Colburn J C, Michnoff C H, Hsu L C, Slaughter C A, Kamm K E, Stull J T
Department of Physiology, University of Texas Southwestern Medical Center, Dallas 75235.
J Biol Chem. 1988 Dec 15;263(35):19166-73.
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.
纯化的平滑肌肌球蛋白轻链可被肌球蛋白轻链激酶和蛋白激酶C在多个位点磷酸化。我们已经确定了完整牛气管平滑肌中肌球蛋白轻链上被磷酸化的位点。用10微摩尔卡巴胆碱刺激1分钟后，肌球蛋白轻链有66±5%为单磷酸化，11±2%为双磷酸化；30分钟后，47±4%为单磷酸化，5±2%为双磷酸化。肌球蛋白重链含0.06±0.01摩尔磷酸盐/摩尔蛋白，其含量不随卡巴胆碱变化。在1分钟和30分钟时，单磷酸化的肌球蛋白轻链仅含磷酸丝氨酸，而双磷酸化的肌球蛋白轻链同时含磷酸丝氨酸和磷酸苏氨酸。从卡巴胆碱刺激的组织中获得的单磷酸化和双磷酸化肌球蛋白轻链的胰蛋白酶消化产物的二维肽图，分别与经肌球蛋白轻链激酶单磷酸化和双磷酸化的纯化轻链的肽图相似；这些肽图与经蛋白激酶C磷酸化的气管轻链的肽图不同。肌球蛋白轻链激酶使气管平滑肌肌球蛋白轻链磷酸化产生胰蛋白酶磷酸肽ATSNVFAMFDQSQIQEFK，在单磷酸化肌球蛋白轻链中S为磷酸丝氨酸，在双磷酸化肌球蛋白轻链中TS为磷酸苏氨酸和磷酸丝氨酸。因此，用高浓度卡巴胆碱刺激气管平滑肌会导致单磷酸化和双磷酸化肌球蛋白轻链的形成，尽管双磷酸化轻链的量远少于单磷酸化轻链。在完整肌肉中，肌球蛋白轻链在与肌球蛋白轻链激酶磷酸化相对应的位点被磷酸化。
