PASK (proline-alanine-rich STE20-related kinase), a regulatory kinase of the Na-K-Cl cotransporter (NKCC1).
Although the phosphorylation-dependent activation of the Na-K-Cl cotransporter (NKCC1) has been previously well documented, the identity of the kinase(s) responsible for this regulation has proven elusive. Recently, Piechotta et al. (Piechotta, K., Lu, J., and Delpire, E. (2002) J. Biol. Chem. 277, 50812-50819) reported the binding of PASK (also referred as SPAK (STE20/SPS1-related proline-alanine-rich kinase)) and OSR1 (oxidative stress response kinase) to cation-chloride cotransporters KCC3, NKCC1, and NKCC2. In this report, we show that overexpression of a kinase inactive, dominant negative (DN) PASK mutant drastically reduces both shark (60 +/- 5%) and human (80 +/- 3%) NKCC1 activation. Overexpression of wild type PASK causes a small (sNKCC1 22 +/- 8% p < 0.05, hNKCC1 12 +/- 3% p < 0.01) but significant increase in shark and human cotransporter activity in HEK cells. Importantly, DNPASK also inhibits the phosphorylation of two threonines, contained in the previously described N-terminal regulatory domain. We additionally show the near complete restoration of NKCC1 activity in the presence of the protein phosphatase type 1 inhibitor calyculin A, demonstrating that DNPASK inhibition results from an alteration in kinase/phosphatase dynamics rather than from a decrease in functional cotransporter expression. Coimmunoprecipitation assays confirm PASK binding to NKCC1 in transfected HEK cells and further suggest that this binding is not a regulated event; neither PASK nor NKCC1 activity affects the association. In cells preloaded with 32Pi, the phosphorylation of PASK, but not DNPASK, coincides with that of NKCC1 and increases 5.5 +/- 0.36-fold in low [Cl]e. These data conclusively link PASK with the phosphorylation and activation of NKCC1.
Yale University, School of Medicine, Department of Cellular and Molecular Physiology, New Haven, Connecticut 06520-0826, USA. brian.dowd@yale.edu
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D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005799:Genes, Dominant; D005822:Genetic Vectors; D006801:Humans; D008387:Marine Toxins; D010080:Oxazoles / Q000737:chemistry; D018384:Oxidative Stress; D010766:Phosphorylation; D011233:Precipitin Tests; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry* / Q000502:physiology*; D017434:Protein Structure, Tertiary; D051381:Rats; D012413:Rubidium / Q000378:metabolism; D012754:Sharks; D028021:Sodium-Potassium-Chloride Symporters / Q000378:metabolism*; D064506:Solute Carrier Family 12, Member 2; D013912:Threonine / Q000737:chemistry; D013997:Time Factors
D018076:DNA, Complementary; D008387:Marine Toxins; D010080:Oxazoles; C578877:SLC12A2 protein, human; C578879:Slc12a2 protein, rat; D028021:Sodium-Potassium-Chloride Symporters; D064506:Solute Carrier Family 12, Member 2; D013912:Threonine; C059041:calyculin A; C116037:PAS domain kinases; D017346:Protein Serine-Threonine Kinases; D012413:Rubidium