Okadaic acid inhibits secretion from mast cells, suggesting a regulatory role for protein Ser/Thr phosphatases type I (PP1) and/or 2A (PP2A) in the secretory process. In unstimulated RBL-2H3 cells, okadaic acid pretreatment inhibited PP2A activity in both cytosol and membrane fractions, but inhibition of secretion correlated with inhibition of membrane-bound rather than cytosolic PP2A activity. Okadaic acid had very little effect on PP1 activity. Stimulation of RBL-2H3 cells by antigen led to the activity and amount of PP2A in the membrane fraction increasing nearly 2-fold. In contrast, there was little change in the activity or distribution of PP1. Importantly, the translocation of PP2A was transient, coinciding with or marginally preceding the peak rate of secretion, suggesting a link between PP2A translocation, activity, and secretion. Phorbol 12-myristate 13-acetate plus the calcium ionophore A23187 induced a slower, prolonged rate of secretion that coincided with a similarly protracted translocation of PP2A to the membrane fraction. PP2A translocation is not the only event required for secretion as translocation was also induced by phorbol 12-myristate 13-acetate, without resulting in secretion. These results indicate that increased protein dephosphorylation in the membrane fraction mediated by PP2A is required for mast cell secretion. To our knowledge, this is the first demonstration of a signalmediated, rapid, transient translocation and activation of PP2A in membranes in any system.

The study addressed the functional link between remodelling of the actomyosin cytoskeleton in pancreatic L L-cells and the regulation of insulin secretion. Confocal microscopy revealed that myosin heavy chain (MHC) IIA co-localized very well with filamentous (F)-actin in RINm5F cells but MHCIIB did not. Subcellular localization of MHCIIB was not altered by stimulation with 30 mM KCl (which evokes Ca 2+ -dependent insulin secretion). In contrast MHCIIA redistributed in a manner similar to F-actin, especially towards the apical surface, but also away from peripheral regions towards cell contact points on the basal surface. Finally, Ca 2+ -dependent insulin secretion was inhibited by stabilization of actin filaments with jasplakinolide. The results support a role for the MHCIIA/actin cytoskeleton in regulating insulin secretion. ß

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 PKCα or PKCβ. Phorbol ester-stimulated Ser1917 phosphorylation was reconstituted in HEK-293 cells (which lack endogenous PKCβ) by overexpression of both wild-type and constitutively active PKCβII but not the corresponding PKCβI or PKCα constructs. A similar selectivity for PKCβII 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 PKCβII activation in diverse cell types.

Modulation of exocytosis is integral to the regulation of cellular signalling, and a variety of disorders (such as epilepsy, hypertension, diabetes and asthma) are closely associated with pathological modulation of exocytosis. Emerging evidence points to protein phosphatases as key regulators of exocytosis in many cells and, therefore, as potential targets for the design of novel therapies to treat these diseases. Diverse yet exquisite regulatory mechanisms have evolved to direct the specificity of these enzymes in controlling particular cell processes, and functionally driven studies have demonstrated differential regulation of exocytosis by individual protein phosphatases. This Review discusses the evidence for the regulation of exocytosis by protein phosphatases in three major secretory systems, (1) mast cells, in which the regulation of exocytosis of inflammatory mediators plays a major role in the respiratory response to antigens, (2) insulin-secreting cells in which regulation of exocytosis is essential for metabolic control, and (3) neurons, in which regulation of exocytosis is perhaps the most complex and is essential for effective neurotransmission.