Proteomic, functional, and domain-based analysis of in vivo 14-3-3 binding proteins involved in cytoskeletal regulation and cellular organization

Jing Jin, F. Donelson Smith, Chris Stark, Clark D. Wells, James P. Fawcett, Sarang Kulkarni, Pavel Metalnikov, Paul O'Donnell, Paul Taylor, Lorne Taylor, Alexandre Zougman, James R. Woodgett, Lorene K. Langeberg, John D. Scott, Tony Pawson

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354 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)1436-1450
Number of pages15
JournalCurrent Biology
Volume14
Issue number16
DOIs
StatePublished - Aug 24 2004

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ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Agricultural and Biological Sciences(all)

Cite this

Jin, J., Smith, F. D., Stark, C., Wells, C. D., Fawcett, J. P., Kulkarni, S., Metalnikov, P., O'Donnell, P., Taylor, P., Taylor, L., Zougman, A., Woodgett, J. R., Langeberg, L. K., Scott, J. D., & Pawson, T. (2004). Proteomic, functional, and domain-based analysis of in vivo 14-3-3 binding proteins involved in cytoskeletal regulation and cellular organization. Current Biology, 14(16), 1436-1450. https://doi.org/10.1016/j.cub.2004.07.051