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 (approximately 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 (approximately 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 (approximately 3-fold and approximately 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 Ca(2+)-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.
Tanimura
A
A
Clinical Investigations and Patient Care Branch, NIDR, National Institutes of Health, Bethesda, Maryland 20892, USA.
Kurihara
K
K
Reshkin
S J
SJ
Turner
R J
RJ
D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D002712:Chlorides / Q000378:metabolism*; D000242:Cyclic AMP / Q000502:physiology; D007545:Isoproterenol / Q000494:pharmacology; D008297:Male; D010306:Parotid Gland / Q000378:metabolism*; D010449:Peptide Mapping; D010766:Phosphorylation; D011188:Potassium / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D012964:Sodium / Q000378:metabolism*; D028021:Sodium-Potassium-Chloride Symporters; D015854:Up-Regulation
D002352:Carrier Proteins; D002712:Chlorides; D028021:Sodium-Potassium-Chloride Symporters; D012964:Sodium; D000242:Cyclic AMP; D007545:Isoproterenol; D011188:Potassium