Proteomic, functional, and domain-based analysis of in vivo 14-3-3 binding proteins involved in cytoskeletal regulation and cellular organization.
BACKGROUND
14-3-3 proteins are abundant and conserved polypeptides that mediate the cellular effects of basophilic protein kinases through their ability to bind specific peptide motifs phosphorylated on serine or threonine.


RESULTS
We have used mass spectrometry to analyze proteins that associate with 14-3-3 isoforms in HEK293 cells. This identified 170 unique 14-3-3-associated proteins, which show only modest overlap with previous 14-3-3 binding partners isolated by affinity chromatography. To explore this large set of proteins, we developed a domain-based hierarchical clustering technique that distinguishes structurally and functionally related subsets of 14-3-3 target proteins. This analysis revealed a large group of 14-3-3 binding partners that regulate cytoskeletal architecture. Inhibition of 14-3-3 phosphoprotein recognition in vivo indicates the general importance of such interactions in cellular morphology and membrane dynamics. Using tandem proteomic and biochemical approaches, we identify a phospho-dependent 14-3-3 binding site on the A kinase anchoring protein (AKAP)-Lbc, a guanine nucleotide exchange factor (GEF) for the Rho GTPase. 14-3-3 binding to AKAP-Lbc, induced by PKA, suppresses Rho activation in vivo.


CONCLUSIONS
14-3-3 proteins can potentially engage around 0.6% of the human proteome. Domain-based clustering has identified specific subsets of 14-3-3 targets, including numerous proteins involved in the dynamic control of cell architecture. This notion has been validated by the broad inhibition of 14-3-3 phosphorylation-dependent binding in vivo and by the specific analysis of AKAP-Lbc, a RhoGEF that is controlled by its interaction with 14-3-3.
Jin
Jing
J
Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto, Ontario M5G 1X5, Canada.
Smith
F Donelson
FD
Stark
Chris
C
Wells
Clark D
CD
Fawcett
James P
JP
Kulkarni
Sarang
S
Metalnikov
Pavel
P
O'Donnell
Paul
P
Taylor
Paul
P
Taylor
Lorne
L
Zougman
Alexandre
A
Woodgett
James R
JR
Langeberg
Lorene K
LK
Scott
John D
JD
Pawson
Tony
T
D048948:14-3-3 Proteins; D000199:Actins / Q000502:physiology; D000818:Animals; D002454:Cell Differentiation / Q000235:genetics; D048429:Cell Size / Q000235:genetics; D002478:Cells, Cultured; D016000:Cluster Analysis; D019295:Computational Biology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D003599:Cytoskeleton / Q000235:genetics / Q000502:physiology*; D017931:DNA Primers; D018076:DNA, Complementary / Q000235:genetics; D004285:Dogs; D005455:Fluorescent Antibody Technique; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism; D006801:Humans; D013058:Mass Spectrometry; D051379:Mice; D010766:Phosphorylation; D011233:Precipitin Tests; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000502:physiology*; D011506:Proteins / Q000378:metabolism / Q000502:physiology*; D040901:Proteomics / Q000379:methods; D064067:Rho Guanine Nucleotide Exchange Factors; D014162:Transfection; D014446:Tyrosine 3-Monooxygenase / Q000378:metabolism*
D048948:14-3-3 Proteins; D000199:Actins; D017931:DNA Primers; D018076:DNA, Complementary; D020662:Guanine Nucleotide Exchange Factors; D011506:Proteins; D064067:Rho Guanine Nucleotide Exchange Factors; D014446:Tyrosine 3-Monooxygenase; D017346:Protein Serine-Threonine Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D019204:GTP-Binding Proteins