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First published online 10 June 2003
doi: 10.1242/jcs.00632

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Tyrosine phosphorylation of the CrkII adaptor protein modulates cell migration

¹ Craniofacial Developmental Biology and Regeneration Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD 20892-4370, USA
² Department of Molecular Virology and Oncology, Cancer Research Institute, Kanazawa University, 13-1 Takara-machi, Kanazawa 920-0934, Japan

View larger version (27K): [in a new window]   Fig. 1. CrkII protects p130cas from dephosphorylation. (A) 293-EBNA cells were transfected with either pRK-VSV-CrkII (2 µg) or pRK-GFP-PTP1B (2 µg) expression plasmids, or co-transfected with pRK-VSV-CrkII and pRK-GFP-PTP1B. Cell lysates were immunoprecipitated with anti-p130cas (IP: p130cas), anti-paxillin (IP: paxillin) or anti-FAK (IP: FAK) antibodies, and then immunoblotted with anti-phosphotyrosine (Blot: pTyr), anti-p130cas (Blot: p130cas), anti-paxillin (Blot: paxillin) or anti-FAK (Blot: FAK) antibodies. The whole-cell lysates (WCL) were immunoblotted with anti-GFP (Blot: GFP) or anti-VSV (Blot: VSV) antibodies. The numerical values indicate relative ratios as a percentage of the control for each band of phosphoprotein/total protein after scanning and analysis using NIH Image software. (B) pRK-GFP-PTP1B (2 µg) and various quantities of pcDNA-CrkII expression plasmids as indicated were co-transfected in 293-EBNA cells. Cell lysates were immunoprecipitated with anti-p130cas (IP: p130cas) and then immunoblotted with anti-phosphotyrosine (Blot: pTyr), anti-p130cas (Blot: p130cas) or anti-Crk (Blot: Crk) antibodies. The whole-cell lysates (WCL) were immunoblotted with anti-GFP (Blot: GFP) or anti-Crk (Blot: Crk) antibodies. The numbers indicate relative ratios as a percentage of controls for phosphoprotein/total protein or for CrkII bound to p130cas compared with the control.

View larger version (31K): [in a new window]   Fig. 2. PTP1B directly dephosphorylates CrkII. (A) An in-blot tyrosine phosphatase assay was performed as described in Materials and Methods. Immunoprecipitates using anti-VSV antibody (IP: VSV) from lysates of 293-EBNA cells co-transfected with VSV-FAK and VSV-CrkII were subjected to SDS-PAGE and electroblotting. The membranes were incubated with recombinant human PTP1B (20 U/ml) at 37°C for 30 minutes, then immunoblotted with anti-phosphotyrosine (Blot: pTyr) or anti-VSV antibodies (Blot: VSV). (B) 293-EBNA cells were co-transfected with either 0.5 µg of pcDNA CrkII-WT or CrkII-Y221F with or without pRK-GFP-PTP1B (2 µg), and then homogenized in NP-40 buffer. The cell lysates were immunoprecipitated with anti-Crk (IP: Crk) polyclonal antibody, and then immunoblotted with anti-phosphotyrosine (Blot: pTyr) or anti-Crk (Blot: Crk) monoclonal antibodies. The whole-cell lysates (WCL) were immunoblotted with anti-GFP antibody (Blot: GFP). (C) Immunoprecipitates using anti-CrkII antibody (IP: CrkII) from lysates of 293-EBNA cells co-transfected with pcDNA-CrkII and either pRK-GFP-PTP1B or GFP-PTP1B-C215S were subjected to SDS-PAGE and immunoblotting with anti-phosphotyrosine (Blot: pTyr) or anti-CrkII (Blot: CrkII) antibodies. The whole-cell lysates were immunoblotted with anti-GFP-antibody (Blot: GFP). (D) 293-EBNA cells co-transfected with either pcDNA-CrkII-WT or CrkII-Y221F were homogenized, and the lysates were immunoprecipitated with either anti-p130cas (IP: p130cas) or anti-paxillin (IP: paxillin) antibodies, and then immunoblotted with anti-Crk (Blot: Crk), anti-p130cas (Blot: p130cas), or anti-paxillin (Blot: paxillin) antibodies.

View larger version (45K): [in a new window]   Fig. 3. Fibronectin promotes dephosphorylation of CrkII. (A) 293-EBNA and HT1080 cells were serum-starved for 6 hours or 12 hours, respectively. The cells were trypsinized, kept in suspension for 20 minutes, re-plated onto culture dishes coated with 10 µg/ml fibronectin for 10, 30 or 120 minutes, and then homogenized. The 293-EBNA lysates were immunoprecipitated with anti-p130cas (IP: p130cas) or anti-Crk (IP: Crk) antibodies, and then immunoblotted with anti-phosphotyrosine (Blot: pTyr), anti-p130cas (Blot: p130cas) or anti-Crk (Blot: Crk) antibodies. The whole-cell lysates (WCL) from HT1080 cells were immunoblotted with anti-Crk (Blot: Crk) antibody. (B) HT1080 cells were serum-starved for 12 hours, trypsinized, kept in suspension for 20 minutes, then replated onto culture dishes coated with 10 µg/ml fibronectin for 30 or 120 minutes. The cells were homogenized and immunoprecipitated with anti-p130cas (IP: p130cas) or anti-Crk (IP: Crk) antibodies, and then immunoblotted with anti-phosphotyrosine (Blot: pTyr), anti-p130cas (Blot: p130cas) or anti-Crk (Blot: Crk) antibodies. (C) HT1080 cells in 100 mm dishes were co-transfected with 1 µg of pHA262pur and 2 µg of pRK-GFP-PTP1B, and transfectants were selected by puromycin for 36 hours. After puromycin selection, cells were serum-starved for 12 hours and replated onto culture dishes coated with 10 µg/ml fibronectin for 2 hours. The cells were homogenized and immunoprecipitated with anti-p130cas (IP: p130cas) antibody, and then immunoblotted with anti-phosphotyrosine (Blot: pTyr) or anti-p130cas (Blot: p130cas) antibodies. The whole-cell lysates (WCL) were immunoblotted with anti-PTP1B (Blot: PTP1B) or anti-Crk (Blot: Crk) antibodies.

View larger version (34K): [in a new window]   Fig. 4. PTP1B is essential for effective fibronectin-induced CrkII dephosphorylation. (A) 293-EBNA cells were co-transfected with siRNA (2x10-11 or 2x10-10 mol) for either PTP1B or ß-actin and pRK-GFP and pcDNA3 plasmids. The cells were homogenized at 36 hours after transfection and immunoblotted with anti-PTP1B (Blot: PTP1B) or anti-actin (Blot: actin) antibodies. (B) 293-EBNA cells were co-transfected with siRNA for PTP1B, and pRK-GFP and pcDNA3 plasmids. At 36 hours after transfection, cells were trypsinized, kept in suspension for 20 minutes, and replated onto culture dishes coated with 10 µg/ml fibronectin for 30 or 120 minutes. The cells were homogenized and immunoblotted with anti-PTP1B (Blot: PTP1B), anti-Crk (Blot: Crk) or anti-GFP (Blot: GFP) antibodies.

View larger version (101K): [in a new window]   Fig. 5. Subcellular localization of PTP1B and CrkII. HT1080 cells in 60 mm dishes were co-transfected with 0.5 µg of pHA262pur and 1 µg of pRK-GFP-PTP1B, and transfectants were selected by puromycin for 36 hours. After puromycin selection, cells were replated on glass coverslips coated with 10 µg/ml fibronectin for 2 hours. The cells were analyzed by immunofluorescence confocal microscopy using anti-Crk (Crk), anti-paxillin (Paxillin), or anti-ß-tubulin (Tubulin) antibodies, or rhodamine-phalloidin (F-actin). Bars, 20 µm.

View larger version (61K): [in a new window]   Fig. 6. CrkII-Y221F accelerates cell motility on fibronectin. (A) HT1080 cells were co-transfected with 0.5 µg of pHA262pur, 0.5 µg of pRK-GFP, and 0.5 µg or 2 µg of pcDNA-CrkII-WT and pcDNA-CrkII-Y221F, or 2 µg of CrkII-W169L, psSR-DN-p130cas or control plasmids, and transfectants were selected by puromycin for 36 hours. After puromycin selection, cells were replated on 35 mm glass dishes coated with 10 µg/ml fibronectin, and cultured overnight in serum-free DMEM containing 1 mg/ml BSA. Cell movements were monitored for 3 hours by time-lapse video microscopy. Error bars indicate s.d. for at least 30 cells per condition (*P<0.001 versus controls). Parallel cultures of these cells were homogenized and subjected to immunoblotting with anti-Crk (WCL Blot: Crk) antibody. (B) Phase contrast microscopy of HT1080 cells co-transfected with 0.5 µg of pHA262pur, pRK-GFP, and 0.5 µg (low) or 2 µg (high) of pcDNA-CrkII-WT, CrkII-Y221F or control plasmids 12 hours after replating onto fibronectin. Magnification, x200.

View larger version (40K): [in a new window]   Fig. 7. Co-expression of CrkII and PTP1B promotes cell migration. (A) HT1080 cells in 100 mm dishes were co-transfected with 1 µg of pHA262pur and 0.5 µg of pcDNA-CrkII with 0.5 µg or 1.0 µg of pRK-GFP-PTP1B, and transfectants were selected by puromycin for 36 hours. After puromycin selection, cells were serum-starved for 12 hours and then replated on culture dishes coated with 10 µg/ml fibronectin for 2 hours. The cells were homogenized and immunoprecipitated with anti-Crk (IP: Crk), anti-p130cas (IP: p130cas), or anti-paxillin (IP: paxillin) antibodies, and then immunoblotted with anti-phosphotyrosine (Blot: pTyr), anti-Crk (Blot: Crk), anti-p130cas (Blot: p130cas), or anti-paxillin (Blot: paxillin) antibodies. The whole-cell lysates (WCL) were immunoblotted with anti-PTP1B (Blot: PTP1B) antibody. (B) HT1080 cells were co-transfected with 0.5 µg of pHA262pur with 0.3 µg of pcDNA-CrkII-WT or pcDNA-CrkII-Y221F, and 0.5 µg of pRK-GFP-PTP1B, and transfectants were selected by puromycin for 36 hours. Cell migration was measured using 48-well chemotaxis chambers as described in Materials and Methods. Error bars indicate s.d. for at least three experiments (*P<0.05 versus controls).