Transient translocation and activation of protein phosphatase 2A during mast cell secretion.

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 signal-mediated, rapid, transient translocation and activation of PP2A in membranes in any system.

Developmental regulation of protein phosphatase types 1 and 2A in post-hatch chicken brain.

The activity and subcellular distribution of protein phosphatases 1 and 2A were measured in chicken forebrain and cerebellum during post-hatch development. At all post-hatch ages, a large proportion of PP1 and PP2A was membrane bound and these enzymes were less active than their cytosolic counterparts. The protein concentration of PP1 in the membranes increased 40% between 2 and 14 days and a further 60% between 14 days and adult, whereas the PP1 enzyme activity in the membranes progressively decreased. In contrast to PP1, the protein concentration of PP2A remained constant in all fractions during post-hatch development, and the enzyme activity of PP2A did not change except for a decrease in the membrane-bound activity between 2 and 14 days. These results show that the subcellular distribution and activity of PP1 is selectively regulated during post-hatch development and that membrane association and inactivation of PP1 are independent events.

Protein phosphatase activity in cyanobacteria: consequences for microcystin toxicity analysis.

Hepatotoxic microcystin levels in cyanobacteria (blue-green algae) were assessed by an assay based on inhibition of protein phosphatases type 1 (PP1) and type 2A (PP2A) in crude chicken forebrain extracts using 32P-labelled glycogen phosphorylase as substrate. While cyanobacteria are reported to be devoid of phosphorylase phosphatase activity, two samples obtained from cyanobacterial scums, containing predominantly Anabaena circinalis, were found to contain high levels of a phosphorylase phosphatase activity which completely masked the presence of microcystin. Furthermore, samples containing predominantly Microcystis aeruginosa but increasing Anabaena circinalis contained sufficient phosphorylase phosphatase activity to cause a fourfold underestimation of microcystin levels. Thus, protocols for microcystin toxicity analysis should take into account the possible presence of endogenous phosphatase activity, thereby preventing underestimation of toxin levels.