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

Curr Biol. 2004 Aug 24;14(16):1436-50. doi: 10.1016/j.cub.2004.07.051.

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.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • 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*

Substances

  • 14-3-3 Proteins
  • Actins
  • DNA Primers
  • DNA, Complementary
  • Guanine Nucleotide Exchange Factors
  • Proteins
  • Rho Guanine Nucleotide Exchange Factors
  • Tyrosine 3-Monooxygenase
  • Protein-Serine-Threonine Kinases
  • Cyclic AMP-Dependent Protein Kinases
  • GTP-Binding Proteins