PASK (Proline-Alanine-rich STE20-related Kinase), a Regulatory Kinase of the Na-K-Cl Cotransporter (NKCC1)
2003; Elsevier BV; Volume: 278; Issue: 30 Linguagem: Inglês
10.1074/jbc.m301899200
ISSN1083-351X
AutoresBrian F.X. Dowd, Biff Forbush,
Tópico(s)Protein Kinase Regulation and GTPase Signaling
ResumoAlthough 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. 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. The phosphorylation of the N terminus of the secretory or housekeeping isoform of the Na-K-Cl cotransporter (NKCC1) 1The abbreviations used are: NKCC1, Na-K-Cl cotransporter; sNKCC1, shark NKCC1; hNKCC1, human NKCC1; NCC, Na-Cl cotransporter; KCC, K-Cl cotransporter; PASK, proline-alanine-rich STE20-related kinase; DNPASK, dominant-negative (DN) PASK; MST, mammalian sterile twenty-like kinase; sMST1/2, shark MST; [Cl]e, extracellular chloride concentration; [Cl]i, intracellular chloride concentration; HEK, human embryonic kidney; vector 1, pJB20; vector 2, pIRES puro3; GST, glutathione S-transferase; HA, hemagglutinin.1The abbreviations used are: NKCC1, Na-K-Cl cotransporter; sNKCC1, shark NKCC1; hNKCC1, human NKCC1; NCC, Na-Cl cotransporter; KCC, K-Cl cotransporter; PASK, proline-alanine-rich STE20-related kinase; DNPASK, dominant-negative (DN) PASK; MST, mammalian sterile twenty-like kinase; sMST1/2, shark MST; [Cl]e, extracellular chloride concentration; [Cl]i, intracellular chloride concentration; HEK, human embryonic kidney; vector 1, pJB20; vector 2, pIRES puro3; GST, glutathione S-transferase; HA, hemagglutinin. has been previously shown to confer its activation in numerous experimental systems using various stimuli (1Lytle C. Forbush III, B. J. Biol. Chem. 1992; 267: 25438-25443Abstract Full Text PDF PubMed Google Scholar, 2Kurihara K. Moore-Hoon M.L. Saitoh M. Turner R.J. Am. J. Physiol. 1999; 277: C1184-C1193Crossref PubMed Google Scholar). Our current model suggests a low intracellular chloride concentration and/or a reduction in cell volume as the principle stimulus for NKCC1 activation. The kinase(s) mediating this activation, however, has remained obscure. Our laboratory has previously identified three threonines on the N terminus of the Na-K-Cl cotransporter that modulate activity, highlighting Thr189 (numbering in shark) as essential for cotransporter function (3Darman R.B. Forbush B. J. Biol. Chem. 2002; 277: 37542-37550Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). A phosphospecific antibody specifically recognizes two of these phosphothreonines (Thr184, Thr189) and has proven to be a powerful tool in discerning the activation state of NKCC1 in cells and tissue (4Flemmer A.W. Gimenez I. Dowd B.F. Darman R.B. Forbush B. J. Biol. Chem. 2002; 277: 37551-37558Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). This regulatory domain resides in one of three highly conserved regions on the N terminus and was shown to mediate the activation of NKCC1 in species ranging from shark to human (4Flemmer A.W. Gimenez I. Dowd B.F. Darman R.B. Forbush B. J. Biol. Chem. 2002; 277: 37551-37558Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). Another of these N-terminal conserved regions has recently been identified by our laboratory as a protein phosphatase type 1 binding site (RVXFXD) (5Darman R.B. Flemmer A. Forbush B. J. Biol. Chem. 2001; 276: 34359-34362Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). Mutation of this site to either enhance or disrupt protein phosphatase type 1 binding significantly alters the activation profile of NKCC1. Recently, the significance of the third conserved domain in the NKCC1 N terminus was suggested by studies of Piechotta et al. (6Piechotta K. Lu J. Delpire E. J. Biol. Chem. 2002; 277: 50812-50819Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar). This 10-amino acid region contains an (R/K)FX(V/I) sequence that was found to bind the proline alanine-rich STE20-related kinase (PASK) and the highly homologous (67% identical) oxidative stress response kinase (OSR1) in yeast two-hybrid and GST pull-down assays. An additional binding motif is found 54 residues downstream (6Piechotta K. Lu J. Delpire E. J. Biol. Chem. 2002; 277: 50812-50819Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar), overlapping the previously described protein phosphatase type 1 binding site (5Darman R.B. Flemmer A. Forbush B. J. Biol. Chem. 2001; 276: 34359-34362Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). To date no functional effect of PASK upon NKCC1 has been identified. The direct binding of this kinase, possibly with the resultant disruption of its regulatory phosphatase, made PASK an attractive candidate for NKCC regulation. In addition, the high expression of PASK in solute-transporting epithelia (7Ushiro H. Tsutsumi T. Suzuki K. Kayahara T. Nakano K. Arch Biochem. Biophys. 1998; 355: 233-240Crossref PubMed Scopus (72) Google Scholar) parallels that of NKCC1. A number of protein kinases have been previously discussed in conjunction with NKCC1 activation. Human NKCC1 contains known consensus phosphorylation sites for protein kinase C, casein kinase II, and protein kinase A, but none of these has been directly linked to NKCC1 activation. The inhibition of myosin light chain kinase has been shown to inhibit NKCC1 function, but it does not affect NKCC1 phosphorylation and has been proposed to act indirectly through cytoskeletal disruption (8Klein J.D. O'Neill W.C. Am. J. Physiol. 1995; 269: C1524-C1531Crossref PubMed Google Scholar). Serum- and glucocorticoid-dependent kinase has been suggested to mediate an increase in NKCC1 activity via an increase of cotransporter insertion into the cell membrane but has not been shown to directly activate functional NKCC1 (9Fillon S. Warntges S. Matskevitch J. Moschen I. Setiawan I. Gamper N. Feng Y.X. Stegen C. Friedrich B. Waldegger S. Broer S. Wagner C.A. Huber S.M. Klingel K. Vereninov A. Lang F. Comp. Biochem. Physiol. A. Mol. Integr. Physiol. 2001; 130: 367-376Crossref PubMed Scopus (23) Google Scholar). c-Jun N-terminal kinase has also been proposed as an NKCC1 regulatory kinase candidate; to date, however, this has only been supported by in-gel kinase assays (10Klein J.D. Lamitina S.T. O'Neill W.C. Am. J. Physiol. 1999; 277: C425-C431Crossref PubMed Google Scholar). PASK (also referred as SPAK 2Accession number O88506, denoted as PASK in the original publication (7Ushiro H. Tsutsumi T. Suzuki K. Kayahara T. Nakano K. Arch Biochem. Biophys. 1998; 355: 233-240Crossref PubMed Scopus (72) Google Scholar). The human homologue has also been termed SPAK (STE20/SPS1-related, proline alanine-rich kinase) (11Johnston A.M. Naselli G. Gonez L.J. Martin R.M. Harrison L.C. DeAizpurua H.J. Oncogene. 2000; 19: 4290-4297Crossref PubMed Scopus (116) Google Scholar).2Accession number O88506, denoted as PASK in the original publication (7Ushiro H. Tsutsumi T. Suzuki K. Kayahara T. Nakano K. Arch Biochem. Biophys. 1998; 355: 233-240Crossref PubMed Scopus (72) Google Scholar). The human homologue has also been termed SPAK (STE20/SPS1-related, proline alanine-rich kinase) (11Johnston A.M. Naselli G. Gonez L.J. Martin R.M. Harrison L.C. DeAizpurua H.J. Oncogene. 2000; 19: 4290-4297Crossref PubMed Scopus (116) Google Scholar). (STE20/SPS1-related, proline alanine-rich kinase (11Johnston A.M. Naselli G. Gonez L.J. Martin R.M. Harrison L.C. DeAizpurua H.J. Oncogene. 2000; 19: 4290-4297Crossref PubMed Scopus (116) Google Scholar)) is a 55-kDa serine/threonine kinase within the germinal center kinase VI subfamily of STE20 group kinases (12Dan I. Watanabe N.M. Kusumi A. Trends Cell Biol. 2001; 11: 220-230Abstract Full Text Full Text PDF PubMed Scopus (506) Google Scholar). First isolated from rat brain, PASK was found to be heavily expressed in epithelial cells active in ion transport and to associate with the cytoskeleton (7Ushiro H. Tsutsumi T. Suzuki K. Kayahara T. Nakano K. Arch Biochem. Biophys. 1998; 355: 233-240Crossref PubMed Scopus (72) Google Scholar). The only demonstrated biological function for this kinase was reported for Fray, the Drosophila PASK homologue, which was shown to be expressed in glial cells and required for the proper ensheathing neighboring axons (13Leiserson W.M. Harkins E.W. Keshishian H. Neuron. 2000; 28: 793-806Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). In this report, we describe the regulation of NKCC1 by PASK in transfected HEK cells. We show that overexpression of PASK causes a small but significant increase in NKCC1 activity. We also show that a dominant-negative PASK mutant drastically inhibits shark and human NKCC1 activation. This inhibition is via a decrease in NKCC1 regulatory phosphorylation, not a decrease in number of functional cotransporter molecules or a down-regulation in expression. We also show that PASK phosphorylation parallels that of NKCC1 in low [Cl]e and that binding of the kinase does not appear to be regulated by or dependent upon the activation state of PASK or NKCC1. cDNA Constructs and the Production of Stable Cell Lines—The rat PASK clone was HA-tagged on the N terminus and subcloned into the pIRES puro3 vector (Invitrogen) using PCR. Briefly, a forward primer (p1) containing the HA antigen and EcoR1 site (ggaattccatgtacccatacgacgtcccagactacgctATGGCGGAGCCGAGCGGCTCG) (the PASK sequence is in uppercase) and reverse primer (p2) containing a stop codon and BamH1 site (cgggatccTCAGCTCACACTCAACTGGGCGAAC) were used in a PCR reaction with pGST-PASK as a template (a kind gift of William Leiserson) (7Ushiro H. Tsutsumi T. Suzuki K. Kayahara T. Nakano K. Arch Biochem. Biophys. 1998; 355: 233-240Crossref PubMed Scopus (72) Google Scholar). The PCR product was then subcloned into the pIRES puro3 vector using EcoR1 and BamH1. The dominant negative, kinase inactive K101R PASK was created using a two-step PCR approach. First, an internal primer (p3) (CGCGTAGCCATAAgGCGGATCAACTTG) and its reverse complement (p4) (CAAGTTGATCCGCcTTATGGCTACGCG) were created containing a single nucleotide substitution to produce the K101R mutation (mutation in lowercase). PCR reactions were completed using p1/p4 and p2/p3 primer combinations. After gel purification of the two products, equal amounts were subjected to five cycles of PCR with Pfu polymerase but without primers (for extension). The p1 and p2 primers were then added to amplify the full-length mutant. Subcloning into pIRES puro3 (Clontech) was completed as described above. Sequence verification was completed by the Mount Desert Island Biological Laboratory DNA Sequencing Center. For most experiments a shark protein kinase (sMST1/2) 3Accession number AY242842. This protein contains 83 and 79% identity to human MST1 and MST2 respectively. with significant homology to mammalian MST1 (accession number AAA83254) and MST2 (accession number 2204254A) was used as a control in addition to vector-transfected cells. We identified MST1/2 in an expression screen of a λ phage shark kidney library. This kinase identification assay was adapted from a method previously described by Matsuo et al. (14Matsuo R. Ochiai W. Nakashima K. Taga T. J. Immunol. Methods. 2001; 247: 141-151Crossref PubMed Scopus (62) Google Scholar). Using the previously described phosphospecific Thr184/Thr189 NKCC antibody (R5) (4Flemmer A.W. Gimenez I. Dowd B.F. Darman R.B. Forbush B. J. Biol. Chem. 2002; 277: 37551-37558Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar) candidates were selected for their ability to phosphorylate a 15-amino acid GST fusion tandem-repeat protein containing the Thr184/Thr189 residues residing within the previously described N-terminal regulatory domain of NKCC1 (GST-YYLRTFGHNTMDAVP YYLRTFGHNTMDAVP; the Thr residues in shown bold are at positions 184, 189, 184, and 189, respectively). MST1/2 was identified in this screen, but subsequent experiments failed to support an important role for the kinase in NKCC1 regulation, and we have utilized it as a control enzyme in the current study. Interestingly, like PASK, MST1/2 is a member of the germinal center kinase family of STE20 kinases. In addition, the kinase domain of MST1/2 retains a high homology (40%) to PASK, and both kinases contain putative caspase 3 cleavage sites and nuclear localization signals (11Johnston A.M. Naselli G. Gonez L.J. Martin R.M. Harrison L.C. DeAizpurua H.J. Oncogene. 2000; 19: 4290-4297Crossref PubMed Scopus (116) Google Scholar). IRES puro3 vector (Clontech) containing PASK, dominant negative mutant (DNPASK), or shark MST1/2 were transfected with LipofectAMINE 2000 (Invitrogen) into various HEK stable clonal cell lines, all carrying the Geneticin resistance marker and previously described (15Xu J.C. Lytle C. Zhu T.T. Payne J.A. Benz Jr., E. Forbush III, B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2201-2205Crossref PubMed Scopus (370) Google Scholar, 16Payne J.A. Xu J.C. Haas M. Lytle C.Y. Ward D. Forbush III, B. J. Biol. Chem. 1995; 270: 17977-17985Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar). Cells were selected and maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 1 μg/ml puromycin (Clontech), and 0.8 mg/ml Geneticin (Invitrogen). With the use of the IRES vector, clonal selection was not needed in isolation of stable lines. The IRES vector is herein referred to as "vector 2," whereas the pJB20 vector used for sNKCC1 and hNKCC1 expression is referred to as "vector 1." 86Rb + Influx Assays—Influx assays to assess NKCC1 cotransport activity were performed as previously described using an automated 96-well plate flux machine (3Darman R.B. Forbush B. J. Biol. Chem. 2002; 277: 37542-37550Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). For calyculin A time courses, the compound was dissolved in Me2SO and diluted into the appropriate preincubation media with a final concentration of ∼500 nm at specific time points before the flux assay. The flux time was 2 min for exogenously expressed cell lines (sNKCC1 and hNKCC1) and 8 min for the measurement of endogenous NKCC1 activity. 32P Incorporation and Coimmunoprecipitation Experiments—Near confluent 10-cm dishes were rinsed with phosphate-free isotonic media (135 mm NaCl, 5 mm RbCl, 1 mm CaCl2/MgCl2, and 15 mm NaHEPES, pH 7.4) and incubated with 250 μCi of 32Pi for 20 min. Cells were then rinsed 2× with either phosphate-free isotonic or phosphate-free 3 mm Cl– with gluconate replacement (135 mm sodium gluconate, 5 mm potassium gluconate, 1 mm CaCl2, 1 mm MgCl2, 1 mm Na2HPO4, 1 mm NaSO4) media for 1 h at room temperature. Cells were lysed on ice in 1 ml of lysis buffer (300 mm NaCl, 50 mm Hepes, 25 mm β-glycerophosphate, 1% Nonidet P-40, 1 mm sodium vanadate, 5 mm EDTA, 0.5 μm calyculin A, protease inhibitor mixture (Roche Applied Science)) and centrifuged to remove debris. Bovine serum albumin (0.25% final concentration) and 5 μg of antibody were added before a 2-h incubation at 4 °C. Protein G beads (Pierce) were blocked in 5% milk/phosphate-buffered saline, 0.1% Tween 20, washed in lysis buffer, added to lysate, and incubated at 4 °C for an additional hour. Immunocomplexed beads were washed 3× in ice-cold wash buffer (lysis buffer without calyculin A and protease inhibitors) and 1× in ice-cold phosphate-buffered saline. Sample buffer with 2-mercaptoethanol was added before boiling. Samples were then subjected to SDS-PAGE using a 10% acrylamide gel. Western blotting was completed as previously described (4Flemmer A.W. Gimenez I. Dowd B.F. Darman R.B. Forbush B. J. Biol. Chem. 2002; 277: 37551-37558Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). For protein determinations of 96-well flux plates, cells were solubilized with 1% SDS after being exposed to phosphorimaging plates, and the DC-BCA kit (Pierce) was used according to the manufacturer's instructions. In the post-lysis binding experiment, immunoprecipitation was conducted as described above except for the following changes; after lysis and centrifugation, 200 μl of HEK cell lysate was added to 200 μl of vector 1/PASK, sNKCC1/vector 2, or sNKCC1/PASK. In a fourth tube, 200 μl of lysate from vector 1/PASK and sNKCC1/vector 2 were combined before the immunoprecipitation step. Regulatory Phosphorylation Time Course—Transfected HEK cells were grown to confluence in a 96-well polylysine-treated plate. Wells were rinsed with incubation media for specific time points, and the reaction was terminated with the addition of 1 n H3PO4, 1% SDS. Samples were neutralized with 3 m Tris, 1 n NaOH and loaded in duplicate onto two separate gels. SDS-PAGE with Western analysis using the phosphospecific R5 and J3 antibodies was completed in parallel. Comparison of parallel blots was necessary due to selective loss of signal on stripping. Antibodies—The sNKCC1-specific J3 antibody and R5 phospho-specific NKCC have been previously described (1Lytle C. Forbush III, B. J. Biol. Chem. 1992; 267: 25438-25443Abstract Full Text PDF PubMed Google Scholar, 4Flemmer A.W. Gimenez I. Dowd B.F. Darman R.B. Forbush B. J. Biol. Chem. 2002; 277: 37551-37558Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). Polyclonal (MBL) and monoclonal (Roche Applied Science) anti-HA antibodies were purchased. General—All incubations were carried out at room temperature (21–24 °C) unless otherwise noted. Data are presented as the means ± S.E. from replicate experiments except as noted (Fig. 1). Statistical significance was determined for summary data by the paired t test (data related to Figs. 1 and 2) or Wilcoxon paired rank test (data related to Fig. 4).Fig. 2The effect of PASK and DNPASK on NKCC1 regulatory phosphorylation. A, the time course of NKCC1 activation was measured using the anti-phospho-NKCC1 antibody (R5; top four rows). Before lysis, cells were incubated in hypotonic (150 mosmol) 1.5 mm Cl– for the time noted. The lower band on the R5 blots is believed to represent endogenous hNKCC1 (see Flemmer et al. (4Flemmer A.W. Gimenez I. Dowd B.F. Darman R.B. Forbush B. J. Biol. Chem. 2002; 277: 37551-37558Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar)). The sNKCC1 immunoblot (using the J3 antibody) was utilized as a measure of total NKCC1; Coomassie Blue staining illustrates that there are similar protein levels in the control cells. B, regulatory NKCC1 phosphorylation at three time points, analyzed as the R5/J3 ratio normalized to the vector 2 control within each of four experiments. Differences are significant (p < 0.01) for DNPASK as well as (p < 0.05) for PASK at the 10-min time point and sMST1/2 at the 3-min time point.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 4sNKCC1/PASK reciprocal coimmunoprecipitation. Vector 1/PASK or sNKCC1 clonal cell lines transfected with DNPASK, PASK, or vector 2 were preloaded with 32Pi and placed in 3 Cl– (+) isotonic buffer or 140 Cl– (–) isotonic buffer for 1 h before lysis. sNKCC1- or HA-tagged kinases were immunoprecipitated for 3 h at 4 °C using the J3 antibody or anti-HA antibodies, respectively. A, J3 IP, top panel (i), phosphorimage of Western blot. The upper band represents NKCC1, whereas the lower (lighter) band represents PASK. ii, the same image as in i, cropped with increased contrast, to highlight PASK phosphorylation. Middle panel, Western blot of immunoprecipitated samples probed with a polyclonal anti-HA antibody. The HA signal represents the amount of PASK and DNPASK coimmunoprecipitated with sNKCC1. Bottom panel, J3 blot showing expressed sNKCC1 levels in the immunoprecipitates. B, summary of eight experiments similar to that in A. sNKCC1 32P incorporation is reported as the 32P/J3 signal normalized to sNKCC1/vector 2 (+). C, anti-HA IP. Top panel, phosphorimage of Western blot. The upper band represents NKCC1, the middle band represents PASK, and the lowest band represents sMST1/2. Middle panel, HA blot showing immunoprecipitation levels of exogenously expressed PASK, DNPASK, and sMST1/2. Bottom panel, the Western blot in the middle panel was stripped and probed with a monoclonal anti-sNKCC1 antibody (J3). The J3 signal represents the amount of sNKCC1 coimmunoprecipitated with HA-tagged kinases. D, summary of five similar experiments showing the fold stimulation by low Cl– preincubation of 32P incorporation in HA-immunoprecipitated kinases (normalized to the HA signal and the basal activation condition).View Large Image Figure ViewerDownload Hi-res image Download (PPT) To address the effect of PASK activity upon NKCC1 activation, HEK clonal cell lines stably expressing sNKCC1 or hNKCC1 were transfected with PASK or a kinase inactive, DNPASK. This previously described PASK mutation (7Ushiro H. Tsutsumi T. Suzuki K. Kayahara T. Nakano K. Arch Biochem. Biophys. 1998; 355: 233-240Crossref PubMed Scopus (72) Google Scholar, 11Johnston A.M. Naselli G. Gonez L.J. Martin R.M. Harrison L.C. DeAizpurua H.J. Oncogene. 2000; 19: 4290-4297Crossref PubMed Scopus (116) Google Scholar), consisting of a single amino acid substitution (K101R), involves an essential lysine residue found within the catalytic domain of most kinases and has been shown to abolish the positioning of the terminal phosphate of ATP (17Hanks S.K. Hunter T. FASEB J. 1995; 9: 576-596Crossref PubMed Scopus (2281) Google Scholar). sNKCC1 cells were also transfected with the shark homologue of MST1/2 or with a MST1/2 kinase inactive mutant created via a Thr → Ala mutation in the activation loop of its catalytic domain. This mutation has been previously described to suppress the catalytic activity of MST (18Lee K.K. Yonehara S. J. Biol. Chem. 2002; 277: 12351-12358Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). We identified sMST1/2 in a screen for NKCC1-specific phosphorylation activity, but this kinase does not appear to be significantly involved in NKCC1 regulation (see "Experimental Procedures"); it was used here as a kinase control. The expression of DNPASK results in a dramatic inhibition of NKCC1 activation in HEK cells. Fig. 1 illustrates the results of several experiments in which 86Rb influx activity was examined in NKCC1-expressing HEK cells transfected with DNPASK, PASK, or vector 2 alone. In these experiments, cotransport activity was activated by preincubation in media of various Cl– concentrations, as previously described (5Darman R.B. Flemmer A. Forbush B. J. Biol. Chem. 2001; 276: 34359-34362Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). As shown here, DNPASK expression produced a dramatic decrease in the level of NKCC1 activity for both human (Fig. 1, A and B) and shark (Fig. 1C) cotransporters. Under conditions of maximal activation by preincubation in 3 mm Cl–, DNPASK decreased the activity of hNKCC1 by 80 ± 3% in 14 experiments including those of Fig. 1, and it decreased sNKCC1 activity by 60 ± 5% in 6 experiments. This high level of inhibition mediated by the DN mutant strongly suggests that PASK activity is essential for the activation of NKCC1 in HEK cells. As seen in Fig. 1, overexpression of PASK causes an increase in the activity of NKCC1. Under conditions of maximal activation, the differences were not statistically significant when all of the experiments were analyzed, but at intermediate activation levels the differences were larger. For instance, in the 14 experiments with hNKCC1 and 6 with sNKCC1, after preincubation in 30 mm Cl–, the PASK-induced increase in NKCC1 activity was 12 ± 3% (p < 0.01) and 22 ± 8% (p < 0.05), respectively, after each curve was normalized to the maximal activity in 1.5 mm Cl– hypotonic conditions. These results are consistent with a situation in which there is sufficient endogenous PASK to fully regulate NKCC1 but in which overexpression of PASK creates a shift toward higher activation at intermediate levels of stimulation. The present data are the first to directly link PASK with the regulation of NKCC1 activity, complementing an earlier study in which PASK binding to NKCC was demonstrated (6Piechotta K. Lu J. Delpire E. J. Biol. Chem. 2002; 277: 50812-50819Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar). Previous work in our laboratory has identified an N-terminal regulatory domain in which the phosphorylation state of two threonines (Thr184/Thr189 in shark) correlates with NKCC1 activation (3Darman R.B. Forbush B. J. Biol. Chem. 2002; 277: 37542-37550Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). To determine whether DNPASK-mediated NKCC1 inhibition also causes a reduction in regulatory phosphorylation, we used the previously described, phosphospecific antibody (R5) raised against two of these phosphorylated threonines (4Flemmer A.W. Gimenez I. Dowd B.F. Darman R.B. Forbush B. J. Biol. Chem. 2002; 277: 37551-37558Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). As shown in Fig. 2A, overexpression of DNPASK causes a dramatic reduction in the Thr184/Thr189 phosphorylation of sNKCC1 throughout the activation time course; in the four experiments summarized in Fig. 2B, the reduction is 75 ± 8% at the 10-min time point. This result is consistent with the results of 86Rb+ influx assays and it links PASK to both the activation and phosphorylation of NKCC1. As in the 86Rb influx assays, any effect of PASK under conditions of maximal stimulation is small, and statistical significance is achieved in these four experiments only at the 10-min time point (14% increase). The experiments of Fig. 2 rule out nonspecific interactions like that of the myosin light chain kinase inhibitor ML-7, where inhibition of NKCC1 did not alter the level of NKCC1 phosphorylation (8Klein J.D. O'Neill W.C. Am. J. Physiol. 1995; 269: C1524-C1531Crossref PubMed Google Scholar). Importantly, DNPASK-mediated inhibition of NKCC1 is not due to a decrease in cotransporter expression levels, as shown using the shark-specific sNKCC1 antibody, J3. These data are also consistent with the decrease of sNKCC1 32P incorporation under low [Cl]e conditions (see Fig. 4) and indicate that the action of DNPASK is to prevent phosphorylation of NKCC1. To further test the action of DNPASK in affecting the NKCC1 phosphorylation state, we measured 86Rb+ influx in the presence of the protein phosphatase type 1 inhibitor calyculin A. We have previously shown that this compound causes a rapid and dramatic increase in NKCC1 activity via the inhibition of
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