Protein Domains That Interact with Protein Domains That Interact with Receptor Tyrosine Kinases: Receptor Tyrosine Kinases:

Structural Aspects Structural Aspects

Ming-Ming Zhou, Ph.D.MSSM Department of Pharmacology Course on

“Cell Signaling Systems”January 27, 2005

The Role of Protein Domains in Cell SignalingThe Role of Protein Domains in Cell Signaling

PLCPLC--SosSos RafRaf

PI(3)KPI(3)K MEKMEK

MAPKMAPK

c-fos, c-myc, c-junc-fos, c-myc, c-jun

Grb2Grb2

ShcShc

PP

PTBPTB

SH2SH2

CHCH

PP

PP

PP

PP

PP

PP

RTKRTK

Growth FactorsGrowth Factors

RasRas GDPGDP RasRas

GTPGTP

SH2SH2SH3SH3 SH3SH3

-pY-X-X-hy--pY-X-X-hy-

SH2SH2

-hy-X-N-P-X-(p)Y--hy-X-N-P-X-(p)Y- PhospholipidsPhospholipids

PTBPTB PHPH SH3SH3 WWWW

EHEH 14-3-314-3-3

-N-P-F--N-P-F- -R-S-X-pS-X-P--R-S-X-pS-X-P-

-P-X-X-P-X--P-X-X-P-X- -P-P-X-Y--P-P-X-Y-

PDZPDZ

-E-S/T-D/V--E-S/T-D/V-COOHCOOH--

. . .. . .

Protein Domains in Cell Signal TransductionProtein Domains in Cell Signal Transduction

Human TrkA, pY490 HIIENPQpYFSDA 280 0.053

Mouse Polyoma MT, pY250 SLLSNPTpYSVMRS 320 0.140

Human ErbB3, pY1309 SAFDNPDpYWHSRLF 230 0.020

Human ErbB2, pY1248 AENPEpYLGLDV 1290 N.D.

Human EGFR, pY1086 GSVQNPVpYHNQPLN 220 5.30

Human EGFR, pY1148 SLDNPDpYQQDFF 1000 0.200

Human EGFR, pY1114 TAVGNPEpYLNTVQ 290 0.140

Human EGFR, pY992 VADADEpYLIPQQ 130 680

Human EGFR, pY1173 TAENAEpYLRVA 60 770

Human TCR Chain, pY141 GHDGLpYQGLSTATK 50 >10000

ProteinpTyr Sites

pTyrPeptides

Shc SH2 Shc PTBKd (M) Kd (M)

Ligand Recognition of the Shc PTB and SH2 DomainsLigand Recognition of the Shc PTB and SH2 Domains

Ref: Zhou et al (1995) JBC 270, 31119-31123.

Phosphotyrosine Binding DomainsPhosphotyrosine Binding Domains

Ligand SpecificityLigand Specificity Structural Versatility Structural Versatility Functional DiversityFunctional Diversity

SH2 Domain:

---pYpYxxxx--- PTB Domain:

---xxNPNPxxpYpY---

Q:Q:

3D Structue of the Shc SH2 Domain/TCR Peptide Complex3D Structue of the Shc SH2 Domain/TCR Peptide Complex

TCR chain peptide: GHDGLpYQGLSTATK

BG

BC

A

B

N

C

pYpY+3

EF

D’

EF

CB

D

G

AR B5

BG

N

C

pY

pY+3

F

C

D

G

R B5

R A2

V C3

H D4

L D6

A BG7

L D5

T E4

I BG4

Ref: Zhou et al (1995) PNAS 92, 7784-7788.

BG

A

B

N CD’

EF

C

B

D

G

ApY

pY+3

DE

pYpY+3

BG

DE

EF

D’

pY+3pY

BG

DE

EF

D’

pYpY+3

BG

DEE

F D’

Shc Lck

SypPLC-1

Structures of SH2 DomainsStructures of SH2 Domains

Shc

Lck

Syp

PLC-1

Structures of SH2 DomainsStructures of SH2 Domains

3D NMR Structure of the Shc PTB Domain/TrkA Complex3D NMR Structure of the Shc PTB Domain/TrkA Complex

Ref: Zhou et al (1995) Nature 378, 584-592.

XNPXpY Recognition by the Shc PTB DomainXNPXpY Recognition by the Shc PTB Domain

PTB Domain PTB Domain versusversus SH2 Domain SH2 Domain

Shc PTB Domain Shc PTB Domain Shc SH2 DomainShc SH2 Domain

Biological Ligands of PTB DomainsBiological Ligands of PTB Domains

PTB DomainPTB Domain Binding PartnersBinding Partners Binding Sites Binding Sites

ShcShc hTrkA, pY490hTrkA, pY490 HIHIIIEENPNPQQpYpYFSDAFSDAmPolyoma MT, pY250mPolyoma MT, pY250 SLSLLLSSNPNPTTpYpYSVMRSSVMRShErbB3, pY1309hErbB3, pY1309 SASAFFDDNPNPDDpYpYWHSRLWHSRLhEGFR, pY1148hEGFR, pY1148 ISISLLDDNPNPDDpYpYQQDFFQQDFF

IRS-1IRS-1 hIR, pY960hIR, pY960 LYALYASSSSNPNPEEpYpYLSASLSAShIL-4R, pY497hIL-4R, pY497 LVILVIAGAGNPNPAApYpYRSFSRSFShIGFIR, pY950hIGFIR, pY950 LYALYASVSVNPNPEEpYpYFSASFSAS

NumbNumb LNXLNX GGLLDDNPNPAAYYTSSV TSSV NAKNAK GGFFSSNMNMSSFFEDFP EDFP Peptide LibraryPeptide Library YIGPYIGPYYLL

X11, Fe65, Ced-6X11, Fe65, Ced-6 APPAPP GGYYEENPNPTTYYKKFFFFEE

3D Structure of the IRS-1 PTB Domain/IL-4R Complex3D Structure of the IRS-1 PTB Domain/IL-4R Complex

Ref: Zhou et al (1996) Nature Structural Biology 3, 388-393.

Ligand Recognition of the IRS-1 PTB domainLigand Recognition of the IRS-1 PTB domain

Distinct Mechanisms for NPXpY RecognitionDistinct Mechanisms for NPXpY Recognition

IRS-1 PTB Domain IRS-1 PTB Domain Shc PTB Domain Shc PTB Domain

PTB & PH Domains: A Conserved Structural FoldPTB & PH Domains: A Conserved Structural Fold

AA BB

CC DD

FGFRsFGFRs (juxtamembrane region):(juxtamembrane region):

KSDFH SQMAV HKLAK SIPLR RQVTSKSDFH SQMAV HKLAK SIPLR RQVTS

NGFRsNGFRs::•TrkA,TrkA,pY960pY960 HIIEHIIENPNPQQpYpYFSDAFSDA•TrkBTrkB PVIEPVIENPNPQQpYpYFGITFGIT•TrkCTrkC PVIEPVIENPNPQQpYpYFRQGFRQG

RETRET, pY1062pY1062

TWIETWIENNKLKLpYpYGRISGRIS

SNT/FRS2 Binding Sites in Neurotrophic ReceptorsSNT/FRS2 Binding Sites in Neurotrophic Receptors

11

33

4455

66

77

FGFRFGFR

CC

NN

NN

CC

22

11

88

’’

113344

5566

77

FGFRFGFR

CC

NN

NN

CC

22

11

88 ’’

3D Structure of the SNT-1 PTB Domain/FGFR1 3D Structure of the SNT-1 PTB Domain/FGFR1 ComplexComplex

Ref: Dhalluin et al (2000) Molecular Cell 6, 921-929.

SNT-1 SNT-1 versusversus IRS-1 PTB domains IRS-1 PTB domains

R78

FGFR

11

33

4488

55

6677

C

N

22

11

R63

’’

N

C

R212

IL-4R

11

33

44

55

6677

C

N

22

11

R227

N

C

pY-3

pY-6

pY-8

pY

A

B

SNT-1SNT-1

IRS-1IRS-1

Sequence Alignment of the SNT & IRS PTB DomainsSequence Alignment of the SNT & IRS PTB Domains

R78R78

11

33

44

88

5566

77

FGFRFGFR

CC NN NN

CC

22

11

R63R63

’’

Vec

tor

Vec

tor

SN

T-1

PT

B

SN

T-1

PT

B

D

om

ain

Do

mai

n S

NT

-1 P

TB

S

NT

-1 P

TB

D

om

ain

D

om

ain

88

HisHis--, Leu, Leu--, Trp, Trp--LeuLeu--, Trp, Trp--HisHis--, Leu, Leu--, Trp, Trp--LeuLeu--, Trp, Trp--HisHis--, Leu, Leu--, Trp, Trp--LeuLeu--, Trp, Trp--

TRKBTRKBhFGFR1hFGFR1VectorVector

SNT-1 Truncation Effect on FGFR & TRK BindingSNT-1 Truncation Effect on FGFR & TRK Binding

w/FGFRw/FGFR

Yeast Two-Hybrid Binding AssayYeast Two-Hybrid Binding Assay

11

33

44

5566

77 NN

22

11

pYpY

pY-5pY-5

R78R78

R63R63

TrkBTrkB

CC

CC

w/TrkBw/TrkB

Unifying Features of PTB DomainsUnifying Features of PTB Domains

Ref: Yan & Zhou (2002) FEBS Lett. 513, 67-70.

Structure & Function of the SH3 DomainStructure & Function of the SH3 Domain

Ref: Zarrinpar (2003) Sci. STKE 2003(179), re8

CCAA

BB

RTKs: Activation by Receptor Dimerization

Ref: Hubbard, et al (1998) JBC 273, 11987-11990.

Autoinhibition of Insulin and FGF ReceptorsAutoinhibition of Insulin and FGF Receptors

IRKIRK FGFR1KFGFR1K

Unphosphorylated IRK (IRK0P)Unphosphorylated IRK (IRK0P)

Tris-phosphorylated IRK (IRK3P)Tris-phosphorylated IRK (IRK3P)

Unphosphorylated FGFR1KUnphosphorylated FGFR1K

Tris-phosphorylated IRK (IRK3P)Tris-phosphorylated IRK (IRK3P)

- Cis-inhibition & Trans-activation- Cis-inhibition & Trans-activation

AutoRegulation & Activation of AutoRegulation & Activation of SrcSrc Tyrosine Kinase Tyrosine Kinase

3D Structure of Protein Tyrosine Kinase 3D Structure of Protein Tyrosine Kinase HckHck

Ref: Xu, et al (1997) Nature 385, 595-602; Sicheri, et al (1997) Nature 385, 602-609.

Rational Design of Nonpeptide Inhibitors of Rational Design of Nonpeptide Inhibitors of SrcSrc SH2 Domain SH2 Domain

Ref: Sundaramoorthi et al (2003) Biopolymers 71, 717-729.

3D Structure of the Abl Kinase Domain/Drug Complex3D Structure of the Abl Kinase Domain/Drug Complex

Ref: Daub, H. et al (2004) Nature Review Drug Discovery 3, 1001-1010.

Summary

Structure-Function of Protein Domains in RTKs

Conserved 3D structural fold with divergent sequences

Conserved structural fold with different functions

Single protein domain with multiple functions

Cooperative regulation of RTK activity in cell signaling