In vitro phosphorylation of a synthetic collagen peptide by cyclic AMP-dependent protein kinase.

A synthetic tridecapeptide that corresponds closely to amino acid residues 98 to 110 in chick collagen alpha 1(I) contains several determinants of specificity required for recognition and phosphorylation by the cyclic AMP-dependent protein kinase. The peptide Gly-Leu-Hyp-Gly-Nle-Lys-Gly-His-Arg-Gly-Phe-Ser-Gly was predicted to be a substrate for the cyclic AMP-dependent protein kinase because it contained multiple basic amino acids NH2-terminal to a potentially phosphorylatable seryl residue. When tested as a substrate for the enzyme, the peptide was stoichiometrically phosphorylated. Phosphoserine was identified as the only phosphoamino acid in a partial hydrolysate of the phosphorylated peptide. The peptide and several of its analogs were phosphorylated by the cyclic AMP-dependent protein kinase with Km values from 1 to 6 mM and Vmax values from 1 to 3 mumol of phosphate/min/mg of enzyme. Although the Km of the kinase for the collagen peptide was high, these results confirmed the prediction made from knowledge of the substrate specificity of cyclic AMP-dependent protein kinase. The potential for such a phosphorylation reaction to occur in vivo is discussed.