SMAD Phosphorylation and the TGF-βPathwaySMAD Phosphorylation and the TGF-βPathway 1 Joan...

SMAD Phosphorylation and the TGF-β PathwayJoan Massagué, Ph.D.

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SMAD Phosphorylation and the TGF-β Pathway

1

Joan Massagué, Ph.D.Alfred P. Sloan Chair, Cancer Biology and Genetics

Program Investigator, Howard Hughes Medical InstituteMemorial Sloan-Kettering Cancer Center, New York

ALK4TGFβR1

ALK7BMPR1BBMPR1A

ALK1ALK2

ActR2ActR2B

TGFβR2AMHR2

The human kinomeTyr kinases

2

Receptor Ser/Thr kinases:TGFβ receptors

BMPR2

A powerful morphogen

Courtesy A. Brivanlou

th

TGFβ: a paradigm of multifunctionality

BMP

TGFβActivinNodal Neural, dorsal fates

a metastasis promoter

ventral mesoderm

3

a growth suppressor

McPherron & Lee PNAS 1997

a metastasis promoter

Yin et al., J. Clin. Invest. 1999

Osteoclasts

Bonedegradation

TGFβ



TGFβEpitheliaG th i hibiti

Immune SystemInhibits T-cell proliferationImpairs NK, CTL functionsModulates Treg developmentEnforces immune tolerance

EndotheliaMigration

TGFβ in tissue homeostasis

4

Growth inhibitionMigrationPlasticity (EMT)Paracrine networkMatrix, adhesion

FibroblastsMatrix productionProliferationCytokine secretion

MorphogenesisGrowth control

+ 10 pM TGFβ

TGFβ inhibits cell proliferation

Control

5

Lung epithelial cells

ReceptorTGFβCyclinE/A-CDK2CyclinD-CDK4/6

TGFβ induces cell cycle arrest

Observation: TGFβ-treated cells contain a heat-stable CDK inhibitor

X

Discovery purification

G2

S

G1

G2

S

G1

M

6Polyak et al., Cell 1994; Russo et al., Nature 1996

Ni:H6 Cyclin E

CDK2

phospho-pRb

– p27Kip1

X

CDK2 kinase assay

Discovery, purificationand cloning of p27KIP1 M



Two sets of CDK inhibitors in TGFβ action

p21Cip1p27Kip1p57Kip2

p15Ink4bp16Ink4ap18Ink4c

TGFβCyclinE/A-CDK2CyclinD-CDK4/6

Lee et al., Genes Dev 1995Reynisdóttir et al., Genes Dev 1995

Scandura et al., PNAS 2004

G2

S

G1

7

p18Ink4cp19Ink4d M

p57KIP2 c-MYC

Epithelial cells

Neural cells, Astrocytes

Hematopoietic cells

T cells

Cytostatic TGFβ gene responses by cell typep27KIP1p15INK4b p21CIP1

Affinity-label TGFβ binding proteins

WTMutantsR DR

HybridR DR

TGFβ

Discovering TGFβ receptors: biochemistry + genetics

Isolate TGFβ–resistant mutants

Mutagenize(EMS) +TGFβ

125I-TGFβ

Receptors

Y

8

Rec

epto

rs

II –

I –

WT R DR RxDR

III –

Massagué JBC 1985Cheifetz et al., Cell 1987

Laiho et al., JBC 1989

II IIII

Cross-linker(disuccinimidyl

suberate)

SDS-PAGE

Y

Yx x

Y

x x

Receptor IIReceptor I

wt KR* wtwt wt KR*

II –

32P-phosphate

Two pairs of ligand-activated receptor Serine/Threonine kinases

TGFβ

II IIII

Mechanism of TGFβ receptor activation

9

I –

35S-methionine

Lopez-C et al., Cell 1991; Attisano et al., Cell 1992; Wrana et al., Nature 1994

Signal

II

I



TGFβ receptor activation switch

FKBP12

To plasma membrane

To plasma membrane

FKBP12TβRI

GS domainL45 loop

TβRII-mediatedphosphorylation

To plasma membrane R-Smad

10Huse et al., Cell 1999Huse et al., Mol Cell 2001

Locked kinase Basal state Activated kinase(Model)

R-Smad

L3 loop

TGFβ signaling pathway

pTxpSxpSxpSIn the GS region

Receptor I

FKBP12

11

Smad2 (MH2)

Massagué Cell 2008

Massagué Ann Rev Biochem 1998; Shi and Massagué Cell 2003

Massagué Cell 2008

ActivatedSmad complex

(dimer or trimer)

pSxpS groupbound to Smad4

Smad2

Smad4

Massagué Ann Rev Biochem 1998Shi and Massagué Cell 2003

Massagué Cell 2008

TGFβ signaling pathway (2)

12Massagué Cell 2008

Massagué Cell 2008



Smad protein structure and functions

MH1 domain

MH2 domain

MH1 domain binds:• DNA (5’CAGAC3’), but• Affinity too low to act alone

13

MH2 domain binds:• Receptors • Cytoplasmic anchors • Nucleoporins• Co-Smad (Smad4)• Transcription factors

Shi and Massagué Cell 2003

Cofactors

Co-activators,Co-repressors

Identifying a Smad DNA-binding cofactor

Vent-2 -243 GAGCCAACTAAC--GGCAGACATGGTGGAGCAGCTCTTAGTGAGAGGCA -185Vent-1 -186 GAAATCACTAACCTGACAGACTCACTGGAGCCAGGACCAGGGGCATTTG -134

SBE 3’ box

OAZY1H screening with BRE

Vent-2BRE

DNA pull down

WTSBE

t3’

toligo

14GGCAGACATGGTGGAGCAGCTCTCCGTCTGTACCACCTCGTCGAGA

OAZ

a 30 zinc-finger protein

Hata et al., Cell 2000

+-WT mut mut

BMP2

Smad4

oligo

+- +-

Smad DNA-binding cofactors

Cell-type specificity Target specificity

Three levels of specificity

Pathway specificity

Endogenous OAZ

ty trary

uni

ts)

25

30

Vent2 TIx2Smad1 Smad2

OAZ -bound Smads

15Hata et al., Cell 2000

P19 C2C12

None+ BMP2

Luci

fera

se a

ctiv

it

0246

82530

Exog. OAZ

Cell line P19 C2C12

- + - +

Luci

fera

se a

ctiv

ity (a

rbit

0

5

10

15

20

25

Exogenous OAZ

None+ BMP2

BMP2 TGFβ+- +-



Context-dependent Smad transcriptional action

16Massagué Cell 2008

Massagué et al., Genes Dev 2005

Paracrine network• IL11, VEGF, CTGF, Jagged1, Ang-L4• IL1β, BMP4

Signaling network• BMPR-II, VDR, EphB2, CDC42EP2, RhoGEF114, • SGK1, Mek4, LDLR, PGE-R4, βAR-2

Transcriptional network• Ets2, c-Jun, JunB, ATF3, Gadd45β, Pim1• Mad2, Mad4, C/EBPδ, MRG1, TRIP-Br2

A TGFβ transcriptional program

Receptor

TGFβ

SmadCo-activators,

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Cytostatic program• p21Cip1, p15Ink4b• c-Myc, Id1, Id2, Id3

Extracellular matrix• PAI-1, uPA, Col VI-A1, ADAM19• Integrin α5, integrin β6

Other responses• T-box3, MN1, Sialyl transf.4A• Sprouty 2, IAP3, UDPG-ceramide GT

Negative feedback• Smuf1, Smurf2, Smad7, SnoN, LEMD3

FOXO

Seoane et al., Cell 2004Gomis et al., PNAS 2006

ActivationRepression

DNA-binding cofactors

Co-repressors

TGFβ signaling: cytostatic gene responses

G2

S

G1

M

Smad3:Smas4:FOXO

Smad3:Smas4:FOXO:C/EBPβ

Smad3:Smas4:E2F4/5:C/EBPβ

Smad3:Smas4:ATF3

p21CIP1

p15INK4b

MYC

ID1

CDK

CDKII I

18



Smad pathway cofactors and regulators

TGFβ, BMP, Nodal…

• Ligand traps:LAP, Noggin, Chordin, Gremlin

• Inert ligands: Inhibin, Lefty

• Kinase blockers:FKBP12

• Pseudo-Receptors:BAMBI

• Co-receptors:TGFBRIII, Endoglin, Cripto

• Smad anchors:SARA

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BAMBI

• Inhibitory Smads:Smad6, Smad7

• Nuclear exclusion:TAZ

• Smad phosphatases:SCP, PPM1A

• Smad Ub ligases:Smurf, Nedd4L

• Blocking cofactors:FOXG1

SARA

• Nucleoporins:Nup153, Nup214

• Smad phospho-tail binders:Smad4; TRIM33

• DNA-binding cofactors:FOXH1, FOXO, OAZ, E2F4

• Transcriptional coactivators and corepressors:CBP, BRG1, YAP1, TGIF, ATF3 Massagué et al.,

Genes Dev. 2005

Smad linker phosphorylation

Smad1/5 Smad2/3

MAPKs

EGF, FGF, UV

BMPR

BMP

MAPKs

EGF, RAS, UV

TGFβR

TGFβ

MH1 MH2 MH1 MH2

20

Conserved MAPK/CDK sitesConserved GSK3 sites WW domain binding box, PPPXY

Kretzschmar et al., Nature 1997Signaling function

Kretzschmar et al., Genes Dev 1999Signaling function

Smad1Smad5Smad2Smad3

183 PFPHSPNSSYPNSPGSSSSTYPHSPTSSDPGSPFQMPADTPPPAYLPPEDP 233182 PFPLSPNSPYPPSP--ASSTYPNSPASSGPGSPFQLPADTPPPAYMPPDDQ 230

187 196 206 214 222

208 213204179

207 PAGIEPQSNYIPETPPPGYISEDGETSDQQLNQSMDTGSPAELSPTTLSPV 257167 PAGIEPQSN-IPETPPPGYLSEDGETSDHQMNHSMDAGSP-NLSPNPMSPA 215

HECT ubiquitin ligases recognize linker-phosphorylated Smads

Nedd4L C2 HECT1 2 3 4C2

Smad2/3Smad1/5

Smurf1 HECTC2 1 2

WW domains

21Sapkota et al., Mol Cell 2007

Gao et al., Mol Cell 2009

To proteasome for destruction

JNK, Erk, p38 activators:Mitogens, developmental factors, stresses



0 10 20 30BMP, min

Smad1/5 Phospho-Tail

Agonist-induced linker phosphorylation

Smad1/5

Smad1/5 pTail

Smad1 linker (pS206)

TGFβBMP—

Cyt Nuc Cyt Nuc Cyt Nuc

22

p

Smad1 Phospho-Linker

Alarcón et al., Cell 2009

Smad2 pTail

Smad2 linker (pT220)Smad3 linker (pT179)

α-Tubulin

Histone 1B

Smad2Smad3

Smad1 pLinker Smad1 pTail

ventricle

VZMerge Merge

Smad1pLinker

Smad1pTail

Telencephalinc ventricular zone (mouse embryo, E13.5)

Linker phosphorylation in mouse embryo

23

Smad2 pLinker Smad2 pTail

Dorsal root ganglia (mouse embryo, E13.5)Alarcón et al.,

Cell 2009

Immunohistochemical staining Immunofluorescence staining

Smad2 pTailSmad2 pLinker Merge

CDK8/9 mediate agonist-induced linker phosphorylation

Smad1

CyclinC CDK8

Smad3

CyclinC CDK8

BMP

Smad1 pS206

_ _ _ _+ + + +Control CDK7 CDK8 CDK9

RNAi

24

Smurf1

CyclinC-CDK8CyclinT-CDK9

Erk2 MAPK

Nedd4L

CyclinC-CDK8CyclinT-CDK9

Erk2 MAPK

Alarcón et al., Cell 2009; Gao et al., Mol Cell 2009

Smad1/5 pTail

Smad1/5



Smurf1, Nedd4L bind to CDK8/9-phosphorylated Smads

Pull-down of E3 Ub ligases with CDK8/9-phosphorylated GST-Smad baits

GST-Smad3 baitGST-Smad1 bait

– + – + – + – +

Smurf1 Smurf2 Nedd4 Nedd4L

Kinase

HA-tagged E3 Smurf1 Smurf2 Nedd4 Nedd4L

– + – + – + – +

25Alarcón et al., Cell 2009; Gao et al., Mol Cell 2009

CyclinC-CDK8

CyclinT-CDK9

Smad3

HA

Smad3

HA

Smad1

HA

Smad1

HA

Smurf1 and Nedd4L limit length of Smad activation

Post BMP, h

Smad1/5 pTail

Smad1/5 pTail

Smad1/5

ControlRNAi

– 0 0.5 1 2 4 Post TGFβ, h

Smad2 pTail

Smad2 pTail

Smad2/3

ControlRNAi

– 0 0.5 1 2 4

26Cell line: HaCaT human keratinocyte

Alarcón et al., Cell 2009Gao et al., Mol Cell 2009

p

Smad1/5

Smad1/5 pTail

Smad1/5

Smurf1RNAi

MG132

Smad2 pTail

Smad2/3

Smad2 pTail

Smad2/3

Nedd4LRNAi

MG132

Smurf1 C2 HECTWW WW

Agonist-induced linker phosphorylation drives Smad1–YAP interaction

Smad1/5 Interaction requires linker P sites

YAP TX’N

Flag-Smad1 _ _ _ _+

27

BMP-dependent YAP-Smad1 interaction

Alarcón et al., Cell 2009; Gao et al., Mol Cell 2009

BMP_ + _ + _ +

Input IP:IgG IP:Smad1/5

Smad1/5

YAP

gFlag-Smad1(AP)Flag-Smad3Flag-Smad3(V/AP)

_ _ _ _+_ _ _ _ +

_ +_ _ _

WB YAP

WB YAP

WB Flag

WB FlagIP Flag



CDK8/9 driving the Smad cycle

RSmad

P

P

Antagonistic signals

MAPKs Mitogens, stresses

PP

Ubiquitin Ligases:Smurf1 for Smad1/5Nedd4L for Smad2/3

Agonist signals

ReceptorsTGFβ, BMP

28

P

Phosphatases(linker, C-tail)

Smadtranscriptional

complex

P

Linker Coactivators:YAP for Smad1(X for Smad2/3)

P

Alarcón et al., Cell 2009

Linker Kinases: CDK8/9

P

P

Transcriptional CDKs Adapted from:Malik & Roeder,

TIBS, 2005Sims & Reinberg,Genes Dev, 2004

Enhancer-binding transcription factors (e.g., Smad)

CyclinH-CDK7CyclinC-CDK8

29

Facilitated Pol II recruitment Stimulation of basal activity

Transcriptional elongation

SCPs

CyclinT-CDK9

CyclinH-CDK7

CyclinC-CDK8

Inherited human disorders in TGFβ and BMP pathways

Gene

NOGGIN

AMH

GDF5

Component

BMP trap

Ligand

Ligand

Hereditary disorder

Proximal synphalangism

Persistent Muellerian duct syndrome

Hereditary chondrodysplasia

30

Endoglin

ALK1

AMHRII

TGFBRI, II

BMPBRII

SMAD4

BMP9 coreceptor

BMP9 receptor

AMH receptor

�TGFβ receptors

BMP receptor

Co-Smad

Hereditary hemorrhagic telangiectasia

Hereditary hemorrhagic telangiectasia

Persistent Muellerian duct syndrome

Loeys-Dietz Syndrome

Primary pulmonary hypertension

Juvenile polyposis



Loeys-Dietz syndrome

• A new syndrome characterized by: – Hypertelorism

– Cleft palate / bifid uvula,

– Arterial tortuosity, aneurysm (aortic dissection)

31

6/10 families: TGFBR2 mutation

Loeys et al., NEJM 2006

TGFβ and cancer: suppressor of pre-malignant progression

Massagué Cell 2008

32

Somatic mutations disable the TGFβ-Smad pathway in cancer

TGFBRII (3p22)• RER+ GI cancers (90%)• Other gastric and colon cancers• Head & neck cancer

kinaseTGFBRI (9q22)• Ovarian cancer

kinase

Missense mutations

Non-sense mutations

33

kinase • Ovarian cancer• T-cell lymphoma

MH1 MH2SMAD2 (18q21)• Colon cancer

MH1 MH2

SMAD4/DPC4 (18q21)• Pancreatic cancer (50%)• Metastatic colon cancer (35%)• Biliary, endometrial, ovarian,

lung, head & neck cancers



• Gene responses: tumor suppression• Cytostasis: CDK inhibition, Myc inhibition

• Differentiation: ID1 regulation

TGFβ in cancer: loss of entire pathway versus selective loss of suppressor arm

34

• Apoptosis: cell death signaling

• Gene responses: other effects• Phenotypic plasticity: EMT inducers

• Environment: ECM, cytokines, proteases

• Signaling: receptors, transducers, TFs

Massagué Cell 2008

Cytostatic gene responses lost in cancer

G2

S

G1

Smad4-Smas3-FOXO

Smad4-Smas3-FOXO-C/EBPβ

p21CIP1

p15INK4b CDK

CDK

II I

Glioblastoma

Breast CAPancreatic CA

Colorectal CA

35

G2G1

MSmad4-Smas3-E2F4/5-C/EBPβ

Smad4-Smas3-ATF3

II I

Smad4-Smas3-Xxx

Smad4-Smas3-Yyy

Smad4-Smas3-Zzz

Many other gene responses

MYCID1

Seoane et al., Cell 2004Gomis et al., Cancer Cell 2006

Stromal TGFβ

The TGFβ-Smad pathway as promoter of metastasis

ANGPTL4

36

Lung metastasis

Bone metastasis

ANGPTL4

Priming of cancer cells for lung metastasis

Chiang and Massagué NEJM 2008

TGFβ

IL11, CTGF,PTHrP



In summary• A biochemically contiguous pathway can be traced

from TGFβ ligand to cellular responses:– A framework for target gene selectivity

– A basis for context-dependent responses

– Mechanisms for pathway regulation and integration

• The TGFβ transcriptional response b d t t d i t di ti t

37

can be deconstructed into distinct programs:– Cytostatic program (p15, p21, MYC, ID1)

– FOXO synexpession group

• The Smad tumor suppressor pathway may turn into a tumor progression pathway by:

– Selective loss of cytostatic gene responses

– Gain of metastatic gene responses

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SMAD Phosphorylation and the TGF-βPathwaySMAD Phosphorylation and the TGF-βPathway 1 Joan...

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