The Na-K-Cl cotransport protein of shark rectal gland. II. Regulation by direct phosphorylation.

摘要: We determined the relationship between activation state and phosphorylation of Na-K-Cl cotransport protein in tubules isolated from shark rectal gland, a prototypic chloride-secreting epithelium. In response to cAMP-dependent secretagogues (e.g. vasoactive intestinal peptide, adenosine, forskolin) or osmotically induced changes cell volume, (assessed measurements loop diuretic binding) increased 5-10 fold. The was temporally associated with comparable increase (3-9 fold) phosphorylation. Graded cotransporter evoked proportional Under conditions our experiments, 195-kDa only membrane whose conspicuously both cAMP shrinkage. Both stimuli promoted at serine threonine residues. One cAMP-sensitive phosphoacceptors found within segment comprised sequence (Phe-Gly-His-Asn-Thr*-Ile-Asp-Ala-Val-Pro) that corresponds predicted by cDNA analysis, where phosphoacceptor (Thr*) is 189. Incubation gland K-252a H-8, structurally different kinase inhibitors, rendered insensitive conclude regulated direct reversible sites.