Details

Autor(en) / Beteiligte

• 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

Titel

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

Ist Teil von

• Current biology, 2004-08, Vol.14 (16), p.1436-1450

Ort / Verlag

England: Elsevier Inc

Erscheinungsjahr

2004

Quelle

ScienceDirect

Beschreibungen/Notizen

• 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.