Ca2+/Calmodulin-dependent Protein Kinase IV-mediated LIM Kinase Activation Is Critical for Calcium Signal-induced Neurite Outgrowth
2009; Elsevier BV; Volume: 284; Issue: 42 Linguagem: Inglês
10.1074/jbc.m109.006296
ISSN1083-351X
AutoresMiyohiko Takemura, Toshiaki Mishima, Yan Wang, Jiro Kasahara, Kohji Fukunaga, Kazumasa Ohashi, Kensaku Mizuno,
Tópico(s)Axon Guidance and Neuronal Signaling
ResumoActin cytoskeletal remodeling is essential for neurite outgrowth. LIM kinase 1 (LIMK1) regulates actin cytoskeletal remodeling by phosphorylating and inactivating cofilin, an actin filament-disassembling factor. In this study, we investigated the role of LIMK1 in calcium signal-induced neurite outgrowth. The calcium ionophore ionomycin induced LIMK1 activation and cofilin phosphorylation in Neuro-2a neuroblastoma cells. Knockdown of LIMK1 or expression of a kinase-dead mutant of LIMK1 suppressed ionomycin-induced cofilin phosphorylation and neurite outgrowth in Neuro-2a cells. Ionomycin-induced cofilin phosphorylation and neurite outgrowth were also blocked by KN-93, an inhibitor of Ca2+/calmodulin-dependent protein kinases (CaMKs), and STO-609, an inhibitor of CaMK kinase. An active form of CaMKIV but not CaMKI enhanced Thr-508 phosphorylation of LIMK1 and increased the kinase activity of LIMK1. Moreover, the active form of CaMKIV induced cofilin phosphorylation and neurite outgrowth, and a dominant negative form of CaMKIV suppressed ionomycin-induced neurite outgrowth. Taken together, our results suggest that LIMK1-mediated cofilin phosphorylation is critical for ionomycin-induced neurite outgrowth and that CaMKIV mediates ionomycin-induced LIMK1 activation. Actin cytoskeletal remodeling is essential for neurite outgrowth. LIM kinase 1 (LIMK1) regulates actin cytoskeletal remodeling by phosphorylating and inactivating cofilin, an actin filament-disassembling factor. In this study, we investigated the role of LIMK1 in calcium signal-induced neurite outgrowth. The calcium ionophore ionomycin induced LIMK1 activation and cofilin phosphorylation in Neuro-2a neuroblastoma cells. Knockdown of LIMK1 or expression of a kinase-dead mutant of LIMK1 suppressed ionomycin-induced cofilin phosphorylation and neurite outgrowth in Neuro-2a cells. Ionomycin-induced cofilin phosphorylation and neurite outgrowth were also blocked by KN-93, an inhibitor of Ca2+/calmodulin-dependent protein kinases (CaMKs), and STO-609, an inhibitor of CaMK kinase. An active form of CaMKIV but not CaMKI enhanced Thr-508 phosphorylation of LIMK1 and increased the kinase activity of LIMK1. Moreover, the active form of CaMKIV induced cofilin phosphorylation and neurite outgrowth, and a dominant negative form of CaMKIV suppressed ionomycin-induced neurite outgrowth. Taken together, our results suggest that LIMK1-mediated cofilin phosphorylation is critical for ionomycin-induced neurite outgrowth and that CaMKIV mediates ionomycin-induced LIMK1 activation. Actin cytoskeletal remodeling is essential for cell morphological changes, motility, and migration. In neurons, actin filament remodeling plays a critical role in the control of neurite outgrowth and guidance (1Luo L. Nat. Rev. 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Cell Biol. 2007; 178: 107-119Crossref PubMed Scopus (153) Google Scholar). LIM kinase Ca2+/calmodulin-dependent protein kinase Ca2+/calmodulin-dependent protein kinase kinase cyan fluorescent protein green fluorescent protein hemagglutinin microtubule affinity-regulating kinase 2 Thr-177-phosphorylated CaMKI Thr-196-phosphorylated CaMKIV Ser-3-phosphorylated cofilin Thr-508-phosphorylated LIMK1 short hairpin RNA Slingshot cAMP-responsive element-binding protein wild type glutathione S-transferase. Calcium ion is a critical second messenger that mediates a variety of neuronal functions, including neurite outgrowth, axonal guidance, neuronal differentiation, synaptic plasticity, and memory formation. Numerous Ca2+-evoked responses are mediated by the Ca2+-binding protein calmodulin and its downstream Ca2+/calmodulin-dependent protein kinases (CaMKs), including CaMKI, CaMKII, CaMKIV, and CaMK kinase (CaMKK) (22Wayman G.A. Lee Y.S. Tokumitsu H. Silva A. Soderling T.R. Neuron. 2008; 59: 914-931Abstract Full Text Full Text PDF PubMed Scopus (457) Google Scholar). CaMKs are activated by the binding of the Ca2+/calmodulin complex as well as by phosphorylation; CaMKI and CaMKIV are fully activated by CaMKK-mediated phosphorylation (22Wayman G.A. Lee Y.S. Tokumitsu H. Silva A. Soderling T.R. Neuron. 2008; 59: 914-931Abstract Full Text Full Text PDF PubMed Scopus (457) Google Scholar). Calcium ionophores, such as ionomycin and A23187, which promote calcium ion influx, stimulate neuritogenesis in neuroblastoma cells (23Wu G. Fang Y. Lu Z.H. Ledeen R.W. J. Neurocytol. 1998; 27: 1-14Crossref PubMed Scopus (96) Google Scholar, 24Uboha N.V. Flajolet M. Nairn A.C. Picciotto M.R. J. Neurosci. 2007; 27: 4413-4423Crossref PubMed Scopus (57) Google Scholar). CaMKI mediates calcium signal-induced neurite outgrowth through activation of extracellular signal-regulated kinase or microtubule affinity-regulating kinase 2 (MARK2) (24Uboha N.V. Flajolet M. Nairn A.C. Picciotto M.R. J. Neurosci. 2007; 27: 4413-4423Crossref PubMed Scopus (57) Google Scholar, 25Wayman G.A. Kaech S. Grant W.F. Davare M. Impey S. Tokumitsu H. Nozaki N. Banker G. Soderling T.R. J. Neurosci. 2004; 24: 3786-3794Crossref PubMed Scopus (153) Google Scholar, 26Schmitt J.M. Wayman G.A. Nozaki N. Soderling T.R. J. Biol. Chem. 2004; 279: 24064-24072Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar), and CaMKIV mediates neuritogenesis by phosphorylation of a transcription factor, cAMP-responsive element binding protein (CREB), and subsequent activation of CREB-dependent gene transcription (27Enslen H. Sun P. Brickey D. Soderling S.H. Klamo E. Soderling T.R. J. Biol. Chem. 1994; 269: 15520-15527Abstract Full Text PDF PubMed Google Scholar, 28Bito H. Deisseroth K. Tsien R.W. Curr. Opin. Neurobiol. 1997; 7: 419-429Crossref PubMed Scopus (249) Google Scholar, 29Redmond L. Kashani A.H. Ghosh A. Neuron. 2002; 34: 999-1010Abstract Full Text Full Text PDF PubMed Scopus (383) Google Scholar, 30Tai Y. Feng S. Ge R. Du W. Zhang X. He Z. Wang Y. J. Cell Sci. 2008; 121: 2301-2307Crossref PubMed Scopus (131) Google Scholar, 31Spencer T.K. Mellado W. Filbin M.T. Mol. Cell. Neurosci. 2008; 38: 110-116Crossref PubMed Scopus (51) Google Scholar). However, little is known about the signaling pathways that transduce the intracellular Ca2+ elevation into actin cytoskeletal remodeling for neurite outgrowth. In this study, we investigated the role of LIMK1 in the calcium signal-induced neurite outgrowth in Neuro-2a mouse neuroblastoma cells. We provide evidence that LIMK1-mediated cofilin phosphorylation plays a crucial role in ionomycin-induced neurite outgrowth and that CaMKIV mediates the ionomycin-induced LIMK1 activation. Ionomycin and puromycin were purchased from Sigma. KN-93 and STO-609 were purchased from Wako (Osaka, Japan). Rabbit polyclonal antibodies against cofilin, P-cofilin, and LIMK1 (C-10) were prepared as described previously (32Okano I. Hiraoka J. Otera H. Nunoue K. Ohashi K. Iwashita S. Hirai M. Mizuno K. J. Biol. Chem. 1995; 270: 31321-31330Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar, 33Toshima J. Toshima J.Y. Amano T. Yang N. Narumiya S. Mizuno K. Mol. Biol. Cell. 2001; 12: 1131-1145Crossref PubMed Scopus (222) Google Scholar). Antibodies against Myc (9E10) and hemagglutinin (HA) (3F10) epitopes were purchased from Roche Applied Science. Antibodies against green fluorescent protein (GFP) and β-actin were purchased from Invitrogen and Sigma, respectively. Polyclonal antibodies against Thr-508-phosphorylated LIMK1 (anti-P-LIMK1(T508) antibody) were generated against the phosphopeptide Cys-Asp-Arg-Lys-Lys-Arg-Tyr-phospho-Thr-Val-Val-Gly-Asn-Pro-Tyr, corresponding to the sequence of human LIMK1 amino acids 502–514, as described (33Toshima J. Toshima J.Y. Amano T. Yang N. Narumiya S. Mizuno K. Mol. Biol. Cell. 2001; 12: 1131-1145Crossref PubMed Scopus (222) Google Scholar). The antibodies were purified with an antigenic peptide-conjugated column (33Toshima J. Toshima J.Y. Amano T. Yang N. Narumiya S. Mizuno K. Mol. Biol. Cell. 2001; 12: 1131-1145Crossref PubMed Scopus (222) Google Scholar). Antibodies against Thr-196-phosphorylated CaMKIV (anti-P-CaMKIV(T196) antibody) and Thr-177-phosphorylated CaMKI (anti-P-CaMKI(T177) antibody) were prepared as described (34Kasahara J. Fukunaga K. Miyamoto E. J. Biol. Chem. 1999; 26: 9061-9067Abstract Full Text Full Text PDF Scopus (40) Google Scholar, 35Han F. Nakano T. Yamamoto Y. Shioda N. Lu Y.M. Fukunaga K. Brain Res. 2009; 1265: 205-214Crossref PubMed Scopus (22) Google Scholar). Expression plasmids coding for Myc-LIMK1, Myc-LIMK1(D460A), and LIMK1(D460A)-cyan fluorescent protein (CFP) were constructed as described previously (32Okano I. Hiraoka J. Otera H. Nunoue K. Ohashi K. Iwashita S. Hirai M. Mizuno K. J. Biol. Chem. 1995; 270: 31321-31330Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar, 36Amano T. Kaji N. Ohashi K. Mizuno K. J. Biol. Chem. 2002; 277: 22093-22102Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). Expression plasmids for GFP-tagged CaMKIα-(1–293), CaMKIα-(K49E), CaMKIV-(wild type (WT)), CaMKIV- (1–313), and CaMKIV-(K71E) were constructed as reported (37Tokumitsu H. Muramatsu M. Ikura M. Kobayashi R. J. Biol. Chem. 2000; 275: 20090-20095Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 38Chatila T. Anderson K.A. Ho N. Means A.R. J. Biol. Chem. 1996; 271: 21542-21548Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). A plasmid encoding HA-tagged human MARK2 was constructed by subcloning the PCR-amplified MARK2 cDNA into the pUCD2-2HA vector. The cDNA for the kinase-inactive MARK2(K49A) mutant was constructed using a site-directed mutagenesis kit (Clontech). The shRNA-targeting constructs were generated using pSUPER or pSUPER.retro.puro vector plasmids (OligoEngine, Seattle, WA), as described previously (39Horita Y. Ohashi K. Mukai M. Inoue M. Mizuno K. J. Biol. Chem. 2008; 283: 6013-6021Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). The 19-base sequences targeting mouse LIMK1 were 5′-GAAGGACTACTGGGCCCGC-3′ for the pSUPER-LIMK1 shRNA and 5′-GCTGGAACAATGGCTAGAA-3′ for the pSUPER. retro.puro-LIMK1 shRNA. As a control, we used a nontargeting sequence, 5′-TCTTCCCCCAAGAAAGATA-3′, which does not exist in the mouse genome. Neuro-2a cells were obtained from the American Type Culture Collection (CCL-131) and cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. Cells were transfected with expression plasmids or shRNA plasmids using FuGENE6 transfection reagent (Roche Applied Science). For testing the effect of LIMK1 knockdown on ionomycin-induced cofilin phosphorylation, Neuro-2a cells plated in 100-mm dishes (1.5 × 106 cells/dish) were cultured for 20 h and then transfected with pSUPER.retro.puro-LIMK1 shRNA. Transfected cells were cultured for 24 h and selected by culturing for an additional 48 h with 7 μg/ml puromycin. Then cells were replated on 60-mm dishes (2.0 × 106 cells/dish), cultured for 16 h, serum-starved for 4 h, and then cultured with or without 1.5 μm ionomycin for 30 min. For neurite outgrowth assays, Neuro-2a cells were plated on coverslips (2.5 × 104 cells/35-mm dish), cultured for 24 h, and transfected with pSUPER-LIMK1 shRNA. After incubation for 24 h, the cells were treated with 1.5 μm ionomycin, cultured for 48 h, and then fixed. Cells were lysed with lysis buffer (25 mm Tris-HCl (pH 7.5), 150 mm NaCl, 1% Nonidet P-40, 5% glycerol, 1 mm MgCl2, 1 mm MnCl2, 20 mm NaF, 1 mm Na3VO4, 1 mm dithiothreitol, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin). Cell lysates were subjected to immunoprecipitation or immunoblot analyses, as described previously (32Okano I. Hiraoka J. Otera H. Nunoue K. Ohashi K. Iwashita S. Hirai M. Mizuno K. J. Biol. Chem. 1995; 270: 31321-31330Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). Endogenous LIMK1 or Myc-LIMK1 expressed in Neuro-2a cells was immunoprecipitated with an anti-LIMK1 or anti-Myc antibody and then subjected to an in vitro kinase reaction using His6-cofilin as a substrate, as described previously (12Ohashi 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 (421) Google Scholar). CaMKI- or CaMKIV-catalyzed LIMK1 activation was analyzed in cell-free assays as follows. GFP-tagged CaMKI or CaMKIV mutants were expressed in Neuro-2a cells, immunoprecipitated with an anti-GFP antibody, and incubated in 20 μl of lysis buffer containing 50 μm ATP and 185 kBq of [γ-32P]ATP (110 TBq/mmol; PerkinElmer Life Sciences) with 1 μg/ml GST-LIMK1 and 2 μg/ml His6-cofilin at 30 °C for 30 min. GST-LIMK1 and His6-cofilin were expressed in Sf21 insect cells, using the Bac-to-Bac baculovirus expression system (Invitrogen), and purified by glutathione-Sepharose (GE Healthcare), as described (40Kurita S. Watanabe Y. Gunji E. Ohashi K. Mizuno K. J. Biol. Chem. 2008; 283: 32542-32552Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). The reaction mixture was separated with SDS-PAGE and analyzed with autoradiography to measure 32P-labeled cofilin and immunoblotting with an anti-P-LIMK1(T508) antibody. Neuro-2a cells (2.5 × 104 cells/35-mm dish) were plated on glass coverslips pretreated with 1 μg/ml poly-l-lysine, cultured for 24 h, and transfected with expression plasmids or pSUPER shRNA plasmids. After incubation for 24 h, cells were treated or not treated with 1.5 μm ionomycin and further cultured for 48 h. Then the cells were fixed with 4% formaldehyde in phosphate-buffered saline for 15 min. The CFP-positive cells with neurites longer than two cell body lengths were scored as neurite-bearing cells. Previous studies showed that ionomycin promotes neurite outgrowth in Neuro-2a cells (23Wu G. Fang Y. Lu Z.H. Ledeen R.W. J. Neurocytol. 1998; 27: 1-14Crossref PubMed Scopus (96) Google Scholar, 24Uboha N.V. Flajolet M. Nairn A.C. Picciotto M.R. J. Neurosci. 2007; 27: 4413-4423Crossref PubMed Scopus (57) Google Scholar). To examine whether LIMK1 is involved in ionomycin-induced neurite outgrowth, we first analyzed changes in the kinase activity of LIMK1 and the level of P-cofilin in ionomycin-stimulated Neuro-2a cells. In an in vitro kinase assay using cofilin as a substrate, the kinase activity of LIMK1 increased 1.6-fold by 30 min after ionomycin stimulation and then gradually decreased (Fig. 1A). The level of P-cofilin, measured by immunoblotting with an anti-P-cofilin antibody, increased 2.2-fold by 30 min after ionomycin treatment and then gradually decreased (Fig. 1B). These results suggest that ionomycin treatment increases both LIMK1 activity and cofilin phosphorylation in Neuro-2a cells. To assess whether LIMK1 is involved in ionomycin-induced cofilin phosphorylation, we knocked down LIMK1 by transfecting Neuro-2a cells with shRNA targeting LIMK1. Neuro-2a cells were transfected with pSUPER.retro.puro plasmids coding for control or LIMK1 shRNA target sequences and selected with puromycin. An immunoblot analysis showed that the LIMK1 shRNA suppressed expression of endogenous LIMK1 in Neuro-2a cells (Fig. 2A). The LIMK1 shRNA but not the control shRNA blocked ionomycin-induced cofilin phosphorylation (Fig. 2B). Furthermore, overexpression of a kinase-dead form of LIMK1, LIMK1(D460A), in which the catalytic Asp-460 was replaced by Ala, significantly suppressed the ionomycin-induced elevation of the P-cofilin level in Neuro-2a cells (Fig. 2C). These results suggest that LIMK1 plays a critical role in ionomycin-induced cofilin phosphorylation in Neuro-2a cells. To examine the role of LIMK1 in ionomycin-induced neurite outgrowth, we analyzed the effect of LIMK1 knockdown on neurite outgrowth in Neuro-2a cells. Neuro-2a cells were cotransfected with CFP and control or LIMK1 shRNA plasmids, cultured for 24 h, and then stimulated with ionomycin for 48 h. The cells were fixed, and the CFP fluorescence of the shRNA-transfected cells was visualized (Fig. 3A, top). We then scored the percentage of neurite-bearing cells with neurites longer than two cell body lengths in CFP-positive cells. Knockdown of LIMK1 almost completely blocked ionomycin-induced neurite outgrowth (Fig. 3A, bottom). Overexpression of kinase-dead LIMK1(D460A) also inhibited ionomycin-induced neurite outgrowth (Fig. 3B). These results suggest that LIMK1 plays a crucial role in ionomycin-induced neurite outgrowth in Neuro-2a cells and that LIMK1(D460A) functions as a dominant negative form. To determine whether CaMKs and CaMKK are involved in ionomycin-induced cofilin phosphorylation and neurite outgrowth, we examined the effects of KN-93 and STO-609. KN-93 inhibits the kinase activities of CaMKI, CaMKII, and CaMKIV, and STO-609 inhibits CaMKK, which activates CaMKI and CaMKIV but not CaMKII. When Neuro-2a cells were pretreated with KN-93 or STO-609, ionomycin-induced cofilin phosphorylation was almost completely blocked (Fig. 4A), and ionomycin-induced neurite outgrowth was significantly suppressed (Fig. 4, B and C). Thus, CaMKK and one or both of its downstream kinases, CaMKI and CaMKIV, are involved in ionomycin-induced cofilin phosphorylation and neurite outgrowth. We next examined whether CaMKI and CaMKIV have the potential to activate LIMK1. We tested the effects of coexpressing constitutively active or kinase-dead forms of CaMKs on the kinase activity of Myc-LIMK1 in Neuro-2a cells. Coexpression of an active form of CaMKIV, CaMKIV-(1–313), increased the kinase activity of Myc-LIMK1 2.7-fold (Fig. 5A). In contrast, coexpression of an active form of CaMKI, CaMKI-(1–293), had no apparent effect on the kinase activity of Myc-LIMK1 (Fig. 5A). Expression of a kinase-dead form of CaMKI, CaMKI-(K49E), or CaMKIV, CaMKIV-(K71E), also did not affect Myc-LIMK1 activity. The kinase-inactive Myc-LIMK1(D460A), when coexpressed with active CaMKs, did not exhibit any cofilin-phosphorylating activity (data not shown). These results indicate that CaMKIV but not CaMKI has the potential to activate LIMK1. To determine whether CaMKIV directly activates LIMK1, GST-LIMK1 was expressed with a baculovirus expression system, purified with glutathione-Sepharose, and incubated with active or inactive CaMKIV. The kinase activity of GST-LIMK1 was measured using cofilin as a substrate. CaMKIV-(1–313) but not CaMKIV-(K71E) increased the kinase activity of GST-LIMK1 (Fig. 5B). Because ROCK and PAK activate LIMK1 by phosphorylating Thr-508 (10Edwards D.C. Sanders L.C. Bokoch G.M. Gill G.N. Nat. Cell Biol. 1999; 1: 253-259Crossref PubMed Scopus (846) Google Scholar, 11Maekawa 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 (1288) Google Scholar, 12Ohashi 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 (421) Google Scholar), we used immunoblotting with an anti-P-LIMK1(T508) antibody to examine whether active CaMKIV phosphorylates LIMK1 at Thr-508. CaMKIV-(1–313) but not CaMKIV-(K71E) increased the level of P-LIMK1(T508) (Fig. 5B, third panel), indicating that CaMKIV phosphorylates LIMK1 at Thr-508. In contrast, neither CaMKI-(1–293) nor CaMKI-(K49E) affected LIMK1 kinase activity or the level of P-LIMK1 (Fig. 5C). These results further indicate that CaMKIV but not CaMKI has the potential to activate LIMK1. CaMKIV is activated by CaMKK-catalyzed phosphorylation at Thr-196 (34Kasahara J. Fukunaga K. Miyamoto E. J. Biol. Chem. 1999; 26: 9061-9067Abstract Full Text Full Text PDF Scopus (40) Google Scholar). To examine whether CaMKIV is activated by ionomycin stimulation, we analyzed changes in the level of Thr-196 phosphorylation of CaMKIV in ionomycin-stimulated Neuro-2a cells by immunoblotting with an anti-P-CaMKIV(T196) antibody. The level of P-CaMKIV(T196) increased about 2.0-fold by 10 min after ionomycin stimulation (Fig. 6A), which suggests that ionomycin induces CaMKIV activation in Neuro-2a cells. We also analyzed whether active CaMKIV induces cofilin phosphorylation in Neuro-2a cells. Expression of CaMKIV-(1–313) but not CaMKIV-(K71E) increased the level of P-cofilin (Fig. 6B). These results further support the notion that CaMKIV mediates ionomycin-induced cofilin phosphorylation. In addition, we also examined whether CaMKI is activated by ionomycin by measuring the level of Thr-177 phosphorylation of CaMKI (35Han F. Nakano T. Yamamoto Y. Shioda N. Lu Y.M. Fukunaga K. Brain Res. 2009; 1265: 205-214Crossref PubMed Scopus (22) Google Scholar). The level of P-CaMKI(T177) increased 5–10 min after ionomycin treatment (Fig. 6C). Thus, CaMKI is also activated by ionomycin treatment in Neuro-2a cells. Previous studies showed that CaMKI is involved in neurite outgrowth (24Uboha N.V. Flajolet M. Nairn A.C. Picciotto M.R. J. Neurosci. 2007; 27: 4413-4423Crossref PubMed Scopus (57) Google Scholar, 25Wayman G.A. Kaech S. Grant W.F. Davare M. Impey S. Tokumitsu H. Nozaki N. Banker G. Soderling T.R. J. Neurosci. 2004; 24: 3786-3794Crossref PubMed Scopus (153) Google Scholar, 26Schmitt J.M. Wayman G.A. Nozaki N. Soderling T.R. J. Biol. Chem. 2004; 279: 24064-24072Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). To further examine the roles of CaMKI and CaMKIV in ionomycin-induced neurite outgrowth in Neuro-2a cells, we analyzed the effects of expressing the constitutively active and dominant negative forms of these CaMKs. Expression of a dominant negative form of either CaMKI or CaMKIV (CaMKI-(K49E) or CaMKIV-(K71E)) significantly suppressed ionomycin-induced neurite outgrowth (Fig. 7). In addition, expression of a constitutively active mutant of CaMKI or CaMKIV (CaMKI-(1–293) or CaMKIV-(1–313)) promoted neurite outgrowth in Neuro-2a cells (Fig. 7). These results suggest that both CaMKI and CaMKIV are involved in ionomycin-induced neurite outgrowth in Neuro-2a cells. A previous study showed that CaMKI induced neurite outgrowth by activating MARK2 (24Uboha N.V. Flajolet M. Nairn A.C. Picciotto M.R. J. Neurosci. 2007; 27: 4413-4423Crossref PubMed Scopus (57) Google Scholar). In order to understand the mechanism by which active CaMKI induces neurite outgrowth, we examined the effect of a kinase-dead form of MARK2 (MARK2(K49A)) on CaMKI-(1–293)-induced neuritogenesis. Coexpression of MARK2(K49A) significantly suppressed CaMKI-(1–293)-induced neurite outgrowth in Neuro-2a cells (Fig. 8), which suggests that CaMKI-induced neurite outgrowth is mediated by MARK2. In this study, we identified a novel signaling pathway that is crucial for calcium signal-induced neurite outgrowth. We showed that ionomycin induces LIMK1 activation and cofilin phosphorylation and that depletion of LIMK1 or expression of kinase-dead LIMK1 blocks ionomycin-induced cofilin phosphorylation and neurite outgrowth. This indicates that LIMK1 mediates calcium signal-induced cofilin phosphorylation and neurite outgrowth. The findings that ionomycin-induced cofilin phosphorylation and neurite outgrowth were blocked by STO-609 and KN-93 indicate that CaMKK-mediated activation of CaMKI and/or CaMKIV is involved in calcium signal-induced cofilin phosphorylation and neurite outgrowth. Interestingly, an active form of CaMKIV but not CaMKI increased the kinase activity of LIMK1 in both cell-free and coexpression assays. Furthermore, expression of active CaMKIV enhanced cofilin phosphorylation and neurite outgrowth, and a dominant negative form of CaMKIV inhibited ionomycin-induced neurite outgrowth. Based on these observations, we propose a novel signaling pathway composed of CaMKK-CaMKIV-LIMK1-cofilin that plays a crucial role in calcium signal-induced neurite outgrowth in Neuro-2a cells. The level of LIMK1 activation in response to ionomycin is small relative to the effect on neurite extension. This may reflect that ionomycin induces LIMK1 activation in the limited region of the cell, such as the site where neurites start to grow. It has been proposed that the CaMKK-CaMKIV cascade is involved in calcium-induced neurite growth through phosphorylation and activation of the transcription factor CREB (27Enslen H. Sun P. Brickey D. Soderling S.H. Klamo E. Soderling T.R. 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These results suggest that CaMKIV plays dual roles in calcium-induced neurite outgrowth: the short term role of promoting the initiation of neurite outgrowth via LIMK1 activation and actin reorganization and the long term role of contributing to neurite extension and maintenance via activation of CREB-dependent gene transcription. Although CaMKIV is predominantly localized in the nucleus, it is also distributed in the cytoplasm and has the potential to shuttle between the nucleus and cytoplasm (41Jensen K.F. Ohmstede C.A. Fisher R.S. Sahyoun N. Proc. Natl. Acad. Sci. U.S.A. 1991; 88: 2850-2853Crossref PubMed Scopus (183) Google Scholar, 42Wu J.Y. Gonzalez-Robayna I.J. Richards J.S. Means A.R. Endocrinology. 2000; 141: 4777-4783Crossref PubMed Scopus (50) Google Scholar, 43Kotera I. Sekimoto T. Miyamoto Y. Saiwaki T. Nagoshi E. Sakagami H. Kondo H. Yoneda Y. EMBO J. 2005; 24: 942-951Crossref PubMed Scopus (78) Google Scholar). Thus, it is possible that LIMK1 is activated by CaMKIV in the cytoplasm. On the other hand, given that LIMK1 also shuttles between the nucleus and cytoplasm using its intrinsic nuclear export and nuclear localization signals (44Yang N. Higuchi O. Mizuno K. Exp. Cell Res. 1998; 241: 242-252Crossref PubMed Scopus (34) Google Scholar, 45Yang N. Mizuno K. Biochem. J. 1999; 338: 793-798Crossref PubMed Scopus (47) Google Scholar), LIMK1 may be activated in the nucleus and then transported to the cytoplasm to regulate actin cytoskeleton in this region. Similar to the effects of CaMKIV mutants, expression of an active form of CaMKI induced neurite outgrowth, and a dominant negative form of CaMKI suppressed ionomycin-induced neurite outgrowth in Neuro-2a cells, thus indicating that CaMKI, like CaMKIV, plays a crucial role in calcium signal-induced neurite outgrowth. Although CaMKI has been shown to activate Rac (an upstream regulator of LIMK1) through activation of Rac-guanine nucleotide exchange factors, such as STEF and βPIX (46Takemoto-Kimura S. Ageta-Ishihara N. Nonaka M. Adachi-Morishima A. Mano T. Okamura M. Fujii H. Fuse T. Hoshino M. Suzuki S. Kojima M. Mishina M. Okuno H. Bito H. Neuron. 2007; 54: 755-770Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 47Saneyoshi T. Wayman G. Fortin D. Davare M. Hoshi N. Nozaki N. Natsume T. Soderling T.R. Neuron. 2008; 57: 94-107Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar), CaMKI failed to activate LIMK1 in our coexpression experiments in Neuro-2a cells (Fig. 5A). Thus, CaMKI acts in neurite outgrowth by a mechanism distinct from Rac-mediated LIMK1 activation, at least in Neuro-2a cells. In this respect, previous studies showed that CaMKI induced neurite outgrowth by activating extracellular signal-regulated kinase, which in turn stimulated CREB-dependent transcription (22Wayman G.A. Lee Y.S. Tokumitsu H. Silva A. Soderling T.R. Neuron. 2008; 59: 914-931Abstract Full Text Full Text PDF PubMed Scopus (457) Google Scholar, 26Schmitt J.M. Wayman G.A. Nozaki N. Soderling T.R. J. Biol. Chem. 2004; 279: 24064-24072Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar), or by activating MARK2 (24Uboha N.V. Flajolet M. Nairn A.C. Picciotto M.R. J. Neurosci. 2007; 27: 4413-4423Crossref PubMed Scopus (57) Google Scholar). We showed that expression of kinase-dead MARK2 suppressed CaMKI-induced neurite outgrowth, indicating that MARK2 mediates CaMKI-induced neuritogenesis. Because MARK2 regulates microtubule reorganization by phosphorylating microtubule-associated proteins (48Biernat J. Wu Y.Z. Timm T. Zheng-Fischhöfer Q. Mandelkow E. Meijer L. Mandelkow E.M. Mol. 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We and other investigators previously showed that ROCK and PAK activate LIMK1 by phosphorylation at Thr-508, which is in the activation loop within the kinase domain of LIMK1 (10Edwards D.C. Sanders L.C. Bokoch G.M. Gill G.N. Nat. Cell Biol. 1999; 1: 253-259Crossref PubMed Scopus (846) Google Scholar, 11Maekawa 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 (1288) Google Scholar, 12Ohashi 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 (421) Google Scholar). Thus, various signaling pathways, such as Rho-ROCK, Rac-PAK1, and CaMKK-CaMKIV, activate LIMK1 by the same mechanism, Thr-508 phosphorylation. This suggests that LIMK1 activation is a point of convergence that links various signaling pathways to the regulation of actin cytoskeletal reorganization in cells. Previous studies showed that knockdown of LIMK1 or inhibition of LIMK1 activity suppresses neurite outgrowth in PC12 rat pheochromocytoma cells, chick dorsal root ganglion neurons, and hippocampal pyramidal neurons (18Rosso S. Bollati F. Bisbal M. Peretti D. Sumi T. Nakamura T. Quiroga S. Ferreira A. Cáceres A. Mol. Biol. Cell. 2004; 15: 3433-3449Crossref PubMed Scopus (112) Google Scholar, 19Tursun B. Schlüter A. Peters M.A. Viehweger B. Ostendorff H.P. Soosairajah J. Drung A. Bossenz M. Johnsen S.A. Schweizer M. Bernard O. Bach I. Genes Dev. 2005; 19: 2307-2319Crossref PubMed Scopus (97) Google Scholar, 20Endo M. Ohashi K. Mizuno K. J. Biol. Chem. 2007; 282: 13692-13702Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar), which suggests that LIMK1 has a critical role in neurite outgrowth. On the other hand, overexpression of LIMK1 or depletion of SSH1/SSH2 also suppressed neurite outgrowth in PC12 cells and chick dorsal root ganglion neurons (16Endo M. Ohashi K. Sasaki Y. Goshima Y. Niwa R. Uemura T. Mizuno K. J. Neurosci. 2003; 23: 2527-2537Crossref PubMed Google Scholar, 20Endo M. Ohashi K. Mizuno K. J. Biol. Chem. 2007; 282: 13692-13702Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). These results indicate that a precise level of LIMK1 activation and a proper balance of cofilin phosphorylation and dephosphorylation by LIMK and SSH activities are necessary for neurite outgrowth. We previously showed that calcium signals induce SSH1 activation and cofilin dephosphorylation via calcineurin in other types of cells (49Wang Y. Shibasaki F. Mizuno K. J. Biol. Chem. 2005; 280: 12683-12689Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). Thus, it is possible that calcium signals stimulate both LIMK1 and SSH1 to regulate the actin cytoskeleton and that the spatial and temporal regulation of LIMK1 and SSH1 activities and the balance between them are important for the control of neurite outgrowth. Because LIMK1 depletion inhibits stimulus-induced actin filament assembly (20Endo M. Ohashi K. Mizuno K. J. Biol. Chem. 2007; 282: 13692-13702Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 50Nishita M. Tomizawa C. Yamamoto M. Horita Y. Ohashi K. Mizuno K. J. Cell Biol. 2005; 171: 349-359Crossref PubMed Scopus (173) Google Scholar), LIMK1 is probably involved in the actin filament assembly and stabilization required for the initial phase of neurite outgrowth. In contrast, cofilin is required for neurite extension because it stimulates actin filament disassembly and thereby supplies actin monomers to the leading edge of the extending growth cones (5Pollard T.D. Borisy G.G. Cell. 2003; 112: 453-465Abstract Full Text Full Text PDF PubMed Scopus (3278) Google Scholar, 51Kiuchi T. Ohashi K. Kurita S. Mizuno K. J. Cell Biol. 2007; 177: 465-476Crossref PubMed Scopus (143) Google Scholar); therefore, SSH is likely involved in the neurite extension by promoting actin filament turnover via cofilin dephosphorylation and reactivation. In conclusion, we have identified a novel signaling pathway that transduces calcium elevations into LIMK1 activation and actin cytoskeletal reorganization via CaMKK and CaMKIV. We have the data that brain-derived neurotrophic factor induces LIMK1 activation in primary rat cortical neurons and that expression of dominant negative CaMKIV or depletion of LIMK1 blocked brain-derived neurotrophic factor-induced dendritogenesis, which suggests that the CaMKIV-LIMK1 pathway is probably involved in brain-derived neurotrophic factor-induced dendritogenesis in primary neurons. In further studies, we will elucidate the roles of this signaling pathway in a variety of calcium-induced actin cytoskeletal responses in neuronal and non-neuronal cells. Calcium signals regulate various important neural functions and development, including axon guidance, dendrite arborization, spine morphology, synaptic plasticity, and learning and memory formation. Actin cytoskeletal remodeling mediated by the LIMK1-cofilin pathway plays an important role in the dynamics of dendritic spine structures, persistence of long term potentiation, and synaptic plasticity (52Meng Y. Zhang Y. Tregoubov V. Janus C. Cruz L. Jackson M. Lu W.Y. MacDonald J.F. Wang J.Y. Falls D.L. Jia Z. Neuron. 2002; 35: 121-133Abstract Full Text Full Text PDF PubMed Scopus (551) Google Scholar, 53Fukazawa Y. Saitoh Y. Ozawa F. Ohta Y. Mizuno K. Inokuchi K. Neuron. 2003; 38: 447-460Abstract Full Text Full Text PDF PubMed Scopus (578) Google Scholar, 54Eaton B.A. Davis G.W. Neuron. 2005; 47: 695-708Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). Therefore, CaMKIV-mediated LIMK1 activation and cofilin phosphorylation may be among the important mechanisms by which calcium signals regulate synaptic plasticity and higher order brain functions, in addition to those needed for neurite outgrowth. Our identification of the CaMKIV-LIMK1 pathway will help to elucidate the mechanisms by which actin cytoskeletal remodeling is regulated in response to various stimuli mediated by calcium signaling. We thank Dr. Y. Ohta for providing the CaMKI cDNA and Dr. S. Kurita for helpful advice.
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