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First published online February 22, 2006
doi: 10.1242/10.1242/jcs.02797

Journal of Cell Science 119, 923-932 (2006)
Published by The Company of Biologists 2006
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Elmo1 inhibits ubiquitylation of Dock180

Yoshinori Makino1, Masumi Tsuda1, Shin Ichihara1, Takuya Watanabe1, Mieko Sakai1, Hirofumi Sawa1,*, Kazuo Nagashima1, Shigetsugu Hatakeyama2 and Shinya Tanaka1,{ddagger}

1 Laboratory of Molecular and Cellular Pathology, Hokkaido University Graduate School of Medicine, N15, W7, Sapporo 060-8638, Japan
2 Department of Molecular Biochemistry, Hokkaido University Graduate School of Medicine, N15, W7, Sapporo 060-8638, Japan


Figure 1
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  		Fig. 1. Elmo1 increases the stability of Dock180 protein. (A) Left panels; HEK293T cells were transiently transfected with mammalian expression plasmids for Flag-Dock180 alone or Flag-Dock180 and myc-Elmo1. After 48 hours, cells were lysed and analysed by immunoblotting (IB) with anti-Flag tag, anti-myc tag and anti-actin antibodies. The active form of Rac was detected in a pull-down assay. Right panels: HEK293T cells were transfected with mammalian expression plasmids for Flag-Crk II alone or Flag-Crk II and myc-Elmo1, and cells were analysed in the same way as for the left panels. (B) For pulse-chase analysis for Dock180, HEK293T cells transiently expressing Dock180 alone (-) or Dock180 and Elmo1 (+) were labelled for 1 hour and then chased for the time indicated at the top of the panel. (C) The signal intensity of Dock180 was measured and shown as a bar graph with the average and standard errors of three independent experiments; **P<0.05. (D) mRNA levels of exogenous Dock180. RT-PCR analysis was performed using mRNA extracted from HEK293T cells transiently expressing Dock180 alone or Dock180 and Elmo1. GAPDH is a control for the amounts of PCR templates. T/F, transfection; RT, reverse transcriptase. (E) HT1080 cells were transfected with siRNAs for negative control, scramble control and Elmo1#3. After incubation for 96 hours, cell lysates were subjected to immunoblotting for detection of Dock180, Elmo1 and actin. (F) The signal intensity of Dock180, normalised by the intensity of actin, is shown as a bar graph with the average and standard errors of three independent experiments; **P<0.05. (G) mRNA levels of endogenous Dock180. RT-PCR analysis was performed using the same mRNA extracts as E. GAPDH is a control for the amount of PCR template. (H) HEK293T cells were transfected with negative control and Elmo1#2 and cells were analysed in the same manner as E.

 

Figure 2
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  		Fig. 2. Ubiquitylation of Dock180 in HEK293T cells. (A) Results of an in vivo ubiquitylation assay. HEK293T cells were transfected with the indicated plasmids, and after treatment of 10 µM MG-132 for 12 hours, cells were lysed and subjected to immunoprecipitation (IP) and immunoblotting (IB). MIG, normal mouse immunoglobulin; TCL, total cell lysate; Ub, ubiquitin. (B) For the urea-reversal immunoprecipitation, the initial precipitates from the anti-Dock180 mAb were treated with 8 M urea buffer for 1 minute and then immunoprecipitated again using the same antibody. (C) MCAS and HEK293T cells were treated with 20 µg/ml cyclohexamide and 10 µg/ml MG-132 for 24 hours as indicated at the top of the panel. Cell lysates were immunoblotted with anti-Dock180 antibody to detect endogenous Dock180.

 

Figure 3
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  		Fig. 3. Elmo1 suppresses ubiquitylation of Dock180. (A) A schematic representation of wild-type and mutant forms of Elmo1 used in the study. DUF609, domain of unknown function; FL, full length; LZ, leucine zipper; PH, pleckstrin homology; PxxP, polyproline-rich motif. (B) Association between Dock180 and Elmo1 in HEK293T cells. Cells were transiently transfected with the indicated plasmids, and lysates were immunoprecipitated with anti-myc tag antibody, and then probed with anti-myc tag and anti-Flag tag antibodies. TCL, total cell lysate. (C) Ubiquitylation of Dock180 in HEK293T cells in the presence of Elmo1 and its mutants. 24 hours after transfection with the indicated plasmids, cells were lysed and subjected to in vivo ubiquitylation assay. (D) The urea-reversal immunoprecipitation was performed using the same lysates as C. The IP ratio represents the relative signal intensity of immunoprecipitated Dock180 in lane 1 as 1.0 (bottom of the panel). (E) The ubiquitylation of Dock180 in the absence or presence of Elmo1 and GST. The immunoprecipitation was performed using anti-Flag tag antibody. (F) The ubiquitylation of Dock180 and its mutant. Lysates from HEK293T cells expressing the indicated plasmids were immunoprecipitated with anti-Dock180 antibody at in vivo ubiquitylation assay. The IP ratio represents the relative signal intensity of immunoprecipitated Dock180 in lane 1 as 1.0.

 

Figure 4
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  		Fig. 4. Ubiquitylation of Dock180 on the plasma membrane. (A) Ubiquitylation of Dock180 in the membrane fraction. Cell lysates of HEK293T cells expressing the indicated plasmids were separated into cytosolic and membrane fractions. The samples were subjected to an in vivo ubiquitylation assay. The expression levels of transfected proteins were analysed using cell lysates of the cytosol and membrane fractions. Anti-E-cadherin antibody was used as a marker for the membrane fraction. CL, cell lysate. (B) Localisation of Dock180 and ubiquitin in cells. HEK293T cells (upper panels) and Cos-7 cells (lower panels) were transiently transfected with Flag-Dock180 or Dock180-RFP and HA-ubiquitin expression plasmids. After incubation for 36 hours, cells were stained with anti-HA tag antibody and analysed by confocal microscopy. (C) Ubiquitylation of Dock180 in the membrane fraction under EGF stimulation. HEK293T cells expressing the indicated plasmids were stimulated by EGF or not. Cell lysates were subjected to in vivo ubiquitylation assay.

 

Figure 5
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  		Fig. 5. The enhancement of ubiquitylation of Dock180 by Crk. An in vivo ubiquitylation assay was performed using lysates of the membrane fraction of HEK293T cells expressing the indicated plasmids (left panels). The IP ratio represents the relative signal intensity of immunoprecipitated Dock180 in lane 1 as 1.0 (bottom of the left panel). The expression levels of Dock180, Crk I and Crk II in the membrane fraction of cell lysates (membrane) and in total cell lysates (total) were determined by immunoblotting (right upper and middle panels). Anti-E-cadherin antibody was used as a marker for the membrane fraction (right lower panel).

 

Figure 6
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  		Fig. 6. Enhancement of ubiquitylation of Dock180 in HEK293T cells re-plated on fibronectin-coated dishes. (A) HEK293T cells were transiently transfected with the indicated plasmids for Dock180 and HA-ubiquitin and re-plated on fibronectin-coated dishes for the indicated duration (top of the panel); lysates in the membrane fraction were subjected to in vivo ubiquitylation assay. Sus, suspended cells; FN, fibronectin. Ratio represents the relative signal intensity of Dock180 in the membrane fraction in lane 1 as 1.0 (bottom of the panel). (B) Inhibition of ubiquitylation of Dock180 by Elmo1. HEK293T cells were transiently transfected with the indicated plasmids, and the re-plating assay and in vivo ubiquitylation assay were performed as in A.

 

Figure 7
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  		Fig. 7. Model of Dock180 regulation. (A,B) Following integrin stimulation by their ligands (including fibronectin), tyrosine kinases are activated and phosphorylate several components of focal adhesion such as p130Cas, and both Crk and Dock180 are recruited from the cytoplasm. (C) Recruited Dock180 is known to activate Rac, and we speculate that recruited Dock180 is ubiquitylated near the plasma membrane by an ubiquitin ligase. (D) Furthermore, ubiquitylated Dock180 might be removed from the complex comprising Crk and p130Cas, and is degraded by the proteasome.

 

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© The Company of Biologists Ltd 2006