Characterization of the Interaction of the Stress Kinase SPAK with the Na+-K+-2Cl– Cotransporter in the Nervous System

Abstract

Activity of heterologously expressed NKCC1 was analyzed under basal and activated conditions in the presence and absence of binding of Ste20-related proline-alanine-rich kinase (SPAK). Mutant NKCC1 that lacks the ability to bind to this kinase showed K+ transport function identical to wild-type NKCC1. Thus, preventing the binding of the kinase to the cotransporter does not affect cotransporter function. In contrast, several experiments suggest a possible role for SPAK as a scaffolding protein. First, Western blot analysis revealed the presence, and in some tissues abundance, of truncated forms of SPAK and OSR1 in which the kinase domains are affected and thus lack kinase activity. Second, a yeast two-hybrid screen of proteins that interact with the regulatory (binding) domain of SPAK identified several proteins all involved in cellular stress pathways. Third, p38, one of the three major MAPKs, can be coimmunoprecipitated with SPAK and with NKCC1 in an activity-dependent manner. The amount of p38 coimmunoprecipitated with the kinase and the cotransporter significantly decreases upon cellular stress, whereas the interaction of the kinase with NKCC1 remains unchanged. These findings suggest that cation-chloride cotransporters might act as “sensors” for cellular stress, and SPAK, by interacting with the cotransporter, serves as an intermediate in the response to cellular stress. Activity of heterologously expressed NKCC1 was analyzed under basal and activated conditions in the presence and absence of binding of Ste20-related proline-alanine-rich kinase (SPAK). Mutant NKCC1 that lacks the ability to bind to this kinase showed K+ transport function identical to wild-type NKCC1. Thus, preventing the binding of the kinase to the cotransporter does not affect cotransporter function. In contrast, several experiments suggest a possible role for SPAK as a scaffolding protein. First, Western blot analysis revealed the presence, and in some tissues abundance, of truncated forms of SPAK and OSR1 in which the kinase domains are affected and thus lack kinase activity. Second, a yeast two-hybrid screen of proteins that interact with the regulatory (binding) domain of SPAK identified several proteins all involved in cellular stress pathways. Third, p38, one of the three major MAPKs, can be coimmunoprecipitated with SPAK and with NKCC1 in an activity-dependent manner. The amount of p38 coimmunoprecipitated with the kinase and the cotransporter significantly decreases upon cellular stress, whereas the interaction of the kinase with NKCC1 remains unchanged. These findings suggest that cation-chloride cotransporters might act as “sensors” for cellular stress, and SPAK, by interacting with the cotransporter, serves as an intermediate in the response to cellular stress. Cation-chloride cotransporters, which mediate the tightly coupled, electroneutral movement of cations (Na+ and K+) together with Cl–, can be divided into Na+-dependent transporters, such as Na-K-2Cl cotransporters (NKCC1–2) and the Na-Cl cotransporter (NCC), and Na+-independent K-Cl cotransporters (KCC1–4). All of these transporters have a well conserved topology with large intracellular amino-terminal and carboxyl-terminal tails and 12 transmembrane spanning domains. The core protein shares some homology to amino acid permeases (for reviews see Refs. 1Delpire E. Mount D.B. Annu. Rev. Physiol. 2002; 64: 803-843Crossref PubMed Scopus (188) Google Scholar and 2Hebert, S. C., Mount, D. B., and Gamba, G. Pfluegers Arch., in pressGoogle Scholar). A variety of stimuli regulates these transporters, including hormonal (3Kaplan M.R. Mount D.B. Delpire E. Gamba G. Hebert S.C. Annu. Rev. Physiol. 1996; 58: 649-668Crossref PubMed Google Scholar), cytokines (4Topper J.N. Wasserman S.M. Anderson K.R. Cai J. Falb D. Gimbrone Jr., M.A. J. Clin. Investig. 1997; 15: 2941-2949Crossref Scopus (56) Google Scholar, 5Sun D. Lytle C. O'Donnell M.E. Am. J. Physiol. 1997; 272: C1829-C1835Crossref PubMed Google Scholar), cell volume (6Russell J.M. Physiol. Rev. 2000; 80: 211-276Crossref PubMed Scopus (737) Google Scholar, 7Lauf P.K. Bauer J. Adragna N.C. Fujise H. Zade-Oppen A.A.M. Ryu K. Delpire E. Am. J. Physiol. 1992; 263: C917-C932Crossref PubMed Google Scholar), oxidative stress (8Flatman P.W. Creanor J. J. Physiol. (Lond.). 1999; 519: 143-152Crossref Scopus (16) Google Scholar), etc. There is also accumulating evidence that cation-chloride cotransporters participate in pathways leading to cell differentiation, growth and proliferation (9Panet R. Marcus M. Atlan H. J. Cell Physiol. 2000; 182: 109-118Crossref PubMed Scopus (65) Google Scholar, 10Shen M.-R. Chou C.-Y. Hsu K.-F. Liu H.-S. Dunham P.B. Holtzman E.J. Ellory J.C. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14714-14719Crossref PubMed Scopus (83) Google Scholar), and apoptosis (11Loffing J. Loffing-Cueni D. Hegyi I. Kaplan M.R. Hebert S.C. Le Hir M. Kaissling B. Kidney Int. 1996; 50: 1180-1190Abstract Full Text PDF PubMed Scopus (146) Google Scholar, 12Kim J.A. Kang Y.S. Park S.H. Kim H.W. Cho S.Y. Lee Y.S. Eur. J. Pharmacol. 2001; 433: 1-6Crossref PubMed Scopus (8) Google Scholar). At the molecular level, the activation-deactivation of these cotransporters mostly involves phosphorylation/dephosphorylation mechanisms, the details of which are still the subject of intense investigation. To identify proteins that directly interact and regulate cation-chloride cotransporters, we recently performed a yeast two-hybrid screen using the cytosolic amino-terminal tail of KCC3. We identified two closely related kinases that bind to KCC3, NKCC1 and NKCC2 (13Piechotta K. Lu J. Delpire E. J. Biol. Chem. 2002; 277: 50812-50819Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar). These kinases belong to the group of Ste20 kinases that function as regulators of MAPK 1The abbreviations used are: MAPKmitogen-activated protein kinaseMAP2microtubule-associated protein 2PVDFpolyvinylidene difluoridePBSphosphate-buffered salineGFAPglial fibrillary acidic proteinAATYKapoptosis-associated tyrosine kinase. cascades (14Dan I. Watanabe N.M. Kusumi A. Trends Cell Biol. 2001; 11: 220-230Abstract Full Text Full Text PDF PubMed Scopus (514) Google Scholar). The first kinase, SPAK (Ste-20 related proline-alanine-rich kinase, or PASK, as the rat homologue), is highly expressed in epithelia and neurons (15Ushiro H. Tsutsumi T. Suzuki K. Kayahara T. Nakano K. Arch. Biochem. Biophys. 1998; 355: 233-240Crossref PubMed Scopus (72) Google Scholar). Its gene is located on human chromosome 2q31.1. The second kinase, OSR1, which is named based on a 30% homology to an oxidative stress response kinase, has much higher homology to SPAK (67%). The OSR1 gene is located on human chromosome 3p22-p21.3 (16Tamari M. Daigo Y. Nakamura Y. J. Hum. Genet. 1999; 44: 116-120Crossref PubMed Scopus (53) Google Scholar). There are orthologues of OSR1 in Caenorhabditis elegans and Drosophila. Inactivation of the Drosophila ortholog, fray, results in a phenotype with axonal ensheathment deficits (17Leiserson W.M. Harkins E.W. Keshishian H. Neuron. 2000; 28: 793-806Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). mitogen-activated protein kinase microtubule-associated protein 2 polyvinylidene difluoride phosphate-buffered saline glial fibrillary acidic protein apoptosis-associated tyrosine kinase. The catalytic (kinase) domains of SPAK and OSR1 are located at the amino terminus, and the regulatory domains are at the carboxyl terminus (14Dan I. Watanabe N.M. Kusumi A. Trends Cell Biol. 2001; 11: 220-230Abstract Full Text Full Text PDF PubMed Scopus (514) Google Scholar). We demonstrated that the binding with cation-chloride cotransporters takes place at the extreme carboxyl termini of SPAK and OSR1 (13Piechotta K. Lu J. Delpire E. J. Biol. Chem. 2002; 277: 50812-50819Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar). The two kinases bind to peptides with a minimum of 9 residues with the following initial consensus sequence: (R/K)FX(V/I). This motif was found twice in the amino terminus of NKCC1, and each sequence was shown to interact with the kinases. Recently, Dowd and Forbush (18Dowd B.F. Forbush B. J. Biol. Chem. 2003; 278: 27347-27353Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar) proposed a regulatory role of SPAK (PASK) on the activity of NKCC1. They showed that overexpression of a dominant negative PASK (inactive kinase) resulted in a lack of activation of NKCC1 upon the intracellular They also demonstrated that SPAK to NKCC1 upon or not the kinase directly and activated the cotransporter still remains In the we that preventing the interaction of SPAK and OSR1 with NKCC1 to the activity of the cotransporter under basal or activated results suggest that binding of the kinase to the two SPAK binding domain is not for NKCC1 We that SPAK, with cation-chloride cotransporters, with proteins that are involved in the response of the cell to stress. we that the ability of SPAK to p38 upon stress. Thus, we that the kinase serves as a scaffolding proteins in the of the NKCC1 in of the NKCC1 was into a for in The was with and into using the was by of at and was by A was into the for and and used to the in The was with a that and a of the was into and and to sequence and The was into the NKCC1 in and as K. R. Delpire E. Am. J. Physiol. 2000; PubMed Google Scholar) and at in with to a of and with and of the following with of performed of with of saline 2 and for in of saline The was and with or with of The of the was identical to the with an of of at the of the and used as of the was and the with of or experiments have shown that the is a NKCC1 is expressed in of analysis was performed using and analysis of of NKCC1 in of with of wild-type NKCC1 or NKCC1 and at for in with saline and for in of saline the three with of saline and in a and was by the for on in the by with a an of on and a of for of the was for protein and an amount of protein was with to a volume of was and for 2 at under The and three the into of at for and to and Western blot of an of SPAK and OSR1 proteins a the carboxyl terminus with amino acid see of (13Piechotta K. Lu J. Delpire E. J. Biol. Chem. 2002; 277: 50812-50819Abstract Full Text Full Text PDF PubMed Scopus (308) Google Thus, we a that to an A acid of the OSR1 was and to The protein was in E. and with by of the protein into two The the was as M. Lu J. Mount D.B. Delpire E. 2001; Scopus Google Scholar). binding domain of SPAK to cotransporters was by using a an and an a by a The was of the binding domain in two-hybrid and into J. 1996; PubMed Google Scholar). yeast with a in and a of as (13Piechotta K. Lu J. Delpire E. J. Biol. Chem. 2002; 277: 50812-50819Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar). To for interaction SPAK and the amino terminus of NKCC1, the amino terminus of the cotransporter was and into the NKCC1 in and with the wild-type in with and with for and with for and in for at in 30% for at and on for and at on a and as J. M. Delpire E. J. 1999; PubMed Scopus Google Scholar). in in with and at in for of see K. Lu J. Delpire E. J. Biol. Chem. 2002; 277: 50812-50819Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar). in and and in to the at a of for in and with of the in and at at a of with fibrillary acidic protein to identify or protein 2 to identify These with The as for was We have demonstrated recently (13Piechotta K. Lu J. Delpire E. J. Biol. Chem. 2002; 277: 50812-50819Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar) the of the SPAK by Western blot negative of was performed the first or The revealed a in the of some which we as a for a analyzed on a with a to a two and in of was in of 2 with a The at for for and for The of protein was in a and 2 protein using a of protein on and to Western blot and Western on and into two The first was in of 2 and with a and on and the second was into and in for The of was as the of the the as demonstrated by the activation of the in and on for at for and the was performed by of with of NKCC1 M.R. D. Hebert S.C. Delpire E. J. Clin. Investig. 1996; 98: PubMed Scopus Google Scholar), SPAK (13Piechotta K. Lu J. Delpire E. J. Biol. Chem. 2002; 277: 50812-50819Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar), p38 residues of human p38 MAPK and MAPK under at by the of of protein for 2 at three in the in and at for the by and polyvinylidene difluoride for 2 at with in to NKCC1, SPAK in at in and for with in protein by NKCC1 in two SPAK binding domains can be found in the amino terminus of NKCC1. The first is located residues of the The second is located residues of the first motif and with a motif A. Forbush J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). using the yeast two-hybrid we demonstrated that these two of interaction with the kinases SPAK and OSR1 (13Piechotta K. Lu J. Delpire E. J. Biol. Chem. 2002; 277: 50812-50819Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar). We also demonstrated that of the at 2 or the at to the that the are located of the for for transport Forbush B. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). using we several NKCC1 and function in The first the first SPAK binding domain by the with that this not affect NKCC1 as as in or the second we into a that SPAK interaction and also binding A. Forbush J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). This second also to affect NKCC1 transport which was in this in and conditions the binding of SPAK to a motif might be to regulate NKCC1, we a and in the first and second SPAK interaction with NKCC1 is the in and are still identical to the with wild-type NKCC1 To that the results in a of we performed yeast two-hybrid with SPAK and the wild-type the two and the shown in all the showed interaction with the stress kinase. In to the a of a large binding domains as well as a significantly NKCC1 activity under and the activation by and with was To the in NKCC1 activity results a in NKCC1 we of with wild-type NKCC1 and NKCC1. the and Western blot analysis of protein with NKCC1 revealed a higher of cotransporter in the for the cotransporter, with wild-type NKCC1 of the and also SPAK in to that the of OSR1 is not a with the shown in the activity of and wild-type NKCC1 proteins in SPAK not These that preventing interaction with NKCC1 by preventing the binding has on NKCC1 transport activity. to a function for interaction with NKCC1, of cotransporter of NKCC1 and NKCC1 in and in or and in or and NKCC1 in or K+ in wild-type and NKCC1 in or a and K+ in is significantly the one in of K+ in is significantly in are expressed in of K+ the a and each was at K+ to of NKCC1 on SPAK as revealed by yeast two-hybrid the interaction SPAK and the amino terminus of NKCC1 growth of the yeast in and and and interaction SPAK and each NKCC1 absence of growth in yeast with SPAK and NKCC1 blot analysis of NKCC1 protein of wild-type and NKCC1 with and proteins using and on a was with identical of SPAK and of OSR1 and SPAK was by Western blot analysis using an and a SPAK (13Piechotta K. Lu J. Delpire E. J. Biol. Chem. 2002; 277: 50812-50819Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar). shown in SPAK and OSR1 are expressed in all tissues with the of the that to OSR1 can be this and with two major a of molecular and a of The SPAK is the SPAK and the is for OSR1 with the molecular the higher such as and SPAK in higher and In contrast, the tissues with OSR1 protein and with and The molecular of OSR1 is the molecular of SPAK To that the presence of two major on Western with the SPAK and is not to we proteins on and the in the of the was with one in the of the large molecular and molecular are for SPAK and This that are two SPAK and two OSR1 of the SPAK and OSR1 revealed the presence of second consensus in proteins at the first proteins of and for SPAK and OSR1, In contrast, at the second results in truncated proteins of and results of and are with the of two the Western analysis that the large and is for SPAK for the second for OSR1 is a consensus the first with the higher of the protein These results suggest that truncated SPAK and OSR1 proteins are expressed in of SPAK in the and was in and using the which we (13Piechotta K. Lu J. Delpire E. J. Biol. Chem. 2002; 277: 50812-50819Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar). negative the was the on a of to the with SPAK is shown in The SPAK was found on the of as (13Piechotta K. Lu J. Delpire E. J. Biol. Chem. 2002; 277: 50812-50819Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar). We also found for SPAK at the of in to the of NKCC1 M. J. B. R. 2002; Scopus Google Scholar). In the in the and the of with and that neurons as well as are for to neurons in the in the we with the by the We the for SPAK and found in the The in the of shown in and and the of the on a A was in the of the and in the The in the was found in the of the and and In we found in the neurons of the in of the as well as and in neurons A was in the The significantly still the this the was in the The was not to the and was of in the of the for SPAK and identified as neurons by with and that the of the the as well as the SPAK was also found in neurons of the In to the which was was found in the as shown by the lack of in and by the of the with SPAK and in These results suggest that SPAK is expressed in the and of the the of in the and in several of for the SPAK was shown in and glial of with was on the of SPAK at the of of in the in the and of neurons in the of the with and the glial to the and of the of shown in the following in and the the of the on a SPAK was as neurons of the the of the neurons of the the the the in A and and and in and of a with the of the cation-chloride cotransporters to SPAK, we to identify binding of the kinase. We a yeast two-hybrid in by using the binding domain of SPAK as a on growth and we that several of these we identified several some of which identified apoptosis-associated tyrosine kinase was identified one of which was several was identified as using the and as a tyrosine kinase using the This protein a tyrosine kinase Its with revealed an homology with several of a the two of the amino acid sequence of these revealed the presence of for the apoptosis-associated tyrosine kinase, we identified three the carboxyl terminus that the These and We all three for interaction with SPAK by using the yeast two-hybrid and the 2 which to the motif that of these are involved in the response of to and of yeast two-hybrid with the binding domain of SPAK in a of SPAK, NKCC1, and p38 the NKCC1, SPAK, and cellular stress, we proteins and which was to and performed for stress was by the activation of The also that the amount of NKCC1 and SPAK with decreases with cellular stress. In contrast, the amount of SPAK with NKCC1 does not significantly with stress. is the that the molecular of NKCC1, which NKCC1, with two NKCC1 in the NKCC1 with the molecular much the higher molecular the is in a in the in an These suggest that NKCC1, SPAK, and p38 a and that p38 or not SPAK is upon cellular stress. In an to the we have shown by on and NKCC2 that a of the at 2 or the at into SPAK interaction with these cotransporters (13Piechotta K. Lu J. Delpire E. J. Biol. Chem. 2002; 277: 50812-50819Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar). The absence of interaction was also by In the we several NKCC1 one or binding that we identified at the amino terminus of NKCC1, and we transport activity in was as the kinase and NKCC1, thus leading to a in preventing the kinase interacting with the cotransporter activation of that the two are binding of one does not whereas the a absence of experiments that a cotransporter that is to interact with the kinase is and to the by and stimuli as wild-type NKCC1. is of a expressed SPAK in heterologously SPAK, the binding preventing is on the In the activity of NKCC1 and can be directly the In which Dowd and Forbush (18Dowd B.F. Forbush B. J. Biol. Chem. 2003; 278: 27347-27353Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar), we thus to These recently a of human and NKCC1 activity by the kinase dominant negative PASK the two of and this suggest that regulatory proteins and regulatory pathways identical activation identical stimuli in cell also that the binding and the are in which the kinase NKCC1 in the absence of one has to that the binding of the kinase to the cotransporter serves a function that is of is that in PASK overexpression Dowd and Forbush (18Dowd B.F. Forbush B. J. Biol. Chem. 2003; 278: 27347-27353Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar) a at an intermediate activation in not at or of Thus, at activation the of Dowd and Forbush (18Dowd B.F. Forbush B. J. Biol. Chem. 2003; 278: 27347-27353Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar) is in with of of transport activity. the overexpression of the dominant negative SPAK in in the by Dowd and Forbush (18Dowd B.F. Forbush B. J. Biol. Chem. 2003; 278: 27347-27353Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar) might have on NKCC1 using on cotransporters are to with is possible that the or have kinase that directly NKCC1. We have evidence in this of kinases that interact with SPAK these kinases are into with the cotransporter SPAK the interaction of the scaffolding protein the of the kinase and thus affect the activity of the is to this and these revealed that SPAK in the is OSR1 OSR1 is found in to SPAK, at much not shown in SPAK is in on the with NKCC1 (13Piechotta K. Lu J. Delpire E. J. Biol. Chem. 2002; 277: 50812-50819Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar, Kaplan M.R. Hebert S.C. Delpire E. Am. J. 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A. 2003; PubMed Scopus Google Scholar). with the by G. 2000; PubMed Scopus Google Scholar) and by that SPAK with p38, these identified of SPAK suggest a role for this Ste20 kinase in cellular stress. with that SPAK and OSR1 function as scaffolding proteins is the that the two proteins in two forms in a large molecular that the catalytic domain of the kinase, and a molecular with a truncated catalytic SPAK, is in that the large molecular is the molecular OSR1, which is SPAK, the truncated is the This an regulatory role as a kinase or a function of the protein that is of kinase activity. is of to that the second is conserved in and in Drosophila is by or residues in C. elegans and The of the is to an of OSR1 in higher such as and for the that SPAK and OSR1 might function as scaffolding proteins that SPAK interaction with NKCC1 of SPAK coimmunoprecipitated with is not affected by cellular stress, such as stress, whereas the interaction of p38 with SPAK coimmunoprecipitated with and with NKCC1 coimmunoprecipitated with is significantly upon stress. with the that SPAK was shown to highly with NKCC1 the of on the of at the of and NKCC2 of of not under suggest that SPAK might a scaffolding We suggest that proteins in the of cation-chloride cotransporters as we have shown for p38 and SPAK, proteins upon stress is to that the cotransporters might a role in the response of to stress. We for the