phosphoserine/threoninebinding domains: molecular

Phosphoserine/Threonine Binding Domains:Molecular Integrators of Protein Kinase

Signaling in Cell Cycle Control and Cancer

Prof. Michael B. Yaffe

The screen versions of these slides have full details of copyright and acknowledgements 1

1

Prof. Michael B. YaffeCenter for Cancer Research

Biology & Biological Engineering

MIT

Phosphoserine/Threonine

Binding Domains

Molecular Integrators of Protein Kinase


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Checkpo int

Cell cycle

DNA damage

signalKinase Substrate Molecular

effect

Phospho-binding domains

Cell cycle

checkpoint

DNA repair

apoptosis/

senensence

Kinase 3Kinase 1

Kinase 2

Systemsapproaches

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• Centr al contr ol mechanism in eukar yotes

• One-thir d of all protei ns phosphor yl ated

• 1-2% of human genes encode

protein ki nases

• Abnormaliti es in phosphor yl ati on

are cause or conseq uence of cancer,

di abetes, i nfl ammation

• Defec ts in genes

for kinases

and phosphatases

underli e some

lymphomas, leukemi as,

and immunodefi ciency disor ders

Protein phosphorylation

Serine, threonineor tyrosine side chain





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PDGF-R

Kinase

Y

Y

Y

P

P

P

P

P

P

Membrane

Extracellular space

Intracellular space

Sos

Ras-GDP Ras-

GTP

+DAG

PIP2

IP3

**PKCs** **MAPKs**

PIP3

Tyrosine kinases signal

by recruit ing modular

phosphot yrosine-binding

domains in a sequence

specific manner

Y

Y

Y

Kinase

PDGF-R

AKT**

Historical perspective on signaling by tyrosine kinases

PLC -γγγγ SH2

PI 3-K

SH2SH3

SH3

SH2

Grb2

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SH2 domains bind ligands via pTyr and the pTyr+3 residue

Example:

Src SH2 binds

pY-X-X-I

SH2 domains bind ligands via pTyr

and the pTyr+3 residue

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The new ‘dogma’ of signal transduction by Serine/Threonine kinases

P

P

Tradit ion

al v iew

P

New v iew

pSer/Thr-bindingdomain

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Cdc2 kinase

Cyclin B

Cak

Wee1Myt 1mik1p

Mitosis

Polo-like

Kinase 1

+

X

Concept: Ser/Thr kinase SubstrateP Binding

domainExampleATM, ATR , BRCA1

Chk1, Chk2T68

DNA damagemodulates

activity

Mitotic

exit

Cyclin B

14-3-3

-RSXpSXP

Pin1-

PP1

MAPKAP-K2

, p38

Cdc25





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14-3-3

Yaffe et al, Cell 91, 961-71 (1997)

Rittinger et al, Molecular Cell 4, 153-66 (1999)

Brunet et al, J. Cell Biol. 156, 817-28 (2002)

Yaffe, Febs Lett. 513, 53-7 (2002)

Nguyen et al., Nature Biotech. 22, 993-1000 (2004)

14-3-3 Proteins are pSer/pThr-binding proteins

Pin1 WW Rotamase

Yaffe et al, Science 278, 1957-60 (1997) Lu et al, Science 283, 1325-1328 (1998)

Pin1-A pSer/pThr-Pro isomerase

FHA KinaseChk2

Durocher et al, Molecular Cell 6, 1169-92 (2000); Li et al, Molecular Cell 9, 1045-54 (2002)

FHA domains are pThr-binding modules

pS-P pS-PpS-P

Pin114-3-3 Proteins are pSer/pThr-binding proteins

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A functional proteomic approach to identifynew phospho-binding domains involved

in cell cycle regulation

A functional proteomic approach to identifynew phospho-binding domains involved

in cell cycle regulation

Cdks

Motif

Step 1

Step 2

Phospho

motif

Unknown

pSer/Thr- bind ing

domain

P

Cyclin

Step 3

Cell

cycle

progression

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biotin-Z-G-Z-G-G-X-X-B-X-pT-P-X-X-X-X

Cdk/MAPKmotif

Cdk/MAPK

Motif

Andy Elia

Phosphopeptide binding domains and protein kinases have co-evolved to recognize

overlapping consensus motifs

pSer/pThrbinding motif

Kinasephosphor ylation

motif

LxRXXSFP

14-3-3

Phosphopeptide binding domains and protein kinases have co-evolved to recognize overlapping consensus motifs

LxRXXSFP

P

LxRXXSFP

Use this concept to dev ise a proteome-wide screen

for phosphopeptide-binding domains

Linker

Step 1

A Hit !C-terminus of Polo-likeKinase 1

...x- T-P- x…

Chk2

LxRXXSFP

Step 2In vitro translate

cDNA library with 100 cDNAsper pool

Run prote ins on gel and analyze protein bound to compare phospho vs. non-phospho binding

Split each pool

and pull down 35S labeled

proteins with

peptide-bound

beads

Phospho peptide library bound to beads

P

Non-phospho peptide library bound to beads





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The biology supports the biochemistry

Early mitosis

The biology supports the biochemistry

Plk1, Plx1

Cdc 25C Cdc 25CP

Phosphat ase activit y ↑↑↑↑

Nuclear Localization

CohesinCdc 25

CohesinSeparase

Coh esin

e.g. MEN, FEAR Network

P

Polo, Plk1 P

P

PP

P

P

P

Polo family kinases together with cyclin-dependent kinases,

play many roles throughout mitosis

Mitotic entry

Centrosome maturat ion

Chromatid separation

Mitotic exit

Late mitosis

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Architecture of polo-like kinases

KinasePoloBox 1

PoloBox 2

Plk1, 2, 3

A single new domain

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Optimal motif: S-[pS/pT]-[P/X]

Polo-box domain ( PBD)

KinasePolo

Box 1PoloBox 2

Plk1

Structural basis for phospho-dependent binding

Partial Cdkmotif overlap

X-(pS/pT)-P





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Pro

Met Gln

Ser

pThr

Pro

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KinasePoloBox 1

PoloBox 2Plk1

1 - Phosphopeptide-binding to the PBD localizes

Plk1 to centrosomes

PBD

H538 K540

What does the Polo-box domain do in cells?

Plk1 PBD γγγγ-tubulin DAPI merge

W t

H538A

K540M

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Kinase PoloBox 1

PoloBox 2Plk1

2 - The PBD targe ts Plk1 to mitotic substrates

Cdc25

Polo-like

Kinase 1

+

Cdk

Cycli n

Cdc25C: 129S-pT-P131 Mitotic cyclin/Cdks

AG1/S arrest

NM arrest

P’aseM-inducerCdc25C

PBD

http://scansite.mit.edu





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Kinase activ

ity

3 - In the absence of a ligand, the PBD inhibi ts the kinase

activity of Plk1

basal activit y

Phosphorylation of casein by Plk1

Minutes

+ PBD peptide

3 - In the absence of a ligand, the PBD inhibi ts the kinase

activity of Plk1

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Synergy between Cdk activity and the Plk1 PBD explains the auto-amplification loop

for Cdc2/Cyclin-B activation

Nucleus

Centrosome

X

1 2

Wee-1

Mitosis

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Cdk/MAPK

Motif

A proteomic approach to identifying new phospho-Ser/Thr-binding domains

Kinase PoloBox 1

PoloBox 2Plk1

C-terminus of Po lo-l ike kinases mitosis

ATM/ATR Checkpoint

arre st

Andy Elia

ATM/ATR

MotifIsaac Manke

“PBD”

A phospho-Ser/Thr-b inding domain that recogn ized

Cdk-phosphorylat ed sit es

A molecular basis for phosphorylation-dependent checkpoint function?





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The DNA damage response pathway

IR UV

T-TDSBDNA

ATM ATR

? Phospho-

bind ing domains

that recognize…

X-X-X-X-pS-Q-X-X -X-X

Motif:

X-X-X-X-pS-Q-X-X -X-X

The DNA damage response pathway

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A proteome-wide screen for phosphopeptide-binding domains adapted

to “high-throughput” analysis

A proteome-wide screen for phosphopeptide-binding domains adapted

to “high-throughput” analysis

96,000 IVT ‘proteins’

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25PTIP: Pax2-transactiv ation domain interacting prot ein

C. elegans homologue - PIS- 1

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BRCA1

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A subset of tandem BRCT domains, including those

in BRCA1 are phospho-ser/thr-binding domains





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Tandem BRCT domains select phosphopeptidesbased on the pS+3 position

pS-X-X-F/I/L (PTIP)

F/Y (BRCA1)

Bach1 - DNA helicase

…associates with BRCA1

BRCT domains via motif

STpSPTFNK

Cantor et al., 2001 (Livingston),Yu et al., 2003 (Chen)

Tandem BRCT domains select phosphopeptides

based on the pS+3 position

BRCT-binding peptide: A-Y-D-L-pS-Q-V-F-P-F

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Structural basis for tandem BRCT-phosphopeptide binding

30Asn 1774

Il 1680

Gln 1779

Ser 1655

Leu 1657

Gly 1656

Phe 1704

Met 1775

Leu 1839

Glu 1836

Asp 1840

Arg 1699

Arg 1699

Glu 1698

Lys 1702

Leu 1701

-extraction + extraction

Ser-1655 and Lys-1702 mediate phosphate interactions

Anti-myc Anti-pS-Q Merge

WT

S1655AK1702M

-IR +IRa b





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Cancer-associated mutations in BRCA1 BRCT domains compromise phosphopeptide-binding

Cancer-associated mutations in BRCA1 BRCT

domains compromise phosphopeptide-binding

and Bach1-binding

Y1853XR1699Q

C1697R

D1692Y

S1655FM1775R

S1715R

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Cancer mutation M1775→→→→R

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Foster & Downs, FEBS J. 2005

In addition to BRCA1… amplification of the ATM/Chk2 signal after IR involves

phosphorylation of H2AX and recruitment of MDC1

In addition to BRCA1… amplification of the ATM/Chk2 signal after IR involves

phosphorylation of H2AX and recruitment of MDC1

H2AX C-terminus: KKATQApSQEY

MDC1

Stewart et al, Nature 421 (2003)





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The phosphorylated C-terminus of H2AX binds to the BRCT domains of MDC1

The phosphorylated C-terminus of H2AX binds to the BRCT domains of MDC1

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The MDC1 BRCT domains are ‘tuned’ to bind to γγγγH2AX

Peptide libraries:

XXXX-(pS/pT)-XXXX

XXXX-pS-XXFXX

XXXX-pS-XXX-COOH

The MDC1 BRCT domains are ‘tuned’ to bind to γγγγH2AX

Optimal moti f:

pS-(PIV)-(EIV)-Y-COOH

γγγγH2AX:pS-Q-E-Y- COOH

IVT

+

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Structural basis for MDC1 BRCT: γγγγH2AX binding

Structural basis for MDC1 BRCT: γγγγH2AX binding





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Mutants that disrupt MDC1 BRCT function or the γγγγH2AX motif cause loss of MDC1

foci after IR

Mutants that disrupt MDC1 BRCT function

or the γγγγH2AX motif cause

loss of MDC1 foci after IR

MDC1

mutants

γγγγH2AX mutants

γγγγH2AX

GFP-MDC1

- WT S136/139A Y142A

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A proteomic approach to identifying

new phospho-ser/thr-binding domains

Cdk/MAPK

Motif

Kinase PoloBox 1

PoloBox 2Plk1

C-termi nus of Polo-li ke kinases mitosis

Andy Elia“PBD”

A phospho-Ser/Thr-b inding domain that recogn ized Cdk-

phosphorylated sit es to coord inate mitotic progression

ATM/ATR Tandem BRCT domains

Cell cycle checkpoint arrest

ATM/ATR

Motif

Isaac Manke

A molecular basis for phosphorylation-dependent checkpoint function

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WW Rotamase

FHA Kinase

BRCT BRCTRing

KinasePolo

Box 1

Polo

Box 2

14-3-3

Pin1

Chk2

Plk1

Brca1

(MDC1 also)

= pSer/pThr

Binding Domain





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14-3-3

MDC1

Cdc25

Cdc2 kinase

Cyclin B

Cak

Wee1Myt 1mik1p

Mitosis

Polo-like

Kinase 1

+

-

X

Concept: Kinase SubstrateP Binding

domainExample

T68

DNA damage

modulates

activity

Pin1-

Mitotic exit

PDGF receptor

Intracellular tail

pTyr-SH2 Domaininteractions

pSer/pThr-binding domains

coordinate mitotic signaling

complexes

Pin1-

PP1

, p38

MAPKAP-K2

ATM, BRCA1

Chk2

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Cell cycle cont rol

Tyrosine kinase signaling

and pTyr-binding domains

Limited to multicellular eukaryotes

Ser/Thr kinase signaling

and pSer/Thr-binding domains

Found in even single cell eukaryotes

Homo sapiens Budding yeast Fission yeast

Brca1, 53BP1 Rad9p Rhp9 ?

Chk2 Rad53p Cds1

Plk1, 2, 3 Cdc5p Plo1

Pin1 Pin1Ess1p

14-3-3s (Bmh1, 2) Rad24, 25

Evolutionary difference

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Phosphoserine/threonine binding domainsControl the cell cycle and DNA damage response

14-3- 3

WD40 repeat s

of F-box prot eins

Polo-box domains

Tandem BRCTs

WW domain of Pin1

FHA domains





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Conclusions

• All of cell cycle progression and checkpoint signaling

in eukaryotes involves the coordination of Ser/Thr kinases

with pSer/pThr-binding domains to establish networks

of phosphorylation-dependent protein-protein interactions

• There is partial overlap of mitotic and checkpoint kinase

phosphorylation motifs with the optimal sequences recognized

by pSer/pThr-binding domains to provide spatial and temporal

control of protein-protein complexes

• Examples of these domains include 14-3-3 proteins, WW

domains, FHA domains, WD40 repeats, Polo-box domains

and tandem BRCT domains

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Acknowledgements

Duaa Mohammad

Isaac Manke

NIMR

Julie Clapperton

Steve Smerdon

Drew Lowery

Andy Elia

Katja Hoepfer

Coky Nguyen

Timmy Ho

Irma Rangel

MIT

Cambridge, UK

Manny Stucki

Steve Jackson

HMS

David Livingston

Ralph Sculley

Lew Cantley

Christian Reinhardt

Andy Elia

Acknowledgements

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phosphoserine/threoninebinding domains: molecular

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