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Phosphorylation of Nonmuscle Myosin Heavy Chain IIA on Ser¹⁹¹⁷ Is Mediated by Protein Kinase CII and Coincides with the Onset of Stimulated Degranulation of RBL-2H3 Mast Cells¹

Russell I. Ludowyke^2,, Zehra Elgundi^2,*, Tanya Kranenburg, Justine R. Stehn^3,*, Carsten Schmitz-Peiffer^*, William E. Hughes^* and Trevor J. Biden^4,*

^* Garvan Institute of Medical Research and Centre for Immunology, St. Vincent’s Hospital, Sydney, Australia

Dynamic remodeling of the actinomyosin cytoskeleton is integral to many biological processes. It is regulated, in part, by myosin phosphorylation. Nonmuscle myosin H chain IIA is phosphorylated by protein kinase C (PKC) on Ser¹⁹¹⁷. Our aim was to determine the PKC isoform specificity of this phosphorylation event and to evaluate its potential role in regulated secretion. Using an Ab against the phosphorylated form of Ser¹⁹¹⁷, we show that this site is not phosphorylated in unstimulated RBL-2H3 mast cells. The physiological stimulus, Ag, or the pharmacological activators, PMA plus A23187, induced Ser¹⁹¹⁷ phosphorylation with a time course coincident with the onset of granule mediator secretion. Dephosphorylation at this site occurred as Ag-stimulated secretion declined from its peak, but dephosphorylation was delayed in cells activated with PMA plus A23187. Phosphate incorporation was also enhanced by PMA alone and by inhibition of protein phosphatase 2A. Gö6976, an inhibitor of conventional PKC isoforms, abolished secretion and Ser¹⁹¹⁷ phosphorylation with similar dose dependencies consistent with involvement of either PKC or PKC. Phorbol ester-stimulated Ser¹⁹¹⁷ phosphorylation was reconstituted in HEK-293 cells (which lack endogenous PKC) by overexpression of both wild-type and constitutively active PKCII but not the corresponding PKCI or PKC constructs. A similar selectivity for PKCII overexpression was also observed in MIN6 insulinoma cells infected with recombinant PKC wild-type adenoviruses. Our results implicate PKC-dependent phosphorylation of myosin H chain IIA in the regulation of secretion in mast cells and suggest that Ser¹⁹¹⁷ phosphorylation might be a marker of PKCII activation in diverse cell types.

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