Calcium-dependent threonine phosphorylation of nonmuscle myosin in stimulated RBL-2H3 mast cells.
Stimulation of RBL-2H3 m1 mast cells through the IgE receptor with antigen, or through a G protein-coupled receptor with carbachol, leads to the rapid appearance of phosphothreonine in nonmuscle myosin heavy chain II-A (NMHC-IIA). We demonstrate that this results from phosphorylation of Thr-1940 by calcium/calmodulin-dependent protein kinase II (CaM kinase II), activated by increased intracellular calcium. The phosphorylation site in rodent NMHC-IIA was localized to the carboxyl terminus of NMHC-IIA distal to the coiled-coil region, and identified as Thr-1940 by site-directed mutagenesis. A fusion protein containing the NMHC-IIA carboxyl terminus was phosphorylated by CaM kinase II in vitro, while mutation of Thr-1940 to Ala eliminated phosphorylation. In contrast to rodents, in humans Thr-1940 is replaced by Ala, and human NMHC-IIA fusion protein was not phosphorylated by CaM kinase II unless Ala-1940 was mutated to Thr. Similarly, co-transfected Ala --> Thr-1940 human NMHC-IIA was phosphorylated by activated CaM kinase II in HeLa cells, while wild type was not. In RBL-2H3 m1 cells, inhibition of CaM kinase II decreased Thr-1940 phosphorylation, and inhibited release of the secretory granule marker hexosaminidase in response to carbachol but not to antigen. These data indicate a role for CaM kinase stimulation and resultant threonine phosphorylation of NMHC-IIA in RBL-2H3 m1 cell activation.
Buxton
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Laboratory of Molecular Cardiology, NHLBI, National Institutes of Health, Bethesda, Maryland 20892, USA. db225a@nih.gov
Adelstein
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism*; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D002460:Cell Line; D017931:DNA Primers; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D015759:Ionomycin / Q000494:pharmacology; D008407:Mast Cells / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D009218:Myosins / Q000378:metabolism*; D010766:Phosphorylation; D013912:Threonine / Q000378:metabolism*
D017931:DNA Primers; D004791:Enzyme Inhibitors; D013912:Threonine; D015759:Ionomycin; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D009218:Myosins; D002118:Calcium