The Na+-K+-2Cl– cotransporter plays important roles in regulating cell K+ and Cl– concentrations and cell volume. Total cotransporter phosphorylation (measured with 32P) correlates with cotransport rate under a variety of conditions (Haas & Forbush, 2000). In this communication we examine how phosphorylation of the transporter on threonine residues is affected by inhibitors of protein kinases and phosphatases. We used ferret red cells as a convenient model in which to explore the properties of the mammalian cotransporter.
Blood was taken by cardiac puncture from terminally anaesthetised ferrets (sodium pentobarbitone, 120 mg kg-1 I.P.). Membranes were prepared by hypotonic lysis of control red cells or cells incubated with 20 nM calyculin A (protein phosphatase 1 inhibitor), 1 mM sodium arsenite or 50 µM 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimadine (PP1, Src kinase inhibitor). Cotransporter was immunoprecipitated from membranes with monoclonal antibody T4 (Developmental Studies Hybridoma Bank, University of Iowa). Proteins from immunoprecipitates were separated by SDS-PAGE, blotted from the gels, and immunodetected with antibodies to phospho-threonine (Cell Signalling) or phospho-tyrosine (Zymed) using ECL detection (Amersham Biosciences). Signal intensities were measured by densitometry.
Phosphothreonine was detected in two smeared bands corresponding to the cotransporter existing as a monomer (137-163 kDa) and in a high molecular weight complex (320-407 kDa) under all conditions. Similar bands were detected with T4 (138-163, 327-402 kDa). The cotransporter is phosphorylated on threonine in control cells, corresponding to the substantial resting cotransport rate seen in these cells. Threonine phosphorylation (total in both bands) increases significantly (two-tailed paired t test, P < 0.05) in cells treated with calyculin or sodium arsenite (phosphorylation normalized with respect to controls (mean ± S.E.M., n = 5): 1.69 ± 0.22 and 2.62 ± 0.38, respectively), reflecting increased transport rate under these conditions (Flatman & Creanor, 1999a, b). Importantly, phosphorylation was significantly reduced, but still detectable, in cells treated with PP1 (0.66 ± 0.10), corresponding to the reduced transport rate seen here (Flatman & Creanor, 1999a). This finding supports the idea that treatment of cells with kinase inhibitors traps the cotransporter in a partially phosphorylated, and thus still active state. We did not observed any difference in tyrosine phosphorylation under all experimental conditions used.
This work was supported by The Wellcome Trust.