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Identification of mitogen‐activated protein kinase phosphorylation sequences in mammalian h‐Caldesmon
Author(s) -
Adam Leonard P.,
Hathaway David R.
Publication year - 1993
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(93)81110-l
Subject(s) - caldesmon , phosphorylation , protein kinase a , kinase , microbiology and biotechnology , mapk/erk pathway , biochemistry , biology , vascular smooth muscle , chemistry , calmodulin , enzyme , smooth muscle , endocrinology
h‐Caldesmon in vascular smooth muscle is phosphorylated in response to pharmacologie 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.