QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 4 December 2002
doi: 10.1242/jcs.00244

This Article Summary Full Text Full Text (PDF) Movie Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Related articles in JCS Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Scaife, R. M. Articles by Langdon, W. Y. Search for Related Content PubMed PubMed Citation Articles by Scaife, R. M. Articles by Langdon, W. Y.

The multi-adaptor proto-oncoprotein Cbl is a key regulator of Rac and actin assembly

¹ Department of Pathology, University of Western Australia, QE II Medical Centre, Crawley WA 6009, Australia
² Van Andel Research Institute, Grand Rapids, MI 49503, USA

View larger version (49K): [in a new window]   Fig. 1. Effect of dominant-negative Cbl expression on RTK-induced cytoskeletal dynamics. (A) Truncation of c-Cbl at amino-acid residue 480 uncouples the N-terminal TKB and RING domains from the numerous C-terminal docking sites. (B) Wild-type, HA-c-Cbl-and HA-480-Cbl-expressing NIH 3T3 cells were lysed and aliquots of the lysates were subjected to SDS-PAGE and western blotting with anti-c-Cbl and anti-HA antibodies as indicated. (C) Serum-starved wild-type (panels 1, 2 and 3) and 480-Cbl-expressing NIH 3T3 cells (panels 4, 5 and 6) were treated with PDGF for 5 minutes. Merged confocal fluorescence microscopy Z-section stacks of phalloidin (red) and anti-tubulin (green) staining are shown in panels 1 and 3, whereas 1.5 µm thick apical sections are shown in panels 2 and 5 for phalloidin staining and panels 3 and 6 for anti-tubulin staining. A PDGF-induced actin aggregate in wild-type cells is indicated by the narrow arrowhead in panel 1. An actin dorsal ruffle is indicated by the broad arrowhead in panel 4. The scale bar represents 20 µm.

View larger version (91K): [in a new window]   Fig. 2. Cbl-mediated regulation of PDGF-induced actin dorsal ruffle assembly. (A) Serum-starved wild-type (panels 1 and 3), c-Cbl (panel 5), 480-Cbl (panels 2 and 4) and G306E 480-Cbl- (panel 6) expressing NIH 3T3 cells were either left untreated (panels 1 and 2) or treated with PDGF for 5 minutes (panels 3-6). Microfilaments were visualized by confocal fluorescence microscopy (the scale bar represents 100 µm), and (B) the percentage of cells that form actin dorsal ruffles at 0, 5 and 15 minutes of PDGF treatment was quantified (see Materials and Methods).

View larger version (62K): [in a new window]   Fig. 3. Expression of 480-Cbl does not affect PDGF receptor activation or signaling to MAP kinase and PI 3-kinase. Wild-type, c-Cbl- and 480-Cbl expressing NIH 3T3 cells were lysed following serum starvation or after 1 and 5 minutes of PDGF activation as indicated. Aliquots of the lysates were subjected to SDS-PAGE and western blotting with anti-phosphotyrosine (A), anti-phospho-Shc (Tyr317) and anti-Shc (B), anti-phospho-MAP kinase (Thr202/Tyr204) and anti-MAPK (C) and anti-phospho-Akt (Ser473) and anti-Akt (D), as indicated.

View larger version (56K): [in a new window]   Fig. 4. Expression of 480-Cbl does not affect PDGF receptor phosphorylation and polyubiquitylation. Wild-type, c-Cbl- and 480-Cbl-expressing NIH 3T3 cells were lysed following serum starvation or after 3 and 10 minutes of PDGF activation, as indicated. Aliquots of the lysates were immunoprecipitated with anti-PDGF receptor antibodies and probed by western blot with anti-phosphotyrosine (A), anti-ubiquitin (B) or anti-PDGF receptor (C) following SDS-PAGE.

View larger version (54K): [in a new window]   Fig. 5. Enhancement of actin dorsal ruffle formation by 480-Cbl requires Src, PI 3-kinase and Rac. (A) Following serum starvation, 480-Cbl-expressing NIH 3T3 cells were treated for 20 minutes with either DMSO carrier, PKC inhibitor BIM I (5 µM), Rac inhibitor SCH 51344 (75 µM), Src inhibitors SU6656 (5 µM) or PP2 (5 µM), PI 3-kinase inhibitors Wortmannin (0.5 µM) or LY294002 (30 µM), or left untreated, as indicated. Cells were fixed and stained with TRITC phalloidin to quantify actin dorsal ruffle formation after 5 minutes of activation by FCS (10%), LPA (2.5 µM) or PDGF (10 ng/ml) as indicated. (B) 480-Cbl expressing cells were transiently transfected with NLS-GFP (panel 1), NLS-GFP and R296/F528-Src (panel 2), NLS-GFP and N17-Rac1 (panel 3) and NLS-GFP and p85iSH2N (panel 4). The cells were treated with PDGF for 5 minutes following serum starvation and the NLS-GFP and TRITC phalloidin staining of representative samples were visualized by confocal fluorescence microscopy. The number of NLS-GFP-positive cells forming distinct actin dorsal ruffles is indicated in each panel. Bar, 30 µm.

View larger version (34K): [in a new window]   Fig. 6. Effect of 480-Cbl expression on PDGF-mediated activation of Src. Wild-type, c-Cbl- and 480-Cbl-expressing NIH 3T3 cells were lysed following serum starvation or after 1 and 5 minutes of treatment with PDGF. Aliquots of the lysates were directly subjected to SDS-PAGE and probed by western blotting with anti-phospho-Shc (Y239) and anti-Shc (A) or anti-phospho-Stat3 (Y704) and anti-Stat3 (B) as indicated.

View larger version (40K): [in a new window]   Fig. 7. Effect of 480-Cbl expression on PDGF-mediated activation of Rac. Wild-type, c-Cbl- and 480-Cbl-expressing NIH 3T3 cells were lysed following serum starvation or after 1 and 5 minutes of treatment with PDGF. (A) Aliquots of the lysates were either directly subjected to SDS-PAGE and western blotting with anti-Rac antibodies or incubated with sepharose-bound GST-PAK-CD and bound proteins subjected to SDS-PAGE and western blotting with anti-Rac antibodies as indicated. (B) Densitometric scans for anti-Rac blots of GST-PAK-CD bound proteins obtained from five experiments were averaged and plotted. Error bars represent the standard deviations. (C) Aliquots of serum starved and PDGF treated wild-type, c-Cbl and 480-Cbl expressing NIH 3T3 cells were subjected to SDS-PAGE and Western blotting with anti-phospho-p38 MAPK (Thr180/Tyr182) and anti-p38 MAPK specific antibodies, as indicated.

View larger version (27K): [in a new window]   Fig. 8. Src and PI 3-kinase mediate the increased Rac-activation by 480-Cbl expression. (A) Following serum starvation, 480-Cbl-expressing NIH 3T3 cells were treated for 20 minutes with either DMSO carrier, PKC inhibitor BIM I (3 µM), Src inhibitors SU6656 (5 µM) or PP2 (10 µM) or PI 3-kinase inhibitor LY294002 (30 µM), as indicated. Cells were lysed either after no further treatment or after 1 minute of activation by PDGF (10 ng/ml). Aliquots of the lysates were either directly subjected to SDS-PAGE and probed by western blotting with anti-Rac antibodies or were incubated with sepharose-bound GST-PAK-CD and bound proteins subjected to SDS-PAGE and western blotting with anti-Rac antibodies. (B) Serum-starved NIH 3T3 expressing either full-length c-Cbl or truncations at amino acids 655, 563, 528, 480 (with and without the TKB domain inactivating G306E mutation) and 388 were treated with PDGF for 5 minutes. Microfilaments were visualized by confocal fluorescence microscopy following Tritc-phalloidin staining, and the percentage of cells that form actin dorsal ruffles was quantified (see Materials and Methods).

View larger version (86K): [in a new window]   Fig. 9. RTK-induced actin dorsal ruffles are formed from c-Cbl containing actin-rich nucleation sites. (A) Confocal fluorescence images of serum-starved NIH 3T3 cells expressing EGFP-actin were recorded every minute following addition of PDGF to the culture medium. The arrowhead indicates a site of actin dorsal ruffle assembly in selected images. Bar, 30 µm. An animated compilation of the individual images is represented in Movie 1 (available at jcs.biologists.org/supplemental). (B) Serum-starved HA-c-Cbl-expressing NIH 3T3 cells were treated with PDGF for 2, 5 or 10 minutes. Single 1 µm apical sections of TRITC-phalloidin (red) and anti-HA staining (green) were visualized individually and as merged images by immunofluorescence confocal microscopy. The arrowheads indicate putative actin nucleation sites. Bar, 30 µm.