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. 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.
Mesh Terms:
14-3-3 Proteins, Actins, Animals, Cell Differentiation, Cell Size, Cells, Cultured, Cluster Analysis, Computational Biology, Cyclic AMP-Dependent Protein Kinases, Cytoskeleton, DNA Primers, DNA, Complementary, Dogs, Fluorescent Antibody Technique, GTP-Binding Proteins, Guanine Nucleotide Exchange Factors, Humans, Mass Spectrometry, Mice, Phosphorylation, Precipitin Tests, Protein Structure, Tertiary, Protein-Serine-Threonine Kinases, Proteins, Proteomics, Transfection, Tyrosine 3-Monooxygenase
14-3-3 Proteins, Actins, Animals, Cell Differentiation, Cell Size, Cells, Cultured, Cluster Analysis, Computational Biology, Cyclic AMP-Dependent Protein Kinases, Cytoskeleton, DNA Primers, DNA, Complementary, Dogs, Fluorescent Antibody Technique, GTP-Binding Proteins, Guanine Nucleotide Exchange Factors, Humans, Mass Spectrometry, Mice, Phosphorylation, Precipitin Tests, Protein Structure, Tertiary, Protein-Serine-Threonine Kinases, Proteins, Proteomics, Transfection, Tyrosine 3-Monooxygenase
Curr. Biol.
Date: Aug. 24, 2004
PubMed ID: 15324660
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