Okadaic acid indicates a major function for protein phosphatases in stimulus-response coupling of RINm5F rat insulinoma cells

Summary

Stimulus-induced insulin secretion involves the activation of several protein kinases within the beta cell. Most prominent are protein kinase A, protein kinase C and calcium/calmodulin-dependent protein kinases. Protein kinase action is functionally antagonized by protein phosphatases. The four ubiquious serine/threonine protein phosphatases are termed PP-1, PP-2A, -2B and -2C. PP-1 and PP-2A are in vivo parts of major protein complexes. These complexes presumably regulate the phosphatase activity and direct the enzyme to its site of action. Therefore, PP-1 and -2A could play an important role in controlling intracellular signal transmission. Two different toxins, okadaic acid and calyculin A, both from marine invertebrates, were recently discovered and identified as potent and highly specific inhibitors of PP-1 and PP-2A. Both compounds emerged as very useful tools for studying intracellular phosphorylation events. We took advantage of these substances to investigate the significance of protein phosphatase action in stimulus-induced insulin secretion. To avoid major complexity, we confined our study to the cAMP and the phosphoinositide signal pathway. Okadaic acid alone evoked virtually no secretory response. cAMP-de-pendent secretion was markedly enhanced by 1 uM okadaic acid. The stimulatory effect of okadaic acid was strongly dependent on the concentration of cAMP análoga. In contrast, insulin release caused by the cholinergic agonist carbachol was not influenced by okadaic acid. Calyculin A (10 nM) slightly increased cAMP-induced secretion, but its high toxicity prohibited accurate interpretation of the data.

Our findings support the idea that serine/threonine phosphatases act as important regulators in stimulus response coupling. Firstly, by affecting cAMP-induced insulin liberation, protein phosphatases could, theoretically, modify the magnitude of response to a given stimulus. Secondly, the cAMP pathway is obviously controlled by phosphatases other than the phosphoinositide pathway since the latter is not influenced by okadaic acid. This argues for a more differentiated function of protein phosphatases than just switching off any protein kinase activation.

Key words

insulin secretion - protein phosphatases - okadaic acid - RINm5F cells