kinome Fábio M Marques Madeira Supervisor Professor Carol MacKintosh 1 th February 2013 1433s dock onto pairs of tandem phosphoSer Thr P P Kinase 1 Kinase 2 Hundreds of structurally and functionally diverse targets ID: 335744
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Slide1
Mapping the 14-3-3-binding 2R-ohnologue protein families of the human kinome
Fábio M. Marques MadeiraSupervisor: Professor Carol MacKintosh
1
th
February 2013Slide2
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
1Slide3
The human 14-3-3 interactome is highly enriched in 2R-ohnologues
2R-ohnologues
Invertebrate chordates
Mammals
1R-WGD
2R-WGD
Selection/Loss
2Slide4
2R-Ohnologues and the ‘lynchpin’
phosphosites
P
P
P
P
3
Lynchpin site
Lynchpin siteSlide5
2R-Ohnologues and the ‘lynchpin’
phosphosites
Evolving site
(different kinase)
P
P
P
P
Lynchpin site
Lynchpin 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
)Slide6
Aims
Develop a web resource on the 14-3-3 interactome and a predictor of 14-3-3-binding phosphositesUse the resource to map experimental/candidate 14-3-3-binding
2R-ohnologue protein families of the human
kinome
Biochemically validate high priority candidate 14-3-3
binders
4Slide7
Why?
Huge amount of dispersed data on the 14-3-3 interactomeThe gold standard 14-3-3 binders (>200)High-throughput (HT) 14-3-3 capture experiments (thousands of candidate 14-3-3 binders)
No reported resource to store, analyse and display this complex information
ANIA:
ANnotation
and Integrated Analysis of the 14-3-3
interactome
5Slide8
ANIA:
ANnotation and Integrated Analysis of the 14-3-3 interactome
Predictions
Database
2R-ohnologue human kinase families
T
he gold standard 14
-3-3 binders
HT 14-3-3 capture experiments
HT contaminants (in-house)
Maps for mouse and rat homologous proteinsUniProt, GO terms and GAD
Conservation
Phosphorylation
Prediction PSSM
Prediction NN
Disorder
IntracellularSlide9
Homepage of ANIA
ANIA:
ANnotation
and Integrated Analysis of the 14-3-3
interactome
7Slide10
Tabular view of results
ANIA:
ANnotation
and Integrated Analysis of the 14-3-3
interactome
7Slide11
Detailed view of each protein queried
ANIA:
ANnotation
and Integrated Analysis of the 14-3-3
interactome
7Slide12
Tabular view of results
ANIA:
ANnotation
and Integrated Analysis of the 14-3-3
interactome
7Slide13
Detailed analysis of candidate 14-3-3-binding phosphosites
ANIA:
ANnotation
and Integrated Analysis of the 14-3-3
interactome
7Slide14
8
2R-ohnologue families of the Human
Kinome
Total
GD
Families
142
20
Members
355
23Slide15
Total
Lynchpins
TP
Families
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
KinomeSlide16
Total
Lynchpins
Families
142
57
Members
355
158
Lynchpin sites for
~45
% members of the human
kinome
PAK4
8
2R-ohnologue families of the Human
KinomeSlide17
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?
All members are 14-3-3-binding candidates PAK4 was identified in an in-house HT 14-3-3 capture exp. and in several published HT experiments
9Slide18
Candidate 14-3-3-binding phosphosites of PAK4
Ser99
Ser162
Ser181
Ser474
...
...
...
...
...
10Slide19
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-PAK4
S99A
S162A
S181A
S474A
S162/181A
GFP-PFKFB
2
S466/483A
14-3-3
GFP-PAK4
S99A
S162A
S181A
S474A
Calyculin
A
Second site that is phosphorylated
Decreased binding
11Slide20
Phorbol esters regulate the phosphorylation of PAK4
BI-D1870 + PMA
Serum Starved
IGF1
PI-103 + IGF1
EGF
PMA
Forskolin
H-89 +
Forskolin
A769662
A23187
Calyculin
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
12Slide21
Phorbol esters regulate the phosphorylation of PAK4
BI-D1870 + PMA
Serum Starved
IGF1
PI-103 + IGF1
EGF
PMA
Forskolin
H-89 +
Forskolin
A769662
A23187
Calyculin
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
12Slide22
‘Signalling signatures’ of PAK4
PKC, PKD or p90RSK
13
?
S181Slide23
Conclusions
We developed a user friendly web resource for the annotation and prediction of the 14-3-3 interactomeOur projections indicate that 14-3-3s may dock onto ~45% of 2R-ohnologue human kinase family membersWe 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
14Slide24
Future work
Site-directed mutagenesis of S181A double mutants and loss of Calyculin A-/PMA-stimulated 14-3-3 bindingStimuli/inhibitor experiments to investigate different patterns of ‘signalling signatures’ of PAK4In vivo
phosphorylation (using SILAC) of endogenous
PAK4
Further investigate the effects of 14-3-3 binding on PAK4
Extend studies to all the human 2R-ohnologue families
15Slide25
Acknowledgements
Professor Carol MacKintoshDr 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)Slide26
14-3-3s
Family of regulatory proteins found in all eukaryotesMammals have 7 isoforms ubiquitously expressed in all tissues
Small
proteins (~
30
kDa
) that form homo- or heterodimers
Presenting an highly helical structureSlide27
Human kinome
Set of proteins kinases in the human genome
Extensively studied
PK create 14-3-3-binding
phosphosites
and are also 14-3-3 bindersSlide28
Human kinome
Why?Discover new 14-3-3-based kinase cascades and mechanisms of signalling ‘cross talk’
Understand 14-3-3-based regulatory interplay between members of 2R-ohnologue families
Expertise and resources for biochemical validation of candidate 14-3-3 bindersSlide29
Prediction
Search
Predictor
Alignment
Web Interface
Intracellular
Disorder
Prediction NN
Prediction PSSM
Phosphorylation
Database
Feature
Conservation
User Input
Output
Diagram of the Predictor integrated in ANIASlide30
Phorbol esters regulate the phosphorylation of PAK4
BI-D1870 + PMA
Serum Starved
IGF1
PI-103 + IGF1
EGF
PMA
Forskolin
H-89 +
Forskolin
A769662
A23187
Calyculin
A
GFP pull-downs
14-3-3 Overlay
α
-GFP
14-3-3
Cell lysates by Dr
Gerta
Hoxhaj
α
-GFP
pS473 PKB
pS79 ACC
pS157
VASP
pT202/204 ERK1/2
Response to
phorbol
ester stimulation
‘Panel’ of
stimulli
/inhibitors that activate or inhibit AGC and CAMK kinases Slide31
Olsson O
.
pS473 PKB *
*
pS157 VASP
pT172 AMPK
pS79
ACC
* pT202/204 ERK1/2
Signalling pathways activated/inhibited in the ‘panel’ exp.Slide32
Sluchanko
, N. N., and Gusev, N. B. (2010) 14-3-3 Proteins and regulation of cytoskeleton. Biochemistry (Moscow) 75, 1528–1546.
PK C
PAK4 and 14-3-3s are involved in the regulation of cytoskeletonSlide33
Koh
, W., Mahan, R. D., and Davis, G. E. (2008) Cdc42- and Rac1-mediated endothelial lumen formation requires Pak2, Pak4 and Par3, and PKC-dependent signaling. Journal of cell science 121, 989–1001.Slide34
Johnson
, C., Tinti, M., Wood, N. T., Campbell, D. G., Toth, R., Dubois, F., Geraghty, K. M., Wong, B. H. C., Brown, L. J., Tyler, J., Gernez, A., Chen, S., Synowsky
, S., and MacKintosh, C. (2011) Visualization and biochemical analyses of the emerging mammalian 14-3-3-phosphoproteome.
Molecular & cellular proteomics
10
, M110.005751.
Mode I: RSX(pS
)XPMode II: RXXX(pS)XP Slide35