Protein kinase C (PKC) is involved in cell-matrix and cell-cell adhesion, and the cytoplasmic domain of syndecan-2 contains two serines (residues 197 and 198) which lie in a consensus sequence for phosphorylation by PKC. Other serine and threonine residues are present but not in a consensus sequence. We investigated phosphorylation of syndecan-2 cytoplasmic domain by PKC, using purified GST-syndecan-2 fusion proteins and synthetic peptides corresponding to regions of the cytoplasmic domain. A synthetic peptide encompassing the entire cytoplasmic domain of syndecan-2 was phosphorylated by PKC with high affinity. Peptide mapping and substitution studies showed that both serines were phosphoacceptors, but each had slightly different affinity, with that of serine-197 being higher than serine-198. The efficiency of phosphorylation was concentration-dependent. At low concentrations, the cytoplasmic domain peptides were monomeric, with 2 mol/mol serine phosphorylation. At higher concentrations, however, the peptides formed dimers, with only 0.5 mol/mol phosphorylation. Concentration-dependent dimerization was not altered by phosphorylation. Phosphorylation is, therefore, dependent on the conformation of syndecan-2 cytoplasmic domain, but does not affect its oligomeric status.
Keywords: Amino Acid Sequence; Blotting, Western; Chromatography, Gel; Chromatography, Thin Layer; Cytoplasm; Electrophoresis, Polyacrylamide Gel; Escherichia coli; Glutathione Transferase; Kinetics; Membrane Glycoproteins; Molecular Sequence Data; Peptide Fragments; Phosphates; Phosphopeptides; Phosphorylation; Phosphoserine; Protein Kinase C; Proteoglycans; Recombinant Fusion Proteins; Syndecan-2