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Mapping the 14-3-3-binding 2R-ohnologue protein families of the human kinome. Fábio M. Marques Madeira Supervisor: Professor Carol MacKintosh. 1 th February 2013. 14-3-3s dock onto pairs of tandem phosphoSer / Thr. P. P. Kinase 1. 14-3-3. Kinase 2. - PowerPoint PPT Presentation
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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
7
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 GDFamilies 142 20
Members 355 23
Total Lynchpins TPFamilies 20 12 10
Members 23 15 13
8
Lynchpin sites for ~65% of the gold-standard 14-3-3 binders
87% true-positives
2R-ohnologue families of the Human Kinome
Total LynchpinsFamilies 142 57
Members 355 158
Lynchpin sites for ~45% members of the human kinome
PAK4
8
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
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
S99/
162/
181/
474A
14-3-3 Overlay
α-GFP
GFP pull-downs
GFP
-PAK
4S9
9A
S162
A
S181
A
S474
A
S162
/181
A
GFP
-PFK
FB2
S466
/483
A
14-3-3
GFP
-PAK
4S9
9A
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
+ PM
A
Seru
m S
tarv
edIG
F1PI
-103
+ IG
F1EG
FPM
A
Fors
kolin
H-8
9 +
Fors
kolin
A769
662
A231
87Ca
lycu
lin 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
+ PM
A
Seru
m S
tarv
edIG
F1PI
-103
+ IG
F1EG
FPM
A
Fors
kolin
H-8
9 +
Fors
kolin
A769
662
A231
87Ca
lycu
lin 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
12
‘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
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)