The Motor Protein Kinesin-1 Links Neurofibromin and Merlin in a Common Cellular Pathway of Neurofibromatosis
2002; Elsevier BV; Volume: 277; Issue: 40 Linguagem: Inglês
10.1074/jbc.c200434200
ISSN1083-351X
AutoresMohamed‐Ali Hakimi, David W. Speicher, Ramin Shiekhattar,
Tópico(s)Microtubule and mitosis dynamics
ResumoMutations in either of the two tumor suppressor genes NF1 (neurofibromin) and NF2 (merlin) result in Neurofibromatosis, a condition predisposing individuals to developing a variety of benign and malignant tumors of the central and peripheral nervous systems. Here we report the identification of two distinct NF1-containing complexes, one in the soluble and the other in the particulate fraction of HeLa extract. We show that the soluble NF1 complex delineates a large holo-NF1 complex (2 MDa) encompassing the components of a smaller particulate core-NF1 complex (400 kDa). Purification of the core-NF1 complex followed by mass spectrometric analysis revealed the motor protein, kinesin-1 heavy chain (HsuKHC/KIF5B), as a catalytic subunit of both NF-1-containing complexes. Importantly, although NF1 and NF2 are not in a stable association, NF2 is also a component of a distinct kinesin-1-containing complex. These results point to kinesin-1 as a common denominator between NF1 and NF2. Mutations in either of the two tumor suppressor genes NF1 (neurofibromin) and NF2 (merlin) result in Neurofibromatosis, a condition predisposing individuals to developing a variety of benign and malignant tumors of the central and peripheral nervous systems. Here we report the identification of two distinct NF1-containing complexes, one in the soluble and the other in the particulate fraction of HeLa extract. We show that the soluble NF1 complex delineates a large holo-NF1 complex (2 MDa) encompassing the components of a smaller particulate core-NF1 complex (400 kDa). Purification of the core-NF1 complex followed by mass spectrometric analysis revealed the motor protein, kinesin-1 heavy chain (HsuKHC/KIF5B), as a catalytic subunit of both NF-1-containing complexes. Importantly, although NF1 and NF2 are not in a stable association, NF2 is also a component of a distinct kinesin-1-containing complex. These results point to kinesin-1 as a common denominator between NF1 and NF2. neurofibromatosis types 1 and 2 GTPase-activating protein GAP-related domain mass spectrometry dithiothreitol phenylmethylsulfonyl fluoride β-mercaptoethanol 120-kDa heavy chain 64-kDa light chain amyloid precursor protein Neurofibromatosis type 1 (NF1)1 or von Recklinghausen disease is a common neurological genetic disease that affects 1 in 3500 individuals world wide (1Zhu Y. Parada L.F. Exp. Cell Res. 2001; 264: 19-28Crossref PubMed Scopus (66) Google Scholar, 2Cichowski K. Jacks T. Cell. 2001; 104: 593-604Abstract Full Text Full Text PDF PubMed Scopus (486) Google Scholar). Mutations in the human NF1 gene lead to a common neurocutaneous disorder characterized by benign tumors (neurofibromas and giomas), abnormal distribution of melanocytes (cafe-au-lait spots), and malignant tumors, including neurofibrosarcomas, pheochromocytomas, rhabdomyosarcomas, astrocytomas, and juvenile myeloid leukemias. NF1 patients also exhibit cognitive deficits and other symptoms unrelated to cancer, affecting neural crest-derived tissues outside of the nervous system reflective of a role for NF1 in developmental control (2Cichowski K. Jacks T. Cell. 2001; 104: 593-604Abstract Full Text Full Text PDF PubMed Scopus (486) Google Scholar, 3Costa R.M. Federov N.B. Kogan J.H. Murphy G.G. Stern J. Ohno M. Kucherlapati R. Jacks T. Silva A.J. Nature. 2002; 415: 526-530Crossref PubMed Scopus (458) Google Scholar).NF1 encodes a large protein of 2818 amino acids designated neurofibromin (4Buchberg A.M. Cleveland L.S. Jenkins N.A. Copeland N.G. Nature. 1990; 347: 291-294Crossref PubMed Scopus (178) Google Scholar, 5DeClue J.E. Cohen B.D. Lowy D.R. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9914-9918Crossref PubMed Scopus (175) Google Scholar, 6Gutmann D.H. Wood D.L. Collins F.S. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9658-9662Crossref PubMed Scopus (230) Google Scholar). The protein is highly conserved from yeast to human. Neurofibromin is expressed ubiquitously in human, with the highest expression in adult peripheral and central nervous systems (7Upadhyaya M. Cooper D.N. Neurofibromatosis Type 1; From Genotype to Phenotype. BIOS Scientific Publishers Limited, Oxford1998: 65-88Google Scholar). The protein contains a GAP-related domain (GRD) that shares homology to known GTPase-activating proteins (GAPs). NF1-GRD has been shown to act as a GAP for the Ras family of small GTPases (8Ballester R. Marchuk D. Boguski M. Saulino A. Letcher R. Wigler M. Collins F. Cell. 1990; 63: 851-859Abstract Full Text PDF PubMed Scopus (648) Google Scholar, 9Martin G.A. Viskochil D. Bollag G. McCabe P.C. Crosier W.J. Haubruck H. Conroy L. Clark R. O'Connell P. Cawthon R.M. Cell. 1990; 63: 843-849Abstract Full Text PDF PubMed Scopus (728) Google Scholar, 10Xu G.F. Lin B. Tanaka K. Dunn D. Wood D. Gesteland R. White R. Weiss R. Tamanoi F. Cell. 1990; 63: 835-841Abstract Full Text PDF PubMed Scopus (548) Google Scholar). Thus, several studies suggest that the tumor suppressor activity of neurofibromin depends on its ability to negatively regulate theras-mediated signaling pathway that regulate cell growth and differentiation in a variety of cell types (11Bourne H.R. Sanders D.A. McCormick F. Nature. 1990; 348: 125-132Crossref PubMed Scopus (1822) Google Scholar). Neurofibromatosis type 2 (NF2) is an autosomal dominant disorder implicated in the development of sporadic schwannomas, meningiomas, ependymonas, and astrocytomas (12Mautner V.F. Lindenau M. Baser M.E. Hazim W. Tatagiba M. Haase W. Samii M. Wais R. Pulst S.M. Neurosurgery. 1996; 38: 880-885Crossref PubMed Scopus (233) Google Scholar, 13Evans D.G. Huson S.M. Donnai D. Neary W. Blair V. Teare D. Newton V. Strachan T. Ramsden R. Harris R. J. Med. Genet. 1992; 29: 841-846Crossref PubMed Scopus (403) Google Scholar, 14Rouleau G.A. Merel P. Lutchman M. Sanson M. Zucman J. Marineau C. Hoang-Xuan K. Demczuk S. Desmaze C. Plougastel B. Pulst S.M. Lenoir G. Bijlsma E. Fashold R. Dumanski J. et al.Nature. 1993; 363: 515-521Crossref PubMed Scopus (1180) Google Scholar). The NF2 gene encodes a 595-amino acid protein termed merlin belonging to the ERM (ezrin, radixin andmoesin) family that link the actin cytoskeleton to cell surface glycoproteins (15Trofatter J.A. MacCollin M.M. Rutter J.L. Murrell J.R. Duyao M.P. Parry D.M. Eldridge R. Kley N. Menon A.G. Pulaski K. Cell. 1993; 72: 791-800Abstract Full Text PDF PubMed Scopus (1088) Google Scholar).We have initiated the biochemical characterization of NF1- and NF2-containing complexes from mammalian cells. These experiments led to the identification of two distinct NF1-containing complexes. We show that while NF1 purified from the soluble fraction reside in a large complex of ∼2MDa, NF1 in the particulate fraction is a component of a smaller complex of 400 kDa. To gain insights into the functions of these complexes, we used a combination of conventional and affinity chromatography to purify the smaller core-NF1 complex from the particulate fraction. We have identified the catalytic subunit of this complex as the motor protein kinesin-1. Importantly, we show that although NF1 and NF2 proteins are not stably associated, NF2 is also a component of a distinct kinesin-1-containing complex.MATERIALS AND METHODSWestern Blot AnalysisFor detection of the NF1 protein, affinity-purified polyclonal antibodies sc-68 (NF1GRP-D) raised against synthetic peptides corresponding to the carboxyl terminal domain of the human NF1 gene product were used (Santa Cruz Biotechnology). For detection of the NF2 protein, affinity-purified polyclonal antibodies sc331 (A-19) and sc332 (C-18) raised against synthetic peptides corresponding to the NH2 terminus and the COOH terminus of the NF2 protein were used (Santa Cruz Biotechnology). For detection of the kinesin-1 protein, one polyclonal antibody raised against the insert 1 region of the head of human uKHC (KIF5B) (gift from Ronald D. Vale's laboratory) and two monoclonal antibodies H1 and H2 raised against bovine brain kinesin (Chemicon International, Inc.) were used.Protein Identification Using Liquid Chromatography-MS/MSGel bands were excised from colloidal Coomassie-stained gels, and bands were destained, alkylated with iodoacetamide, and digested using modified trypsin (Promega) for 16 h at 37 °C essentially. A portion of the extracted peptides were loaded to a nanocapillary reverse-phase 75-μm column terminating in a nanospray 15-μm tip (New Objective) packed with Porous R2 resin (Applied Biosystems). The nanocolumn was directly coupled to a ThermoFinnigan LCQ quadrupole ion trap mass spectrometer, and peptides were eluted into the mass spectrometer using an acetic acid-acetonitrile gradient. Data were acquired using triple play mode to automatically obtain peptide masses, peptide charge states, and MS/MS spectra. The resulting data were searched against the non-redundant NCBI using the TurboSEQUEST Browser to identify proteins.Preparation of the Soluble and Particulate Fractions from HeLa Cells or Calf BrainThe method of Dignam et al. (16Dignam J.D. Lebovitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9140) Google Scholar) was used to prepare soluble or particulate fractions from HeLa cells and Calf brain. First, viable cells are prepared and collected in a conical test tube by centrifuging. Next, cells are resuspended in hypotonic buffer A (10 mm Tris-HCl, pH 7.9, 1.5 mm MgCl2, 10 mm KCl, 0.5 mm DTT, 0.2 mm PMSF) that causes them to swell, thus making them easy to lyse. The outer membranes are disrupted by homogenization, and the soluble fraction is then collected after pelleting membrane debris. The particulate fraction is carefully resuspended in buffer B (20 mmTris-HCl, pH 7.9, 25% glycerol, 420 mm NaCl, 1.5 mm MgCl2, 0.2 mm EDTA, 0.5 mm DTT, 0.5 mm PMSF). Following further homogenization and pelleting of the nuclear membrane debris, the particulate fraction is collected.Chromatographic Purification of NF1 Complex from HeLa Cells or Calf BrainHeLa Particulate FractionHeLa particulate extract (3 g) was loaded on a 500-ml column of phosphocellulose (P11, Whatman) and fractionated stepwise by the indicated KCl concentration in buffer A (20 mm Tris-HCl, pH 7.9, 0.2 mm EDTA, 10 mm βME, 10% glycerol, 0.2 mm PMSF). The P11 0.3 m KCl fraction (700 mg) was loaded on a 80-ml DEAE-Sephacel column (Amersham Biosciences) and eluted with 0.5 m KCl in buffer A. The 0.5 mKCl elution (500 mg) was dialyzed to 10 mmKxPO4 in buffer B (5 mm Hepes, pH 7.6, 1 mm DTT, 0.5 mm PMSF, 10 μmCaCl2, 10% glycerol, 40 mm KCl) and loaded on a 70-ml Bio-Gel HT column (hydroxyapatite, Bio-Rad). The column was resolved by using a linear 10-column volume gradient of 50–500 mm KxPO4. A pool of the fractions 11–13 were dialyzed to 700 mm NH4SO4 in buffer HB (20 mm Hepes, pH 7.6, 4 mm DTT, 0.5 mm EDTA, 10% glycerol, 0.5 mm PMSF) and loaded on a butyl-Sepharose (Amersham Biosciences). The column was resolved using a linear 10-column volume gradient of 700 to 0 mm NH4SO4 in buffer HB. NF1-containing fractions 11–15 were dialyzed to 100 mm KCl in buffer A and loaded on Heparine-5PW (Tosohaas). The column was resolved using a linear 20-column volume gradient of 100–500 mm KCl in buffer A. The fractions 12–14 were used for the immunoaffinity purification of the NF1-containing complex.Calf Brain Particulate FractionCalf brain particulate fraction (1 g) was loaded on a 500-ml column of phosphocellulose (P11, Whatman) and fractionated stepwise by the indicated KCl concentration in buffer A (20 mm Tris-HCl, pH 7.9, 0.2 mmEDTA, 10 mm βME, 10% glycerol, 0.2 mm PMSF). The P11 0.5 m KCl fraction (700 mg) was loaded on an 80-ml DEAE-Sephacel column (Amersham Biosciences) and eluted with 0.5m KCl. 60 mg of the 0.5 m KCl elution was dialyzed to 700 mm NH4SO4 in buffer HB (20 mm Hepes, pH 7.6, 4 mm DTT, 0.5 mm EDTA, 10% glycerol, 0.5 mm PMSF) and loaded on a butyl-Sepharose (Amersham Biosciences). The column was resolved using a linear 10-column volume gradient of 700 to 0 mm NH4SO4 in buffer HB. NF1-containing fractions 10–14 were dialyzed to 100 mm KCl in buffer A and loaded on Heparine-5PW (Tosohaas). The column was resolved using a linear 20-column volume gradient of 100–500 mm KCl in buffer A. The fractions 10–16 were used for the immunoaffinity purification of the NF1-containing complex.Immunoaffinity Purification of the NF1-containing ComplexAnti-NF1 antibodies (500 μg, COOH-terminal, Santa Cruz Biotechnology, sc-68) were cross-linked to protein A-Sepharose (1 ml, Repligen) using standard techniques for affinity purification. The heparin fractions from HeLa cells and calf brain were incubated with 1 ml of antibody-protein A beads for 4–5 h at 4 °C in buffer A. The beads were washed with 1 m KCl and 1% Nonidet P-40 in buffer A. The beads were then washed with 100 mm KCl in buffer A, and the proteins were eluted with 0.1 m glycine, pH 2.5, and neutralized with 0.10 volume 1.0 m Tris-HCl, pH 8.0.DISCUSSSIONKinesin-1 is a tetramer consisting of two 120-kDa heavy chains (KHC) and two 64-kDa light chains (KLC). Kinesin-1 heavy chain HsuKHC/KIF5B belongs to the kinesin protein superfamily (KIF) (19Miki H. Setou M. Kaneshiro K. Hirokawa N. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7004-7011Crossref PubMed Scopus (461) Google Scholar). This family has been shown to transport protein complexes, organelles, and mRNA to specific destinations in an ATP- and microtubule-dependent manner (20Hirokawa N. Science. 1998; 279: 519-526Crossref PubMed Scopus (1360) Google Scholar, 21Brendza R.P. Serbus L.R. Duffy J.B. Saxton W.M. Science. 2000; 289: 2120-2122Crossref PubMed Scopus (290) Google Scholar). Furthermore, some members of this family are also involved in chromosomal and spindle movements during mitosis and meiosis (22Sharp D.J. Rogers G.C. Scholey J.M. Nature. 2000; 407: 41-47Crossref PubMed Scopus (470) Google Scholar, 23Hirokawa N. Noda Y. Okada Y. Curr. Opin. Cell Biol. 1998; 10: 60-73Crossref PubMed Scopus (271) Google Scholar). Although a stable association of kinesin-1 and NF1 or NF2 was an unexpected finding, it is consistent with previous microscopy studies indicating the subcellular localization of NF1 and NF2 with the cytoskeleton (17Li C. Cheng Y. Gutmann D.A. Mangoura D. Brain Res. Dev. Brain Res. 2001; 130: 231-248Crossref PubMed Scopus (63) Google Scholar,24Gregory P.E. Gutmann D.H. Mitchell A. Park S. Boguski M. Jacks T. Wood D.L. Jove R. Collins F.S. Somat. Cell Mol. Genet. 1993; 19: 265-274Crossref PubMed Scopus (114) Google Scholar, 25Xu H. Gutmann D.H. Brain Res. 1997; 759: 149-152Crossref PubMed Scopus (56) Google Scholar, 26Gautreau A. Louvard D. Arpin M. Curr. Opin. Cell Biol. 2002; 14: 104-109Crossref PubMed Scopus (160) Google Scholar, 27Xu H.M. Gutmann D.H. J. Neurosci. Res. 1998; 51: 403-415Crossref PubMed Scopus (139) Google Scholar). Taken together, the association of NF1 and NF2 with the motor protein kinesin-1 suggests a role for these proteins in microtubule-mediated intracellular signal transduction pathways.Recent studies have shown that the axonal transport of amyloid precursor protein (APP) in neurons is mediated by the direct biochemical interaction between APP and KLC, the light chain subunit of kinesin-1 (28Kamal A. Stokin G.B. Yang Z. Xia C.H. Goldstein L.S. Neuron. 2000; 28: 449-459Abstract Full Text Full Text PDF PubMed Scopus (443) Google Scholar, 29Kamal A. Almenar-Queralt A. LeBlanc J.F. Roberts E.A. Goldstein L.S. Nature. 2001; 414: 643-648Crossref PubMed Scopus (494) Google Scholar). Considering that microtubule-dependent trafficking requires at least two entities, a cargo-bound receptor and the motor proteins, the authors proposed that APP may be a membrane cargo receptor for a kinesin-mediated axonal transport of β-secretase and presenilin-1 (29Kamal A. Almenar-Queralt A. LeBlanc J.F. Roberts E.A. Goldstein L.S. Nature. 2001; 414: 643-648Crossref PubMed Scopus (494) Google Scholar). In analogy with this model, the association between kinesin-1 and NF1 or NF2 might reflect a new function for these proteins in transport of vesicular cargoes within cells. Although NF1 has several known functions, including Ras GTPase-activating protein activity (8Ballester R. Marchuk D. Boguski M. Saulino A. Letcher R. Wigler M. Collins F. Cell. 1990; 63: 851-859Abstract Full Text PDF PubMed Scopus (648) Google Scholar, 9Martin G.A. Viskochil D. Bollag G. McCabe P.C. Crosier W.J. Haubruck H. Conroy L. Clark R. O'Connell P. Cawthon R.M. Cell. 1990; 63: 843-849Abstract Full Text PDF PubMed Scopus (728) Google Scholar, 10Xu G.F. Lin B. Tanaka K. Dunn D. Wood D. Gesteland R. White R. Weiss R. Tamanoi F. Cell. 1990; 63: 835-841Abstract Full Text PDF PubMed Scopus (548) Google Scholar) or adenylyl cyclase modulation (30Guo H.F. The I. Hannan F. Bernards A. Zhong Y. Science. 1997; 276: 795-798Crossref PubMed Scopus (170) Google Scholar, 31Guo H.F. Tong J. Hannan F. Luo L. Zhong Y. Nature. 2000; 403: 895-898Crossref PubMed Scopus (204) Google Scholar), this new function might explain the high incidence of learning disabilities and cognitive problems related to Nf1 mutations (1Zhu Y. Parada L.F. Exp. Cell Res. 2001; 264: 19-28Crossref PubMed Scopus (66) Google Scholar, 3Costa R.M. Federov N.B. Kogan J.H. Murphy G.G. Stern J. Ohno M. Kucherlapati R. Jacks T. Silva A.J. Nature. 2002; 415: 526-530Crossref PubMed Scopus (458) Google Scholar, 31Guo H.F. Tong J. Hannan F. Luo L. Zhong Y. Nature. 2000; 403: 895-898Crossref PubMed Scopus (204) Google Scholar, 32Silva A.J. Frankland P.W. Marowitz Z. Friedman E. Lazlo G. Cioffi D. Jacks T. Bourtchuladze R. Nat. Genet. 1997; 15: 281-284Crossref PubMed Scopus (289) Google Scholar, 33Zhu Y. Romero M.I. Ghosh P., Ye, Z. Charnay P. Rushing E.J. Marth J.D. Parada L.F. Genes Dev. 2001; 15: 859-876Crossref PubMed Scopus (427) Google Scholar). Thus aberrant kinesin-1/NF1-mediated trafficking or transport of neurotransmitter containing vesicle may affect the normal development of the cerebral cortex. Future studies are needed to test this hypothesis rigorously. In conclusion, our data through the demonstration of a stable association of NF1 and NF2 proteins with the motor protein kinesin-1 identifies a common pathway underlying the mechanism of neurofibromatosis. Neurofibromatosis type 1 (NF1)1 or von Recklinghausen disease is a common neurological genetic disease that affects 1 in 3500 individuals world wide (1Zhu Y. Parada L.F. Exp. Cell Res. 2001; 264: 19-28Crossref PubMed Scopus (66) Google Scholar, 2Cichowski K. Jacks T. Cell. 2001; 104: 593-604Abstract Full Text Full Text PDF PubMed Scopus (486) Google Scholar). Mutations in the human NF1 gene lead to a common neurocutaneous disorder characterized by benign tumors (neurofibromas and giomas), abnormal distribution of melanocytes (cafe-au-lait spots), and malignant tumors, including neurofibrosarcomas, pheochromocytomas, rhabdomyosarcomas, astrocytomas, and juvenile myeloid leukemias. NF1 patients also exhibit cognitive deficits and other symptoms unrelated to cancer, affecting neural crest-derived tissues outside of the nervous system reflective of a role for NF1 in developmental control (2Cichowski K. Jacks T. Cell. 2001; 104: 593-604Abstract Full Text Full Text PDF PubMed Scopus (486) Google Scholar, 3Costa R.M. Federov N.B. Kogan J.H. Murphy G.G. Stern J. Ohno M. Kucherlapati R. Jacks T. Silva A.J. Nature. 2002; 415: 526-530Crossref PubMed Scopus (458) Google Scholar). NF1 encodes a large protein of 2818 amino acids designated neurofibromin (4Buchberg A.M. Cleveland L.S. Jenkins N.A. Copeland N.G. Nature. 1990; 347: 291-294Crossref PubMed Scopus (178) Google Scholar, 5DeClue J.E. Cohen B.D. Lowy D.R. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9914-9918Crossref PubMed Scopus (175) Google Scholar, 6Gutmann D.H. Wood D.L. Collins F.S. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9658-9662Crossref PubMed Scopus (230) Google Scholar). The protein is highly conserved from yeast to human. Neurofibromin is expressed ubiquitously in human, with the highest expression in adult peripheral and central nervous systems (7Upadhyaya M. Cooper D.N. Neurofibromatosis Type 1; From Genotype to Phenotype. BIOS Scientific Publishers Limited, Oxford1998: 65-88Google Scholar). The protein contains a GAP-related domain (GRD) that shares homology to known GTPase-activating proteins (GAPs). NF1-GRD has been shown to act as a GAP for the Ras family of small GTPases (8Ballester R. Marchuk D. Boguski M. Saulino A. Letcher R. Wigler M. Collins F. Cell. 1990; 63: 851-859Abstract Full Text PDF PubMed Scopus (648) Google Scholar, 9Martin G.A. Viskochil D. Bollag G. McCabe P.C. Crosier W.J. Haubruck H. Conroy L. Clark R. O'Connell P. Cawthon R.M. Cell. 1990; 63: 843-849Abstract Full Text PDF PubMed Scopus (728) Google Scholar, 10Xu G.F. Lin B. Tanaka K. Dunn D. Wood D. Gesteland R. White R. Weiss R. Tamanoi F. Cell. 1990; 63: 835-841Abstract Full Text PDF PubMed Scopus (548) Google Scholar). Thus, several studies suggest that the tumor suppressor activity of neurofibromin depends on its ability to negatively regulate theras-mediated signaling pathway that regulate cell growth and differentiation in a variety of cell types (11Bourne H.R. Sanders D.A. McCormick F. Nature. 1990; 348: 125-132Crossref PubMed Scopus (1822) Google Scholar). Neurofibromatosis type 2 (NF2) is an autosomal dominant disorder implicated in the development of sporadic schwannomas, meningiomas, ependymonas, and astrocytomas (12Mautner V.F. Lindenau M. Baser M.E. Hazim W. Tatagiba M. Haase W. Samii M. Wais R. Pulst S.M. Neurosurgery. 1996; 38: 880-885Crossref PubMed Scopus (233) Google Scholar, 13Evans D.G. Huson S.M. Donnai D. Neary W. Blair V. Teare D. Newton V. Strachan T. Ramsden R. Harris R. J. Med. Genet. 1992; 29: 841-846Crossref PubMed Scopus (403) Google Scholar, 14Rouleau G.A. Merel P. Lutchman M. Sanson M. Zucman J. Marineau C. Hoang-Xuan K. Demczuk S. Desmaze C. Plougastel B. Pulst S.M. Lenoir G. Bijlsma E. Fashold R. Dumanski J. et al.Nature. 1993; 363: 515-521Crossref PubMed Scopus (1180) Google Scholar). The NF2 gene encodes a 595-amino acid protein termed merlin belonging to the ERM (ezrin, radixin andmoesin) family that link the actin cytoskeleton to cell surface glycoproteins (15Trofatter J.A. MacCollin M.M. Rutter J.L. Murrell J.R. Duyao M.P. Parry D.M. Eldridge R. Kley N. Menon A.G. Pulaski K. Cell. 1993; 72: 791-800Abstract Full Text PDF PubMed Scopus (1088) Google Scholar). We have initiated the biochemical characterization of NF1- and NF2-containing complexes from mammalian cells. These experiments led to the identification of two distinct NF1-containing complexes. We show that while NF1 purified from the soluble fraction reside in a large complex of ∼2MDa, NF1 in the particulate fraction is a component of a smaller complex of 400 kDa. To gain insights into the functions of these complexes, we used a combination of conventional and affinity chromatography to purify the smaller core-NF1 complex from the particulate fraction. We have identified the catalytic subunit of this complex as the motor protein kinesin-1. Importantly, we show that although NF1 and NF2 proteins are not stably associated, NF2 is also a component of a distinct kinesin-1-containing complex. MATERIALS AND METHODSWestern Blot AnalysisFor detection of the NF1 protein, affinity-purified polyclonal antibodies sc-68 (NF1GRP-D) raised against synthetic peptides corresponding to the carboxyl terminal domain of the human NF1 gene product were used (Santa Cruz Biotechnology). For detection of the NF2 protein, affinity-purified polyclonal antibodies sc331 (A-19) and sc332 (C-18) raised against synthetic peptides corresponding to the NH2 terminus and the COOH terminus of the NF2 protein were used (Santa Cruz Biotechnology). For detection of the kinesin-1 protein, one polyclonal antibody raised against the insert 1 region of the head of human uKHC (KIF5B) (gift from Ronald D. Vale's laboratory) and two monoclonal antibodies H1 and H2 raised against bovine brain kinesin (Chemicon International, Inc.) were used.Protein Identification Using Liquid Chromatography-MS/MSGel bands were excised from colloidal Coomassie-stained gels, and bands were destained, alkylated with iodoacetamide, and digested using modified trypsin (Promega) for 16 h at 37 °C essentially. A portion of the extracted peptides were loaded to a nanocapillary reverse-phase 75-μm column terminating in a nanospray 15-μm tip (New Objective) packed with Porous R2 resin (Applied Biosystems). The nanocolumn was directly coupled to a ThermoFinnigan LCQ quadrupole ion trap mass spectrometer, and peptides were eluted into the mass spectrometer using an acetic acid-acetonitrile gradient. Data were acquired using triple play mode to automatically obtain peptide masses, peptide charge states, and MS/MS spectra. The resulting data were searched against the non-redundant NCBI using the TurboSEQUEST Browser to identify proteins.Preparation of the Soluble and Particulate Fractions from HeLa Cells or Calf BrainThe method of Dignam et al. (16Dignam J.D. Lebovitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9140) Google Scholar) was used to prepare soluble or particulate fractions from HeLa cells and Calf brain. First, viable cells are prepared and collected in a conical test tube by centrifuging. Next, cells are resuspended in hypotonic buffer A (10 mm Tris-HCl, pH 7.9, 1.5 mm MgCl2, 10 mm KCl, 0.5 mm DTT, 0.2 mm PMSF) that causes them to swell, thus making them easy to lyse. The outer membranes are disrupted by homogenization, and the soluble fraction is then collected after pelleting membrane debris. The particulate fraction is carefully resuspended in buffer B (20 mmTris-HCl, pH 7.9, 25% glycerol, 420 mm NaCl, 1.5 mm MgCl2, 0.2 mm EDTA, 0.5 mm DTT, 0.5 mm PMSF). Following further homogenization and pelleting of the nuclear membrane debris, the particulate fraction is collected.Chromatographic Purification of NF1 Complex from HeLa Cells or Calf BrainHeLa Particulate FractionHeLa particulate extract (3 g) was loaded on a 500-ml column of phosphocellulose (P11, Whatman) and fractionated stepwise by the indicated KCl concentration in buffer A (20 mm Tris-HCl, pH 7.9, 0.2 mm EDTA, 10 mm βME, 10% glycerol, 0.2 mm PMSF). The P11 0.3 m KCl fraction (700 mg) was loaded on a 80-ml DEAE-Sephacel column (Amersham Biosciences) and eluted with 0.5 m KCl in buffer A. The 0.5 mKCl elution (500 mg) was dialyzed to 10 mmKxPO4 in buffer B (5 mm Hepes, pH 7.6, 1 mm DTT, 0.5 mm PMSF, 10 μmCaCl2, 10% glycerol, 40 mm KCl) and loaded on a 70-ml Bio-Gel HT column (hydroxyapatite, Bio-Rad). The column was resolved by using a linear 10-column volume gradient of 50–500 mm KxPO4. A pool of the fractions 11–13 were dialyzed to 700 mm NH4SO4 in buffer HB (20 mm Hepes, pH 7.6, 4 mm DTT, 0.5 mm EDTA, 10% glycerol, 0.5 mm PMSF) and loaded on a butyl-Sepharose (Amersham Biosciences). The column was resolved using a linear 10-column volume gradient of 700 to 0 mm NH4SO4 in buffer HB. NF1-containing fractions 11–15 were dialyzed to 100 mm KCl in buffer A and loaded on Heparine-5PW (Tosohaas). The column was resolved using a linear 20-column volume gradient of 100–500 mm KCl in buffer A. The fractions 12–14 were used for the immunoaffinity purification of the NF1-containing complex.Calf Brain Particulate FractionCalf brain particulate fraction (1 g) was loaded on a 500-ml column of phosphocellulose (P11, Whatman) and fractionated stepwise by the indicated KCl concentration in buffer A (20 mm Tris-HCl, pH 7.9, 0.2 mmEDTA, 10 mm βME, 10% glycerol, 0.2 mm PMSF). The P11 0.5 m KCl fraction (700 mg) was loaded on an 80-ml DEAE-Sephacel column (Amersham Biosciences) and eluted with 0.5m KCl. 60 mg of the 0.5 m KCl elution was dialyzed to 700 mm NH4SO4 in buffer HB (20 mm Hepes, pH 7.6, 4 mm DTT, 0.5 mm EDTA, 10% glycerol, 0.5 mm PMSF) and loaded on a butyl-Sepharose (Amersham Biosciences). The column was resolved using a linear 10-column volume gradient of 700 to 0 mm NH4SO4 in buffer HB. NF1-containing fractions 10–14 were dialyzed to 100 mm KCl in buffer A and loaded on Heparine-5PW (Tosohaas). The column was resolved using a linear 20-column volume gradient of 100–500 mm KCl in buffer A. The fractions 10–16 were used for the immunoaffinity purification of the NF1-containing complex.Immunoaffinity Purification of the NF1-containing ComplexAnti-NF1 antibodies (500 μg, COOH-terminal, Santa Cruz Biotechnology, sc-68) were cross-linked to protein A-Sepharose (1 ml, Repligen) using standard techniques for affinity purification. The heparin fractions from HeLa cells and calf brain were incubated with 1 ml of antibody-protein A beads for 4–5 h at 4 °C in buffer A. The beads were washed with 1 m KCl and 1% Nonidet P-40 in buffer A. The beads were then washed with 100 mm KCl in buffer A, and the proteins were eluted with 0.1 m glycine, pH 2.5, and neutralized with 0.10 volume 1.0 m Tris-HCl, pH 8.0. Western Blot AnalysisFor detection of the NF1 protein, affinity-purified polyclonal antibodies sc-68 (NF1GRP-D) raised against synthetic peptides corresponding to the carboxyl terminal domain of the human NF1 gene product were used (Santa Cruz Biotechnology). For detection of the NF2 protein, affinity-purified polyclonal antibodies sc331 (A-19) and sc332 (C-18) raised against synthetic peptides corresponding to the NH2 terminus and the COOH terminus of the NF2 protein were used (Santa Cruz Biotechnology). For detection of the kinesin-1 protein, one polyclonal antibody raised against the insert 1 region of the head of human uKHC (KIF5B) (gift from Ronald D. Vale's laboratory) and two monoclonal antibodies H1 and H2 raised against bovine brain kinesin (Chemicon International, Inc.) were used. For detection of the NF1 protein, affinity-purified polyclonal antibodies sc-68 (NF1GRP-D) raised against synthetic peptides corresponding to the carboxyl terminal domain of the human
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