mapping the 14-3-3-binding 2r-ohnologue protein families of the human kinome
DESCRIPTION
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 PresentationTRANSCRIPT
![Page 1: Mapping the 14-3-3-binding 2R-ohnologue protein families of the human kinome](https://reader035.vdocuments.mx/reader035/viewer/2022062813/5681650c550346895dd7837f/html5/thumbnails/1.jpg)
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
![Page 2: Mapping the 14-3-3-binding 2R-ohnologue protein families of the human kinome](https://reader035.vdocuments.mx/reader035/viewer/2022062813/5681650c550346895dd7837f/html5/thumbnails/2.jpg)
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
![Page 3: Mapping the 14-3-3-binding 2R-ohnologue protein families of the human kinome](https://reader035.vdocuments.mx/reader035/viewer/2022062813/5681650c550346895dd7837f/html5/thumbnails/3.jpg)
The human 14-3-3 interactome is highly enriched in 2R-ohnologues
2R-ohnologues
Invertebrate chordates Mammals
1R-WGD 2R-WGD
Selection/Loss
2
![Page 4: Mapping the 14-3-3-binding 2R-ohnologue protein families of the human kinome](https://reader035.vdocuments.mx/reader035/viewer/2022062813/5681650c550346895dd7837f/html5/thumbnails/4.jpg)
2R-Ohnologues and the ‘lynchpin’ phosphosites
P P P P
3
Lynchpin siteLynchpin site
![Page 5: Mapping the 14-3-3-binding 2R-ohnologue protein families of the human kinome](https://reader035.vdocuments.mx/reader035/viewer/2022062813/5681650c550346895dd7837f/html5/thumbnails/5.jpg)
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)
![Page 6: Mapping the 14-3-3-binding 2R-ohnologue protein families of the human kinome](https://reader035.vdocuments.mx/reader035/viewer/2022062813/5681650c550346895dd7837f/html5/thumbnails/6.jpg)
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
![Page 7: Mapping the 14-3-3-binding 2R-ohnologue protein families of the human kinome](https://reader035.vdocuments.mx/reader035/viewer/2022062813/5681650c550346895dd7837f/html5/thumbnails/7.jpg)
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
![Page 8: Mapping the 14-3-3-binding 2R-ohnologue protein families of the human kinome](https://reader035.vdocuments.mx/reader035/viewer/2022062813/5681650c550346895dd7837f/html5/thumbnails/8.jpg)
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
![Page 9: Mapping the 14-3-3-binding 2R-ohnologue protein families of the human kinome](https://reader035.vdocuments.mx/reader035/viewer/2022062813/5681650c550346895dd7837f/html5/thumbnails/9.jpg)
Homepage of ANIA
ANIA: ANnotation and Integrated Analysis of the 14-3-3 interactome
7
![Page 10: Mapping the 14-3-3-binding 2R-ohnologue protein families of the human kinome](https://reader035.vdocuments.mx/reader035/viewer/2022062813/5681650c550346895dd7837f/html5/thumbnails/10.jpg)
Tabular view of results
ANIA: ANnotation and Integrated Analysis of the 14-3-3 interactome
7
![Page 11: Mapping the 14-3-3-binding 2R-ohnologue protein families of the human kinome](https://reader035.vdocuments.mx/reader035/viewer/2022062813/5681650c550346895dd7837f/html5/thumbnails/11.jpg)
Detailed view of each protein queried
ANIA: ANnotation and Integrated Analysis of the 14-3-3 interactome
7
![Page 12: Mapping the 14-3-3-binding 2R-ohnologue protein families of the human kinome](https://reader035.vdocuments.mx/reader035/viewer/2022062813/5681650c550346895dd7837f/html5/thumbnails/12.jpg)
Tabular view of results
ANIA: ANnotation and Integrated Analysis of the 14-3-3 interactome
7
![Page 13: Mapping the 14-3-3-binding 2R-ohnologue protein families of the human kinome](https://reader035.vdocuments.mx/reader035/viewer/2022062813/5681650c550346895dd7837f/html5/thumbnails/13.jpg)
Detailed analysis of candidate 14-3-3-binding phosphosites
ANIA: ANnotation and Integrated Analysis of the 14-3-3 interactome
7
![Page 14: Mapping the 14-3-3-binding 2R-ohnologue protein families of the human kinome](https://reader035.vdocuments.mx/reader035/viewer/2022062813/5681650c550346895dd7837f/html5/thumbnails/14.jpg)
8
2R-ohnologue families of the Human Kinome
Total GDFamilies 142 20
Members 355 23
![Page 15: Mapping the 14-3-3-binding 2R-ohnologue protein families of the human kinome](https://reader035.vdocuments.mx/reader035/viewer/2022062813/5681650c550346895dd7837f/html5/thumbnails/15.jpg)
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
![Page 16: Mapping the 14-3-3-binding 2R-ohnologue protein families of the human kinome](https://reader035.vdocuments.mx/reader035/viewer/2022062813/5681650c550346895dd7837f/html5/thumbnails/16.jpg)
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
![Page 17: Mapping the 14-3-3-binding 2R-ohnologue protein families of the human kinome](https://reader035.vdocuments.mx/reader035/viewer/2022062813/5681650c550346895dd7837f/html5/thumbnails/17.jpg)
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
![Page 18: Mapping the 14-3-3-binding 2R-ohnologue protein families of the human kinome](https://reader035.vdocuments.mx/reader035/viewer/2022062813/5681650c550346895dd7837f/html5/thumbnails/18.jpg)
Candidate 14-3-3-binding phosphosites of PAK4
Ser99 Ser162 Ser181 Ser474
... ... ... ......
10
![Page 19: Mapping the 14-3-3-binding 2R-ohnologue protein families of the human kinome](https://reader035.vdocuments.mx/reader035/viewer/2022062813/5681650c550346895dd7837f/html5/thumbnails/19.jpg)
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
![Page 20: Mapping the 14-3-3-binding 2R-ohnologue protein families of the human kinome](https://reader035.vdocuments.mx/reader035/viewer/2022062813/5681650c550346895dd7837f/html5/thumbnails/20.jpg)
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
![Page 21: Mapping the 14-3-3-binding 2R-ohnologue protein families of the human kinome](https://reader035.vdocuments.mx/reader035/viewer/2022062813/5681650c550346895dd7837f/html5/thumbnails/21.jpg)
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
![Page 22: Mapping the 14-3-3-binding 2R-ohnologue protein families of the human kinome](https://reader035.vdocuments.mx/reader035/viewer/2022062813/5681650c550346895dd7837f/html5/thumbnails/22.jpg)
‘Signalling signatures’ of PAK4
PKC, PKD or p90RSK
13
? S181
![Page 23: Mapping the 14-3-3-binding 2R-ohnologue protein families of the human kinome](https://reader035.vdocuments.mx/reader035/viewer/2022062813/5681650c550346895dd7837f/html5/thumbnails/23.jpg)
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
![Page 24: Mapping the 14-3-3-binding 2R-ohnologue protein families of the human kinome](https://reader035.vdocuments.mx/reader035/viewer/2022062813/5681650c550346895dd7837f/html5/thumbnails/24.jpg)
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
![Page 25: Mapping the 14-3-3-binding 2R-ohnologue protein families of the human kinome](https://reader035.vdocuments.mx/reader035/viewer/2022062813/5681650c550346895dd7837f/html5/thumbnails/25.jpg)
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)