1. Proteomic, functional, and domain-based analysis of in vivo 14-3-3 binding proteins involved in cytoskeletal regulation and cellular organization.
- Author
-
Jin J, Smith FD, Stark C, Wells CD, Fawcett JP, Kulkarni S, Metalnikov P, O'Donnell P, Taylor P, Taylor L, Zougman A, Woodgett JR, Langeberg LK, Scott JD, and Pawson T
- Subjects
- 14-3-3 Proteins, Actins physiology, Animals, Cell Differentiation genetics, Cell Size genetics, Cells, Cultured, Cluster Analysis, Computational Biology, Cyclic AMP-Dependent Protein Kinases metabolism, Cytoskeleton genetics, DNA Primers, DNA, Complementary genetics, Dogs, Fluorescent Antibody Technique, GTP-Binding Proteins genetics, GTP-Binding Proteins metabolism, Guanine Nucleotide Exchange Factors metabolism, Humans, Mass Spectrometry, Mice, Phosphorylation, Precipitin Tests, Protein Serine-Threonine Kinases genetics, Protein Serine-Threonine Kinases metabolism, Proteins metabolism, Proteomics methods, Rho Guanine Nucleotide Exchange Factors, Transfection, Cytoskeleton physiology, Protein Structure, Tertiary physiology, Proteins physiology, Tyrosine 3-Monooxygenase metabolism
- Abstract
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., Conclusion: 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.
- Published
- 2004
- Full Text
- View/download PDF