Identification of mitogen-activated protein kinase phosphorylation sequences in mammalian h-Caldesmon.
h-Caldesmon in vascular smooth muscle is phosphorylated in response to pharmacologic stimulation. Although many kinases phosphorylate h-caldesmon, in vitro, the responsible kinase in intact tissue is unknown. The sites of phosphorylation in caldesmon from intact canine aortas have recently been identified and are consensus sequences for a proline-directed protein kinase. In this study, we investigated the phosphorylation of h-caldesmon by mitogen-activated protein kinase (MAPK). Purified, recombinant MAPK phosphorylated porcine stomach h-caldesmon to a stoichiometry approaching 2 mol phosphate/mol protein. Phosphorylated h-caldesmon was subjected to proteolysis and the phosphopeptides were purified by high performance liquid chromatography. Two major phosphopeptides were identified and sequenced. These two peptides, VTS*PTKV and S*PAPK, were identical to the sequences of the sites phosphorylated in intact tissue. Antibodies to several enzymes implicated in the cascade of activation of MAPK were used to evaluate vascular smooth muscle by Western blotting. All components were found to be present. These data suggest that MAPK can function as a 'caldesmon kinase' in vascular smooth muscle.
Adam
L P
LP
Department of Medicine, Indiana University School of Medicine, Indianapolis 46202.
Hathaway
D R
DR
D000595:Amino Acid Sequence; D000818:Animals; D016203:CDC2 Protein Kinase / Q000378:metabolism; D002148:Calmodulin-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D004789:Enzyme Activation; D006367:HeLa Cells; D006801:Humans; D019950:Mitogen-Activated Protein Kinase 1; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000737:chemistry / Q000378:metabolism*; D051381:Rats; D013552:Swine
D002148:Calmodulin-Binding Proteins; D011494:Protein Kinases; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; C056959:caldesmon kinase; D016203:CDC2 Protein Kinase; D019950:Mitogen-Activated Protein Kinase 1