Proline- and Alanine-rich Ste20-related Kinase Associates with F-actin and Translocates from the Cytosol to Cytoskeleton upon Cellular Stresses

Proline- and alanine-rich Ste20-related kinase (PASK) is a Ste20-related protein kinase isolated from rat brain. Cell fractionation studies showed that PASK was present both in the cytosol and in Triton X-100-insoluble cytoskeletal fraction in rat tissues. In brain, PASK associated with protein complexes that contained actin and tubulin, confirming the association of PASK with the cytoskeletonin vivo. Glutathione S-transferase-PASK fusion protein cosedimented with F-actin, indicating that PASK binds to F-actin. In contrast to rat tissues, PASK was detected only in the Triton X-100-soluble cytosolic fraction in cultured PC12 and NIH 3T3 cells. Cytosolic PASK translocated to the cytoskeleton when these cells were stimulated with severe cellular stresses such as hypertonic sodium chloride, hydrogen peroxide, and heat shock at 45 °C. Our results suggest that PASK may be involved in the regulation of the cytoskeleton in response to cellular stresses such as hyperosmotic shock. Proline- and alanine-rich Ste20-related kinase (PASK) is a Ste20-related protein kinase isolated from rat brain. Cell fractionation studies showed that PASK was present both in the cytosol and in Triton X-100-insoluble cytoskeletal fraction in rat tissues. In brain, PASK associated with protein complexes that contained actin and tubulin, confirming the association of PASK with the cytoskeletonin vivo. Glutathione S-transferase-PASK fusion protein cosedimented with F-actin, indicating that PASK binds to F-actin. In contrast to rat tissues, PASK was detected only in the Triton X-100-soluble cytosolic fraction in cultured PC12 and NIH 3T3 cells. Cytosolic PASK translocated to the cytoskeleton when these cells were stimulated with severe cellular stresses such as hypertonic sodium chloride, hydrogen peroxide, and heat shock at 45 °C. Our results suggest that PASK may be involved in the regulation of the cytoskeleton in response to cellular stresses such as hyperosmotic shock. p21-activated protein kinase Jun N-terminal kinase polyvinylidene difluoride glutathione S-transferase polyacrylamide gel electrophoresis proline- and alanine-rich Ste20-related kinase Ste20 is a yeast protein kinase that acts upstream of the pheromone-responsive mitogen-activated protein kinase cascade (1.Wu C. Whiteway M. Thomas D.Y. Leberer E. J. Biol. Chem. 1995; 270: 15984-15992Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar, 2.Leberer E. Dignard D. Harcus D. Thomas D.Y. Whiteway M. EMBO J. 1992; 11: 4815-4824Crossref PubMed Scopus (345) Google Scholar). Ste20-related protein kinases, which have a catalytic domain highly homologous to that of Ste20, have been identified in various eukaryotes, and their family is expanding. Members of this family can be divided into two groups based on their structure and regulation. The kinases of the first group, including PAKs1 and Ste20, have a catalytic domain at the carboxyl terminus and a regulatory domain at the amino terminus, which contains a binding site for Rac1 and Cdc42 (3.Manser E. Leung T. Salihuddin H. Zhao Z.S. Lim L. Nature. 1994; 367: 40-46Crossref PubMed Scopus (1298) Google Scholar). The kinases of the second group have a catalytic domain at the amino terminus and a putative regulatory domain at the carboxyl terminus and are structurally related to yeast Sps1. Some of the Sps1 subfamily members, including germinal center kinase, hematopoietic progenitor kinase, Nck-interacting kinase, and kinase homologous to SPS1/STE20, have been shown to activate the stress-activated protein kinase/JNK pathway in transfection experiments (4–8; for review, see Ref. 9.Kyriakis J.M. J. Biol. Chem. 1999; 274: 5259-5262Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). In addition to their roles as upstream activators of mitogen-activated protein kinase pathways, members of the Ste20 family have been implicated in the regulation of cytoskeletal reorganization. The small GTPase Rac1 and Cdc42, upstream activators of PAK/Ste20 subfamily members, are considered key regulatory molecules that link surface receptors to the organization of the actin cytoskeleton (10.Hall A. Science. 1998; 279: 509-514Crossref PubMed Scopus (5216) Google Scholar). ADrosophila homolog of PAK, DPAK, has been reported to colocalize with focal adhesion and focal complexes (11.Harden N. Lee J. Loh H.Y. Ong Y.M. Tan I. Leung T. Manser E. Lim L. Mol. Cell. Biol. 1996; 16: 1896-1908Crossref PubMed Scopus (174) Google Scholar). PAK1 in fibroblast cell lines has been shown to translocate from the cytosol to Rac- and Cdc42-dependent actin structures when cells were stimulated with platelet-derived growth factor (12.Dharmawardhane S. Sanders L.C. Martin S.S. Daniels R.H. Bokoch G.M. J. Cell Biol. 1997; 138: 1265-1278Crossref PubMed Scopus (196) Google Scholar). It has been reported that expression of activated mutants of PAK1 in mammalian cells induces actin reorganization (13.Manser E. Huang H.Y. Loo T.H. Chen X.Q. Dong J.M. Leung T. Lim L. Mol. Cell. Biol. 1997; 17: 1129-1143Crossref PubMed Google Scholar, 14.Sells M.A. Knaus U.G. Bagrodia S. Ambrose D.M. Bokoch G.M. Chernoff J. Curr. Biol. 1997; 7: 202-210Abstract Full Text Full Text PDF PubMed Scopus (576) Google Scholar). For Sps1/germinal center kinase subfamily members, there are no reports concerning their roles in the regulation of cytoskeletal proteins except thatDictyostelium Severin kinase has been shown to phosphorylate actin binding protein severin (15.Eichinger L. Bahler M. Dietz M. Eckerskorn C. Schleicher M. J. Biol. Chem. 1998; 273: 12952-12959Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). We have recently cloned a rat Ste20-related protein kinase of the Sps1/germinal center kinase subfamily and named it PASK (proline- and alanine-richSte20-related kinase; Ref. 16.Ushiro H. Tsutsumi T. Suzuki K. Kayahara T. Nakano K. Arch. Biochem. Biophys. 1998; 355: 233-240Crossref PubMed Scopus (72) Google Scholar). PASK was present in both the cytosol and particulate fraction in the rat brain. The particulate PASK was not solubilized by extraction with Triton X-100, suggesting that it was associated with complexes of high density such as cytoskeleton. In this report, we identified actin and tubulin as the major constituents of PASK-associated proteins in the brain, confirming the association of PASK with the cytoskeleton in vivo. In addition, we examined changes in the subcellular distribution of PASK in cultured PC12 and NIH 3T3 cells in response to extracellular stimuli. We demonstrated that PASK translocates from the cytosol to the cytoskeleton upon stimulation of the cells with severe cellular stresses. PASK is the first example of Sps1/germinal center kinase subfamily kinase that is shown to associate with the cytoskeleton. All procedures were carried out at 0–4 °C. The experimental protocol was approved by the Committee for Animal Research of Mie University. Tissues from adult Harlan Sprague Dawley rats (13 weeks old) were homogenized with a Polytron in 10 volumes of extraction buffer containing 20 mm Tris-HCl, pH 7.5, 10 mm EDTA, 10 mm 2-mercaptoethanol, and 1 mm p-phenylmethylsulfonyl fluoride (buffer A). To the homogenates, we added 1 volume of buffer A containing 4% Triton X-100. After incubation on ice for 10 min, the homogenates were centrifuged at 10,000 × g for 10 min. The supernatants (Triton X-100-soluble fraction) were saved, and the pellets were washed with buffer A containing 1% Triton X-100 to obtain the Triton X-100-insoluble cytoskeletal fractions. Antibody against the carboxyl-terminal region of PASK (amino acid residues 424–553) was prepared as described previously (16.Ushiro H. Tsutsumi T. Suzuki K. Kayahara T. Nakano K. Arch. Biochem. Biophys. 1998; 355: 233-240Crossref PubMed Scopus (72) Google Scholar). This antibody recognized a protein of 66 kDa on Western blot in COS-7 cells transfected with a full-length PASK cDNA but not in cells transfected with an empty vector. The antibody also detected a single protein of 66 kDa in many rat tissues, indicating that the antibody specifically recognizes PASK. In addition, the antibody immunoprecipitated PASK from the lysate of the transfected cells. Anti-PASK antibody (0.3 mg) was covalently bound to 1 ml ofN-hydroxysuccimide-activated Sepharose (HiTrap affinity column; Amersham Pharmacia Biotech) to prepare an anti-PASK immunoaffinity column. Frozen rat brains (4 g) were homogenized with Polytron in 20 ml of extraction buffer containing 20 mmTris-HCl, pH 7.5, 1 mm EDTA, 10 mm2-mercaptoethanol, and 1 mm p-phenylmethylsulfonyl fluoride. The homogenate was centrifuged at 30,000 × g for 15 min, and the resulting supernatant (210 mg of protein) was applied to the anti-PASK antibody column. After washing the column with phosphate-buffered saline and 10 mm Tris-HCl, pH 7.5, the bound proteins were eluted with 0.1 m glycine-HCl, pH 2.5. Rat brain extract (14 mg of protein) prepared as described above was incubated with 10 μg of anti-PASK antibody for 1 h at 4 °C, and the immune complex was precipitated with 10 μl of protein A-Sepharose CL-4B (Amersham Pharmacia Biotech). After washing the gel with phosphate-buffered saline containing 0.1% Nonidet P-40, 10 mm Tris-HCl, pH 7.5, containing 0.5 m NaCl, and 10 mm Tris-HCl, pH 7.5, bound proteins were eluted with 0.1m glycine-HCl, pH 2.5. After electrophoresis on 10% SDS-polyacrylamide gels, proteins were transferred onto PVDF membranes (Immobilon; Millipore, Bedford, MA) using a semidry electroblot apparatus. PVDF membranes were probed for 1 h with anti-PASK antibody (1 μg/ml). Anti-JNK1 antibody (catalog no. sc-571; Santa Cruz Biotechnology, Santa Cruz, CA) was used at 0.5 μg/ml to detect JNK1 and JNK2. Bound antibodies were detected by alkaline phosphatase-conjugated goat anti-rabbit IgG antibody (Zymed, San Francisco, CA) with nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate as substrate. Actin and tubulin in the immune complex were detected with anti-actin (Roche Molecular Biochemicals) and anti-α-tubulin (Cedarlane Laboratories, Hornby, Ontario, Canada) antibodies, respectively: bound antibodies were visualized by the use of peroxidase-anti-peroxidase complex and diaminobenzidine. Recombinant GST-PASK and -truncated PASK fusion proteins were prepared as described previously (16.Ushiro H. Tsutsumi T. Suzuki K. Kayahara T. Nakano K. Arch. Biochem. Biophys. 1998; 355: 233-240Crossref PubMed Scopus (72) Google Scholar). GST-JNK2 protein was purchased from Santa Cruz Biotechnology. pGEX-4T-xynCΔC, which encodes GST-tagged Clostridium thermocellum xylanase XynC (amino acids 33–547), was a generous gift from Dr. T. Kimura and Dr. K. Ohmiya (Mie University; Ref. 17.Hayashi H. Takagi K.I. Fukumura M. Kimura T. Karita S. Sakka K. Ohmiya K. J. Bacteriol. 1997; 179: 4246-4253Crossref PubMed Google Scholar). GST-xylanase (33–547) was expressed from this plasmid inEscherichia coli strain BL21 and purified using glutathione-Sepharose CL-4B (Amersham Pharmacia Biotech). Actin sedimentation assay was performed according to den Hartigh et al. (18.den Hartigh J.C. van Bergen en Henegouwen P.M. Verkleij A.J. Boonstra J. J. Cell Biol. 1992; 119: 349-355Crossref PubMed Scopus (228) Google Scholar). Briefly, 2 μg of GST fusion proteins were incubated with 10 μg of rabbit muscle G-actin (Worthington Biochemical, Lakewood, NJ) for 10 min in 200 μl of buffer containing 2 mm Tris-HCl, pH 7.4, 0.2 mm CaCl2, 0.2 mm dithiothreitol, and 0.5 mm ATP. Actin polymerization was induced by adding 75 mm KCl and 2 mm MgCl2, and after 1 h at room temperature, the samples were centrifuged at 100,000 × g for 1 h at 25 °C. Proteins in the supernatant and the pellet were separated on a 10% SDS-polyacrylamide gel and analyzed by silver staining. To determine whether PASK could bind to unpolymerized G-actin, 2 μg of glutathione-Sepharose-bound GST-PASK fusion protein were incubated with 10 μg of G-actin under the same conditions as for the cosedimentation assay, except that KCl and MgCl2 were omitted. After a 1-h incubation at room temperature with gentle agitation, the Sepharose beads were washed three times with the incubation buffer containing 1% Triton X-100 and boiled in SDS-PAGE loading buffer. Proteins were separated on a 10% SDS-polyacrylamide gel and stained with Coomassie brilliant blue. PC12 cells were obtained from the Riken Cell Bank (Tsukuba, Japan) and grown in RPMI 1640 medium (Nissui Pharmaceutical Co., Tokyo, Japan) supplemented with 5% horse serum (Life Technologies), 5% fetal calf serum (Cansera Ontario, and in a 5% NIH 3T3 cells were obtained from the Research Bank Japan) and cultured in medium supplemented with 10% calf serum (Life Technologies), and μg/ml). PC12 cells grown on San CA) and NIH 3T3 cells grown on were with various as After were on The cells were washed with phosphate-buffered saline and in μl of extraction buffer containing 20 mm Tris-HCl, pH 7.4, 1% Triton X-100, 2 mm EDTA, 1 mm p-phenylmethylsulfonyl 10 mm2-mercaptoethanol, mm 10 mm sodium 1 mm and using a After incubation on ice for 10 min, the were centrifuged at 10,000 × g for 10 min. The supernatants (Triton X-100-soluble fraction) were saved, and the pellets were washed with ml of extraction buffer to obtain Triton X-100-insoluble fractions. were to with anti-PASK antibody as described The of PASK protein in these was by of the using NIH PC12 cells grown on were washed three times with and RPMI 1640 medium (Life and incubated in this medium containing of CA) for 2 the cells were stimulated for 1 h with hypertonic by the addition of ml of m After the cells were washed with medium and in μl of extraction buffer. Triton X-100-soluble supernatants and pellets were prepared as described PASK was performed according to the of and K. in Molecular with Triton X-100-insoluble pellets were in μl of buffer mm Tris-HCl, pH and 1 mm and boiled for min. To the 200 μl of assay buffer Nonidet P-40, sodium mm sodium pH 2 mm EDTA, mm mm mm sodium and mm were and the supernatants were obtained by supernatants were incubated for h with rabbit IgG on protein A-Sepharose beads to proteins that bind to the and IgG and by PASK was immunoprecipitated by incubation with μg of anti-PASK antibody for 1 h at 4 by protein A-Sepharose Sepharose beads were washed times with buffer Nonidet P-40, 1% sodium 0.1% m NaCl, 10 pH 2 mm EDTA, mm mm 10 mm sodium 1 mm 1 mm p-phenylmethylsulfonyl and 10 mm and boiled in SDS-PAGE loading buffer. were separated on a 10% SDS-polyacrylamide gel and transferred onto a PVDF of proteins was by on a The same was to with anti-PASK We performed fractionation studies to determine whether PASK with the cytoskeleton in rat and cultured PC12 and NIH 3T3 cells. of rat brain, PC12 and NIH 3T3 cells prepared in Triton X-100 were centrifuged at 10,000 × g for 10 min to obtain Triton X-100-soluble and fractions. The of PASK protein in fraction was analyzed by with anti-PASK with the results of a (16.Ushiro H. Tsutsumi T. Suzuki K. Kayahara T. Nakano K. Arch. Biochem. Biophys. 1998; 355: 233-240Crossref PubMed Scopus (72) Google of PASK in rat brain was in the Triton X-100-insoluble cytoskeletal shown in PASK was detected in the Triton X-100-insoluble of tissues, the of PASK protein in the Triton X-100-insoluble fraction to that in the fraction these and were that of brain. In contrast to these tissues, PASK was not detected in the Triton X-100-insoluble of PC12 NIH 3T3 cells. results that at a of PASK with the cytoskeleton in rat but not in PC12 NIH 3T3 cells. To that PASK with the proteins associated with PASK in rat brain were analyzed by immunoaffinity and with anti-PASK were applied to the anti-PASK immunoaffinity column. After washing and of bound the proteins were analyzed by 2 silver of the proteins eluted from the anti-PASK antibody affinity column. A at 45 kDa and a at kDa were the major proteins with PASK. shown in 2 these proteins were identified as actin and tubulin by with anti-actin and anti-α-tubulin antibodies, Actin and tubulin were also with PASK by anti-PASK antibody and protein of these proteins was precipitated by IgG 2 results that PASK complexes with actin and To the of PASK and to determine which region of PASK binds the of various GST-PASK and PASK fusion proteins in an actin sedimentation assay was muscle actin was incubated with GST-PASK fusion proteins for 1 h in the of KCl and the actin were by pellet and supernatant were analyzed by shown in when actin was by the addition of KCl and MgCl2, GST-PASK GST-PASK and GST-PASK cosedimented with and were in the GST-PASK also cosedimented with F-actin, a of it in the GST-PASK and GST-PASK not with and were in the actin was unpolymerized 2 the fusion proteins in the To determine the of the association PASK and F-actin, experiments were performed with GST-JNK2 and GST-xylanase which are in to proteins not with under the assay conditions results that PASK with in and that PASK with is by the region amino acid residues To determine whether PASK could bind to unpolymerized G-actin, GST-PASK and GST bound to glutathione-Sepharose were incubated with Sepharose beads were washed with incubation buffer containing 1% Triton X-100. proteins were analyzed by shown in no actin was detected in association with GST-PASK indicating that PASK not bind to G-actin under the assay It is that protein kinases, including and with cytoskeletal proteins and translocate from the cytosol to cytoskeleton in response to extracellular D. S. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, A. J. 1998; PubMed Scopus Google Scholar, A. T. M. K. S. Biochem. J. 1997; PubMed Scopus Google Scholar). We whether of PASK from the cytosol to the cytoskeleton was induced upon stimulation of PC12 and NIH 3T3 cells. For this PASK distribution was analyzed by cell fractionation after of these cells with various stimuli. shown in A and two PASK was detected in the Triton X-100-insoluble cytoskeletal fraction when PC12 and NIH 3T3 cells were with with at 45 °C. In these the of PASK in the Triton X-100-insoluble fraction in a To the changes in the distribution of of the Triton X-100-soluble and were in the same gel and to In cells stimulated with m for 1 h and in cells at 45 for 1 and of PASK for PC12 cells and 20 and of it for NIH 3T3 were in the Triton X-100-insoluble was a in the of PASK. In the of PASK is the of of the were to the results that PASK translocated from the cytosol to the cytoskeleton in response to cellular stresses. The was to PASK and not to an of cell the distribution of JNK1 and in these stimulated cells was from that of PASK and two there was no in in the fraction by of these JNK1 not translocate by hypertonic in NIH 3T3 in the fraction by the stresses. In contrast to the by these PASK in PC12 cells not translocate by stimulation of with 1 mm of protein with μg/ml of the cells to growth factor for 1 h 4 and not stresses such as m NaCl, 0.5 mm and heat shock at also to PASK 4 and not results that PASK was specifically induced by severe cellular stresses. shown in hypertonic PASK was the of PASK in the Triton X-100-insoluble fraction to a when cells were incubated in an medium for 4 h after hypertonic To the of hypertonic PASK we examined whether such a involved of PASK. PC12 cells were with and stimulated with a hypertonic medium m for 1 The cells were to obtain Triton X-100-soluble and fractions. PASK was immunoprecipitated with anti-PASK antibody from the Triton X-100-soluble fraction and after extraction in from the Triton X-100-insoluble were by transferred to a PVDF and to The same was probed with anti-PASK antibody to determine the of PASK protein in shown in PASK was immunoprecipitated from the fraction of cells. This that PASK is in PC12 cells. The of PASK in the Triton X-100-insoluble fraction of the stimulated cells was to and of that in the fraction of the and the stimulated In cells stimulated with the hypertonic a that PASK was detected in the Triton X-100-soluble This was not it was by with anti-PASK results that PASK to the cytoskeleton with PASK We examined the of two cytoskeletal on hypertonic PASK PC12 cells were first with 20 for 1 h to the polymerization of actin tubulin, This was by stimulation of cells with the hypertonic medium m for 1 shown in these no on PASK distribution in both the cells and cells. results suggest that of the cytoskeleton is not involved in PASK of the structure of PASK suggest that it is a protein present in the cell fractionation studies showed that PASK not only in the cytosol but also as protein complexes of high density such as the cytoskeleton. and using anti-PASK antibody that actin and tubulin were the major constituents of PASK-associated protein results that PASK with the cytoskeleton. In cosedimentation PASK was shown to bind in it is not whether PASK binds to it is that the actin used in experiments was by an protein that binds PASK to In addition, PASK has no actin binding Ste20 has been reported to associate protein with actin cytoskeleton T. C. J. K. Whiteway M. Thomas D.Y. Leberer E. Science. 1995; 270: PubMed Scopus Google Scholar). It is that PASK with the cytoskeleton such an is to the cytoskeletal to which PASK Cell fractionation of rat showed that the distribution of PASK and cytoskeletal on the of PASK was present in the Triton X-100-insoluble cytoskeletal fraction of brain and only 10% of PASK was in this fraction of and PASK was in the cytoskeletal fraction of PC12 and NIH 3T3 cells. the that the association of PASK with the cytoskeleton to be these in the distribution of PASK suggest that the PASK and the cytoskeleton may be by factor and may of such in tissues. Our results showed that of PASK from the cytosol to cytoskeleton in PC12 and NIH 3T3 cells when these cells were stimulated with severe cellular stresses. The that hypertonic PASK was by that PASK is not induced by of the cytoskeleton. Cytosolic PASK not associate with actin tubulin of these proteins was with PASK from a 100,000 × g supernatant of brain extract by anti-PASK antibody not This is also by the that GST-PASK not bind to unpolymerized G-actin in we not that cytosolic PASK associated with actin tubulin translocates to the cytoskeleton by polymerization of these cytoskeletal PASK was also not by of protein with not suggesting that PASK is not induced by of which bind PASK to the cytoskeleton. In of PC12 cells showed that the of PASK translocated to the cytoskeleton to of that in the it is that the of PASK may the affinity of PASK for the of the cytoskeleton. this it is whether PASK is induced by PASK experiments using mutants the be to a It has been reported that response and mammalian by Ste20 family members to which PASK is are activated by cellular stresses A. J. T. EMBO J. 1996; PubMed Scopus Google Scholar, S. A. 1996; PubMed Scopus Google Scholar). To whether PASK is activated by cellular PASK was immunoprecipitated from Triton X-100-soluble of hypertonic PC12 and was in with protein as an substrate. we could not detect changes in PASK it was under the assay conditions used not with the that cytosolic PASK was these results suggest that the PASK in the cytosol may be has been reported to be activated by Chernoff J. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). PASK in the Triton X-100-insoluble fraction was not of in under it is that only translocated PASK is activated at the cytoskeleton. PASK is to a of including and such as cells of brain and of the of and cells of the (16.Ushiro H. Tsutsumi T. Suzuki K. Kayahara T. Nakano K. Arch. Biochem. Biophys. 1998; 355: 233-240Crossref PubMed Scopus (72) Google Scholar). cells and to the and of such as and shown cytosolic PASK translocates to the cytoskeleton upon stimulation with hypertonic sodium It is that PASK may be involved in the regulation of cytoskeleton when these cells to changes in of We Dr. T. and Dr. T. for

Fetched live from OpenAlex and de-inverted. Abstracts are not stored in this database: the inverted indexes are 8.6 GB of the frame’s 9.3 GB of text, and the host has 13 GB free.