Insulin-induced myosin light-chain phosphorylation during receptor capping in IM-9 human B-lymphoblasts.
We have examined further the interaction between insulin surface receptors and the cytoskeleton of IM-9 human lymphoblasts. Using immunocytochemical techniques, we determined that actin, myosin, calmodulin and myosin light-chain kinase (MLCK) are all accumulated directly underneath insulin-receptor caps. In addition, we have now established that the concentration of intracellular Ca2+ (as measured by fura-2 fluorescence) increases just before insulin-induced receptor capping. Most importantly, we found that the binding of insulin to its receptor induces phosphorylation of myosin light chain in vivo. Furthermore, a number of drugs known to abolish the activation properties of calmodulin, such as trifluoperazine (TFP) or W-7, strongly inhibit insulin-receptor capping and myosin light-chain phosphorylation. These data imply that an actomyosin cytoskeletal contraction, regulated by Ca2+/calmodulin and MLCK, is involved in insulin-receptor capping. Biochemical analysis in vitro has revealed that IM-9 insulin receptors are physically associated with actin and myosin; and most interestingly, the binding of insulin-receptor/cytoskeletal complex significantly enhances the phosphorylation of the 20 kDa myosin light chain. This insulin-induced phosphorylation is inhibited by calmodulin antagonists (e.g. TFP and W-7), suggesting that the phosphorylation is catalysed by MLCK. Together, these results strongly suggest that MLCK-mediated myosin light-chain phosphorylation plays an important role in regulating the membrane-associated actomyosin contraction required for the collection of insulin receptors into caps.
D000199:Actins / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism; D002460:Cell Line; D003599:Cytoskeleton / Q000378:metabolism; D006801:Humans; D007122:Immunoelectrophoresis; D007328:Insulin / Q000494:pharmacology*; D007424:Intracellular Fluid / Q000378:metabolism; D008214:Lymphocytes / Q000378:metabolism*; D046911:Macromolecular Substances; D008856:Microscopy, Fluorescence; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism; D009218:Myosins / Q000378:metabolism*; D010766:Phosphorylation; D011972:Receptor, Insulin / Q000378:metabolism*
D000199:Actins; D002147:Calmodulin; D007328:Insulin; D046911:Macromolecular Substances; D011972:Receptor, Insulin; D009219:Myosin-Light-Chain Kinase; D009218:Myosins; D002118:Calcium