mapping the 14-3-3-binding 2r-ohnologue protein families of the human kinome fábio m. marques...

Mapping the 14-3-3-binding 2R-ohnologue protein families of the human kinome

Fábio M. Marques Madeira

Supervisor: Professor Carol MacKintosh

1th February 2013

14-3-3s dock onto pairs of tandem phosphoSer/Thr

P P

Kinase 1 Kinase 2

Hundreds of structurally and functionally diverse targets

14-3-3

1

The human 14-3-3 interactome is highly enriched in 2R-ohnologues

2R-ohnologues

Invertebrate chordates Mammals

1R-WGD 2R-WGD

Selection/Loss

2

2R-Ohnologues and the ‘lynchpin’ phosphosites

P P P P

3

Lynchpin siteLynchpin site

2R-Ohnologues and the ‘lynchpin’ phosphosites

Evolving site(different kinase)

P P P P

Lynchpin siteLynchpin site

3

14-3-3-binding motif: RXX(pS)XP

Conserved across family members back to the single pro-orthologue in invertebrate chordates (Branchiostoma and Ciona)

Aims

1. Develop a web resource on the 14-3-3 interactome and a predictor of

14-3-3-binding phosphosites

2. Use the resource to map experimental/candidate 14-3-3-binding 2R-

ohnologue protein families of the human kinome

3. Biochemically validate high priority candidate 14-3-3 binders

4

Why?

1. Huge amount of dispersed data on the 14-3-3 interactome

2. The gold standard 14-3-3 binders (>200)

3. High-throughput (HT) 14-3-3 capture experiments (thousands of

candidate 14-3-3 binders)

4. No reported resource to store, analyse and display this complex

information

ANIA: ANnotation and Integrated Analysis of the 14-3-3 interactome

5

ANIA: ANnotation and Integrated Analysis of the 14-3-3 interactome

PredictionsDatabase

2R-ohnologue human kinase families

The gold standard 14-3-3 binders

HT 14-3-3 capture experiments

HT contaminants (in-house)

Maps for mouse and rat homologous proteins

UniProt, GO terms and GAD

Conservation

Phosphorylation

Prediction PSSM

Prediction NN

Disorder

Intracellular

Homepage of ANIA

ANIA: ANnotation and Integrated Analysis of the 14-3-3 interactome

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Tabular view of results

ANIA: ANnotation and Integrated Analysis of the 14-3-3 interactome

7

Detailed view of each protein queried

ANIA: ANnotation and Integrated Analysis of the 14-3-3 interactome

7

Tabular view of results

ANIA: ANnotation and Integrated Analysis of the 14-3-3 interactome

7

Detailed analysis of candidate 14-3-3-binding phosphosites

ANIA: ANnotation and Integrated Analysis of the 14-3-3 interactome

7

8

2R-ohnologue families of the Human Kinome

Total GD

Families 142 20

Members 355 23

Total Lynchpins TP

Families 20 12 10

Members 23 15 13

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Lynchpin sites for ~65% of the gold-standard 14-3-3 binders

87% true-positives

2R-ohnologue families of the Human Kinome

Total Lynchpins

Families 142 57

Members 355 158

Lynchpin sites for ~45% members of the human kinome

PAK4

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2R-ohnologue families of the Human Kinome

p21-activated protein kinase 4 (PAK4)

PAKs comprise 2R-ohnologue families composed of 2 groups

(group I: PAK1-3, and group II: PAK4-6) PAK 4 is a Ser/Thr kinase activated by Rho-family GTPases Cdc42

and Rac, regulators of actin cytoskeleton dynamics

Why?

1. All members are 14-3-3-binding candidates

2. PAK4 was identified in an in-house HT 14-3-3 capture exp. and in

several published HT experiments

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Candidate 14-3-3-binding phosphosites of PAK4

Ser99 Ser162 Ser181 Ser474

... ... ... ......

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phosphoSer181 of PAK4 participates in the binding to 14-3-3

S99/

162/

181/

474A

14-3-3 Overlay

α-GFP

GFP pull-downs

GFP

-PA

K4

S99A

S162

A

S181

A

S474

A

S162

/181

A

GFP

-PFK

FB2

S466

/483

A

14-3-3

GFP

-PA

K4

S99A

S162

A

S181

A

S474

A

Calyculin A

Second site that is phosphorylated

Decreased binding

11

Phorbol esters regulate the phosphorylation of PAK4

BI-D

1870

+ P

MA

Seru

m S

tarv

edIG

F1PI

-103

+ I

GF1

EG

F

PMA

Fors

kolin

H-8

9 +

Fors

kolin

A76

9662

A23

187

Cal

ycul

in A

GFP pull-downs

Cell lysates

pT202/204 ERK1/2

pS157 VASP

pS473 PKB

pT172 AMPK

14-3-3 Overlay

α-GFP

14-3-3

14-3-3

α-GFP

Abnormal patterns of phosphorylation

‘Panel’ of stimulli/inhibitors that activate or inhibit AGC and CAMK kinases

An outcome of PAK4 overexpression

12

Phorbol esters regulate the phosphorylation of PAK4

BI-D

1870

+ P

MA

Seru

m S

tarv

edIG

F1PI

-103

+ I

GF1

EG

F

PMA

Fors

kolin

H-8

9 +

Fors

kolin

A76

9662

A23

187

Cal

ycul

in A

GFP pull-downs

14-3-3 Overlay

α-GFP

14-3-3

Response to phorbol ester stimulation

‘Panel’ of stimulli/inhibitors that activate or inhibit AGC and CAMK kinases

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‘Signalling signatures’ of PAK4

PKC, PKD or p90RSK

13

? S181

Conclusions

We developed a user friendly web resource for the annotation and prediction

of the 14-3-3 interactome

Our projections indicate that 14-3-3s may dock onto ~45% of 2R-ohnologue

human kinase family members

We validated PAK4 as a novel 14-3-3-binding target, and pinpointed

phosphoSer181 as one of the lynchpin sites

We identified phorbol ester as a stimulus that promotes phosphorylation-

dependent binding of 14-3-3 to PAK4

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Future work

1. Site-directed mutagenesis of S181A double mutants and loss of

Calyculin A-/PMA-stimulated 14-3-3 binding

2. Stimuli/inhibitor experiments to investigate different patterns of

‘signalling signatures’ of PAK4

3. In vivo phosphorylation (using SILAC) of endogenous PAK4

4. Further investigate the effects of 14-3-3 binding on PAK4

5. Extend studies to all the human 2R-ohnologue families

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Acknowledgements

Professor Carol MacKintosh

Dr Michele Tinti (Bioinformatics)

Dr Gerta Hoxhaj and Dr Catherine Johnson (Laboratory)

All members in Carol’s group

MRC PPU and DSST (tissue culture, cloning and sequencing)