Abstract
Purified smooth muscle myosin light chain can be phosphorylated at multiple sites by myosin light chain kinase and protein kinase C. We have determined the sites phosphorylated on myosin light chain in intact bovine tracheal smooth muscle. Stimulation with 10 microM carbachol resulted in 66 +/- 5% monophosphorylated and 11 +/- 2% diphosphorylated myosin light chain after 1 min, and 47 +/- 4% monophosphorylated and 5 +/- 2% diphosphorylated myosin light chain after 30 min. Myosin heavy chain contained 0.06 +/- 0.01 mol of phosphate/mol of protein which did not change with carbachol. At both 1 and 30 min the monophosphorylated myosin light chain contained only phosphoserine whereas the diphosphorylated myosin light chain contained both phosphoserine and phosphothreonine. Two-dimensional peptide mapping of tryptic digests of monophosphorylated and diphosphorylated myosin light chain obtained from carbachol-stimulated tissue was similar to the peptide maps of purified light chain monophosphorylated and diphosphorylated, respectively, by myosin light chain kinase; these maps were distinct from the map obtained with tracheal light chain phosphorylated by protein kinase C. Phosphorylation of tracheal smooth muscle myosin light chain by myosin light chain kinase yields the tryptic phosphopeptide ATSNVFAMFDQSQIQEFK with S the phosphoserine in the monophosphorylated myosin light chain and TS the phosphotreonine and phosphoserine in the diphosphorylated myosin light chain. Thus, stimulation of tracheal smooth muscle with a high concentration of carbachol results in formation of both monophosphorylated and diphosphorylated myosin light chain although the amount of diphosphorylated light chain is substantially less than monophosphorylated light chain. In the intact muscle, myosin light chain is phosphorylated at sites corresponding to myosin light chain kinase phosphorylation.
Highlights
An important reaction for the initiation of smooth muscle phosphorylated at multiple sites by myosin light chain contraction is the rapid phosphorylation of the 20,000-Da kinase and protein kinaseC
Myosin heavy chain contained 0.06 f 0.01 mol of phosphate/mol of protein which did not change with carbachol. At both 1 and 30 min the monophosphorylated myosin lighcthaincontainedonlyphosphoserine demonstrated that gizzard smooth muscle myosin light chain kinase can phosphorylate threonine 18in purified gizzard myosin light chain, but with 32-fold more myosin light chain kinase than theamount required for phosphorylation of serine 19 (Ikebe and Hartshorne, 1985; Ikebe et al, 1986).Phosphowhereas the diphosphorylatedmyosin light chaincon- rylation of the threonine furtherincreases the actin-activated tainedboth phosphoserine and phosphothreonine
The phosphorylation of purified tracheal smooth muscle myosin light chain by myosin light chain kinase in vitro was qualitatively similar to results obtained with gizzard myosin light chain and gizzard myosin light chain kinase (Ikebe and Hartshorne, 1985; Ikebe et al, 1986)
Summary
StullS** From the $Department of Physiology and Moss Heart Centerand the 11Department of Biochemistry and Howard Hughes Medical Institute, University of Teras Southwestern Medical Center at Dallas, Dallas, Teras 75235
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