Substrate Specificity of Phospholipid / Ca 2 + - dependent Protein Kinase as Probed with Synthetic Peptide Fragments of the Bovine Myelin

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 K,,, values of 14 and 10 BM, respectively. Neither MBP(111-118) nor MBP( 11 1-123) 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, [Ala1""] MBP( 104-1 18) was comparable to the parent peptide, whereas [Ala107]MBP(10~-llS~ and [Ala'13]MBP(104-118) were much poorer substrates. These findings indicated that lysine 105 was not essential, but both arginine 107 and arginine 113 were importBnt specificity determinants. Initial studies revealed that [Ala''31MBP( 104-1 18) 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 [Alalo"] MBP(104-118) and [Ala'"71MBP(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 1121, further supporting the concept that arginine residues act as essential substrate specificity determinants for phospholipid/Caa+-dependent protein kinase. quences around the phospho~lation sites have been identified for cyclic AMP-dependent protein kinase (9,101, cyclic GMPdependent protein kinase (111, and myosin light chain kinase (a calmodulinfCa2+-dependent protein kinase) (12). A common feature of synthetic peptide substrates for these enzymes is that they contain basic amino acid residues N-terminal to the phosphorylation sites (serine or threonine) separated by one or more neutral amino acids. Little is known about the identities of endogenous substrate proteins for phospholipid/Ca2+-dependent protein kinase. We have recently found that myelin basic protein (MBP') from brain (13) and troponin I and troponin T from cardiac (14) and skeletal (15) muscle are good substrates for this enzyme, Of the many purified proteins examined, bovine MBP (dephosphorylated species) was found to be the most effective substrate for the enzyme, as indicated by favorable kinetic constants and multiple sites (5 mol of P/mol) of phosphorylation (16). Several synthetic peptides, such as histone H2B(2935), histone H2B(29-39) (16), and histone Hl(34-42) (I?'), have been shown to be phosphorylated by the enzyme. These peptides, however, are rather poor substrates. In the present study, we investigated phosphorylation of a number of peptides and their alanine-substituted analogs containing amino acid sequences around several serine residues in bovine MBP. We found that certain peptides containing serine 115, the preferred phosphorylation site in bovine MBP (18), were effective substrates. Moreover, arginine 107 and arginine 113 were found to be essential for the substrate activity of these peptides.