INVOLVEMENT OF DIRECT PHOSPHORYLATION IN THE REGULATION OF THE RAT PAROTID NA+-K+-2CL(-) COTRANSPORTER

We identify a 175-kDa membrane phosphoprotein (pp175) in rat parotid acini whose properties correlate well with the Na+-K+-2Cl(-) cotransporter previously characterized functionally and biochemically in this tissue. pp175 was the only phosphoprotein immunoprecipitated by an anti-Na+-K+-2Cl(-) cotransporter antibody and the only membrane protein whose phosphorylation state was conspicuously altered after a brief (45-s) exposure of acini to the beta-adrenergic agonist isoproterenol. Phosphopeptide mapping provided evidence for three phosphorylation sites on pp175, only one of which was labeled in response to isoproterenol treatment. The half-maximal effect of isoproterenol on phosphorylation of pp175 (approximate to 20 nM) was in excellent agreement with its previously demonstrated up regulatory effect on cotransport activity. Increased phosphorylation of pp175 was also seen following acinar treatment with a permeant cAMP analogue and with forskolin, conditions that have likewise been shown to up-regulate the cotransporter. Combined with earlier results from our laboratory, these data provide strong evidence that the up-regulation of the cotransporter by these agents is due to direct phosphorylation mediated by protein kinase A. ALF(4)(-) treatment, which results in an up regulation of cotransport activity comparable with that observed with isoproterenol (similar to 6-fold), caused a similar increase in phosphorylation of pp175. However, hypertonic shrinkage and treatment with the protein phosphatase inhibitor calyculin A, which also up-regulate the cotransporter (similar to 3-fold and similar to 6-fold, respectively) caused no change in the phosphorylation level. Furthermore, although acinar treatment with the muscarinic agonist carbachol results in a dramatic up-regulation of cotransport activity and a concomitant phosphorylation of pp175, no phosphorylation of pp175 was seen with the Ca2+-mobilizing agent thapsigargin, which is able to fully mimic the up-regulatory effect of carbachol on transport activity. Taken together, these results indicate that direct phosphorylation is only one of the mechanisms involved in secretagogue-induced regulation of the rat parotid Na+K+-2Cl(-) cotransporter.