Substrate specificity of phospholipid/Ca2+-dependent protein kinase as probed with synthetic peptide fragments of the bovine myelin basic protein.
The substrate specificity of phospholipid/Ca2+-dependent protein kinase (protein kinase C) was studied using synthetic peptides, in particular those corresponding to the amino acid sequence around serine 115 in bovine myelin basic protein (MBP). It was found that MBP (104-118) and MBP (104-123) were substrates for the enzyme, with apparent Km values of 14 and 10 microM, respectively. Neither MBP (111-118) nor MBP (111-123) were phosphorylated, indicating that an additional segment of sequence extending toward the N terminus, but not toward the C terminus, was essential for the substrate activity of the peptides. Of the alanine-substituted analogs examined, [Ala 105] MBP (104-118) was comparable to the parent peptide, whereas [Ala 107] MBP (104-118) and [Ala 113] MBP-(104-118) were much poorer substrates. These findings indicated that lysine 105 was not essential, but both arginine 107 and arginine 113 were important specificity determinants. Initial studies revealed that [Ala 113] MBP (104-118) inhibited phosphorylation by the enzyme of the parent peptide and, to a lesser extent, the intact MBP(1-170). Serine 115 was the only site phosphorylated in the analog peptides [Ala 105] MBP (104-118) and [Ala 107]MBP (104-118). In the parent peptide, serine 115 was the initial site of phosphorylation but after prolonged phosphorylation other sites became phosphorylated (serine 110 and/or serine 112), further supporting the concept that arginine residues act as essential substrate specificity determinants for phospholipid/Ca2+-dependent protein kinase.
R S
RS
Turner
B E
BE
Kemp
H D
HD
Su
J F
JF
Kuo
D000818:Animals; D002417:Cattle; D007700:Kinetics; D004676:Myelin Basic Protein / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000494:pharmacology*; D013379:Substrate Specificity
D004676:Myelin Basic Protein; D010446:Peptide Fragments; D011493:Protein Kinase C