Direct binding of syndecan-4 cytoplasmic domain to the catalytic domain of protein kinase Cα (PKCα) increases focal adhesion localization of PKCα

Abstract

Syndecan-4 is a transmembrane heparan sulfate proteoglycan that acts as a coreceptor with integrins in focal adhesion formation. The central region of syndecan-4 cytoplasmic domain (4V; LGKKPIYKK) binds phosphatidylinositol 4,5-bisphosphate, and together they regulate protein kinase Cα (PKCα) activity. Syndecan 4V peptide directly potentiates PKCα activity, leading to "superactivation" of the enzyme, apparently through an interaction with its catalytic domain. We now have performed yeast two-hybrid and in vitro binding assays to determine the interaction sites between 4V and PKCα. Full-length PKCα weakly interacted with 4V by yeast two-hybrid assays, but PKCα constructs that lack the pseudosubstrate region or constructs of the whole catalytic domain interacted more strongly. A mutated 4V sequence (4V(YF): LGKKPIFKK) did not interact with PKCα, indicating that tyrosine 192 in the syndecan-4 cytoplasmic domain might be critical for this interaction. Further assays identified a novel interaction site in the C terminus of the catalytic domain of PKC α (amino acid sequence 513-672). This encompasses the autophosphorylation sites, which are implicated in activation and stability. Yeast two-hybrid data were confirmed by in vitro binding and coimmunoprecipitation assays. The interaction of syndecan-4 with PKCα appears unique since PKCδ and ε did not interact with 4V in yeast two-hybrid assays or coimmunoprecipitate with syndecan-4. Finally, overexpression of syndecan-4 in rat embryo fibroblast cells, but not expression of the YF mutant, increased PKCα localization to focal adhesions. The data support a mechanism where syndecan-4 binds PKCα and localizes it to focal adhesions, whose assembly may be regulated by the kinase.