The Journal of Neuroscience, January 3, 2007 • 27(1):15–26 • 15 Development/Plasticity/Repair DISC1 Regulates the Transport of the NUDEL/LIS1/14-3-3␧ Complex through Kinesin-1 Shinichiro Taya,1 Tomoyasu Shinoda,1 Daisuke Tsuboi,1 Junko Asaki,1 Kumiko Nagai,1 Takao Hikita,1 Setsuko Kuroda,1 Keisuke Kuroda,1 Mariko Shimizu,1 Shinji Hirotsune,2 Akihiro Iwamatsu,3 and Kozo Kaibuchi1 1Department of Cell Pharmacology, Graduate School of Medicine, Nagoya University, 65 Tsurumai, Showa, Nagoya 466-8550, Japan, 2Department of Genetic Disease Research, Graduate School of Medicine, Osaka City University, Abeno, Osaka 545-8585, Japan, and 3Protein Research Network, Yokohama 236-0004, Japan Disrupted-In-Schizophrenia 1 (DISC1) is a candidate gene for susceptibility to schizophrenia. DISC1 is reported to interact with NudE-like (NUDEL), which forms a complex with lissencephaly-1 (LIS1) and 14-3-3␧. 14-3-3␧ is involved in the proper localization of NUDEL and LIS1inaxons.Althoughthefunctionalsignificanceofthiscomplexinneuronaldevelopmenthasbeenreported,thetransportmechanism of the complex into axons and their functions in axon formation remain essentially unknown. Here we report that Kinesin-1, a motor protein of anterograde axonal transport, was identified as a novel DISC1-interacting molecule. DISC1 directly interacted with kinesin heavy chain of Kinesin-1. Kinesin-1 interacted with the NUDEL/LIS1/14-3-3␧ complex through DISC1, and these molecules localized mainly at cell bodies and partially in the distal part of the axons. DISC1 partially colocalized with Kinesin family member 5A, NUDEL, LIS1, and 14-3-3␧ in the growth cones. The knockdown of DISC1 by RNA interference or the dominant-negative form of DISC1 inhibited the accumulation of NUDEL, LIS1, and 14-3-3␧ at the axons and axon elongation. The knockdown or the dominant-negative form of Kinesin-1 inhibited the accumulation of DISC1 at the axons and axon elongation. Furthermore, the knockdown of NUDEL or LIS1 inhibited axon elongation. Together, these results indicate that DISC1 regulates the localization of NUDEL/LIS1/14-3-3␧ complex into the axons as a cargo receptor for axon elongation. Key words: DISC1; Kinesin-1; axonal transport; axon elongation; schizophrenia; NUDEL Introduction (Millar et al., 2000; Blackwood et al., 2001; Craddock et al., 2005). Schizophrenia is a complex genetic disorder with fairly high her- In a Scottish family, the chromosome translocation (1;11)(q42.1; itability. Recently, several genes have been identified as candidate q14.3) was associated with major psychiatric illnesses, with a pre- genes for susceptibility to schizophrenia, including neuregulin 1 dominance of schizophrenic symptomatology (Blackwood et al., (Stefansson et al., 2002), dysbindin (Straub et al., 2002), G72 2001). This translocation interrupts the coding sequence of (Chumakov et al., 2002), catechol-O-methyltransferase (Egan et DISC1, leading to the reduction of DISC1 expression (Millar et al., 2001; Bilder et al., 2002; Shifman et al., 2002), and others al., 2005) or deletion of the C-terminal region. Haplotype trans- (Craddock et al., 2005; Harrison and Weinberger, 2005). Al- mission analysis using single-nucleotide polymorphisms (SNPs) though candidate genes for schizophrenia have been identified, from the 1q42 region indicated that the DISC1 gene plays an the molecular mechanisms underlying the disease are essentially important role in the etiology of schizophrenia (Hennah et al., unknown. 2003; Hodgkinson et al., 2004; Callicott et al., 2005). DISC1 in- Disrupted-In-Schizophrenia 1 (DISC1) is one of the most teracts with several proteins, including NudE-like (NUDEL) probable candidate genes for susceptibility to schizophrenia (Ozeki et al., 2003; Millar et al., 2003; Morris et al., 2003), fascic- ulation and elongation protein zeta 1 (FEZ1) (Miyoshi et al., 2003), lissencephaly-1 (LIS1) (Brandon et al., 2004), and phos- Received Sept. 2, 2006; revised Oct. 31, 2006; accepted Nov. 24, 2006. phodiesterase 4B (PDE4B) (Millar et al., 2005). Among these This work was supported by Grants-in-aid for scientific research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, Special Coordination Funds for Promoting Science and Technology, the Pharma- proteins, the association with NUDEL and LIS1 supports the ceuticals and Medical Devices Agency, Research Fellowships of the Japan Society for the Promotion of Science for notion that DISC1 contributes to the neuronal development and Young Scientists, the 21st Century Centre of Excellence Program from the Ministry of Education, Culture, Sports, morphology (Kholmanskikh et al., 2003). Science, and Technology of Japan, and the Research Grant (15A-2) for Nervous and Mental Disorders from the LIS1 mutation leads to type 1 lissencephaly, which is charac- Ministry of Health, Labour, and Welfare. We thank Dr. T. Nagase and Dr. L. Goldstein for kindly providing cDNAs, Dr. O. Reiner for kindly providing antibody, Dr. N. Ozaki for his critical and helpful discussions, Dr. A. Sawa for helpful terized by profuse migration defects during brain development discussion, Drs. N. Arimura, Y. Fukata, T. Watanabe, and S. Wang for providing materials, N. Hattori, S. Kozawa, M. (Reiner et al., 1993; Hattori et al., 1994). LIS1-deficient cerebellar Yamamoto, and Y. Yamashita for their technical assistance, and T. Ishii for secretarial assistance. granule neurons show defects in neuronal migration and axon CorrespondenceshouldbeaddressedtoDr.KozoKaibuchi,DepartmentofCellPharmacology,GraduateSchoolofMed- extension (Kholmanskikh et al., 2003). NUDEL is identified as a icine, Nagoya University, 65 Tsurumai, Showa, Nagoya 466-8550, Japan. E-mail: [email protected]. DOI:10.1523/JNEUROSCI.3826-06.2006 LIS1-binding protein (Niethammer et al., 2000; Sasaki et al., Copyright © 2007 Society for Neuroscience 0270-6474/07/270015-12$15.00/0 2000). The NUDEL/LIS1 complex is initially distributed in the 16 • J. Neurosci., January 3, 2007 • 27(1):15–26 Taya et al. • DISC1 Links Kinesin-1 to the NUDEL Complex centrosome or microtubule organizing center (MTOC) and then time-of-flight mass spectrometry and theoretical peptide masses from transported into the axonal growth cones (Niethammer et al., the proteins registered in the National Center for Biotechnology Infor- 2000; Sasaki et al., 2000). It is also known that the NUDEL/LIS1 mation database. complex accumulates with plus-end tracking proteins, including In vitro binding assay. GST or MBP fusion proteins were expressed in cytoplasmic linker protein 170 (CLIP-170) and end-binding pro- Escherichia coli BL21(DE3) and purified according to the instructions of the manufacturer. MBP or MBP–DISC1 (400 pmol) was mixed with tein 1 (EB1) near the plus ends of microtubules, which terminate glutathione–Sepharose 4B beads (Amersham Biosciences) coated with in growth cones (Coquelle et al., 2002). ␧ 100 pmol of GST alone, GST–KLC1, or GST–KIF5A in buffer (20 mM 14-3-3 binds to NUDEL phosphorylated by cyclin-dependent Tris-HCl, 1 mM EDTA, and 1 mM DTT). The bound MBP fusion proteins kinase (cdk5) and maintains NUDEL phosphorylation (Toyo-oka et were coeluted with GST fusion proteins. Portions of the eluates were al., 2003). Mice deficient in 14-3-3␧ show defects in brain develop- subjected to SDS-PAGE, followed by immunoblot analysis with anti- ment and neuronal migration (Toyo-oka et al., 2003), similar to MBP antibody. mice deficient in LIS1 (Hirotsune et al., 1998; Gambello et al., 2003). GST pull-down assay. COS7 cells were transfected in various combi- Deficiency of 14-3-3␧ causes mislocalization of the NUDEL/LIS1 nations (supplemental Fig. 1B,D, available at www.jneurosci.org as sup- complex from axons, suggesting that 14-3-3␧ regulates the axonal plemental material) with Lipofectamine (Invitrogen, Carlsbad, CA) and cultured for 48 h. Cells were lysed with buffer [20 mM Tris-HCl, 1 mM targeting of the NUDEL/LIS1 complex by sustaining NUDEL phos- ␮ phorylation (Toyo-oka et al., 2003). However, it remains unclear EDTA, 150 mM NaCl, 1 mM DTT, 0.1% (w/v) Triton X-100, 10 M ␧ p-amidinophenyl methanesulfonyl fluoride hydrochloride, and 10 how 14-3-3 or other component regulates the transport of the ␮g/ml leupeptin]. The lysate was sonicated and then clarified by centrif- NUDEL/LIS1 complex into axons. ugation at 12,000 ϫ g for 10 min at 4°C. The soluble supernatant was In this study, we identified Kinesin-1, a microtubule- incubated with glutathione–Sepharose 4B beads (supplemental Fig. 1B, dependent and plus-end directed motor, as a DISC1-interacting available at www.jneurosci.org as supplemental material) or glutathio- molecule. Our results show that DISC1 links Kinesin-1 to the ne–Sepharose 4B beads coated with 50 pmol of GST alone or GST–KIF5A NUDEL/LIS1/14-3-3␧ complex, serves as the cargo receptor, and (supplemental Fig. 1D, available at www.jneurosci.org as supplemental regulates the transport of the complex to axons, leading to axon material). Portions of the eluates were subjected to SDS-PAGE, followed elongation. by immunoblot analyses with the indicated antibodies. Coimmunoprecipitation assay. COS7 cells were transfected in various combinations (see Fig. 2B) (supplemental Fig. 1A,C, available at www. Materials and Methods jneurosci.org as supplemental material) with Lipofectamine and cul- Materials and chemicals. The fragment of DISC1–wild type (WT) (1– tured for 48 h. Preparations of the soluble supernatant were described 832) was inserted into pMAL-c2 (New England Laboratories, Beverly, above.