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Am J Physiol Heart Circ Physiol. 2002 Mar;282(3):H1166-71.

Molecular conformation dictates signaling module formation: example of PKCepsilon and Src tyrosine kinase.

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1
Department of Physiology and Biophysics, University of Louisville, Louisville, Kentucky 40202, USA.

Abstract

Our laboratory has conducted multiple functional proteomic analyses to characterize the components of protein kinase C (PKC)epsilon cardioprotective signaling complexes and found that activation of PKCepsilon induces dynamic modulation of these complexes. In addition, it is known that signal transduction within a complex involves the formation of modules, one of which has been shown to include PKCepsilon and Src tyrosine kinase in the rabbit heart. However, the cellular mechanisms that define the assembly of PKCepsilon modules remain largely unknown. To address this issue, the interactions between PKCepsilon and Src were studied. We used recombinant proteins of wild-type PKCepsilon (PKCepsilon-WT) and open conformation mutants of the kinase (PKCepsilon-AE5 and PKCepsilon-AN59), the regulatory and catalytic domains of PKCepsilon, along with glutathione-S-transferase (GST) fusion proteins of Src (GST-Src) and two domains of Src (GST-SH2 and GST-SH3). GST pulldown assays demonstrated that Src and PKCepsilon are binding partners and that the interaction between PKCepsilon and Src appears to involve multiple sites. This finding was supported for endogenous PKCepsilon and Src in the murine heart using immunofluorescence-based confocal microscopy and coimmunoprecipitation. Furthermore, PKCepsilon-WT and GST-Src interactions were significantly enhanced in the presence of phosphatidyl-L-serine, an activator of PKC, indicating that Src favors interaction with activated PKCepsilon. This finding was confirmed when the PKCepsilon-WT was replaced with PKCepsilon-AE5 or PKCepsilon-AN59, demonstrating that the conformation of PKCepsilon is a critical determinant of its interactions with Src. Together, these results illustrate that formation of a signaling module between PKCepsilon and Src involves specific domains within the two molecules and is governed by the molecular conformation of PKCepsilon.

PMID:
11834516
DOI:
10.1152/ajpheart.00830.2001
[Indexed for MEDLINE]
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