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 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.
Verghese
G M
GM
Howard Hughes Medical Institute Laboratories, Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710.
Johnson
J D
JD
Vasulka
C
C
Haupt
D M
DM
Stumpo
D J
DJ
Blackshear
P J
PJ
D000595:Amino Acid Sequence; D000818:Animals; D002147:Calmodulin / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D051379:Mice; D008840:Microfilament Proteins; D008969:Molecular Sequence Data; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D010449:Peptide Mapping; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D011994:Recombinant Proteins
D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D047908:Intracellular Signaling Peptides and Proteins; C000619710:MARCKS protein, human; C070177:MARCKSL1 protein, human; C000619711:Marcks protein, mouse; C512945:Marcksl1 protein, mouse; D008565:Membrane Proteins; D008840:Microfilament Proteins; D010455:Peptides; D011506:Proteins; D011994:Recombinant Proteins; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D011493:Protein Kinase C