Mapping the 14-3-3-binding 2R-ohnologue protein

Report
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
14-3-3
Kinase 1
P
Kinase 2
Hundreds of structurally and
functionally diverse targets
1
The human 14-3-3 interactome is highly enriched in
2R-ohnologues
1R-WGD
2R-WGD
Selection/Loss
2R-ohnologues
Invertebrate
chordates
Mammals
2
2R-Ohnologues and the ‘lynchpin’ phosphosites
P
P
P
Lynchpin site
P
Lynchpin site
3
2R-Ohnologues and the ‘lynchpin’ phosphosites
P
Evolving site
(different kinase)
P
P
Lynchpin site
P
Lynchpin site
 Conserved across family members back to the single proorthologue in invertebrate chordates (Branchiostoma and
Ciona)
 14-3-3-binding motif: RXX(pS)XP
3
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 2Rohnologue protein families of the human kinome
3.
Biochemically validate high priority candidate 14-3-3 binders
4
ANIA: ANnotation and Integrated Analysis of the 14-3-3
interactome
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
5
ANIA: ANnotation and Integrated Analysis of the 14-3-3
interactome
Database
 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
Predictions
 Conservation
 Phosphorylation
 Prediction PSSM
 Prediction NN
 Disorder
 Intracellular
ANIA: ANnotation and Integrated Analysis of the 14-3-3
interactome
Homepage of ANIA
7
ANIA: ANnotation and Integrated Analysis of the 14-3-3
interactome
Tabular view of results
7
ANIA: ANnotation and Integrated Analysis of the 14-3-3
interactome
Detailed view of each protein queried
7
ANIA: ANnotation and Integrated Analysis of the 14-3-3
interactome
Tabular view of results
7
ANIA: ANnotation and Integrated Analysis of the 14-3-3
interactome
Detailed analysis of candidate 14-3-3-binding phosphosites
7
2R-ohnologue families of
the Human Kinome
Total
GD
Families
142
20
Members
355
23
8
2R-ohnologue families of
the Human Kinome
Lynchpin sites for ~65%
of the gold-standard
14-3-3 binders
87% true-positives
Total
Lynchpins
TP
Families
20
12
10
Members
23
15
13
8
2R-ohnologue families of
the Human Kinome
Lynchpin sites for ~45%
members of the human
kinome
PAK4
Total
Lynchpins
Families
142
57
Members
355
158
8
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
9
Candidate 14-3-3-binding phosphosites of PAK4
Ser99
...
Ser162
...
Ser181
...
Ser474
...
...
10
phosphoSer181 of PAK4 participates in the binding to 14-3-3
Calyculin A
14-3-3
Overlay
GFP
pull-downs
14-3-3
α-GFP
Decreased binding
Second site that is
phosphorylated
11
Phorbol esters regulate the phosphorylation of PAK4
‘Panel’ of stimulli/inhibitors
that activate or inhibit AGC
and CAMK kinases
14-3-3 Overlay
14-3-3
GFP pull-downs
α-GFP
Abnormal patterns
of phosphorylation
pT202/204 ERK1/2
pS157 VASP
pS473 PKB
An outcome of PAK4
overexpression
Cell lysates
pT172 AMPK
14-3-3
α-GFP
12
Phorbol esters regulate the phosphorylation of PAK4
‘Panel’ of stimulli/inhibitors
that activate or inhibit AGC
and CAMK kinases
14-3-3 Overlay
14-3-3
GFP pull-downs
α-GFP
Response to phorbol ester stimulation
12
‘Signalling signatures’ of PAK4
?
S181
PKC, PKD or p90RSK
13
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 phosphorylationdependent binding of 14-3-3 to PAK4
14
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
15
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)

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