Mapping the 14-3-3-binding 2R-ohnologue protein
Download
Report
Transcript Mapping the 14-3-3-binding 2R-ohnologue protein
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)