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Cofilin Phosphorylation and Actin Reorganization Activities of Testicular Protein Kinase 2 and Its Predominant Expression in Testicular Sertoli Cells

2001; Elsevier BV; Volume: 276; Issue: 33 Linguagem: Inglês

10.1074/jbc.m102988200

1083-351X

Jiro Toshima, Junko Y. Toshima, Kazuhide Takeuchi, Reiko Mori, Kensaku Mizuno,

14-3-3 protein interactions

We previously identified testicular protein kinase 1 (TESK1), which phosphorylates cofilin and induces actin cytoskeletal reorganization. We now report identification and characterization of another member of a TESK family, testicular protein kinase 2 (TESK2), with 48% amino acid identity with TESK1. Like TESK1, TESK2 phosphorylated cofilin specifically at Ser-3 and induced formation of actin stress fibers and focal adhesions. Both TESK1 and TESK2 are highly expressed in the testis, but in contrast to TESK1, which is predominantly expressed in testicular germ cells, TESK2 is expressed predominantly in nongerminal Sertoli cells. Thus, TESK1 and TESK2 seem to play distinct roles in spermatogenesis. In HeLa cells, TESK1 was localized mainly in the cytoplasm, whereas TESK2 was localized mainly in the nucleus, which means that TESK1 and TESK2 likely have distinct cellular functions. Because the kinase-inactive mutant of TESK2 was localized in the cytoplasm, nuclear/cytoplasmic localization of TESK2 depends on its kinase activity. A TESK2 mutant lacking the C-terminal noncatalytic region had about a 10-fold higher kinase activity in vitro and, when expressed in HeLa cells, induced punctate actin aggregates in the cytoplasm and unusual condensation and fragmentation of nuclei, followed by apoptosis. Thus, we propose that the C-terminal region plays important roles in regulating the kinase activity and cellular functions of TESK2. We previously identified testicular protein kinase 1 (TESK1), which phosphorylates cofilin and induces actin cytoskeletal reorganization. We now report identification and characterization of another member of a TESK family, testicular protein kinase 2 (TESK2), with 48% amino acid identity with TESK1. Like TESK1, TESK2 phosphorylated cofilin specifically at Ser-3 and induced formation of actin stress fibers and focal adhesions. Both TESK1 and TESK2 are highly expressed in the testis, but in contrast to TESK1, which is predominantly expressed in testicular germ cells, TESK2 is expressed predominantly in nongerminal Sertoli cells. Thus, TESK1 and TESK2 seem to play distinct roles in spermatogenesis. In HeLa cells, TESK1 was localized mainly in the cytoplasm, whereas TESK2 was localized mainly in the nucleus, which means that TESK1 and TESK2 likely have distinct cellular functions. Because the kinase-inactive mutant of TESK2 was localized in the cytoplasm, nuclear/cytoplasmic localization of TESK2 depends on its kinase activity. A TESK2 mutant lacking the C-terminal noncatalytic region had about a 10-fold higher kinase activity in vitro and, when expressed in HeLa cells, induced punctate actin aggregates in the cytoplasm and unusual condensation and fragmentation of nuclei, followed by apoptosis. Thus, we propose that the C-terminal region plays important roles in regulating the kinase activity and cellular functions of TESK2. actin-depolymerizing factor conserved region 4′,6-diamidino-2-phenylindole LIM motif-containing protein kinase nucleotide(s) p21-activated kinase polyacrylamide gel electrophoresis phosphate-buffered saline reverse transcription-polymerase chain reaction testicular protein kinase kilobase(s) base pair The dynamics of polymerization/depolymerization of actin filaments and their remodeling are essential for cell movement, adhesion, and division (1Mitchison T.J. Cramer L.P. Cell. 1997; 84: 371-379Abstract Full Text Full Text PDF Scopus (1299) Google Scholar). Cofilin and actin-depolymerizing factor (ADF)1 play an essential role in the rapid turnover of actin filaments and actin-based cytoskeletal reorganization by stimulating depolymerization and severance of actin filaments (2Moon A. Drubin D.G. Mol. Biol. Cell. 1995; 6: 1423-1431Crossref PubMed Scopus (226) Google Scholar, 3Therriot J.A. J. Cell Biol. 1997; 136: 1165-1168Crossref PubMed Scopus (138) Google Scholar, 4Bamburg J.R. Annu. Rev. Cell Dev. Biol. 1999; 14: 305-338Google Scholar). As the activity of cofilin/ADF is negatively regulated by phosphorylation at Ser-3 (5Agnew B.J. Minamide L.S. Bamburg J.R. J. Biol. Chem. 1995; 270: 17582-17587Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar), enzymes phosphorylating cofilin/ADF seem to play important roles in actin filament dynamics. We and other investigators provide evidence that LIM kinase 1 (LIMK1) and LIM kinase 2 (LIMK2) (6Mizuno K. Okano I. Ohashi K. Nunoue K. Kuma K. Miyata T. Nakamura T. Oncogene. 1994; 9: 1605-1612PubMed Google Scholar, 7Okano I. Hiraoka J. Otera H. Nunoue K. Ohashi K. Iwashita S. Hirai M. Mizuno K. J. Biol. Chem. 1995; 270: 31321-31330Crossref PubMed Scopus (169) Google Scholar, 8Nunoue K. Ohashi K. Okano I. Mizuno K. Oncogene. 1995; 11: 701-710PubMed Google Scholar) phosphorylate cofilin/ADF specifically at Ser-3 and induce actin cytoskeletal reorganization by phosphorylating and inactivating cofilin (9Yang N. Higuchi O. Ohashi K. Nagata K. Wada A. Kangawa K. Nishida E. Mizuno K. Nature. 1998; 393: 809-812Crossref PubMed Scopus (1049) Google Scholar, 10Arber S. Barbayannis F.A. Hanser H. Schneider C. Stanyon C.A. Bernard O. Caroni P. Nature. 1998; 393: 805-809Crossref PubMed Scopus (1148) Google Scholar). LIM kinases are activated in cultured cells by Rho family small GTPases Rac, Rho, and Cdc42 (9Yang N. Higuchi O. Ohashi K. Nagata K. Wada A. Kangawa K. Nishida E. Mizuno K. Nature. 1998; 393: 809-812Crossref PubMed Scopus (1049) Google Scholar, 10Arber S. Barbayannis F.A. Hanser H. Schneider C. Stanyon C.A. Bernard O. Caroni P. Nature. 1998; 393: 805-809Crossref PubMed Scopus (1148) Google Scholar, 11Sumi T. Matsumoto K. Takai Y. Nakamura T. J. Cell Biol. 1999; 147: 1519-1532Crossref PubMed Scopus (310) Google Scholar), this activation mediated by downstream effectors p21-activated kinase (PAK) and Rho-associated kinase, by phosphorylation of Thr-508 of LIMK1 or Thr-505 of LIMK2 (12Edwards D.C. Sanders L.C. Bokoch G.M. Gill G.N. Nat. Cell Biol. 1999; 1: 253-259Crossref PubMed Scopus (835) Google Scholar, 13Maekawa M. Ishizaki T. Boku S. Watanabe N. Fujita A. Iwamatsu A. Obinata T. Ohashi K. Mizuno K. Narumiya S. Science. 1999; 285: 895-898Crossref PubMed Scopus (1269) Google Scholar, 14Ohashi K. Nagata K. Maekawa M. Ishizaki T. Narumiya S. Mizuno K. J. Biol. Chem. 2000; 275: 3577-3582Abstract Full Text Full Text PDF PubMed Scopus (413) Google Scholar, 15Amano T. Tanabe K. Eto T. Narumiya S. Mizuno K. Biochem. J. 2001; 354: 149-159Crossref PubMed Scopus (136) Google Scholar, 16Sumi T. Matsumoto K. Nakamura T. J. Biol. Chem. 2001; 276: 670-676Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar).Testicular protein kinase 1 (TESK1) is a serine/threonine kinase with a structure composed of an N-terminal protein kinase domain and a C-terminal proline-rich region (17Toshima J. Ohashi K. Okano I. Nunoue K. Kishioka M. Kuma K. Miyata T. Hirai M. Baba T. Mizuno K. J. Biol. Chem. 1995; 270: 31331-31337Crossref PubMed Scopus (67) Google Scholar). The kinase domain of TESK1 is closely related to those of LIM kinases (17Toshima J. Ohashi K. Okano I. Nunoue K. Kishioka M. Kuma K. Miyata T. Hirai M. Baba T. Mizuno K. J. Biol. Chem. 1995; 270: 31331-31337Crossref PubMed Scopus (67) Google Scholar). We recently obtained evidence that TESK1, like LIM kinases, has the potential to phosphorylate cofilin/ADF specifically at Ser-3 and induces the formation of actin stress fibers and focal adhesions by phosphorylating cofilin/ADF (18Toshima J. Toshima J.Y. Amano T. Yang N. Narumiya S. Mizuno K. Mol. Biol. Cell. 2001; 12: 1131-1145Crossref PubMed Scopus (220) Google Scholar). In contrast to LIM kinases, the kinase activity of TESK1 is not stimulated by either PAK or Rho-associated kinase but can be stimulated by the integrin-mediated signaling pathway (18Toshima J. Toshima J.Y. Amano T. Yang N. Narumiya S. Mizuno K. Mol. Biol. Cell. 2001; 12: 1131-1145Crossref PubMed Scopus (220) Google Scholar). These results suggest that cofilin phosphorylation is regulated by at least three protein kinases, LIMK1, LIMK2, and TESK1, and upstream regulators for TESK1 seem to differ from those of LIM kinases. TESK1 was named after its high expression in the testis (17Toshima J. Ohashi K. Okano I. Nunoue K. Kishioka M. Kuma K. Miyata T. Hirai M. Baba T. Mizuno K. J. Biol. Chem. 1995; 270: 31331-31337Crossref PubMed Scopus (67) Google Scholar, 19Toshima J. Koji T. Mizuno K. Biochem. Biophys. Res. Commun. 1998; 249: 107-112Crossref PubMed Scopus (32) Google Scholar), but it is also expressed in various tissues and cell lines, albeit at a relatively low level (18Toshima J. Toshima J.Y. Amano T. Yang N. Narumiya S. Mizuno K. Mol. Biol. Cell. 2001; 12: 1131-1145Crossref PubMed Scopus (220) Google Scholar, 20Toshima J. Tanaka T. Mizuno K. J. Biol. Chem. 1999; 274: 12171-12176Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar); hence we assumed that TESK1 has general cellular functions rather than specific ones in the testis.Rosøk et al. (21Rosøk Ø. Pedeutour F. Ree A.H. Aasheim H.-C. Genomics. 1999; 61: 4-54Crossref Scopus (38) Google Scholar) report the cloning of human cDNA coding for a novel member of a TESK family, testicular protein kinase 2 (TESK2), with an overall structure closely related to that of TESK1. However, the deduced amino acid sequence of TESK2 by Rosøk et al. (21Rosøk Ø. Pedeutour F. Ree A.H. Aasheim H.-C. Genomics. 1999; 61: 4-54Crossref Scopus (38) Google Scholar) had 16 amino acids deleted within the protein kinase consensus sequence compared with sequences of TESK1 and LIM kinases, and the kinase activity, substrate(s), and cellular functions of TESK2 have remained to be determined.We report here identification and characterization of full-length of rat and human TESK2 with no deletion of 16 amino acids in the kinase domain. TESK2 has activity to phosphorylate cofilin/ADF and to induce actin cytoskeletal changes. We also show the predominant expression of TESK2 protein in rat testicular Sertoli cells and the subcellular distribution mainly in the nucleus, differing from previous findings in case of TESK1, which is expressed predominantly in the testicular germ cells and localizes primarily in the cytoplasm (17Toshima J. Ohashi K. Okano I. Nunoue K. Kishioka M. Kuma K. Miyata T. Hirai M. Baba T. Mizuno K. J. Biol. Chem. 1995; 270: 31331-31337Crossref PubMed Scopus (67) Google Scholar, 18Toshima J. Toshima J.Y. Amano T. Yang N. Narumiya S. Mizuno K. Mol. Biol. Cell. 2001; 12: 1131-1145Crossref PubMed Scopus (220) Google Scholar, 19Toshima J. Koji T. Mizuno K. Biochem. Biophys. Res. Commun. 1998; 249: 107-112Crossref PubMed Scopus (32) Google Scholar, 20Toshima J. Tanaka T. Mizuno K. J. Biol. Chem. 1999; 274: 12171-12176Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). We also characterized the role of the C-terminal noncatalytic region of TESK2 with regard to regulation of its kinase catalytic activity and cellular activity.DISCUSSIONIn this study we identified cDNAs coding for full-length rat and human TESK2. RT-PCR analysis revealed the existence of two isoforms of rat TESK2 transcripts with or without a 49-nt deletion within the protein kinase domain and one transcript for human TESK2 without a 49-nt deletion. Expression of the 49-nt-deleted TESK2 isoform was detected only in rat testis as a minor component, and it codes for the short truncated protein due to a shift in the reading frame. Rosøket al. (21Rosøk Ø. Pedeutour F. Ree A.H. Aasheim H.-C. Genomics. 1999; 61: 4-54Crossref Scopus (38) Google Scholar) report the cDNA sequence for human TESK2, but their reported sequence has a 48-nt (not 49-nt) deletion at the same position within the kinase domain. This sequence seems incorrect when compared with the human genome data bases. We were unable to detect the expression of the 48- or 49-nt deleted isoform of human TESK2 mRNA in several cell lines examined. These findings suggest that TESK2 mRNA without a 49-nt deletion is the major transcript that codes for the full-length functional TESK2 protein, and TESK2 mRNA with the 49-nt deletion seems to play, if any, a minor role. The exon/intron boundaries of TESK1 and TESK2 genes are almost conserved (28Toshima J. Nakagawara K. Mori M. Noda T. Mizuno K. Gene. 1998; 206: 237-245Crossref PubMed Scopus (14) Google Scholar), suggesting that these genes are generated from a common ancestral gene by gene duplication.Both TESK1 and TESK2 are highly expressed in the testis, which suggests a role for TESK family kinases in spermatogenesis. However, expression patterns of these kinases in the testis significantly differ; TESK1 is predominantly expressed in germ cells, particularly at stages of pachytene spermatocytes to early spermatids (17Toshima J. Ohashi K. Okano I. Nunoue K. Kishioka M. Kuma K. Miyata T. Hirai M. Baba T. Mizuno K. J. Biol. Chem. 1995; 270: 31331-31337Crossref PubMed Scopus (67) Google Scholar, 19Toshima J. Koji T. Mizuno K. Biochem. Biophys. Res. Commun. 1998; 249: 107-112Crossref PubMed Scopus (32) Google Scholar), whereas TESK2 is predominantly expressed in somatic Sertoli cells. Northern analysis revealed that expression of TESK1 mRNA (3.6 kb) significantly increases after postnatal days 20–24 (19Toshima J. Koji T. Mizuno K. Biochem. Biophys. Res. Commun. 1998; 249: 107-112Crossref PubMed Scopus (32) Google Scholar), but expression of TESK2 mRNA (3.0 kb) is practically constant throughout 7–24 postnatal days. Rat spermatocytes begin to undergo meiosis and to generate haploid round spermatids on postnatal days 25–30 (29Yang Z.W. Wreford N.G. de Kretser D.M. Biol. Reprod. 1990; 43: 629-635Crossref PubMed Scopus (101) Google Scholar). Based on developmental changes in expression of TESK1 mRNA and protein, we suggested a role for TESK1 in germ cells at and/or after meiotic stages of spermatogenesis (19Toshima J. Koji T. Mizuno K. Biochem. Biophys. Res. Commun. 1998; 249: 107-112Crossref PubMed Scopus (32) Google Scholar). In contrast, expression patterns of TESK2 mRNA and protein suggest its primary role in somatic Sertoli cells. Together these findings suggest that TESK1 and TESK2 play distinct roles in spermatogenesis in the testis. Rosøk et al. (21Rosøk Ø. Pedeutour F. Ree A.H. Aasheim H.-C. Genomics. 1999; 61: 4-54Crossref Scopus (38) Google Scholar) report the expression of TESK2 mRNA with the size of 3.5 kb in rat testis only after the 30th day of postnatal development (21Rosøk Ø. Pedeutour F. Ree A.H. Aasheim H.-C. Genomics. 1999; 61: 4-54Crossref Scopus (38) Google Scholar). Although the reason for the differences between the sizes and expression patterns of TESK2 mRNA is not clear, it may be because Rosøk et al. (21Rosøk Ø. Pedeutour F. Ree A.H. Aasheim H.-C. Genomics. 1999; 61: 4-54Crossref Scopus (38) Google Scholar) used human TESK2 cDNA or a rat EST cDNA clone corresponding to the 3′-noncoding region of rat TESK2 (nt 2529–3003) as the probe for Northern analysis, whereas our probe was full-length rat TESK2 cDNA.The present study provides evidence that TESK2 can phosphorylate cofilin and ADF specifically at Ser-3. Since actin-depolymerizing and -severing activities of cofilin/ADF are abrogated by phosphorylation at Ser-3, TESK2 seems to play an important role in actin filament dynamics by inhibiting cofilin/ADF activity. Actually, expression of TESK2 in HeLa cells induced assembly of actin stress fibers and focal adhesions. Thus, TESK2, similar to other members of a LIMK/TESK family, appears to be involved in actin cytoskeletal reorganization by phosphorylating and regulating cofilin/ADF. Previous studies revealed that LIMK1 and LIMK2 are activated by Rho-associated kinase and PAK, downstream effectors of Rho and Rac, respectively, by phosphorylation of conserved Thr-508 (in LIMK1) and Thr-505 (in LIMK2) in the activation loop of the kinase domain (12Edwards D.C. Sanders L.C. Bokoch G.M. Gill G.N. Nat. Cell Biol. 1999; 1: 253-259Crossref PubMed Scopus (835) Google Scholar, 13Maekawa M. Ishizaki T. Boku S. Watanabe N. Fujita A. Iwamatsu A. Obinata T. Ohashi K. Mizuno K. Narumiya S. Science. 1999; 285: 895-898Crossref PubMed Scopus (1269) Google Scholar, 14Ohashi K. Nagata K. Maekawa M. Ishizaki T. Narumiya S. Mizuno K. J. Biol. Chem. 2000; 275: 3577-3582Abstract Full Text Full Text PDF PubMed Scopus (413) Google Scholar, 15Amano T. Tanabe K. Eto T. Narumiya S. Mizuno K. Biochem. J. 2001; 354: 149-159Crossref PubMed Scopus (136) Google Scholar, 16Sumi T. Matsumoto K. Nakamura T. J. Biol. Chem. 2001; 276: 670-676Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar). Although the protein kinase domains of TESK1 and TESK2 are related to those of LIMKs, they have an alanine residue at the position corresponding to Thr-508 of LIMK1 or Thr-505 of LIMK2, within the activation loops of the kinase domains. Rho-associated kinase and PAK did not affect the kinase activity of TESK1 (18Toshima J. Toshima J.Y. Amano T. Yang N. Narumiya S. Mizuno K. Mol. Biol. Cell. 2001; 12: 1131-1145Crossref PubMed Scopus (220) Google Scholar) and TESK2 2J. Toshima, J. Y. Toshima, K. Takeuchi, R. Mori, and K. Mizuno, unpublished data. in vitro orin vivo. These results suggest that TESK1/TESK2 and LIMK1/LIMK2 commonly phosphorylate cofilin/ADF, but their upstream regulatory pathways differ significantly. As shown in this study, TESK1 and TESK2 have related structures, but their subcellular localization and distribution in cell lines differ significantly; hence, they have distinct modes of regulation and distinct functions in a cell type-specific manner.It remains to be known how nuclear TESK2 can regulate the actin cytoskeleton in the cytoplasm. Cofilin contains a well-characterized nuclear localization signal and has been reported to translocate to the nucleus in response to stimuli such as heat shock, treatment with dimethyl sulfoxide and T-cell costimulation and in a manner dependent on the dephosphorylation of Ser-3 (4Bamburg J.R. Annu. Rev. Cell Dev. Biol. 1999; 14: 305-338Google Scholar). It could be that the nuclear TESK2 phosphorylates cofilin translocated into the nucleus and thereby down-regulates the level of active (non-phosphorylated) cofilin in the cytoplasm and induces actin reorganization. Alternatively, the small amount of TESK2 localized in the cytoplasm may be enough to induce the changes in actin organization.We found that the Δ4 mutant of TESK2 with deletion of the C-terminal half has about a 10-fold higher kinase activity compared with wild-type TESK2. Because other truncated mutants, Δ1, Δ2, and Δ3, have kinase activity similar to that of wild-type TESK2, the region of amino acid sequence (327) seems to be involved in the negative regulation of TESK2 kinase activity. Many protein kinases contain an autoinhibitory domain within their noncatalytic region that inhibits catalytic activity by interacting with the catalytic domain. Among them, the N-terminal autoinhibitory domain of PAK is well characterized (30Manser 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, 31Frost J.A. Khokhlatchev A. Stippec S. White M.A. Cobb M.H. J. Biol. Chem. 1998; 273: 28191-28198Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar, 32Zhao Z.-S. Manser E. Chen X.-Q. Chong C. Leung T. Lim L. Mol. Cell. Biol. 1998; 18: 2153-2163Crossref PubMed Google Scholar). The PAK autoinhibitory domain partially overlaps with the Rac/Cdc42 binding domain and associates with the catalytic site to block substrate binding. When PAK associates with an active GTP binding form of Rac or Cdc42, the autoinhibitory domain dissociates from the catalytic site and sets the kinase domain free, leading to the promotion of autophosphorylation for activation (30Manser 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). The kinase activity of TESK2 might be similarly regulated by binding of upstream regulators to the C-terminal region near residues (327) of TESK2. Identification of proteins that associate with this region may reveal mechanisms regulating TESK2 activation. The Δ4 mutant induced condensation and fragmentation of the nucleus and subsequent cell death. Because a kinase-inactive form of Δ4 had no such effect, the altered nuclear morphology and cell death are probably caused by excess phosphorylation of substrates by abnormally activated TESK2. Recent studies suggest that γPAK (PAK2) is constitutively activated via CPP32 (caspase 3)-catalyzed cleavage at the site between the N-terminal regulatory domain and the C-terminal kinase domain and thereby induces apoptosis in Jurkat cells (33Rudel T. Bokoch G.M. Science. 1997; 276: 1571-1574Crossref PubMed Scopus (602) Google Scholar, 34Lee N. MacDonald H. Reinhard C. Halenbeck R. Roulston A. Shi T. Williams L.T. Proc. Natl. Acad. Sci. 1997; 94: 13642-13647Crossref PubMed Scopus (173) Google Scholar). As the nuclear morphology induced by the Δ4 mutant, condensation and fragmentation of the nucleus, is similar to the typical apoptotic phenotype, TESK2 may play a role in the execution step of apoptosis, where TESK2 may abnormally activated by proteolysis, as in the case of γPAK. The dynamics of polymerization/depolymerization of actin filaments and their remodeling are essential for cell movement, adhesion, and division (1Mitchison T.J. Cramer L.P. Cell. 1997; 84: 371-379Abstract Full Text Full Text PDF Scopus (1299) Google Scholar). Cofilin and actin-depolymerizing factor (ADF)1 play an essential role in the rapid turnover of actin filaments and actin-based cytoskeletal reorganization by stimulating depolymerization and severance of actin filaments (2Moon A. Drubin D.G. Mol. Biol. Cell. 1995; 6: 1423-1431Crossref PubMed Scopus (226) Google Scholar, 3Therriot J.A. J. Cell Biol. 1997; 136: 1165-1168Crossref PubMed Scopus (138) Google Scholar, 4Bamburg J.R. Annu. Rev. Cell Dev. Biol. 1999; 14: 305-338Google Scholar). As the activity of cofilin/ADF is negatively regulated by phosphorylation at Ser-3 (5Agnew B.J. Minamide L.S. Bamburg J.R. J. Biol. Chem. 1995; 270: 17582-17587Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar), enzymes phosphorylating cofilin/ADF seem to play important roles in actin filament dynamics. We and other investigators provide evidence that LIM kinase 1 (LIMK1) and LIM kinase 2 (LIMK2) (6Mizuno K. Okano I. Ohashi K. Nunoue K. Kuma K. Miyata T. Nakamura T. Oncogene. 1994; 9: 1605-1612PubMed Google Scholar, 7Okano I. Hiraoka J. Otera H. Nunoue K. Ohashi K. Iwashita S. Hirai M. Mizuno K. J. Biol. Chem. 1995; 270: 31321-31330Crossref PubMed Scopus (169) Google Scholar, 8Nunoue K. Ohashi K. Okano I. Mizuno K. Oncogene. 1995; 11: 701-710PubMed Google Scholar) phosphorylate cofilin/ADF specifically at Ser-3 and induce actin cytoskeletal reorganization by phosphorylating and inactivating cofilin (9Yang N. Higuchi O. Ohashi K. Nagata K. Wada A. Kangawa K. Nishida E. Mizuno K. Nature. 1998; 393: 809-812Crossref PubMed Scopus (1049) Google Scholar, 10Arber S. Barbayannis F.A. Hanser H. Schneider C. Stanyon C.A. Bernard O. Caroni P. Nature. 1998; 393: 805-809Crossref PubMed Scopus (1148) Google Scholar). LIM kinases are activated in cultured cells by Rho family small GTPases Rac, Rho, and Cdc42 (9Yang N. Higuchi O. Ohashi K. Nagata K. Wada A. Kangawa K. Nishida E. Mizuno K. Nature. 1998; 393: 809-812Crossref PubMed Scopus (1049) Google Scholar, 10Arber S. Barbayannis F.A. Hanser H. Schneider C. Stanyon C.A. Bernard O. Caroni P. Nature. 1998; 393: 805-809Crossref PubMed Scopus (1148) Google Scholar, 11Sumi T. Matsumoto K. Takai Y. Nakamura T. J. Cell Biol. 1999; 147: 1519-1532Crossref PubMed Scopus (310) Google Scholar), this activation mediated by downstream effectors p21-activated kinase (PAK) and Rho-associated kinase, by phosphorylation of Thr-508 of LIMK1 or Thr-505 of LIMK2 (12Edwards D.C. Sanders L.C. Bokoch G.M. Gill G.N. Nat. Cell Biol. 1999; 1: 253-259Crossref PubMed Scopus (835) Google Scholar, 13Maekawa M. Ishizaki T. Boku S. Watanabe N. Fujita A. Iwamatsu A. Obinata T. Ohashi K. Mizuno K. Narumiya S. Science. 1999; 285: 895-898Crossref PubMed Scopus (1269) Google Scholar, 14Ohashi K. Nagata K. Maekawa M. Ishizaki T. Narumiya S. Mizuno K. J. Biol. Chem. 2000; 275: 3577-3582Abstract Full Text Full Text PDF PubMed Scopus (413) Google Scholar, 15Amano T. Tanabe K. Eto T. Narumiya S. Mizuno K. Biochem. J. 2001; 354: 149-159Crossref PubMed Scopus (136) Google Scholar, 16Sumi T. Matsumoto K. Nakamura T. J. Biol. Chem. 2001; 276: 670-676Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar). Testicular protein kinase 1 (TESK1) is a serine/threonine kinase with a structure composed of an N-terminal protein kinase domain and a C-terminal proline-rich region (17Toshima J. Ohashi K. Okano I. Nunoue K. Kishioka M. Kuma K. Miyata T. Hirai M. Baba T. Mizuno K. J. Biol. Chem. 1995; 270: 31331-31337Crossref PubMed Scopus (67) Google Scholar). The kinase domain of TESK1 is closely related to those of LIM kinases (17Toshima J. Ohashi K. Okano I. Nunoue K. Kishioka M. Kuma K. Miyata T. Hirai M. Baba T. Mizuno K. J. Biol. Chem. 1995; 270: 31331-31337Crossref PubMed Scopus (67) Google Scholar). We recently obtained evidence that TESK1, like LIM kinases, has the potential to phosphorylate cofilin/ADF specifically at Ser-3 and induces the formation of actin stress fibers and focal adhesions by phosphorylating cofilin/ADF (18Toshima J. Toshima J.Y. Amano T. Yang N. Narumiya S. Mizuno K. Mol. Biol. Cell. 2001; 12: 1131-1145Crossref PubMed Scopus (220) Google Scholar). In contrast to LIM kinases, the kinase activity of TESK1 is not stimulated by either PAK or Rho-associated kinase but can be stimulated by the integrin-mediated signaling pathway (18Toshima J. Toshima J.Y. Amano T. Yang N. Narumiya S. Mizuno K. Mol. Biol. Cell. 2001; 12: 1131-1145Crossref PubMed Scopus (220) Google Scholar). These results suggest that cofilin phosphorylation is regulated by at least three protein kinases, LIMK1, LIMK2, and TESK1, and upstream regulators for TESK1 seem to differ from those of LIM kinases. TESK1 was named after its high expression in the testis (17Toshima J. Ohashi K. Okano I. Nunoue K. Kishioka M. Kuma K. Miyata T. Hirai M. Baba T. Mizuno K. J. Biol. Chem. 1995; 270: 31331-31337Crossref PubMed Scopus (67) Google Scholar, 19Toshima J. Koji T. Mizuno K. Biochem. Biophys. Res. Commun. 1998; 249: 107-112Crossref PubMed Scopus (32) Google Scholar), but it is also expressed in various tissues and cell lines, albeit at a relatively low level (18Toshima J. Toshima J.Y. Amano T. Yang N. Narumiya S. Mizuno K. Mol. Biol. Cell. 2001; 12: 1131-1145Crossref PubMed Scopus (220) Google Scholar, 20Toshima J. Tanaka T. Mizuno K. J. Biol. Chem. 1999; 274: 12171-12176Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar); hence we assumed that TESK1 has general cellular functions rather than specific ones in the testis. Rosøk et al. (21Rosøk Ø. Pedeutour F. Ree A.H. Aasheim H.-C. Genomics. 1999; 61: 4-54Crossref Scopus (38) Google Scholar) report the cloning of human cDNA coding for a novel member of a TESK family, testicular protein kinase 2 (TESK2), with an overall structure closely related to that of TESK1. However, the deduced amino acid sequence of TESK2 by Rosøk et al. (21Rosøk Ø. Pedeutour F. Ree A.H. Aasheim H.-C. Genomics. 1999; 61: 4-54Crossref Scopus (38) Google Scholar) had 16 amino acids deleted within the protein kinase consensus sequence compared with sequences of TESK1 and LIM kinases, and the kinase activity, substrate(s), and cellular functions of TESK2 have remained to be determined. We report here identification and characterization of full-length of rat and human TESK2 with no deletion of 16 amino acids in the kinase domain. TESK2 has activity to phosphorylate cofilin/ADF and to induce actin cytoskeletal changes. We also show the predominant expression of TESK2 protein in rat testicular Sertoli cells and the subcellular distribution mainly in the nucleus, differing from previous findings in case of TESK1, which is expressed predominantly in the testicular germ cells and localizes primarily in the cytoplasm (17Toshima J. Ohashi K. Okano I. Nunoue K. Kishioka M. Kuma K. Miyata T. Hirai M. Baba T. Mizuno K. J. Biol. Chem. 1995; 270: 31331-31337Crossref PubMed Scopus (67) Google Scholar, 18Toshima J. Toshima J.Y. Amano T. Yang N. Narumiya S. Mizuno K. Mol. Biol. Cell. 2001; 12: 1131-1145Crossref PubMed Scopus (220) Google Scholar, 19Toshima J. Koji T. Mizuno K. Biochem. Biophys. Res. Commun. 1998; 249: 107-112Crossref PubMed Scopus (32) Google Scholar, 20Toshima J. Tanaka T. Mizuno K. J. Biol. Chem. 1999; 274: 12171-12176Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). We also characterized the role of the C-terminal noncatalytic region of TESK2 with regard to regulation of its kinase catalytic activity and cellular activity. DISCUSSIONIn this study we identified cDNAs coding for full-length rat and human TESK2. RT-PCR analysis revealed the existence of two isoforms of rat TESK2 transcripts with or without a 49-nt deletion within the protein kinase domain and one transcript for human TESK2 without a 49-nt deletion. Expression of the 49-nt-deleted TESK2 isoform was detected only in rat testis as a minor component, and it codes for the short truncated protein due to a shift in the reading frame. Rosøket al. (21Rosøk Ø. Pedeutour F. Ree A.H. Aasheim H.-C. Genomics. 1999; 61: 4-54Crossref Scopus (38) Google Scholar) report the cDNA sequence for human TESK2, but their reported sequence has a 48-nt (not 49-nt) deletion at the same position within the kinase domain. This sequence seems incorrect when compared with the human genome data bases. We were unable to detect the expression of the 48- or 49-nt deleted isoform of human TESK2 mRNA in several cell lines examined. These findings suggest that TESK2 mRNA without a 49-nt deletion is the major transcript that codes for the full-length functional TESK2 protein, and TESK2 mRNA with the 49-nt deletion seems to play, if any, a minor role. The exon/intron boundaries of TESK1 and TESK2 genes are almost conserved (28Toshima J. Nakagawara K. Mori M. Noda T. Mizuno K. Gene. 1998; 206: 237-245Crossref PubMed Scopus (14) Google Scholar), suggesting that these genes are generated from a common ancestral gene by gene duplication.Both TESK1 and TESK2 are highly expressed in the testis, which suggests a role for TESK family kinases in spermatogenesis. However, expression patterns of these kinases in the testis significantly differ; TESK1 is predominantly expressed in germ cells, particularly at stages of pachytene spermatocytes to early spermatids (17Toshima J. Ohashi K. Okano I. Nunoue K. Kishioka M. Kuma K. Miyata T. Hirai M. Baba T. Mizuno K. J. Biol. Chem. 1995; 270: 31331-31337Crossref PubMed Scopus (67) Google Scholar, 19Toshima J. Koji T. Mizuno K. Biochem. Biophys. Res. Commun. 1998; 249: 107-112Crossref PubMed Scopus (32) Google Scholar), whereas TESK2 is predominantly expressed in somatic Sertoli cells. Northern analysis revealed that expression of TESK1 mRNA (3.6 kb) significantly increases after postnatal days 20–24 (19Toshima J. Koji T. Mizuno K. Biochem. Biophys. Res. Commun. 1998; 249: 107-112Crossref PubMed Scopus (32) Google Scholar), but expression of TESK2 mRNA (3.0 kb) is practically constant throughout 7–24 postnatal days. Rat spermatocytes begin to undergo meiosis and to generate haploid round spermatids on postnatal days 25–30 (29Yang Z.W. Wreford N.G. de Kretser D.M. Biol. Reprod. 1990; 43: 629-635Crossref PubMed Scopus (101) Google Scholar). Based on developmental changes in expression of TESK1 mRNA and protein, we suggested a role for TESK1 in germ cells at and/or after meiotic stages of spermatogenesis (19Toshima J. Koji T. Mizuno K. Biochem. Biophys. Res. Commun. 1998; 249: 107-112Crossref PubMed Scopus (32) Google Scholar). In contrast, expression patterns of TESK2 mRNA and protein suggest its primary role in somatic Sertoli cells. Together these findings suggest that TESK1 and TESK2 play distinct roles in spermatogenesis in the testis. Rosøk et al. (21Rosøk Ø. Pedeutour F. Ree A.H. Aasheim H.-C. Genomics. 1999; 61: 4-54Crossref Scopus (38) Google Scholar) report the expression of TESK2 mRNA with the size of 3.5 kb in rat testis only after the 30th day of postnatal development (21Rosøk Ø. Pedeutour F. Ree A.H. Aasheim H.-C. Genomics. 1999; 61: 4-54Crossref Scopus (38) Google Scholar). Although the reason for the differences between the sizes and expression patterns of TESK2 mRNA is not clear, it may be because Rosøk et al. (21Rosøk Ø. Pedeutour F. Ree A.H. Aasheim H.-C. Genomics. 1999; 61: 4-54Crossref Scopus (38) Google Scholar) used human TESK2 cDNA or a rat EST cDNA clone corresponding to the 3′-noncoding region of rat TESK2 (nt 2529–3003) as the probe for Northern analysis, whereas our probe was full-length rat TESK2 cDNA.The present study provides evidence that TESK2 can phosphorylate cofilin and ADF specifically at Ser-3. Since actin-depolymerizing and -severing activities of cofilin/ADF are abrogated by phosphorylation at Ser-3, TESK2 seems to play an important role in actin filament dynamics by inhibiting cofilin/ADF activity. Actually, expression of TESK2 in HeLa cells induced assembly of actin stress fibers and focal adhesions. Thus, TESK2, similar to other members of a LIMK/TESK family, appears to be involved in actin cytoskeletal reorganization by phosphorylating and regulating cofilin/ADF. Previous studies revealed that LIMK1 and LIMK2 are activated by Rho-associated kinase and PAK, downstream effectors of Rho and Rac, respectively, by phosphorylation of conserved Thr-508 (in LIMK1) and Thr-505 (in LIMK2) in the activation loop of the kinase domain (12Edwards D.C. Sanders L.C. Bokoch G.M. Gill G.N. Nat. Cell Biol. 1999; 1: 253-259Crossref PubMed Scopus (835) Google Scholar, 13Maekawa M. Ishizaki T. Boku S. Watanabe N. Fujita A. Iwamatsu A. Obinata T. Ohashi K. Mizuno K. Narumiya S. Science. 1999; 285: 895-898Crossref PubMed Scopus (1269) Google Scholar, 14Ohashi K. Nagata K. Maekawa M. Ishizaki T. Narumiya S. Mizuno K. J. Biol. Chem. 2000; 275: 3577-3582Abstract Full Text Full Text PDF PubMed Scopus (413) Google Scholar, 15Amano T. Tanabe K. Eto T. Narumiya S. Mizuno K. Biochem. J. 2001; 354: 149-159Crossref PubMed Scopus (136) Google Scholar, 16Sumi T. Matsumoto K. Nakamura T. J. Biol. Chem. 2001; 276: 670-676Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar). Although the protein kinase domains of TESK1 and TESK2 are related to those of LIMKs, they have an alanine residue at the position corresponding to Thr-508 of LIMK1 or Thr-505 of LIMK2, within the activation loops of the kinase domains. Rho-associated kinase and PAK did not affect the kinase activity of TESK1 (18Toshima J. Toshima J.Y. Amano T. Yang N. Narumiya S. Mizuno K. Mol. Biol. Cell. 2001; 12: 1131-1145Crossref PubMed Scopus (220) Google Scholar) and TESK2 2J. Toshima, J. Y. Toshima, K. Takeuchi, R. Mori, and K. Mizuno, unpublished data. in vitro orin vivo. These results suggest that TESK1/TESK2 and LIMK1/LIMK2 commonly phosphorylate cofilin/ADF, but their upstream regulatory pathways differ significantly. As shown in this study, TESK1 and TESK2 have related structures, but their subcellular localization and distribution in cell lines differ significantly; hence, they have distinct modes of regulation and distinct functions in a cell type-specific manner.It remains to be known how nuclear TESK2 can regulate the actin cytoskeleton in the cytoplasm. Cofilin contains a well-characterized nuclear localization signal and has been reported to translocate to the nucleus in response to stimuli such as heat shock, treatment with dimethyl sulfoxide and T-cell costimulation and in a manner dependent on the dephosphorylation of Ser-3 (4Bamburg J.R. Annu. Rev. Cell Dev. Biol. 1999; 14: 305-338Google Scholar). It could be that the nuclear TESK2 phosphorylates cofilin translocated into the nucleus and thereby down-regulates the level of active (non-phosphorylated) cofilin in the cytoplasm and induces actin reorganization. Alternatively, the small amount of TESK2 localized in the cytoplasm may be enough to induce the changes in actin organization.We found that the Δ4 mutant of TESK2 with deletion of the C-terminal half has about a 10-fold higher kinase activity compared with wild-type TESK2. Because other truncated mutants, Δ1, Δ2, and Δ3, have kinase activity similar to that of wild-type TESK2, the region of amino acid sequence (327) seems to be involved in the negative regulation of TESK2 kinase activity. Many protein kinases contain an autoinhibitory domain within their noncatalytic region that inhibits catalytic activity by interacting with the catalytic domain. Among them, the N-terminal autoinhibitory domain of PAK is well characterized (30Manser 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, 31Frost J.A. Khokhlatchev A. Stippec S. White M.A. Cobb M.H. J. Biol. Chem. 1998; 273: 28191-28198Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar, 32Zhao Z.-S. Manser E. Chen X.-Q. Chong C. Leung T. Lim L. Mol. Cell. Biol. 1998; 18: 2153-2163Crossref PubMed Google Scholar). The PAK autoinhibitory domain partially overlaps with the Rac/Cdc42 binding domain and associates with the catalytic site to block substrate binding. When PAK associates with an active GTP binding form of Rac or Cdc42, the autoinhibitory domain dissociates from the catalytic site and sets the kinase domain free, leading to the promotion of autophosphorylation for activation (30Manser 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). The kinase activity of TESK2 might be similarly regulated by binding of upstream regulators to the C-terminal region near residues (327) of TESK2. Identification of proteins that associate with this region may reveal mechanisms regulating TESK2 activation. The Δ4 mutant induced condensation and fragmentation of the nucleus and subsequent cell death. Because a kinase-inactive form of Δ4 had no such effect, the altered nuclear morphology and cell death are probably caused by excess phosphorylation of substrates by abnormally activated TESK2. Recent studies suggest that γPAK (PAK2) is constitutively activated via CPP32 (caspase 3)-catalyzed cleavage at the site between the N-terminal regulatory domain and the C-terminal kinase domain and thereby induces apoptosis in Jurkat cells (33Rudel T. Bokoch G.M. Science. 1997; 276: 1571-1574Crossref PubMed Scopus (602) Google Scholar, 34Lee N. MacDonald H. Reinhard C. Halenbeck R. Roulston A. Shi T. Williams L.T. Proc. Natl. Acad. Sci. 1997; 94: 13642-13647Crossref PubMed Scopus (173) Google Scholar). As the nuclear morphology induced by the Δ4 mutant, condensation and fragmentation of the nucleus, is similar to the typical apoptotic phenotype, TESK2 may play a role in the execution step of apoptosis, where TESK2 may abnormally activated by proteolysis, as in the case of γPAK. In this study we identified cDNAs coding for full-length rat and human TESK2. RT-PCR analysis revealed the existence of two isoforms of rat TESK2 transcripts with or without a 49-nt deletion within the protein kinase domain and one transcript for human TESK2 without a 49-nt deletion. Expression of the 49-nt-deleted TESK2 isoform was detected only in rat testis as a minor component, and it codes for the short truncated protein due to a shift in the reading frame. Rosøket al. (21Rosøk Ø. Pedeutour F. Ree A.H. Aasheim H.-C. Genomics. 1999; 61: 4-54Crossref Scopus (38) Google Scholar) report the cDNA sequence for human TESK2, but their reported sequence has a 48-nt (not 49-nt) deletion at the same position within the kinase domain. This sequence seems incorrect when compared with the human genome data bases. We were unable to detect the expression of the 48- or 49-nt deleted isoform of human TESK2 mRNA in several cell lines examined. These findings suggest that TESK2 mRNA without a 49-nt deletion is the major transcript that codes for the full-length functional TESK2 protein, and TESK2 mRNA with the 49-nt deletion seems to play, if any, a minor role. The exon/intron boundaries of TESK1 and TESK2 genes are almost conserved (28Toshima J. Nakagawara K. Mori M. Noda T. Mizuno K. Gene. 1998; 206: 237-245Crossref PubMed Scopus (14) Google Scholar), suggesting that these genes are generated from a common ancestral gene by gene duplication. Both TESK1 and TESK2 are highly expressed in the testis, which suggests a role for TESK family kinases in spermatogenesis. However, expression patterns of these kinases in the testis significantly differ; TESK1 is predominantly expressed in germ cells, particularly at stages of pachytene spermatocytes to early spermatids (17Toshima J. Ohashi K. Okano I. Nunoue K. Kishioka M. Kuma K. Miyata T. Hirai M. Baba T. Mizuno K. J. Biol. Chem. 1995; 270: 31331-31337Crossref PubMed Scopus (67) Google Scholar, 19Toshima J. Koji T. Mizuno K. Biochem. Biophys. Res. Commun. 1998; 249: 107-112Crossref PubMed Scopus (32) Google Scholar), whereas TESK2 is predominantly expressed in somatic Sertoli cells. Northern analysis revealed that expression of TESK1 mRNA (3.6 kb) significantly increases after postnatal days 20–24 (19Toshima J. Koji T. Mizuno K. Biochem. Biophys. Res. Commun. 1998; 249: 107-112Crossref PubMed Scopus (32) Google Scholar), but expression of TESK2 mRNA (3.0 kb) is practically constant throughout 7–24 postnatal days. Rat spermatocytes begin to undergo meiosis and to generate haploid round spermatids on postnatal days 25–30 (29Yang Z.W. Wreford N.G. de Kretser D.M. Biol. Reprod. 1990; 43: 629-635Crossref PubMed Scopus (101) Google Scholar). Based on developmental changes in expression of TESK1 mRNA and protein, we suggested a role for TESK1 in germ cells at and/or after meiotic stages of spermatogenesis (19Toshima J. Koji T. Mizuno K. Biochem. Biophys. Res. Commun. 1998; 249: 107-112Crossref PubMed Scopus (32) Google Scholar). In contrast, expression patterns of TESK2 mRNA and protein suggest its primary role in somatic Sertoli cells. Together these findings suggest that TESK1 and TESK2 play distinct roles in spermatogenesis in the testis. Rosøk et al. (21Rosøk Ø. Pedeutour F. Ree A.H. Aasheim H.-C. Genomics. 1999; 61: 4-54Crossref Scopus (38) Google Scholar) report the expression of TESK2 mRNA with the size of 3.5 kb in rat testis only after the 30th day of postnatal development (21Rosøk Ø. Pedeutour F. Ree A.H. Aasheim H.-C. Genomics. 1999; 61: 4-54Crossref Scopus (38) Google Scholar). Although the reason for the differences between the sizes and expression patterns of TESK2 mRNA is not clear, it may be because Rosøk et al. (21Rosøk Ø. Pedeutour F. Ree A.H. Aasheim H.-C. Genomics. 1999; 61: 4-54Crossref Scopus (38) Google Scholar) used human TESK2 cDNA or a rat EST cDNA clone corresponding to the 3′-noncoding region of rat TESK2 (nt 2529–3003) as the probe for Northern analysis, whereas our probe was full-length rat TESK2 cDNA. The present study provides evidence that TESK2 can phosphorylate cofilin and ADF specifically at Ser-3. Since actin-depolymerizing and -severing activities of cofilin/ADF are abrogated by phosphorylation at Ser-3, TESK2 seems to play an important role in actin filament dynamics by inhibiting cofilin/ADF activity. Actually, expression of TESK2 in HeLa cells induced assembly of actin stress fibers and focal adhesions. Thus, TESK2, similar to other members of a LIMK/TESK family, appears to be involved in actin cytoskeletal reorganization by phosphorylating and regulating cofilin/ADF. Previous studies revealed that LIMK1 and LIMK2 are activated by Rho-associated kinase and PAK, downstream effectors of Rho and Rac, respectively, by phosphorylation of conserved Thr-508 (in LIMK1) and Thr-505 (in LIMK2) in the activation loop of the kinase domain (12Edwards D.C. Sanders L.C. Bokoch G.M. Gill G.N. Nat. Cell Biol. 1999; 1: 253-259Crossref PubMed Scopus (835) Google Scholar, 13Maekawa M. Ishizaki T. Boku S. Watanabe N. Fujita A. Iwamatsu A. Obinata T. Ohashi K. Mizuno K. Narumiya S. Science. 1999; 285: 895-898Crossref PubMed Scopus (1269) Google Scholar, 14Ohashi K. Nagata K. Maekawa M. Ishizaki T. Narumiya S. Mizuno K. J. Biol. Chem. 2000; 275: 3577-3582Abstract Full Text Full Text PDF PubMed Scopus (413) Google Scholar, 15Amano T. Tanabe K. Eto T. Narumiya S. Mizuno K. Biochem. J. 2001; 354: 149-159Crossref PubMed Scopus (136) Google Scholar, 16Sumi T. Matsumoto K. Nakamura T. J. Biol. Chem. 2001; 276: 670-676Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar). Although the protein kinase domains of TESK1 and TESK2 are related to those of LIMKs, they have an alanine residue at the position corresponding to Thr-508 of LIMK1 or Thr-505 of LIMK2, within the activation loops of the kinase domains. Rho-associated kinase and PAK did not affect the kinase activity of TESK1 (18Toshima J. Toshima J.Y. Amano T. Yang N. Narumiya S. Mizuno K. Mol. Biol. Cell. 2001; 12: 1131-1145Crossref PubMed Scopus (220) Google Scholar) and TESK2 2J. Toshima, J. Y. Toshima, K. Takeuchi, R. Mori, and K. Mizuno, unpublished data. in vitro orin vivo. These results suggest that TESK1/TESK2 and LIMK1/LIMK2 commonly phosphorylate cofilin/ADF, but their upstream regulatory pathways differ significantly. As shown in this study, TESK1 and TESK2 have related structures, but their subcellular localization and distribution in cell lines differ significantly; hence, they have distinct modes of regulation and distinct functions in a cell type-specific manner. It remains to be known how nuclear TESK2 can regulate the actin cytoskeleton in the cytoplasm. Cofilin contains a well-characterized nuclear localization signal and has been reported to translocate to the nucleus in response to stimuli such as heat shock, treatment with dimethyl sulfoxide and T-cell costimulation and in a manner dependent on the dephosphorylation of Ser-3 (4Bamburg J.R. Annu. Rev. Cell Dev. Biol. 1999; 14: 305-338Google Scholar). It could be that the nuclear TESK2 phosphorylates cofilin translocated into the nucleus and thereby down-regulates the level of active (non-phosphorylated) cofilin in the cytoplasm and induces actin reorganization. Alternatively, the small amount of TESK2 localized in the cytoplasm may be enough to induce the changes in actin organization. We found that the Δ4 mutant of TESK2 with deletion of the C-terminal half has about a 10-fold higher kinase activity compared with wild-type TESK2. Because other truncated mutants, Δ1, Δ2, and Δ3, have kinase activity similar to that of wild-type TESK2, the region of amino acid sequence (327) seems to be involved in the negative regulation of TESK2 kinase activity. Many protein kinases contain an autoinhibitory domain within their noncatalytic region that inhibits catalytic activity by interacting with the catalytic domain. Among them, the N-terminal autoinhibitory domain of PAK is well characterized (30Manser 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, 31Frost J.A. Khokhlatchev A. Stippec S. White M.A. Cobb M.H. J. Biol. Chem. 1998; 273: 28191-28198Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar, 32Zhao Z.-S. Manser E. Chen X.-Q. Chong C. Leung T. Lim L. Mol. Cell. Biol. 1998; 18: 2153-2163Crossref PubMed Google Scholar). The PAK autoinhibitory domain partially overlaps with the Rac/Cdc42 binding domain and associates with the catalytic site to block substrate binding. When PAK associates with an active GTP binding form of Rac or Cdc42, the autoinhibitory domain dissociates from the catalytic site and sets the kinase domain free, leading to the promotion of autophosphorylation for activation (30Manser 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). The kinase activity of TESK2 might be similarly regulated by binding of upstream regulators to the C-terminal region near residues (327) of TESK2. Identification of proteins that associate with this region may reveal mechanisms regulating TESK2 activation. The Δ4 mutant induced condensation and fragmentation of the nucleus and subsequent cell death. Because a kinase-inactive form of Δ4 had no such effect, the altered nuclear morphology and cell death are probably caused by excess phosphorylation of substrates by abnormally activated TESK2. Recent studies suggest that γPAK (PAK2) is constitutively activated via CPP32 (caspase 3)-catalyzed cleavage at the site between the N-terminal regulatory domain and the C-terminal kinase domain and thereby induces apoptosis in Jurkat cells (33Rudel T. Bokoch G.M. Science. 1997; 276: 1571-1574Crossref PubMed Scopus (602) Google Scholar, 34Lee N. MacDonald H. Reinhard C. Halenbeck R. Roulston A. Shi T. Williams L.T. Proc. Natl. Acad. Sci. 1997; 94: 13642-13647Crossref PubMed Scopus (173) Google Scholar). As the nuclear morphology induced by the Δ4 mutant, condensation and fragmentation of the nucleus, is similar to the typical apoptotic phenotype, TESK2 may play a role in the execution step of apoptosis, where TESK2 may abnormally activated by proteolysis, as in the case of γPAK. We thank Dr. Takehiko Watanabe (Tohoku University) and Dr. Yukio Fujiki (Kyushu University) for advice and encouragement.