ADENYLATE

CYCLASE

PHOSPHODI-

ESTERASE

PHOSPHO-

LIPASE C!

PI3 KINASE

CHANNELS

cAMP

cGMP

DERIVATIVES

DERIVATIVES Ca++

PHOSPHATIDYL

INOSITOL

PHOSPHATES

IONS

+DIACYLGLYCEROL

IP3

GsGi

Gt

Go

Gq !"

Gt!"

various incl.!"

ADENYLATE

CYCLASE

PHOSPHO-

DIESTERASE

Gs Gi

Gt

cAMP

cGMP

Note:

There are also

Guanylate Kinase

Receptors

3

SECOND MESSENGER EFFECTORS

cAMP

cGMP

Ca++

DAG PKC Protein Kinases C

PKA Protein Kinase A

Calmodulin (CaM) CaM Kinases

Other Ca++-activated kinases eg MLCK

Other effectors - cAMP phosphodiesterase

- Calcineurin, a Ca++-activated phosphatase

- cytoskeleton

Some but not all Ca++- dependent

Activated by phorbol esters

Channels/Pumps

cGMP-dependent kinases

PM-NUCLEUS

See also

Brivanlou & Darnell

Science 295:813-818

(2002)

4

RTK-P

RTK

PhosphataseLigand

Signaling

GGTP

GGDP

Ligand

Signaling

GEFs GAPs

RGSs

GDIs

Receptor Protein Tyrosine Kinases

Ig

Fn3

HGF/SF

Neurotrophins

Ephrins Gas6

5

Dimerization Trans-phosphorylation

RecruitmentActivation

HGH/HGHR

1 monomeric ligand

2 receptors

R-R interactions

Asymmetric

Also Epo/EpoR

Kosiakoff, De Vos

and Wells

6

VEGF/VEGFR

1 dimeric ligand

2 receptors

R-R interactions

Symmetric

also PDGF, NGF, TGF/BMP

Wiesman et al, Cell 91:695-704 1997

EGF/EGFR

2 monomeric ligands

2 receptors

R-R interactions

Symmetric

7

FGF/FGFR

2 monomeric ligands

2 receptors

2 heparin chains

R-R interactions

Overall symmetric

but L-R binding is

two-site, asymmetric

Schlessinger et al, Mol. Cell 6: 743-750 2000

Mohammadi et al, Curr Opin Struc Biol. 15: 506-516, 2005

Receptor Autoinhibition

• EGFR extracellular domain undergoes conf. change on EGF binding

• BUT EGFR extracellular domain does not dimerize on EGF binding

• Intact EGFR DOES dimerize on EGF binding

• MODEL - ECD conformation blocks dimerization (autoinhibition)

and that is relieved by ligand

• sEGFR is mostly monomeric and low affinity (400-550 nM)

• there is a small population (~5%) with high affinity (2-20 nM)

• deletion of domain IV converts it to high affinity (13-21 nM)

8

Autoinhibition of EGFRs

IV

Cho and Leahy

Science (2002)

EGFEGF

EGF

Autoinhibition of FGFRs

FGFR

FGF

FGF

D3

D2

D3

D2

D1

D1

D1

9

PDGF-Receptor

Recruitment of signal transducers

to tyrosine phosphates

Protein Modules Recruiting Signaling Proteins

to Receptors and to the Membrane

10

Insulin Receptor Substrate (IRS)etc

An alternative way

to recruit

signal transducers-Y-P

P-Y-

-Y-PY-PP-Y

IRS

11

Different Mechanisms for Activating

Signaling Proteins

Recruitment

plus Activation

& Phosphorylation

Recruitment with

Conformational

Change -> Activation

Recruitment plus

Phosphorylation

-> Activation

Receptor -> MAPKinase pathway

MAPK

MAPKK

MAPKKK

MAPKK and MAPK

activations involve

BOTH Y and S/T

phosphorylations

12

Evolutionary Conservation of

MAPKinase Pathways

MAPK

MAPKK

MAPKKK

Effectors

MAP Kinase Pathways/Cascades

Why cascades? How does the cell keep modules separate?

13

switches

Ferrell, TIBS 21: 460-466 (1996)

Cascade as a switch mechanism

Mos -> ->Erk

Is there amplification?

Model Results

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 50

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1Fig. 1: Replica of Fig. 2A - Predicted responses on a linear plot

Input stimulus (E1tot

in multiples of the EC50)

Predicted Steady-State Kinase Activity

MAPKKK

MAPKK

MAPK

MAPKKK

MAPK

10-6

10-5

10-4

10-3

10-2

10-1

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1Fig. 2: Replica of Fig. 2B - Predicted responses on a semi-logarithmic plot

Input stimulus (E1tot

)

Predicted Steady-State Kinase Activity

MAPKKK

MAPKK

MAPK

MAPKKK

MAPK

nH = 1.0

nH = 4.9

Huang C.F. & Ferrell J.E. (1996)

PNAS 93, 10078-83

14

Experimental Validation

Huang C.F. & Ferrell J.E. (1996)

PNAS 93, 10078-83

Scaffolds for MAPK modules

Ste11p Ste7p Fus3p

MAPKKK MAPKK MAPK

Ste5p

MLK MKK7 JNK

JIP1

MLK MKK3 p38

JIP2

kinesin

15

More Scaffolds for MAPK modules

MEK1 ERK1

MAPKKK MAPKK MAPK

MP1late endosomesp14

Focalcontacts

SH2-SH3-SH3

CrkII

p130Cas

paxillinC3G

SOS

DOCK180

(via PPxLPxK)

GEFs

also in JNKMutation blocks

JNK activation by EGF/rac

but not by UV

!-arrestins

GPCR/arr/raf/erk

complexes on

endocytosis

Morrison & Davis

ARCDB 19:91-118

(2003)

RTK-P

RTK

PhosphataseLigand

Signaling

Kinase*

Kinase

RLigand

Signaling

Phosphatase

Receptor-intrinsic

kinases

Receptor-associated

kinases

16

ITAM - YxxLx6-8YxxL

Src family kinase ITAM-binding

(2xSH2) kinase

MHC/peptide

CD3 complexTCR

T Cell Receptor Signaling

P P

Cytokine Receptors

Shared signaling subunits - different ligand-binding subunits

17

JAK/Stat IRS etc

Cytokine Receptors

Signaling Pathways

TGF! Receptor Family - S/T Kinases

II IIII

18

TGF! Receptor Family - SMAD Signaling

TGF! Receptor Family -

Regulators and Feedback Loops

19

Protein Tyrosine Phosphatases

Protein Tyrosine Phosphatases

20

Structure of PTPase domain

D1 from receptor-like

Protein phosphatase-#

Bilwes et al.

Nature 382:555-559

(1996)

wedge

active

site

T Cell Receptor Signaling

Src family kinase ITAM-binding

(2xSH2) kinase

MHC/peptide

CD3 complexTCR

CD45

21

Negative Feedback Regulation