The influence of basic residues on the substrate specificity of protein kinase C.
The substrate specificity of protein kinase C has been examined using a series of synthetic peptide analogs of glycogen synthase, ribosomal protein S6, and the epidermal growth factor receptor. The glycogen synthase analog peptide Pro1-Leu-Ser-Arg-Thr-Leu-Ser-Val-Ala-Ala10 was phosphorylated at Ser7 with a Km of 40.3 microM. Peptide phosphorylation was strongly dependent on Arg4. When lysine was substituted for Arg4 the Km was increased approximately 20-fold. Addition of basic residues on either the NH2-terminal or COOH-terminal side of the phosphorylation site of the glycogen synthase peptide improved the kinetics of peptide phosphorylation. The analog Pro-Leu-Ser-Arg-Thr-Leu-Ser-Val-Ala-Ala-Lys-Lys was phosphorylated with a Km of 4.1 microM. Substitution of Ser7 with threonine increased the apparent Km to 151 microM. The truncated peptide Pro1-Leu-Ser-Arg-Thr-Leu-Ser-Val8 was phosphorylated with similar kinetic constants to the parent peptide, however, deletion of Val8 increased the apparent Km to 761 microM. The ribosomal peptide S6-(229-239) was phosphorylated with a Km of approximately 0.5 microM predominantly on Ser236 and is one of the most potent synthetic peptide substrates reported for a protein kinase. The apparent Km for S6 peptide phosphorylation was increased by either deletion of the NH2-terminal 3 residues Ala229-Arg-231 or by substitution of Arg238 on the COOH-terminal side of the phosphorylation site with alanine. This analog peptide, [Ala238]S6-(229-239) was phosphorylated with an approximate 6-fold reduction in Vmax and a switch in the preferred site of phosphorylation from Ser236 to Ser235. These results support the concept that basic residues on both sides of the phosphorylation site can have an important influence on the kinetics of phosphorylation and site specificity of protein kinase C.
D000818:Animals; D001921:Brain / Q000201:enzymology*; D066246:ErbB Receptors / Q000378:metabolism; D006006:Glycogen Synthase / Q000378:metabolism; D011493:Protein Kinase C / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D038601:Ribosomal Protein S6; D012269:Ribosomal Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D038601:Ribosomal Protein S6; D012269:Ribosomal Proteins; D006006:Glycogen Synthase; D066246:ErbB Receptors; D011493:Protein Kinase C