Abstract
The myristoylated alanine-rich C kinase substrate (MARCKS) and the MARCKS-related protein (MRP) are members of a distinct family of protein kinase C (PKC) substrates that also bind calmodulin in a manner regulated by phosphorylation by PKC. The kinetics of PKC-mediated phosphorylation and the calmodulin binding properties of intact, recombinant MARCKS and MRP were investigated and compared with previous studies of synthetic peptides spanning the PKC phosphorylation site/calmodulin binding domains (PSCBD) of these proteins. Both MARCKS and MRP were high affinity substrates for the catalytic fragment of PKC, and their phosphorylation occurred with positive cooperativity (MARCKS: S0.5 = 100 nM, KH = 1.43; MRP: S0.5 = 238 nM, KH = 1.72). These affinities are similar to the values determined from studies of their respective PSCBD peptides. Two-dimensional mapping of MRP and its synthetic PSCBD peptide yielded identical patterns of tryptic phosphopeptides, indicating that, as in the case of MARCKS, all of the PKC phosphorylation sites in 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 Kapp of 4.6 and 9.5 nM, respectively. Phosphorylation of MARCKS and MRP by PKC disrupted the protein-calmodulin complexes, with half-lives of 4.0 and 3.5 min, respectively. These studies suggest that intact, recombinant MARCKS and MRP are accurately modeled by their synthetic PSCBD peptides with respect to PKC phosphorylation kinetics and their phosphorylation-dependent calmodulin binding properties.
Highlights
The myristoylated alanine-rich C kinase substrate (MARCKS)and theMARCKS-relatedprotein (MRP)are members of a distinct family of protein kinase C (PKC) substrates thatalso bind calmodulin in a manner regulated by phosphorylation by PKC
Phosphorylation of MARCKS and MRPby PKC disrupted the protein-calmodulin complexes, with half-lives of4.0 and 3.5 min, respectively. These studies suggest that intact, recombinant MARCKS and MRP are accurately modeled by their synthetic phosphorylation site/calmodulin binding domains (PSCBD) peptides with respect to PKC phosphorylation kinetics and their phosphorylation-dependent calmodulin binding properties
25-amino acid sequence containing two to four PKC phosphorylation sites as well as the calmodulin binding domain, referred to as the phosphorylation site/calmodulin binding domai(nPSCBDP).reviousstudies of synthetpiceptides representing both the MARCKS PSCBDs and MRP indicate that both peptides are high affinity substrates for PKC and are phosphorylated with positive cooperativity [3, 4]
Summary
The sequence of the peptide is KKKKKFSFKKPFKLSGLSFKRNRK, this is identicalt o the PSCBD sequence of human MRP.S. In Vitro Phosphorylation of MRP and Its PSCBDPeptide-The Mono Q-purified MRP was phosphorylated by protein kinase M (PKM), the tryptic catalytic fragmenotf PKC, essentially as described P(4K)M. was reaction, it was stoppbeyd the addition of (final concentration1) 0%(v.v) acetic acid and placed on ice for 10 min. Following centrifugation to remove acid precipitated proteins (12,000 xg for 10 min a t 4 "C), the reaction mix was chromatographedon a Dowex 1-X8column previously equilibrated with 10% acetic acid, exactly as described [20]. This sample was applied to a C4 reversed phase HPLC column and chromatographed as described [21]. The peak fractions were lyophilizedand taken up in a small volume of 0.1%(w/v)heptafluorobutyric acid and rechromatographed on the same C4 reversed phase column previously equilibrated with0.1% heptafluorobutyric acid, exactly as described [21]. SDS-PolyacrylamideGel Electrophoresis-This was performed as described [23]
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