Mammal-specific, ERK-dependent, caldesmon phosphorylation in smooth muscle. Quantitation using novel anti-phosphopeptide antibodies.
Extracellular signal-regulated kinases (ERKs) phosphorylate the high molecular mass isoform of the actin-binding protein caldesmon (h-CaD) at two sites (Ser(759) and Ser(789)) during smooth muscle stimulation. To investigate the role of phosphorylation at these sites, antibodies were generated against phosphopeptides analogous to the sequences around Ser(759) and Ser(789). Affinity-purified antibodies were phosho- and sequence-specific. The major site of phosphorylation in h-CaD in porcine carotid arterial muscle strips was at Ser(789); however, the amount of phosphate did not vary appreciably with either KCl or phorbol ester stimulation. Phosphorylation at Ser(759) of h-CaD was almost undetectable (<0.005 mol of phosphate/mol of protein). Moreover, phosphorylation of the low molecular mass isoform of the protein (l-CaD) at the site analogous to Ser(789) was greater in serum-stimulated cultured smooth muscle cells than in serum-starved cells. Serum-stimulated l-CaD phosphorylation was attenuated by the protein kinase inhibitor PD98059. These data 1) identify Ser(789) of h-CaD as the major site of ERK-dependent phosphorylation in carotid arteries; 2) show that the level of phosphorylation at Ser(789) is relatively constant following carotid arterial muscle stimulation, despite an increase in total protein phosphate content; and 3) suggest a functional role for ERK-dependent l-CaD phosphorylation in cell division.
D'Angelo
G
G
Boston Biomedical Research Institute, Boston, Massachusetts 02114, USA.
Graceffa
P
P
Wang
C A
CA
Wrangle
J
J
Adam
L P
LP
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology; D002148:Calmodulin-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D066298:In Vitro Techniques; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D008969:Molecular Sequence Data; D009131:Muscle, Smooth, Vascular / Q000378:metabolism*; D010748:Phosphopeptides / Q000276:immunology*; D010766:Phosphorylation; D017386:Sequence Homology, Amino Acid; D013552:Swine
D000906:Antibodies; D002148:Calmodulin-Binding Proteins; D010748:Phosphopeptides; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases