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. 2013 Jan 23;11(1):9.
doi: 10.1186/1478-811X-11-9.

C3G Forms Complexes With Bcr-Abl and p38α MAPK at the Focal Adhesions in Chronic Myeloid Leukemia Cells: Implication in the Regulation of Leukemic Cell Adhesion

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C3G Forms Complexes With Bcr-Abl and p38α MAPK at the Focal Adhesions in Chronic Myeloid Leukemia Cells: Implication in the Regulation of Leukemic Cell Adhesion

Vera Maia et al. Cell Commun Signal. .
Free PMC article

Abstract

Background: Previous studies by our group and others have shown that C3G interacts with Bcr-Abl through its SH3-b domain.

Results: In this work we show that C3G and Bcr-Abl form complexes with the focal adhesion (FA) proteins CrkL, p130Cas, Cbl and Abi1 through SH3/SH3-b interactions. The association between C3G and Bcr-Abl decreased upon Abi1 or p130Cas knock-down in K562 cells, which suggests that Abi1 and p130Cas are essential partners in this interaction. On the other hand, C3G, Abi1 or Cbl knock-down impaired adhesion to fibronectin, while p130Cas silencing enhanced it. C3G, Cbl and p130Cas-SH3-b domains interact directly with common proteins involved in the regulation of cell adhesion and migration. Immunoprecipitation and immunofluorescence studies revealed that C3G form complexes with the FA proteins paxillin and FAK and their phosphorylated forms. Additionally, C3G, Abi1, Cbl and p130Cas regulate the expression and phosphorylation of paxillin and FAK. p38α MAPK also participates in the regulation of adhesion in chronic myeloid leukemia cells. It interacts with C3G, CrkL, FAK and paxillin and regulates the expression of paxillin, CrkL and α5 integrin, as well as paxillin phosphorylation. Moreover, double knock-down of C3G/p38α decreased adhesion to fibronectin, similarly to the single silencing of one of these genes, either C3G or p38α. These suggest that C3G and p38α MAPK are acting through a common pathway to regulate cell adhesion in K562 cells, as previously described for the regulation of apoptosis.

Conclusions: Our results indicate that C3G-p38αMAPK pathway regulates K562 cell adhesion through the interaction with FA proteins and Bcr-Abl, modulating the formation of different protein complexes at FA.

Figures

Figure 1
Figure 1
Bcr-Abl and C3G interact with Cbl, Abi1 and p130Cas. (A) Whole cell extracts from K562 were subjected to pull-down assays using GST-Abl-SH3 construct. The presence of C3G, Abi1, Cbl and p130Cas in the complexes was detected by inmunoblotting with specific antibodies. Representative Western blots of pull-down assays in K562 lysates using (B) Abi1-SH3 and SH3-b domains, (C) p130Cas-SH3, P1 and P2 domains, and (D) Cbl-SH3-b domain fused to GST as baits. The immunoblots were developed with specific antibodies against the indicated proteins. Glutathione-sepharose beads with whole cell lysate (B + K562), lysis buffer with the corresponding GST-fused proteins (LB) and pGEX-4T-1 empty plasmid with K562 whole cell lysate (GST) were used as negative controls. L: whole cell lysate (40 μg). The asterisks indicate unspecific bands.
Figure 2
Figure 2
C3G colocalizes with phospho-p130Cas, Cbl, Abi1 and Bcr-Abl in K562 cells. Confocal microscopy images of K562 cells adhered to fibronectin covered slides (10 μg/ml) and labeled with anti-C3G (G4, mouse monoclonal, rows 1 and 2 or 1008, rabbit polyclonal, rows 3 and 4) and either, anti-phospho-p130Cas (rabbit polyclonal), anti-Cbl (rabbit polyclonal), anti-Abi1 (mouse monoclonal) or anti-Abl (mouse monoclonal) specific antibodies as indicated. Anti-FITC and anti-Cy3 were used as secondary antibodies. Nuclei were labeled with DAPI. Control cells (CT) were incubated with DAPI plus the secondary antibodies. DIC: Differential interference contrast microscopy. p-p130Cas: phospho-p130Cas. The bars represent 10 μm.
Figure 3
Figure 3
Abi1 and p130Cas silencing alter Bcr-Abl/C3G interaction. (A) Western blot analysis of the expression of Abi1, Cbl, p130Cas and p87C3G in whole cell lysates from K562 clones stably transfected with the indicated lentiviral shRNA particles. Relative Abi1/β-tubulin, Cbl/β-tubulin, p130Cas/β-tubulin and C3G/β-tubulin ratios are shown. shAbi1-2, shCbl-1, shp130Cas-5, shC3G-1 and shC3G-8 were selected as representative knock-down clones in the experiments. C: whole cell lysate of a K562 clone expressing control lentiviral shRNA. Pull-down assays with lysates of K562 shAbi1-2, shCbl-1 and shControl (shCT) clones (B) or K562 shp130Cas-5 and shCT clones (C), using Abl-SH3 domain fused to GST as bait. p140C3G, p87C3G, Abi1, CrkL, Cbl and p130Cas expression was detected with specific antibodies. Panels showing p140C3G and p87C3G in Figure 3B correspond to different exposure times (longer for p140 and shorter for p87) for a better visualization of both isoforms. Beads with lysis buffer (B), GST construct with K562 whole cell lysate (GST) and K562 whole cell lysate with lysis buffer (LB) were used as negative controls. L: whole cell lysate (40 μg).
Figure 4
Figure 4
C3G, Cbl, Abi1, p130Cas and p38α MAPK regulate adhesion to fibronectin in K562 cells. (A) Percentage of adhesion to fibronectin of K562 cells upon knock-down of C3G (shC3G-1 and shC3G-8), Cbl (shCbl-1), Abi1 (Abi1-2) or p130Cas (shp130Cas-5). (B) Percentage of adhesion to fibronectin of K562 clones expressing interference RNAs (cloned in pSuper.gfp/neo) for C3G and/or p38α MAPK. The values are the mean ± SEM of at least three independent experiments. All values are relative to K562 cells transfected with control shRNA or empty pSuper.gfp/neo vector (pSuper). *p<0.05; ** p<0,01.
Figure 5
Figure 5
C3G and p38α MAPK interact with FA proteins. (A) Representative immunoprecipitation assays (IP), using the indicated antibodies, performed with protein extracts from K562 cells cultured on 10 μg/ml fibronectin for 24 h. Expression of p33, p46 and p68 phospho-paxillin (p-Pax) and paxillin isoforms, p38α, p87C3G, CrkL, Bcr-Abl, p130Cas, Abi1 and FAK was detected by immunoblotting with specific antibodies. GammaBind G Sepharose beads (B) with either buffer or whole cell lysate (lysate) were used as negative controls. (B) Confocal microscopy of K562 cells adhered for 24 h to slides covered with 10 μg/ml fibronectin and labeled with the indicated antibodies. Nuclei were labeled with DAPI. Control cells (CT) were incubated with DAPI plus the secondary antibodies. C3G-1008 (rabbit polyclonal) was used with Pax (mouse monoclonal) and p-FAK (goat polyclonal). C3G-G4 (mouse monoclonal) was used with p-Pax (rabbit polyclonal). C3G colocalizes with p-Pax and p-FAK in a punctuated pattern (white arrows). DIC: Differential interference contrast microscopy. Pax: paxillin. A488: Alexa Fluor 488. The bars represent 5 μm.
Figure 6
Figure 6
C3G modulates the expression and activation of FA proteins. (A) Western blot analysis of whole cell lysates (50 μg of protein) from K562 cells stably transfected with pLTR2C3G or the empty pLTR2 vector grown in suspension for 24 h. Paxillin (Pax), Cbl, CrkL, FAK, p130Cas and integrin α5 (Int α5) expression and paxillin and CrkL phosphorylation were detected with specific antibodies (B) Comparative analysis of the expression of Int α5, FAK, Pax and p130Cas in the above K562 clones grown with 10 μg/ml fibronectin (FN) for 24 h or in suspension (Susp). β-actin and β-tubulin (ß-Tub) levels were used as loading controls. (C) Western blot analysis of whole cell lysates from K562 cells stably transfected with shC3G (clone 1) or shCT lentivirus, either grown with fibronectin (FN) or in suspension (Susp) for 24 h. FAK, Cbl, paxillin, p130Cas and CrkL expression and paxillin, p130Cas and CrkL phosphorylation were detected. Relative ratios between the levels of these proteins and ß-tubulin are shown. (D) Paxillin expression is decreased in C3G silenced cells. Confocal microscopy of control (shCT) and shC3G-1 K562 cells adhered to fibronectin and labeled with anti-paxillin-Cy3 and anti-phalloidin antibodies. Nuclei were labeled with DAPI. Control cells (CT) were incubated with DAPI plus the secondary antibodies. The bars represent 10 μm (rows 1 and 3) and 7.5 μm (rows 2 and 4). (E) Immunoprecipitation assays (IP) of K562 cell lysates expressing, either shC3G or shCT, with the indicated antibodies followed by Western blot analysis of CrkL, Paxillin, p130Cas and Bcr expression. GammaBind G Sepharose beads (B) with either buffer or whole cell lysate (lysate) were used as negative controls. L: total cell lysate, Pax: paxillin, pY: phospho-tyrosine.
Figure 7
Figure 7
p38α MAPK downregulate the expression and phosphorylation of FA proteins. (A) Representative Western blots showing the expression and phosphorylation (p) of FAK, p130Cas, paxillin (Pax), CrkL, p38α and integrin α5 (Int α5) in K562 clones expressing pSuper-p38α MAPK (p38αi) or the empty vector (pSuper). Cells were cultured in suspension (Susp) or attached to fibronectin (FN). Relative ratios between the levels of the different analyzed proteins and ß-tubulin are shown. (B) The histogram represents the quantification by densitometry of the Western blot bands for each protein, relative to ß-tubulin. Susp: suspension; FN: fibronectin. (C) Western blot showing the expression of p140C3G in K562 cells expressing pSuper-p38α MAPK (p38αi) or empty vector (pSuper) grown on suspension. (D) Representative Western blots showing the expression and phosphorylation (p) of paxillin (Pax), CrkL, p38α and integrinα5 (Int α5) in K562 cells expressing pSuper-p38α MAPK (p38αi) or empty vector (pSuper) grown on suspension and treated or not with 5 μM SB203580 (SB) for 48 h. Relative ratios between the levels of the different analyzed proteins and ß-tubulin or ß-actin are shown.
Figure 8
Figure 8
Abi1, Cbl and p130Cas contribute to FA regulation. (A) Representative Western blots showing the expression and phosphorylation (p) of the indicated proteins in K562 cells upon knock-down of Cbl (clone 1), Abi1 (clone 2) or p130Cas (clone 5), and their corresponding control cells (see Figure  3). ß-actin was used as loading control. Relative ratios between the levels of the different analyzed proteins and ß-actin are shown. (B) The histograms represent the relative values of the levels of expression of the analyzed proteins relative to ß-tubulin. shCT-sc: Control shRNA from Santa Cruz Biotech., shCT-sg: Control shRNA from Sigma. Susp: suspension; FN: fibronectin.

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