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Functional proteomic identification of the interaction of MAPK-activated protein kinase-2 and 14-3-3zeta in human neutrophils

Functional proteomic identification of the interaction of MAPK-activated protein kinase-2 and 14-3-3zeta in human neutrophils

FASEB Journal 16(4): A13, March 20

MAPK activated protein kinase-2 (MK2) mediates p38 MAPK-dependent respiratory burst activity, chemotaxis, and exocytosis in human neutrophils through unknown pathways. To define the pathways leading from MK2 to these functions, we identified potential MK2 substrates using a functional proteomic approach consisting of in vitro phosphorylation of neutrophil lysates by recombinant active MK2, protein separation by SDS-PAGE or two-dimensional gel electrophoresis, and peptide mass fingerprinting of trypsin digested proteins by MALDI-MS. Lysis of neutrophils in buffer containing 7 M urea and 2 M thiourea followed by reduction to 1 M urea prior to an in vitro kinase assay resulted in elimination of endogenous kinase activity, but preserved activity of exogenously added MK2. About 30 phosphorylated proteins were found on gels by autoradiography. Known MK2 substrates, LSP1 and vimentin, and a novel substrate, 14-3-3zeta, were identified. Interaction of MK2 with 14-3-3zeta was confirmed in vitro and in situ. Recombinant active MK2 phosphorylated recombinant 14-3-3zeta. GST pull-down using recombinant proteins confirmed that MK2 and 14-3-3zeta directly interact. Immunoprecipitation of MK2 and GST-MK2 also precipitated 14-3-3zeta from neutrophil lysates. Transfection of HEK293 cells with constitutively active MK2 resulted in phosphorylation of 14-3-3zeta. We conclude that this functional proteomic approach can be used to screen for kinase substrates.

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Accession: 034961677

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