TY - JOUR

T1 - Direct binding of syndecan-4 cytoplasmic domain to the catalytic domain of protein kinase C alpha (PKC alpha) increases focal adhesion localization of PKC alpha.

AU - Lim, Ssang-Taek

AU - Longley, Robert L

AU - Couchman, John R

AU - Woods, Anne

N1 - Keywords: Amino Acid Sequence; Animals; Binding, Competitive; Catalytic Domain; Cell Line; Cells, Cultured; Cytoplasm; Drosophila; Fibroblasts; Focal Adhesions; Immunoblotting; Insects; Membrane Glycoproteins; Molecular Sequence Data; Mutagenesis, Site-Directed; Peptides; Plasmids; Precipitin Tests; Protein Binding; Protein Kinase C; Protein Kinase C-alpha; Protein Structure, Tertiary; Proteoglycans; Rats; Syndecan-4; Transfection; Two-Hybrid System Techniques; Tyrosine

PY - 2003

Y1 - 2003

N2 - 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 C alpha (PKC alpha) activity. Syndecan 4V peptide directly potentiates PKC alpha 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 PKC alpha. Full-length PKC alpha weakly interacted with 4V by yeast two-hybrid assays, but PKC alpha 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 PKC alpha, 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 alpha (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 PKC alpha appears unique since PKC delta and epsilon 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 PKC alpha localization to focal adhesions. The data support a mechanism where syndecan-4 binds PKC alpha and localizes it to focal adhesions, whose assembly may be regulated by the kinase.

AB - 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 C alpha (PKC alpha) activity. Syndecan 4V peptide directly potentiates PKC alpha 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 PKC alpha. Full-length PKC alpha weakly interacted with 4V by yeast two-hybrid assays, but PKC alpha 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 PKC alpha, 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 alpha (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 PKC alpha appears unique since PKC delta and epsilon 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 PKC alpha localization to focal adhesions. The data support a mechanism where syndecan-4 binds PKC alpha and localizes it to focal adhesions, whose assembly may be regulated by the kinase.

U2 - 10.1074/jbc.M208300200

DO - 10.1074/jbc.M208300200

M3 - Journal article

C2 - 12571249

VL - 278

SP - 13795

EP - 13802

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 16

ER -