Proteomic, Functional, and Domain-Based Analysis of In Vivo 14-3-3 Binding Proteins Involved in Cytoskeletal Regulation and Cellular Organization

• Published in: Current biology Vol. 14; no. 16; pp. 1436 - 1450
• Main Authors: Jin, Jing, Smith, F.Donelson, Stark, Chris, Wells, Clark D., Fawcett, James P., Kulkarni, Sarang, Metalnikov, Pavel, O'Donnell, Paul, Taylor, Paul, Taylor, Lorne, Zougman, Alexandre, Woodgett, James R., Langeberg, Lorene K., Scott, John D., Pawson, Tony
• Format: Journal Article
• Language: English
• Published: England Elsevier Inc 24.08.2004
• 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; Cytoskeleton - physiology; 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 Structure, Tertiary - physiology; Protein-Serine-Threonine Kinases - genetics; Protein-Serine-Threonine Kinases - metabolism; Proteins - metabolism; Proteins - physiology; Proteomics - methods; Rho Guanine Nucleotide Exchange Factors; Transfection; Tyrosine 3-Monooxygenase - metabolism
• ISSN: 0960-9822; 1879-0445
• DOI: 10.1016/j.cub.2004.07.051

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.