LIS1 Missense Mutations
2003; Elsevier BV; Volume: 278; Issue: 40 Linguagem: Inglês
10.1074/jbc.m301147200
ISSN1083-351X
AutoresMichal Caspi, Frédéric M. Coquelle, Cynthia Koifman, Talia Levy, Hiroyuki Arai, Junken Aoki, Jan R. De Mey, Orly Reiner,
Tópico(s)Microtubule and mitosis dynamics
ResumoMutations in one allele of the human LIS1 gene cause a severe brain malformation, lissencephaly. Although most LIS1 mutations involve deletions, several point mutations with a single amino acid alteration were described. Patients carrying these mutations reveal variable phenotypic manifestations. We have analyzed the functional importance of these point mutations by examining protein stability, folding, intracellular localization, and protein-protein interactions. Our data suggest that the mutated proteins were affected at different levels, and no single assay could be used to predict the lissencephaly phenotype. Most interesting are those mutant proteins that retain partial folding and interactions. In the case of LIS1 mutated in F31S, the cellular phenotype may be modified by overexpression of specific interacting proteins. Overexpression of the PAF-AH α1 subunit dissolved aggregates induced by this mutant protein and increased its half-life. Overexpression of NudE or NudEL localized this mutant protein to spindle poles and kinetochores but had no effect on protein stability. Our results implicate that there are probably different biochemical and cellular mechanisms obstructed in each patient yielding the varied lissencephaly phenotypes. Mutations in one allele of the human LIS1 gene cause a severe brain malformation, lissencephaly. Although most LIS1 mutations involve deletions, several point mutations with a single amino acid alteration were described. Patients carrying these mutations reveal variable phenotypic manifestations. We have analyzed the functional importance of these point mutations by examining protein stability, folding, intracellular localization, and protein-protein interactions. Our data suggest that the mutated proteins were affected at different levels, and no single assay could be used to predict the lissencephaly phenotype. Most interesting are those mutant proteins that retain partial folding and interactions. In the case of LIS1 mutated in F31S, the cellular phenotype may be modified by overexpression of specific interacting proteins. Overexpression of the PAF-AH α1 subunit dissolved aggregates induced by this mutant protein and increased its half-life. Overexpression of NudE or NudEL localized this mutant protein to spindle poles and kinetochores but had no effect on protein stability. Our results implicate that there are probably different biochemical and cellular mechanisms obstructed in each patient yielding the varied lissencephaly phenotypes. The functional complexity of the vertebrate cerebral cortex is facilitated by a complicated structural organization. The developmental details of these features are therefore of critical interest. A singular feature of cortical development is the generation of neurons at sites distant from their place of terminal differentiation. Abnormalities in the migration of neurons into the embryonic cortex lead to loss of normal convolutions of the human neocortex, known as lissencephaly. One cause for such an acute brain malformation is mutation(s) in the LIS1 gene (1Reiner O. Carrozzo R. Shen Y. Whenert M. Faustinella F. Dobyns W.B. Caskey C.T. Ledbetter D.H. Nature. 1993; 364: 717-721Crossref PubMed Scopus (909) Google Scholar) or in the X-linked doublecortin (DCX) 1The abbreviations used are: DCX, doublecortin; SBH, subcortical band heterotopia; WD, tryptophan-aspartic acid; Lis-H, LIS1 homology domain; BSA, bovine serum albumin; PAF-AH, platelet-activating factor acetylhydrolase; DHC, dynein heavy chain; His6, six-histidine tag; PBS, phosphate-buffered saline; IP, immunoprecipitation; X-gal, 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside; GFP, green fluorescent protein; DAPI, 4,6-diamidino-2-phenylindole; Pipes, 1,4-piperazinediethanesulfonic acid. gene (2des Portes V. Pinard J.M. Billuart P. Vinet M.C. Koulakoff A. Carrie A. Gelot A. Dupuis E. Motte J. Berwald-Netter Y. Catala M. Kahn A. Beldjord C. Chelly J. Cell. 1998; 92: 51-61Abstract Full Text Full Text PDF PubMed Scopus (678) Google Scholar, 3Gleeson J.G. Allen K.M. Fox J.W. Lamperti E.D. Berkovic S. Scheffer I. Cooper E.C. Dobyns W.B. Minnerath S.R. Ross M.E. Walsh C.A. Cell. 1998; 92: 63-72Abstract Full Text Full Text PDF PubMed Scopus (887) Google Scholar, 4Pilz D.T. Matsumoto N. Minnerath S. Mills P. Gleeson J.G. Allen K.M. Walsh C.A. Barkovich A.J. Dobyns W.B. Ledbetter D.H. Ross M.E. Hum. Mol. Genet. 1998; 7: 2029-2037Crossref PubMed Scopus (296) Google Scholar). Lissencephaly (smooth brain) is a severe abnormality of neuronal migration characterized by absent (agyria) or decreased (pachygyria) convolutions, producing a smooth cerebral surface with thickened cortex (5Dobyns W.B. Reiner O. Carrozzo R. Ledbetter D.H. J. Am. Med. Assoc. 1993; 270: 2838-2842Crossref PubMed Google Scholar). Subcortical band heterotopia (SBH) is a related disorder in which there are bilateral bands of gray matter interposed in the white matter between the cortex and the lateral ventricles (6Barkovich A.J. Guerrini R. Battaglia G. Kalifa G. N′Guyen T. Parmeggiani A. Santucci M. Giovanardi-Rossi P. Granata T. D'Incerti L. Ann. Neurol. 1994; 36: 609-617Crossref PubMed Scopus (209) Google Scholar). SBH is very common among females with mutations in DCX (2des Portes V. Pinard J.M. Billuart P. Vinet M.C. Koulakoff A. Carrie A. Gelot A. Dupuis E. Motte J. Berwald-Netter Y. Catala M. Kahn A. Beldjord C. Chelly J. Cell. 1998; 92: 51-61Abstract Full Text Full Text PDF PubMed Scopus (678) Google Scholar, 3Gleeson J.G. Allen K.M. Fox J.W. Lamperti E.D. Berkovic S. Scheffer I. Cooper E.C. Dobyns W.B. Minnerath S.R. Ross M.E. Walsh C.A. Cell. 1998; 92: 63-72Abstract Full Text Full Text PDF PubMed Scopus (887) Google Scholar). Lissencephaly and SBH have been observed in different regions of the same brain, indicating an "agyria-pachygyria-band" spectrum. A grading system characterizes the malformations from grade 1 (most severe) to grade 6 (least severe) (7Dobyns W.B. Truwit C.L. Ross M.E. Matsumoto N. Pilz D.T. Ledbetter D.H. Gleeson J.G. Walsh C.A. Barkovich A.J. Neurology. 1999; 53: 270-277Crossref PubMed Google Scholar). Grades 1–4 are all lissencephalies of decreasing severity. Grade 5 is mixed pachygyria and SBH, whereas grade 6 is SBH alone. Deletions involving LIS1 are far more common than other mutations (8Pilz D.T. Macha M.E. Precht K.S. Smith A.C. Dobyns W.B. Ledbetter D.H. Genet. Med. 1998; 1: 29-33Abstract Full Text PDF PubMed Scopus (46) Google Scholar, 9Cardoso C. Leventer R.J. Dowling J.J. Ward H.L. Chung J. Petras K.S. Roseberry J.A. Weiss A.M. Das S. Martin C.L. Pilz D.T. Dobyns W.B. Ledbetter D.H. Hum. Mutat. 2002; 19: 4-15Crossref PubMed Scopus (94) Google Scholar); 88% result in a truncated or internally deleted protein. Up to now, only five missense mutations have been found in patients (9Cardoso C. Leventer R.J. Dowling J.J. Ward H.L. Chung J. Petras K.S. Roseberry J.A. Weiss A.M. Das S. Martin C.L. Pilz D.T. Dobyns W.B. Ledbetter D.H. Hum. Mutat. 2002; 19: 4-15Crossref PubMed Scopus (94) Google Scholar, 10Cardoso C. Leventer R.J. Matsumoto N. Kuc J.A. Ramocki M.B. Mewborn S.K. Dudlicek L.L. May L.F. Mills P.L. Das S. Pilz D.T. Dobyns W.B. Ledbetter D.H. Hum. Mol. Genet. 2000; 9: 3019-3028Crossref PubMed Scopus (90) Google Scholar). Our previous analysis indicated that truncated or internally deleted LIS1 protein is unlikely to fold correctly (11Sapir T. Eisenstein M. Burgess H.A. Horesh D. Cahana A. Aoki J. Hattori M. Arai H. Inoue K. Reiner O. Eur. J. Biochem. 1999; 266: 1011-1020Crossref PubMed Scopus (28) Google Scholar). This finding was supported by a study done in cells derived from lissencephaly patients, where no protein was detected from mutated alleles that were expected to result in a truncated protein (12Fogli A. Guerrini R. Moro F. Fernandez-Alvarez E. Livet M.O. Renieri A. Cioni M. Pilz D.T. Veggiotti P. Rossi E. Ballabio A. Carrozzo R. Ann. Neurol. 1999; 45: 154-161Crossref PubMed Scopus (51) Google Scholar). The five patients with point mutations that lead to a single amino acid substitution in LIS1 exhibit a variable phenotypic manifestation. Thus, the patients are classified from grade 3a to grade 6 (13Lo Nigro C.L. Chong C.S. Smith A.C. Dobyns W.B. Carrozzo R. Ledbetter D.H. Hum. Mol. Genet. 1997; 6: 157-164Crossref PubMed Scopus (281) Google Scholar, 14Pilz D.T. Kuc J. Matsumoto N. Bodurtha J. Bernadi B. Tassinari C.A. Dobyns W.B. Ledbetter D.H. Hum. Mol. Genet. 1999; 8: 1757-1760Crossref PubMed Scopus (127) Google Scholar, 15Leventer R.J. Cardoso C. Ledbetter D.H. Dobyns W.B. Neurology. 2001; 57: 416-422Crossref PubMed Scopus (43) Google Scholar). LIS1 is a member of the WD (tryptophan-aspartic acid) repeat family of proteins (1Reiner O. Carrozzo R. Shen Y. Whenert M. Faustinella F. Dobyns W.B. Caskey C.T. Ledbetter D.H. Nature. 1993; 364: 717-721Crossref PubMed Scopus (909) Google Scholar), as deduced from its amino acid sequence. A hallmark of this family is their involvement in multiple protein-protein interactions (16Neer E.J. Schmidt C.J. Nambudripad R. Smith T.F. Nature. 1994; 371: 297-300Crossref PubMed Scopus (1292) Google Scholar), and LIS1 is not an exception (reviewed in Ref. 17Reiner O. Neuron. 2000; 28: 633-636Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). LIS1 contains an N-terminal region of 94 amino acids that includes a Lis-H domain (LIS1 homology domain amino acids 8–37) (18Emes R.D. Ponting C.P. Hum. Mol. Genet. 2001; 10: 2813-2820Crossref PubMed Scopus (153) Google Scholar), a coiled-coil domain (amino acids 51–79) important for dimerization (19Cahana A. Escamez T. Nowakowski R.S. Hayes N.L. Giacobini M. von Holst A. Shmueli O. Sapir T. McConnell S.K. Wurst W. Martinez S. Reiner O. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6429-6434Crossref PubMed Scopus (132) Google Scholar), and seven WD repeats. Interestingly, the five reported mutations occur in conserved domains (15Leventer R.J. Cardoso C. Ledbetter D.H. Dobyns W.B. Neurology. 2001; 57: 416-422Crossref PubMed Scopus (43) Google Scholar). The mutation F31S (10Cardoso C. Leventer R.J. Matsumoto N. Kuc J.A. Ramocki M.B. Mewborn S.K. Dudlicek L.L. May L.F. Mills P.L. Das S. Pilz D.T. Dobyns W.B. Ledbetter D.H. Hum. Mol. Genet. 2000; 9: 3019-3028Crossref PubMed Scopus (90) Google Scholar) is located in the Lis-H domain. The patient has grade 4a(1) lissencephaly manifested by generalized pachygyria with the additional feature of moderate hypoplasia of the cerebellar vermis, an unusual finding for patients with LIS1 mutations. Three mutations, H149R (13Lo Nigro C.L. Chong C.S. Smith A.C. Dobyns W.B. Carrozzo R. Ledbetter D.H. Hum. Mol. Genet. 1997; 6: 157-164Crossref PubMed Scopus (281) Google Scholar), G162S (10Cardoso C. Leventer R.J. Matsumoto N. Kuc J.A. Ramocki M.B. Mewborn S.K. Dudlicek L.L. May L.F. Mills P.L. Das S. Pilz D.T. Dobyns W.B. Ledbetter D.H. Hum. Mol. Genet. 2000; 9: 3019-3028Crossref PubMed Scopus (90) Google Scholar), and S169P (14Pilz D.T. Kuc J. Matsumoto N. Bodurtha J. Bernadi B. Tassinari C.A. Dobyns W.B. Ledbetter D.H. Hum. Mol. Genet. 1999; 8: 1757-1760Crossref PubMed Scopus (127) Google Scholar), cluster in WD2 with variable phenotypes. The patient with mutation H149R has the most severe LIS grade and clinical phenotype of the five patients with missense mutations (grade 3a). The patient with mutation G162S has an unusually mild form of LIS (grade 4a(2)), with a normal IQ, infrequent seizures, and mild clumsiness as his only motor deficit. The patient with mutation S169P is the only known patient with subcortical band heterotopia secondary to a LIS1 mutation and is thus classified as grade 6a. It has been suggested that the patient exhibits somatic mosaicism (14Pilz D.T. Kuc J. Matsumoto N. Bodurtha J. Bernadi B. Tassinari C.A. Dobyns W.B. Ledbetter D.H. Hum. Mol. Genet. 1999; 8: 1757-1760Crossref PubMed Scopus (127) Google Scholar). The fifth mutation, D317H (14Pilz D.T. Kuc J. Matsumoto N. Bodurtha J. Bernadi B. Tassinari C.A. Dobyns W.B. Ledbetter D.H. Hum. Mol. Genet. 1999; 8: 1757-1760Crossref PubMed Scopus (127) Google Scholar), is located in WD5 (grade 4a(1)) manifested by generalized pachygyria. LIS1-protein interactions may be grouped conceptually into two classes: evolutionarily conserved and relatively new interactions. Much can be learned from the interactions that are conserved throughout evolution (20Morris N.R. Efimov V.P. Xiang X. Trends Cell Biol. 1998; 8: 467-470Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). NudF, a LIS1 homolog in Aspergillus nidulans, was identified in a screen for mutants defective in nuclear migration (21Xiang X. Osmani A.H. Osmani S.A. Xin M. Morris N.R. Mol. Biol. Cell. 1995; 6: 297-310Crossref PubMed Scopus (290) Google Scholar). NudC, which controls NudF levels (21Xiang X. Osmani A.H. Osmani S.A. Xin M. Morris N.R. Mol. Biol. Cell. 1995; 6: 297-310Crossref PubMed Scopus (290) Google Scholar), and nudE, a multicopy suppressor of mutated nudF (22Efimov V.P. Morris N.R. J. Cell Biol. 2000; 150: 681-688Crossref PubMed Scopus (147) Google Scholar), both have mammalian homologs that interact with LIS1 (22Efimov V.P. 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Other gene products involved in the same pathway are subunits of the microtubule-based motor protein cytoplasmic dynein, or its regulatory complex dynactin (28Beckwith S.M. Roghi C.H. Liu B. Ronald Morris N. J. Cell Biol. 1998; 143: 1239-1247Crossref PubMed Scopus (77) Google Scholar, 29Xiang X. Beckwith S.M. Morris N.R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2100-2104Crossref PubMed Scopus (286) Google Scholar, 30Xiang X. Han G. Winkelmann D.A. Zuo W. Morris N.R. Curr. Biol. 2000; 10: 603-606Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). LIS1 interacts with subunits of the dynein and dynactin complexes in mammalian cells (25Sasaki S. Shionoya A. Ishida M. Gambello M. Yingling J. Wynshaw-Boris A. Hirotsune S. Neuron. 2000; 28: 681-696Abstract Full Text Full Text PDF PubMed Scopus (442) Google Scholar, 31Smith D.S. Niethammer M. Ayala R. Zhou Y. Gambello M.J. Wynshaw-Boris A. Tsai L.H. Nat. 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Neuron. 2000; 28: 681-696Abstract Full Text Full Text PDF PubMed Scopus (442) Google Scholar, 26Niethammer M. Smith D.S. Ayala R. Peng J. Ko J. Lee M.-S. Morabito M. Tsai L.-H. Neuron. 2000; 28: 697-711Abstract Full Text Full Text PDF PubMed Scopus (432) Google Scholar, 36Feng Y. Olson E.C. Stukenberg P.T. Flanagan L.A. Kirschner M.W. Walsh C.A. Neuron. 2000; 28: 665-679Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar). All these results indicate a conserved mechanism from nuclear movement in the fungus and flies to neuronal migration in the brain. An additional LIS1-interacting protein that is functioning in the dynein/dynactin pathway is CLIP-170 (37Coquelle F.M. Caspi M. Cordelieres F.P. Dompierre J.P. Dujardin D.L. Koifman C. Martin P. Hoogenraad C.C. Akhmanova A. Galjart N. De Mey J.R. Reiner O. Mol. Cell. Biol. 2002; 22: 3089-3102Crossref PubMed Scopus (214) Google Scholar). We also detected an interaction between LIS1 and doublecortin (DCX) that mutations in this gene product also result in lissencephaly (38Caspi M. Atlas R. Kantor A. Sapir T. Reiner O. Hum. Mol. Genet. 2000; 9: 2205-2213Crossref PubMed Scopus (126) Google Scholar). Another well documented interaction is between LIS1 and the two catalytic subunits (α1 and α2) of platelet-activating factor acetylhydrolase (PAF-AH) Ib (39Hattori M. Adachi H. Tsujimoto M. Arai N. Inoue K. Nature. 1994; 370: 216-218Crossref PubMed Scopus (456) Google Scholar, 40Manya H. Aoki J. Watanabe M. Adachi T. Asou H. Inoue Y. Arai H. Inoue K. J. Biol. Chem. 1998; 273: 18567-18572Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). LIS1 is believed to act as the regulatory unit of this enzymatic complex. Based on the Drosophila homologs, this interaction is relatively new in evolution, as Drosophila α-subunit lacks both catalytic activity and the ability to interact with Drosophila LIS1 (41Sheffield P. Garrard S. Caspi M. Aoki J. Inoue K. Derewenda U. Suter B. Reiner O. Derewenda Z.S. Proteins. 2000; 39: 1-8Crossref PubMed Scopus (26) Google Scholar). Taking into consideration our current knowledge regarding LIS1 function, we believe it is important to analyze how these five missense mutations affect protein half-life, cellular localization, and protein-protein interaction. That will promote our understanding as to how the disease is manifested in lissencephaly patients. Cell Culture—Culturing and transfection of HeLa and HEK293 cells were performed as described previously (37Coquelle F.M. Caspi M. Cordelieres F.P. Dompierre J.P. Dujardin D.L. Koifman C. Martin P. Hoogenraad C.C. Akhmanova A. Galjart N. De Mey J.R. Reiner O. Mol. Cell. Biol. 2002; 22: 3089-3102Crossref PubMed Scopus (214) Google Scholar). Immunological Methods—Cells were fixed in 100% methanol plus 1 mm EGTA for 10 min at -20 °C, followed by 15 min in 4% paraformaldehyde in PHEM buffer (60 mm Pipes, 25 mm Hepes, 10 mm EGTA, 2 mm magnesium acetate, pH 6.9). They were washed three times for 5 min each in PBS (phosphate-buffered saline) and further permeabilized for 25 min in 0.1% Triton X-100 in PBS. The cells were treated with 50 mm NH4Cl in PBS for 10 min, washed three times for 5 min each in PBS, blocked in PBS-BSA (PBS containing 0.1% BSA), and labeled with the polyclonal anti-CLIP-170 antibody, TA (37Coquelle F.M. Caspi M. Cordelieres F.P. Dompierre J.P. Dujardin D.L. Koifman C. Martin P. Hoogenraad C.C. Akhmanova A. Galjart N. De Mey J.R. Reiner O. Mol. Cell. Biol. 2002; 22: 3089-3102Crossref PubMed Scopus (214) Google Scholar), a monoclonal anti-EB1 antibody (Transduction Laboratories, E46420/Lot 1), or c-Myc antibody 9E10 for 1 h at 37 °C. After being washed in PBS-BSA, the cells were incubated with the Alexa-488 secondary antibodies (Molecular Probes) for 45 min at 37 °C. The cells were post-fixed in 4% formaldehyde in PBS for 16 min and treated with 50 mm NH4Cl for 10 min. The chromosomes were stained with DAPI (Sigma) for 5 min. Coverslips were mounted using PBS plus 15% glycerol containing antifading agent 1,4-diazabicyclo(2.2.2)octane (Sigma) at 100 mg ml-1. Pictures of fixed cells were collected using a three-dimensional deconvolution imaging system, 2J. R. De Mey, unpublished data. or using Olympus IX-70 microscope with a digital camera. Constructs—Site-directed mutagenesis was performed using PCR in LIS1-FLAG-DsRed or pAGA-LIS1 vectors. Based on the patients' mutations (13Lo Nigro C.L. Chong C.S. Smith A.C. Dobyns W.B. Carrozzo R. Ledbetter D.H. Hum. Mol. Genet. 1997; 6: 157-164Crossref PubMed Scopus (281) Google Scholar, 14Pilz D.T. Kuc J. Matsumoto N. Bodurtha J. Bernadi B. Tassinari C.A. Dobyns W.B. Ledbetter D.H. Hum. Mol. Genet. 1999; 8: 1757-1760Crossref PubMed Scopus (127) Google Scholar, 15Leventer R.J. Cardoso C. Ledbetter D.H. Dobyns W.B. Neurology. 2001; 57: 416-422Crossref PubMed Scopus (43) Google Scholar), we synthesized the following oligonucleotides: F31S, 5′-gaggcatattcagtttctaaaaaggaagctgaat; H149R, 5′-cgaactttaaaggacgtacagactctgtacag; G162S, 5′-catttcattcgaccacagcagcaagcttctggcttcc; S169P, 5′-gcttctggcttcctgtcctgcagatatgacc; D317H, 5′-cttgctgtctggatccagacacaagactattaagatgtg. The point mutations were introduced using PCR mutagenesis. A domain of the dynein heavy chain (DHC) previously reported to interact with LIS1 (25Sasaki S. Shionoya A. Ishida M. Gambello M. Yingling J. Wynshaw-Boris A. Hirotsune S. Neuron. 2000; 28: 681-696Abstract Full Text Full Text PDF PubMed Scopus (442) Google Scholar) was produced by RT-PCR using the following primers: forward, 5′-gaggaattcacgcccctcactgaccgttg; reverse, 5′-ctcctcgaggaagagtccgtctgtccac. All PCR products were fully sequenced. PCR products were cloned into the pGEX and pCDNA3 vectors into EcoRI and XhoI sites. All LIS1 mutations were introduced into the bait vector of the two-hybrid system (pEG-202 plasmid; OriGene Technologies, Inc.), using EcoRI and XhoI sites. The interacting proteins were subcloned to the prey vector (pJG4-5 plasmid; OriGene Technologies, Inc.). WD constructs were described previously (38Caspi M. Atlas R. Kantor A. Sapir T. Reiner O. Hum. Mol. Genet. 2000; 9: 2205-2213Crossref PubMed Scopus (126) Google Scholar), as well as c-Myc-NudE, NudEL, and GFP-CLIP-Cter (37Coquelle F.M. Caspi M. Cordelieres F.P. Dompierre J.P. Dujardin D.L. Koifman C. Martin P. Hoogenraad C.C. Akhmanova A. Galjart N. De Mey J.R. Reiner O. Mol. Cell. Biol. 2002; 22: 3089-3102Crossref PubMed Scopus (214) Google Scholar). Protein Half-life Assay—293 cells were transfected by LIS1-FLAG-DsRed (wild type or mutations) in 6-well plates using the CaCl2 method. Half-life was measured using pulse-chase as described (42Bonifacino J.S. Dasso M. Harford J.B. Lippincott-Schwartz J. Yamada K.M. Morgan K.S. Current Protocols in Cell Biology. John Wiley & Sons, Inc., New York1999: 7.1.5Google Scholar) with some modifications. Prior to addition of [35S]methionine, cells were washed and starved. The pulse was for 1 h, followed by addition of an excess of cold methionine. Ten time points were picked (0, 2, 4,6,8,9,10,12,18, and 24 h). The cells were washed and collected in IP buffer (50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 1% Triton X-100), supplemented with protease inhibitors (Sigma). Immunoprecipitations were performed using M2 beads (Sigma), run on 10% PAGE gels. The gels were fixed, and the signal was enhanced by 30-min incubation with sodium salicylate solution (16%), dried, and exposed to phosphoimager (BAS-2500, Fujifilm). The signal was quantified using phosphoimager analysis, and the results were plotted. The half-life was deduced from the plotted results. Trypsin Cleavage—LIS1 protein (wild-type and mutations) was translated in a rabbit reticulocyte TnT system (Promega, Madison, WI) with [35S]methionine. A portion of the labeled reaction (10 μl) was used for tryptic cleavage as described (11Sapir T. Eisenstein M. Burgess H.A. Horesh D. Cahana A. Aoki J. Hattori M. Arai H. Inoue K. Reiner O. Eur. J. Biochem. 1999; 266: 1011-1020Crossref PubMed Scopus (28) Google Scholar). His 6 Pull-down Assay—Recombinant His6 proteins were incubated with transfected 293 cell extract (prepared as described) or rabbit reticulocyte lysate. Pull-down assay was performed as described (38Caspi M. Atlas R. Kantor A. Sapir T. Reiner O. Hum. Mol. Genet. 2000; 9: 2205-2213Crossref PubMed Scopus (126) Google Scholar). Immunoprecipitation Assay—Immunoprecipitation was preformed in 293 cells using M2 beads (Sigma). Cells were co-transfected with LIS1-FLAG-DsRed (wild-type or mutated) and each of the interacting proteins. Transfected cell extracts were prepared in IP buffer, supplemented with protease inhibitors (Sigma). Each 10-cm plate was washed with PBS, put on ice, and scraped with 1 ml of cold IP buffer. The cells were incubated on ice for 15 min and were vortexed for 10 s every 5 min. After centrifugation at 9000 rpm for 15 min at 4 °C, sample buffer was added and the beads were boiled. The samples were separated by SDS-PAGE and Western blotted with the suitable combination of antibodies (α-LIS1 (monoclonal clone 210; Ref. 35Sapir T. Elbaum M. Reiner O. EMBO J. 1997; 16: 6977-6984Crossref PubMed Scopus (266) Google Scholar)), α-FLAG (M2 clone from Sigma), α-myc (9E10 clone from Sigma), α-PAF-AH-α1 antibodies (monoclonal clone 9C9; Ref. 43Koizumi H. Yamaguchi N. Hattori M. Ishikawa T.O. Aoki J. Taketo M.M. Inoue K. Arai H. J. Biol. Chem. 2003; 278: 12489-12494Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar)). Yeast Two-hybrid—Yeast transformation and detection of interactions were performed according to manual (OriGene Technologies, Inc.); briefly, three plasmids were transformed into the yeast strain EGY48; pEG-202-bait, pJG4-5-prey and pSH18-34-LacZ (from the DupLEX-A system, OriGene Technologies, Inc.). The baits were LIS1 wild type or one of the five mutations, and the preys were PAF-AH α1, PAF-AH α2, CLIP-170, and NudEL. The yeast cells were grown to an A 600 of 0.6–0.8, harvested at 1500 × g for 3 min, and resuspended in TE (one third of initial volume). The cells were then pelleted again, resuspended in the same volume of TE/LiOAc, and rotated slowly at 30 °C for 2 h. Afterward, the cells were pelleted and resuspended in TE, 0.1 m LiOAc (100 μl/transformation). The desired bait and prey plasmids plus the LacZ plasmid and 10 μg of salmon sperm DNA were added to the cells. After a 10-min incubation at 30 °C, 500 μl of TE, 0.1 m LiOAc, 50% PEG-3350 was added to each tube and the cells were incubated for 45 more min. Heat shock was performed at 42 °C for 5 min, and the cells were then spun down, resuspended in TE, and plated on glucose-based plates lacking His, Trp, and Ura (SD-H-T-U). The plates were incubated at 30 °C 2–3 days until colonies appeared. From each interaction 4–5 colonies were streaked on galactose-based plates (SG-H-T-U) containing 1 mg/ml X-gal and incubated overnight at 30 °C until the appearance of a blue color. WD Binding to Microtubules—Phosphocellulose-purified tubulin was assembled into microtubules with addition of Taxol® (paclitaxel; final concentration 30 μm) or nocodazole (10 μg/ml) as described (44Horesh D. Sapir T. Francis F. Caspi M. Grayer Wolf S. Elbaum M. Chelly J. Reiner O. Hum. Mol. Genet. 1999; 8: 1599-1610Crossref PubMed Scopus (227) Google Scholar). The assembled microtubules were mixed with 10 μl of [35S]methioninelabeled proteins (LIS1 and the different WD peptides) translated in the rabbit reticulocyte TnT system (Promega). The mixtures were incubated at 37 °C for 10 min and separated into two fractions as described (44Horesh D. Sapir T. Francis F. Caspi M. Grayer Wolf S. Elbaum M. Chelly J. Reiner O. Hum. Mol. Genet. 1999; 8: 1599-1610Crossref PubMed Scopus (227) Google Scholar). The insoluble fractions (pellets) of each experiment were run on 15% SDS-PAGE gels and stained with Coomassie Brilliant Blue (Bio-Rad). After destaining the gels were washed with H2O for 20 min and the signal was enhanced by 30-min incubation with sodium salicylate solution (16%) at room temperature. LIS1 is an essential protein that controls multiple cellular functions through participation in different protein complexes. Deciphering the precise details of LIS1-protein interaction modes under normal and abnormal conditions may allow us to better understand processes of neuronal migration and brain development. For this analysis the existence of several disease causing amino acid substitutions was utilized. Our analysis included several assays to determine whether there were global effects of amino acid substitutions; protein folding was tested by limited trypsin cleavage, LIS1 stability was monitored by examining protein degradation, and its intracellular localization was inspected by immunostaining. To resolve whether LIS1 mutations affect specific protein interactions, we first mapped domains of interaction between LIS1 and some of its protein partners. The described protein mutants were then used to test whether they retain the ability to bind these known interacting proteins. LIS1 Folding—It has been shown that multiple WD-containing proteins (including LIS1) retain the β-propeller-like structure similar to the β-subunits of heterotrimeric G-proteins (45Garcia-Higuera I. Fenoglio J. Li Y. Lewis C. Panchenko M.P. Reiner O. Smith T.F. Neer E.J. Biochemistry. 1996; 35: 13985-13994Crossref PubMed Scopus (164) Google Scholar). Thus, the WD-containing region is protected when these proteins are subjected to limited proteolytic cleavage. In the following assay, LIS1 carrying the different mutations was expressed in vitro in rabbit reticulocytes and labeled with [35S]methionine. When the lysate was subjected to limited trypsin treatment, three protected bands were visible in case of the wild type LIS1 protein (Fig. 1), as has been observed before (45Garcia-Higuera I. Fenoglio J. Li Y. Lewis C. Panchenko M.P. Reiner O. Smith T.F. Neer E.J. Biochemistry. 1996; 35: 13985-13994Crossref PubMed Scopus (164) Google Scholar). Among the mutants, there were two protected bands in the case of the N-terminal mutati
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