In situ phosphorylation of human platelet myosin heavy and light chains by protein kinase C.
Treatment of human platelets with 162 nM 12-O-tetradecanoylphorbol-13-acetate (TPA) resulted in phosphorylation of a number of peptides, including myosin heavy chain and the 20-kDa myosin light chain. The site phosphorylated on the myosin heavy chain was localized by two-dimensional peptide mapping to a serine residue(s) in a single major tryptic phosphopeptide. This phosphopeptide co-migrated with a tryptic peptide that was produced following in vitro phosphorylation of platelet myosin heavy chain using protein kinase C. The sites phosphorylated in the 20-kDa myosin light chain in intact cells were analyzed by two-dimensional mapping of tryptic peptides and found to correspond to Ser1 and Ser2 in the turkey gizzard myosin light chain. In vitro phosphorylation of purified human platelet myosin by protein kinase C showed that in addition to Ser1 and Ser2, a third site corresponding to Thr9 in turkey gizzard myosin light chain is also phosphorylated. The phosphorylatable myosin light chains from human platelets were found to consist of two major isoforms present in approximately equal amounts, but differing in their molecular weights and isoelectric points. A third, minor isoform was also visualized by two-dimensional gel electrophoresis. Following treatment with TPA, both the mono- and diphosphorylated forms of each isoform could be visualized, and the sites of phosphorylation were identified. The phosphate content rose from negligible amounts found prior to treatment with TPA to 1.2 mol of phosphate/mol of myosin light chain and 0.7 mol of phosphate/mol of myosin heavy chain following treatment. These results suggest that TPA mediates phosphorylation of both myosin light and heavy chains in intact platelets by activation of protein kinase C.
Kawamoto
S
S
Laboratory of Molecular Cardiology, National Heart, Lung, and Blood Institute, Bethesda, Maryland 20892.
Bengur
A R
AR
Sellers
J R
JR
Adelstein
R S
RS
D001792:Blood Platelets / Q000201:enzymology*; D006801:Humans; D007700:Kinetics; D015879:Myosin Subfragments; D009218:Myosins / Q000097:blood* / Q000302:isolation & purification; D010446:Peptide Fragments / Q000097:blood* / Q000302:isolation & purification; D010748:Phosphopeptides / Q000302:isolation & purification; D010766:Phosphorylation; D011493:Protein Kinase C / Q000097:blood*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014357:Trypsin
D015879:Myosin Subfragments; D010446:Peptide Fragments; D010748:Phosphopeptides; D011493:Protein Kinase C; D014357:Trypsin; D009218:Myosins; D013755:Tetradecanoylphorbol Acetate