PROTEIN-KINASE C-MEDIATED PHOSPHORYLATION AND CALMODULIN-BINDING OF RECOMBINANT MYRISTOYLATED ALANINE-RICH C-KINASE SUBSTRATE (MARCKS) AND MARCKS-RELATED PROTEIN

The myristoylated alanine-rich C kinase substrate (MARCKS) and the MAR CKS-related protein (MRP) are members of a distinct family of protein kinase C (PKC) substrates that also bind calmodulin in a manner regula ted by phosphorylation by PKC. The kinetics of PKC-mediated phosphoryl ation and the calmodulin binding properties of intact, recombinant MAR CKS and MRP were investigated and compared with previous studies of sy nthetic peptides spanning the PKC phosphorylation site/calmodulin bind ing domains (PSCBD) of these proteins. Both MARCKS and MRP were high a ffinity substrates for the catalytic fragment of PKC, and their phosph orylation occurred with positive cooperativity (MARCKS: S-0.5 = 100 nM , K-H = 1.43; MRP: S-0.5 = 238 nM, K-H = 1.72). These affinities are s imilar to the values determined from studies of their respective PSCBD peptides. Two-dimensional mapping of MRP and its synthetic PSCBD pept ide yielded identical patterns of tryptic phosphopeptides, indicating that, as in the case of MARCKS, all of the PKC phosphorylation sites i n MRP lie within the 24-amino acid PSCBD. Sequence analysis of tryptic phosphopeptides revealed that the first and third, but not the second , serines in the MRP PSCBD were phosphorylated by PKC. Both MARCKS and MRP bound dansyl-calmodulin with high affinity, with a K-app of 4.6 a nd 9.5 nM, respectively. Phosphorylation of MARCKS and MRP by PKC disr upted the protein-calmodulin complexes, with half-lives of 4.0 and 3.5 min, respectively. These studies suggest that intact, recombinant MAR CKS and MRP are accurately modeled by their synthetic PSCBD peptides w ith respect to PKC phosphorylation kinetics and their phosphorylation- dependent calmodulin binding properties.