Src Signaling Regulates Completion of Abscission in Cytokinesis through ERK/MAPK Activation at the Midbody
2006; Elsevier BV; Volume: 282; Issue: 8 Linguagem: Inglês
10.1074/jbc.m608396200
ISSN1083-351X
AutoresKousuke Kasahara, Yuji Nakayama, Yoshimi Nakazato, Kikuko Ikeda, Takahisa Kuga, Naoto Yamaguchi,
Tópico(s)Wnt/β-catenin signaling in development and cancer
ResumoSrc family non-receptor-type tyrosine kinases regulate a wide variety of cellular events including cell cycle progression in G2/M phase. Here, we show that Src signaling regulates the terminal step in cytokinesis called abscission in HeLa cells. Abscission failure with an unusually elongated intercellular bridge containing the midbody is induced by treatment with the chemical Src inhibitors PP2 and SU6656 or expression of membrane-anchored Csk chimeras. By anti-phosphotyrosine immunofluorescence and live cell imaging, completion of abscission requires Src-mediated tyrosine phosphorylation during early stages of mitosis (before cleavage furrow formation), which is subsequently delivered to the midbody through Rab11-driven vesicle transport. Treatment with U0126, a MEK inhibitor, decreases tyrosine phosphorylation levels at the midbody, leading to abscission failure. Activated ERK by MEK-catalyzed dual phosphorylation on threonine and tyrosine residues in the TEY sequence, which is strongly detected by anti-phosphotyrosine antibody, is transported to the midbody in a Rab11-dependent manner. Src kinase activity during the early mitosis mediates ERK activation in late cytokinesis, indicating that Src-mediated signaling for abscission is spatially and temporally transmitted. Thus, these results suggest that recruitment of activated ERK, which is phosphorylated by MEK downstream of Src kinases, to the midbody plays an important role in completion of abscission. Src family non-receptor-type tyrosine kinases regulate a wide variety of cellular events including cell cycle progression in G2/M phase. Here, we show that Src signaling regulates the terminal step in cytokinesis called abscission in HeLa cells. Abscission failure with an unusually elongated intercellular bridge containing the midbody is induced by treatment with the chemical Src inhibitors PP2 and SU6656 or expression of membrane-anchored Csk chimeras. By anti-phosphotyrosine immunofluorescence and live cell imaging, completion of abscission requires Src-mediated tyrosine phosphorylation during early stages of mitosis (before cleavage furrow formation), which is subsequently delivered to the midbody through Rab11-driven vesicle transport. Treatment with U0126, a MEK inhibitor, decreases tyrosine phosphorylation levels at the midbody, leading to abscission failure. Activated ERK by MEK-catalyzed dual phosphorylation on threonine and tyrosine residues in the TEY sequence, which is strongly detected by anti-phosphotyrosine antibody, is transported to the midbody in a Rab11-dependent manner. Src kinase activity during the early mitosis mediates ERK activation in late cytokinesis, indicating that Src-mediated signaling for abscission is spatially and temporally transmitted. Thus, these results suggest that recruitment of activated ERK, which is phosphorylated by MEK downstream of Src kinases, to the midbody plays an important role in completion of abscission. Cytokinesis is the last stage of mitosis when a single cell divides into two daughter cells after chromosome segregation. Defects in cytokinesis can induce cell death and genetic instability that brings about the development of aneuploid malignancies (1Storchova Z. Pellman D. Nat. Rev. Mol. Cell Biol. 2004; 5: 45-54Crossref PubMed Scopus (609) Google Scholar). The process of cytokinesis must be spatially and temporally controlled in a sophisticated manner. During cytokinesis, two daughter cells are connected in a pair with a cytoplasmic bridge containing the midbody, which consists of bundled anti-parallel microtubules and associated proteins. The overlapped region of plus ends of microtubules forms a dense structure called the midbody matrix (2Otegui M.S. Verbrugghe K.J. Skop A.R. Trends Cell Biol. 2005; 15: 404-413Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 3Skop A.R. Liu H. Yates J. II I Meyer B.J. Heald R. Science. 2004; 305: 61-66Crossref PubMed Scopus (384) Google Scholar). These microtubule-based structures play key roles in cytokinesis from initiation to completion. Membrane trafficking ceases during early stages of mitosis, however, it resumes at late mitosis: internalized vesicles are trafficked to the recycling endosome during cleavage furrow ingression, and subsequently to the midbody during late stages of cytokinesis (4Schweitzer J.K. Burke E.E. Goodson H.V. D'Souza-Schorey C. J. Biol. Chem. 2005; 280: 41628-41635Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). There is accumulating evidence that coordinated membrane trafficking to the cleavage furrow and the midbody is essential for completion of cytokinesis (2Otegui M.S. Verbrugghe K.J. Skop A.R. Trends Cell Biol. 2005; 15: 404-413Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 3Skop A.R. Liu H. Yates J. II I Meyer B.J. Heald R. Science. 2004; 305: 61-66Crossref PubMed Scopus (384) Google Scholar, 4Schweitzer J.K. Burke E.E. Goodson H.V. D'Souza-Schorey C. J. Biol. Chem. 2005; 280: 41628-41635Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 5Finger F. White J. Cell. 2002; 108: 727-730Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 6Albertson R. Riggs B. Sullivan W. Trends Cell Biol. 2005; 15: 92-101Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 7Skop A.R. Bergman D. Mohler W.A. White J.G. Curr. Biol. 2001; 11: 735-746Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar, 8Wilson G.M. Fielding A.B. Simon G.C. Yu X. Andrews P.D. Hames R.S. Frey A.M. Peden A.A. Gould G.W. Prekeris R. Mol. Biol. Cell. 2005; 16: 849-860Crossref PubMed Scopus (234) Google Scholar, 9Tomas A. Futter C. Moss S.E. J. Cell Biol. 2004; 165: 813-822Crossref PubMed Scopus (82) Google Scholar, 10Gromley A. Yeaman C. Rosa J. Redick S. Chen C. Mirabelle S. Guha M. Sillibourne J. Doxsey S.J. Cell. 2005; 123: 75-87Abstract Full Text Full Text PDF PubMed Scopus (330) Google Scholar). For instance, inhibition of membrane transport via recycling endosomes (by expression of small interfering RNA for Rab11 or dominant-negative Rab11) or dispersion of the Golgi apparatus (by brefeldin A) causes defects in the late stage of cytokinesis in both Caenorhabditis elegans and mammalian cells (7Skop A.R. Bergman D. Mohler W.A. White J.G. Curr. Biol. 2001; 11: 735-746Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar, 8Wilson G.M. Fielding A.B. Simon G.C. Yu X. Andrews P.D. Hames R.S. Frey A.M. Peden A.A. Gould G.W. Prekeris R. Mol. Biol. Cell. 2005; 16: 849-860Crossref PubMed Scopus (234) Google Scholar, 9Tomas A. Futter C. Moss S.E. J. Cell Biol. 2004; 165: 813-822Crossref PubMed Scopus (82) Google Scholar). However, signal transduction mediated via recycling endosomes at the midbody is unknown. Src family kinases (SFKs), 3The abbreviations used are: SFKs, Src family protein-tyrosine kinases; ERK, extracellular signal-regulated kinase; GFP, green fluorescent protein; MAPK, mitogen-activated protein kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase; HA, hemagglutinin; PBS, phosphate-buffered saline; TRITC, tetramethylrhodamine isothiocyanate; GM1, ganglioside GM1. 3The abbreviations used are: SFKs, Src family protein-tyrosine kinases; ERK, extracellular signal-regulated kinase; GFP, green fluorescent protein; MAPK, mitogen-activated protein kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase; HA, hemagglutinin; PBS, phosphate-buffered saline; TRITC, tetramethylrhodamine isothiocyanate; GM1, ganglioside GM1. which belong to a family of non-receptor-type protein-tyrosine kinases, are activated by various stimuli, and are involved in a wide range of signaling events, such as proliferation, migration, and cytoskeletal reorganization (11Thomas S.M. Brugge J.S. Annu. Rev. Cell Dev. Biol. 1997; 13: 513-609Crossref PubMed Scopus (2134) Google Scholar). SFKs are localized to membranes through their N-terminal myristoylation (11Thomas S.M. Brugge J.S. Annu. Rev. Cell Dev. Biol. 1997; 13: 513-609Crossref PubMed Scopus (2134) Google Scholar). The catalytic activity of SFKs is suppressed by phosphorylation of their C-terminal tyrosine residues by Csk family tyrosine kinases (11Thomas S.M. Brugge J.S. Annu. Rev. Cell Dev. Biol. 1997; 13: 513-609Crossref PubMed Scopus (2134) Google Scholar, 12Blume-Jensen P. Hunter T. Nature. 2001; 411: 355-365Crossref PubMed Scopus (3073) Google Scholar, 13Nada S. Okada M. McAuley A. Cooper J.A. Nakagawa H. Nature. 1991; 351: 69-71Crossref PubMed Scopus (509) Google Scholar, 14Chow L.M. Fournel M. Davidson D. Veillette A. Nature. 1993; 365: 156-160Crossref PubMed Scopus (236) Google Scholar, 15Honda Z. Suzuki T. Hirose N. Aihara M. Shimizu T. Nada S. Okada M. Ra C. Morita Y. Ito K. J. Biol. Chem. 1997; 272: 25753-25760Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 16Ohsato Y. Nada S. Okada M. Nakagawa H. Cancer Sci. 1994; 85: 1023-1028Google Scholar, 17Kasahara K. Nakayama Y. Ikeda K. Fukushima Y. Matsuda D. Horimoto S. Yamaguchi N. J. Cell Biol. 2004; 165: 641-652Crossref PubMed Scopus (68) Google Scholar). Efficient SFK inhibition is induced by Csk translocation from the cytosol to the plasma membrane (14Chow L.M. Fournel M. Davidson D. Veillette A. Nature. 1993; 365: 156-160Crossref PubMed Scopus (236) Google Scholar, 15Honda Z. Suzuki T. Hirose N. Aihara M. Shimizu T. Nada S. Okada M. Ra C. Morita Y. Ito K. J. Biol. Chem. 1997; 272: 25753-25760Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 16Ohsato Y. Nada S. Okada M. Nakagawa H. Cancer Sci. 1994; 85: 1023-1028Google Scholar). Previous studies showed that the catalytic activity of SFKs is required for mitotic progression, because G2/M progression is prevented by microinjection of a neutralizing anti-Src/Fyn/Yes antibody or the SH2 domain of Fyn (18Roche S. Fumagalli S. Courtneidge S.A. Science. 1995; 269: 1567-1569Crossref PubMed Scopus (236) Google Scholar), and by treatment with PD173955, a chemical inhibitor of SFKs (19Moasser M.M. Srethapakdi M. Sachar K.S. Kraker A.J. Rosen N. Cancer Res. 1999; 59: 6145-6152PubMed Google Scholar). The regulation of SFK activity in mitosis is dependent on dephosphorylation of their C-terminal tyrosine residues because c-Src is not activated during mitosis in protein-tyrosine phosphatase α knockout cells (20Zheng X. Shalloway D. EMBO J. 2001; 20: 6037-6049Crossref PubMed Scopus (57) Google Scholar). The activity of c-Src and accessibility of its SH2 domain for protein binding are increased at the initial phase of mitosis (19Moasser M.M. Srethapakdi M. Sachar K.S. Kraker A.J. Rosen N. Cancer Res. 1999; 59: 6145-6152PubMed Google Scholar, 21Bagrodia S. Chackalaparampil I. Kmiecik T.E. Shalloway D. Nature. 1991; 349: 172-175Crossref PubMed Scopus (95) Google Scholar, 22Chackalaparampil I. Shalloway D. Cell. 1988; 52: 801-810Abstract Full Text PDF PubMed Scopus (187) Google Scholar, 23Bagrodia S. Laudano A.P. Shalloway D. J. Biol. Chem. 1994; 269: 10247-10251Abstract Full Text PDF PubMed Google Scholar), suggesting that SFKs may play a regulatory role in the entry into mitosis. However, tyrosine phosphorylation signaling thus far has not been fully characterized in cytokinesis. In this study, we explored SFK-mediated tyrosine phosphorylation in cytokinesis by immunofluorescence staining for phosphotyrosine and live cell imaging experiments using two chemical compounds, PP2 and SU6656, and two membrane-anchored Csk mutants, all of which act as potent inhibitors of SFKs. We show that SFK-mediated tyrosine phosphorylation signaling that is spatially and temporally transmitted is required for completion of abscission in cytokinesis. Plasmids—cDNA encoding human c-Src (1–536; with 1 designating the initiator methionine) (provided by D. J. Fujita; Ref. 24Bjorge J.D. Bellagamba C. Cheng H.C. Tanaka A. Wang J.H. Fujita D.J. J. Biol. Chem. 1995; 270: 24222-24228Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar) and Src-GFP (1–532; green fluorescent protein-fused, constitutively kinase-active) were described (25Kasahara K. Nakayama Y. Sato I. Ikeda K. Hoshino M. Endo T. Yamaguchi N. J. Cell Physiol. 2006; 10.1002/jcp.20931Google Scholar). Src258-GFP (1–258; kinase domain-deleted) was generated from Src-GFP. cDNA encoding human Lyn was provided by T. Yamamoto (26Yamanashi Y. Fukushige S. Semba K. Sukegawa J. Miyajima N. Matsubara K. Yamamoto T. Toyoshima K. Mol. Cell. Biol. 1987; 7: 237-243Crossref PubMed Scopus (164) Google Scholar). GFP- and HA epitope-tagged Lyn constructs lacking the C-terminal negative-regulatory tail, Lyn-GFP (1–506; constitutively kinase-active), Lyn-HA (1–506; constitutively kinase-active), and Lyn-K275A-HA (1–506; kinase-inactive), were previously described (17Kasahara K. Nakayama Y. Ikeda K. Fukushima Y. Matsuda D. Horimoto S. Yamaguchi N. J. Cell Biol. 2004; 165: 641-652Crossref PubMed Scopus (68) Google Scholar). Rat Csk cDNA was provided by M. Okada and S. Nada (13Nada S. Okada M. McAuley A. Cooper J.A. Nakagawa H. Nature. 1991; 351: 69-71Crossref PubMed Scopus (509) Google Scholar). Src16-Csk and Lyn25-Csk were constructed by fusion with the respective N-terminal sequences of c-Src-(1–16) and Lyn-(1–25) at the N terminus of full-length Csk. cDNAs encoding human c-Abl-1b (27Shtivelman E. Lifshitz B. Gale R.P. Canaani E. Nature. 1985; 315: 550-554Crossref PubMed Scopus (1241) Google Scholar) and human Syk (28Law C.L. Sidorenko S.P. Chandran K.A. Draves K.E. Chan A.C. Weiss A. Edelhoff S. Disteche C.M. Clark E.A. J. Biol. Chem. 1994; 269: 12310-12319Abstract Full Text PDF PubMed Google Scholar) tyrosine kinases were provided by E. Canaani and E. A. Clark, respectively. All constructs described were subcloned into the pcDNA4/TO vector (Invitrogen). HA-Rab11S25N subcloned into the pcDNA3.1 vector (Invitrogen) was provided by S. S. G. Ferguson (29Dale L.B. Seachrist J.L. Babwah A.V. Ferguson S.S.G. J. Biol. Chem. 2004; 279: 13110-13118Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). Chemicals—PP2 (30Hanke J.H. Gardner J.P. Dow R.L. Changelian P.S. Brissette W.H. Weringer E.J. Pollok B.A. Connelly P.A. J. Biol. Chem. 1996; 271: 695-701Abstract Full Text Full Text PDF PubMed Scopus (1775) Google Scholar) and SU6656 (31Blake R.A. Broome M.A. Liu X. Wu J. Gishizky M. Sun L. Courtneidge S.A. Mol. Cell. Biol. 2000; 20: 9018-9027Crossref PubMed Scopus (528) Google Scholar) were purchased from Calbiochem. U0126 (Calbiochem), SB203580 (Sigma), and SP600125 (Biomol) were gifts from T. Murayama (32Akiyama N. Shimma N. Takashiro Y. Hatori Y. Hirabayashi T. Horie S. Saito T. Murayama T. Cell. Signal. 2005; 17: 597-604Crossref PubMed Scopus (9) Google Scholar). All chemicals were dissolved in Me2SO at 10 (PP2 and SB203580) or 20 mm (SU6656, U0126, and SP600125). Antibodies—The following antibodies were used: phosphotyrosine (Tyr(P)) (4G10; Upstate Biotechnology, Inc.), Src[pY418] (phospho-Src family; BioSource), Src (clone 327, Oncogene Research Products; and N-16, Santa Cruz Biotechnology), Lyn (H-6 and Lyn44, Santa Cruz Biotechnology), Csk (clone 52, BD Transduction Laboratories; and C-20, Santa Cruz Biotechnology), Abl (8E9, BD Pharmingen), Syk (4D10, Santa Cruz Biotechnology), phospho-p44/42 MAP kinase Thr202/Tyr204 (pERK1/2) (E10, New England Biolabs), ERK2 (C-14, Santa Cruz Biotechnology), HA epitope (Y-11 and F-7, Santa Cruz Biotechnology), actin (CHEMICON International, Inc.), and α-tubulin (YOL-1/34, Serotec). Fluorescein isothiocyanate- or TRITC-conjugated F(ab′)2 fragments of secondary antibodies were obtained from Sigma and BioSource. Horseradish peroxidase-conjugated F(ab′)2 fragments of secondary antibodies were purchased from Amersham Biosciences. Cells and Transient Transfection—HeLa and HeLa S3 (Japanese Collection of Research Bioresources), A431 (provided by M. N. Fukuda), COS-1, MCF-7 (provided by H. Saya), HCT116 (provided by T. Tomonaga), and SYF (provided by M. Okada and S. Nada; Ref. 33Klinghoffer R.A. Sachsenmaier C. Cooper J.A. Soriano P. EMBO J. 1999; 18: 2459-2471Crossref PubMed Scopus (644) Google Scholar) cells were cultured in Isocove's modified Dulbecco's modified essential medium containing 5% fetal bovine serum. Transient transfection was performed using TransIT transfection reagent (Mirus), according to the manufacturer's instructions, as reported previously (17Kasahara K. Nakayama Y. Ikeda K. Fukushima Y. Matsuda D. Horimoto S. Yamaguchi N. J. Cell Biol. 2004; 165: 641-652Crossref PubMed Scopus (68) Google Scholar, 34Yamaguchi N. Nakayama Y. Urakami T. Suzuki S. Nakamura T. Suda T. Oku N. J. Cell Sci. 2001; 114: 1631-1641Crossref PubMed Google Scholar, 35Nakayama Y. Yamaguchi N. Exp. Cell Res. 2005; 304: 570-581Crossref PubMed Scopus (40) Google Scholar). Immunofluorescence—Immunofluorescence staining was detected using a Fluoview FV500 confocal laser scanning microscope with a 40 × 1.00 NA oil objective (Olympus) as described (17Kasahara K. Nakayama Y. Ikeda K. Fukushima Y. Matsuda D. Horimoto S. Yamaguchi N. J. Cell Biol. 2004; 165: 641-652Crossref PubMed Scopus (68) Google Scholar, 34Yamaguchi N. Nakayama Y. Urakami T. Suzuki S. Nakamura T. Suda T. Oku N. J. Cell Sci. 2001; 114: 1631-1641Crossref PubMed Google Scholar, 35Nakayama Y. Yamaguchi N. Exp. Cell Res. 2005; 304: 570-581Crossref PubMed Scopus (40) Google Scholar, 36Matsuda D. Nakayama Y. Horimoto S. Kuga T. Ikeda K. Kasahara K. Yamaguchi N. Exp. Cell Res. 2006; 312: 1205-1217Crossref PubMed Scopus (63) Google Scholar, 37Yamaguchi N. Fukuda M.N. J. Biol. Chem. 1995; 270: 12170-12176Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 38Tada J. Omine M. Suda T. Yamaguchi N. Blood. 1999; 93: 3723-3735Crossref PubMed Google Scholar). Cells were fixed in phosphate-buffered saline (PBS) containing 3% paraformaldehyde for 20 min, and permeabilized in PBS containing 0.1% saponin and 3% bovine serum albumin at room temperature. Cells were subsequently stained with an appropriate primary antibody for 1 h, washed with PBS containing 0.1% saponin, and stained with fluorescein isothiocyanate- or TRITC-conjugated secondary antibody for 1 h. For anti-Tyr(P) (4G10) staining of the midbody, cells were in situ treated with 0.5% Triton X-100 at 4 °C for 3 min before fixation with 3% paraformaldehyde (39Nakayama Y. Kawana A. Igarashi A. Yamaguchi N. Exp. Cell Res. 2006; 312: 2252-3363Crossref PubMed Scopus (26) Google Scholar). DNA was stained with 100 μg/ml propidium iodide for 20 min after treatment with 200 μg/ml RNase A (34Yamaguchi N. Nakayama Y. Urakami T. Suzuki S. Nakamura T. Suda T. Oku N. J. Cell Sci. 2001; 114: 1631-1641Crossref PubMed Google Scholar). Emission signals were detected between 505 and 530 nm for fluorescein, at more than 580 nm for rhodamine and more than 650 nm for propidium iodide. Care was taken to ensure that there was no bleed-through from the fluorescein signal into the red channel (38Tada J. Omine M. Suda T. Yamaguchi N. Blood. 1999; 93: 3723-3735Crossref PubMed Google Scholar). Time Lapse Phase-contrast Imaging—HeLa cells cultured in Isocove's modified Dulbecco's modified essential medium supplemented with 10% fetal bovine serum and 20 mm HEPES (pH 7.4) were placed on a 40 °C preheated stage of an inverted Zeiss Axiovert S100 deconvolution microscope with 10 × 0.30 N.A. and 20 × 0.50 N.A. objectives, and monitored in the presence of various inhibitors. Time lapse monitoring started within 5 min after addition of an inhibitor to living HeLa cells at each stage of mitosis. Each stage is morphologically classified as follows: metaphase (sister chromatids are aligned in the center of cell), anaphase (sister chromatids are separated and moved to opposite poles), telophase (the cleavage furrow is formed between the segregated chromosomes), and cytokinesis (two daughter cells are attached and connected by an intercellular bridge containing the midbody). Images were analyzed with MetaMorph version 4.5 (Universal Imaging). Cell Synchronization—HeLa S3 cells were synchronized using successive aphidicolin and nocodazole blocks, because prolonged treatment with nocodazole for mitotic arrest has adverse effects on synchronous release of cells from nocodazole arrest. HeLa S3 cells grown as monolayers in Isocove's modified Dulbecco's modified essential medium containing 5% fetal bovine serum were blocked at S phase by addition of 1.6 μg/ml aphidicolin. After 16–20 h, the monolayers were washed twice with PBS. The cells were cultured for an additional 6–7 h, then exposed to 40 ng/ml nocodazole for 5–6 h. Round-shaped cells in mitotic stages were gently pipetted and collected by brief centrifugation. Mitotic cells were released from nocodazole treatment by extensively washing with pre-warmed PBS, and subsequently incubating in normal medium at 37 °C in suspension culture. Progression through mitosis was monitored every 10 min by immunofluorescence using anti-α-tubulin antibody and propidium iodide (see Fig. 3, A–C). For immunofluorescence, cells were directly fixed with 2% paraformaldehyde and then attached on coverslips by brief cytocentrifugation. Western Blotting and Immunoprecipitation—Western blotting and immunoprecipitation were performed as described (17Kasahara K. Nakayama Y. Ikeda K. Fukushima Y. Matsuda D. Horimoto S. Yamaguchi N. J. Cell Biol. 2004; 165: 641-652Crossref PubMed Scopus (68) Google Scholar, 35Nakayama Y. Yamaguchi N. Exp. Cell Res. 2005; 304: 570-581Crossref PubMed Scopus (40) Google Scholar, 39Nakayama Y. Kawana A. Igarashi A. Yamaguchi N. Exp. Cell Res. 2006; 312: 2252-3363Crossref PubMed Scopus (26) Google Scholar, 40Hirao A. Hamaguchi I. Suda T. Yamaguchi N. EMBO J. 1997; 16: 2342-2351Crossref PubMed Scopus (66) Google Scholar, 41Hirao A. Huang X-L. Suda T. Yamaguchi N. J. Biol. Chem. 1998; 273: 10004-10010Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 42Mera A. Suga M. Ando M. Suda T. Yamaguchi N. J. Biol. Chem. 1999; 274: 15766-15774Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). In brief, cell lysates were prepared in Triton X-100 lysis buffer (50 mm HEPES, pH 7.4, 10% glycerol, 1% Triton X-100, 4 mm EDTA, 100 mm NaF, and 1 mm Na3VO4) or RIPA lysis buffer (50 mm HEPES, pH 7.4, 10% glycerol, 1% Triton X-100, 0.1% SDS, 1% sodium deoxycholate, 4 mm EDTA, 100 mm NaF, and 1 mm Na3VO4) containing protease inhibitors (2 mm phenylmethylsulfonyl fluoride, 50 μg/ml aprotinin, 100 μm leupeptin, and 25 μm pepstatin A) at 4 °C, subjected to SDS-PAGE, and electrotransferred to polyvinylidene difluoride membranes (Millipore). For immunoprecipitation, cell lysates were incubated with protein G-Sepharose beads (Amersham Biosciences) precoated with anti-Src (clone 327), anti-Lyn (H-6), or anti-ERK2 antibody overnight at 4 °C. The immune complexes were analyzed by SDS-PAGE and Western blotting, as described above. Src Kinase Activity at the Early Phase of Mitosis Is Required for Abscission—There is emerging evidence that Src-mediated tyrosine phosphorylation signaling is involved in cell division, especially at the G2-M transition, cleavage furrow progression, and completion of cytokinesis (18Roche S. Fumagalli S. Courtneidge S.A. Science. 1995; 269: 1567-1569Crossref PubMed Scopus (236) Google Scholar, 43Ng M.M. Chang F. Burgess D.R. Dev. Cell. 2005; 9: 781-790Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 44Tominaga T. Sahai E. Chardin P. McCormick F. Courtneidge S.A. Alberts A.S. Mol. Cell. 2000; 5: 13-25Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar). To investigate the involvement of Src kinase activity in cytokinesis, living HeLa cells were monitored in a time lapse manner in the presence of PP2, a potent SFK inhibitor, under a deconvolution microscope. We observed that prolonged treatment with PP2 prevented cells from entering into M phase (Fig. 1A), consistent with previous reports that the G2-M transition is blocked by inhibition of SFKs (18Roche S. Fumagalli S. Courtneidge S.A. Science. 1995; 269: 1567-1569Crossref PubMed Scopus (236) Google Scholar, 19Moasser M.M. Srethapakdi M. Sachar K.S. Kraker A.J. Rosen N. Cancer Res. 1999; 59: 6145-6152PubMed Google Scholar). As shown in Fig. 1B, control cells completed cytokinesis within 144 ± 35 min after anaphase onset (n = 11, mean ± S.D.). Intriguingly, most cells that escaped from PP2-induced inhibition of G2/M progression formed the cleavage furrow with a normal time course, but remained connected by an unusually elongated intercellular bridge containing the midbody 350 min after anaphase onset (Fig. 1C). These results indicate that a defect in cytokinesis takes place during the terminal step in cytokinesis called abscission. To examine whether the involvement of Src kinase activity in abscission was temporally controlled, living HeLa cells treated with PP2 from a particular stage of M phase were monitored in a time lapse manner under a deconvolution microscope. When PP2 was added to cells before cleavage furrow formation, most cells underwent progression of the cleavage furrow, but remained connected in a pair by an elongated intercellular bridge (Fig. 2, A and C, filled bars, before metaphase and metaphase/anaphase). Additionally, cells sometimes appeared to exhibit a defect in cleavage furrow progression (Fig. 2C, shaded bars, before metaphase and metaphase/anaphase), in agreement with recent findings (43Ng M.M. Chang F. Burgess D.R. Dev. Cell. 2005; 9: 781-790Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). However, when cells were treated with PP2 after cleavage furrow formation, abscission was completed with a normal time course (Fig. 2, B and C, open bars, telophase and cytokinesis). Furthermore, treatment of cells with SU6656 (another selective SFK inhibitor; Ref. 31Blake R.A. Broome M.A. Liu X. Wu J. Gishizky M. Sun L. Courtneidge S.A. Mol. Cell. Biol. 2000; 20: 9018-9027Crossref PubMed Scopus (528) Google Scholar) in metaphase/anaphase induced abscission failure similar to that induced by PP2 (Fig. 2C). These results suggest that before cleavage furrow formation, SFK catalytic activity is required for abscission. PP2 Treatment Induces Abscission Failure in an Adhesion-independent Manner—SFKs play a role in cell adhesion and migration by controlling cytoskeletal reorganization (11Thomas S.M. Brugge J.S. Annu. Rev. Cell Dev. Biol. 1997; 13: 513-609Crossref PubMed Scopus (2134) Google Scholar). However, the possibility that SFK inhibition reduces pulling/traction forces from two separating daughter cells (45Uyeda T.Q. Nagasaki A. Curr. Opin. Cell Biol. 2004; 16: 55-60Crossref PubMed Scopus (44) Google Scholar) may be ruled out, because cell adhesion and motility during M phase was unaffected by PP2 treatment (Fig. 2, A and B, arrows). To further rule out this possibility, we examined mitotic progression in HeLa S3 cells grown in suspension culture. After release from nocodazole arrest (see details under "Experimental Procedures"), normal HeLa S3 cells synchronously progressed through mitosis, and most cells divided into two daughter cells within 180 min after nocodazole release (Fig. 3, A–D, scheme a, DMSO control). PP2 treatment (from 10 to 90 min) had a marginal effect on progression into cytokinesis until 90 min (Fig. 3E). However, when cells were treated with PP2 before furrow formation (10 min after nocodazole release), abscission failure was significantly detected at 180 min (Fig. 3, D and F, scheme b). Intriguingly, PP2 treatment after furrow formation (50 min after nocodazole release) minimally affected completion of abscission at 180 min (Fig. 3, D and F, scheme c). Taken together, SFK kinase activity during early M phase plays an important role in abscission in an adhesion-independent manner. Treatment with PP2 Decreases Tyrosine Phosphorylation Levels at the Midbody—To scrutinize tyrosine phosphorylation signaling specific for M phase, we examined the localization of tyrosine-phosphorylated proteins by in situ detergent extraction and confocal fluorescence microscopy. Immunostaining of Triton X-100-treated cells with anti-phosphotyrosine (Tyr(P)) antibody revealed the localization of tyrosine-phosphorylated proteins to the midbody (Fig. 4A). Because the tyrosine-phosphorylated proteins found at the midbody were in particular resistant to Triton X-100 extraction, they may be tightly associated with cytoskeletal components at the midbody. The levels of tyrosine phosphorylation were higher at the plus-ends (+) of microtubules of the midbody than at the minus-ends (–), and a faint signal was detected at the midbody matrix (Fig. 4A). To investigate whether tyrosine phosphorylation signaling at the midbody was involved in PP2-induced abscission failure, we quantitated the fluorescence intensity of anti-Tyr(P) antibody reacted to the midbody region in PP2-treated cells. 12-h treatment of HeLa cells with PP2 induced a phenotype of cells having an unusually elongated intercellular bridge, as observed in time lapse monitoring of PP2-treated cells (Figs. 1 and 2), and significantly reduced the levels of tyrosine phosphorylation at the midbody (Fig. 4, B and C). These results suggest that PP2-induced abscission failure may be caused by reduced levels of tyrosine phosphorylation at the midbody. In contrast, a 30-min treatment of HeLa cells with PP2 had no effect on increased levels of tyrosine phosphorylation at the midbody and the appearance of a normal, short intercellular bridge (Fig. 4C; data not shown), despite a profound suppression of the kinase activity of c-Src (Fig. 4D). Once the midbody is formed, tyrosine phosphorylation signaling at the midbody may be insensitive to PP2 treatment. In other words, PP2 treatment from metaphase to anaphase, but not after cleavage furrow formation, is critical for induction of abscission failure (Figs. 2 and 3, A–F). Taken together, we suggest that abscission is depen
Referência(s)