CHARACTERIZATION OF PROTEIN SERINE THREONINE PHOSPHATASES IN RAT PANCREAS AND DEVELOPMENT OF AN ENDOGENOUS SUBSTRATE-SPECIFIC PHOSPHATASE ASSAY/

Protein phosphatases have recently been recognized to represent an imp ortant, independently regulated portion of cellular signaling cascades . Although reversible phosphorylation of multiple pancreatic proteins has been described, suggesting a role for these enzymes, little is kno wn about the characteristics of protein phosphatases in this organ. In this work, we have characterized and quantified the serine/threonine phosphatases present in pancreatic cytosol and plasma membranes. Using a sensitive and specific in vitro assay with standard substrates (pho sphorylase a and phosphocasein), the predominant enzymes represented p rotein phosphatase 2A in cytosol and protein phosphatase 1 in plasma m embranes, with both compartments having substantial amounts of both of these enzymes. Both compartments also had protein phosphatase 2B acti vity, whereas protein phosphatase 2C was only measurable in the plasma membrane fraction. Further, a novel assay was developed and validated in which the action of an endogenous protein phosphatase on a specifi c cellular phosphoprotein was studied. For this, we utilized as substr ate the cholecystokinin receptor which had been phosphorylated in resp onse to agonist stimulation of the intact acinar cell. This type of as say will be key for the analysis of the mediation and regulation of de phosphorylation events which actually occur in the cell