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. 2010 Oct 29;401(4):527-32.
doi: 10.1016/j.bbrc.2010.09.087. Epub 2010 Sep 26.

Abba promotes PDGF-mediated membrane ruffling through activation of the small GTPase Rac1

Datong Zheng  1 , Shuqiong Niu, Dan Yu, Xiaoguo H Zhan, Xianchun Zeng, Bota Cui, Yanping Chen, Jennifer Yoon, Stuart S Martin, Xiang Lu, Xi Zhan
Affiliations

Abba promotes PDGF-mediated membrane ruffling through activation of the small GTPase Rac1

Datong Zheng et al. Biochem Biophys Res Commun. .

Abstract

Abba is a member of the I-BAR-domain protein family that is characterized by a convex-shaped membrane-binding motif. Overexpression of GFP-tagged Abba in murine fibroblasts potentiated PDGF-mediated formation of membrane ruffles and lamellipodia. Immunofluorescent microscopy and pull-down analysis revealed that GFP-Abba colocalized with an active form of Rac1 in the membrane ruffles and enhanced the Rac GTPase activity in response to PDGF stimulation. Further immunoprecipitation assays demonstrated that GFP-Abba bound to both wild-type and constitutively active Rac1 and that the binding to either of the Rac1 forms was significantly enhanced upon PDGF stimulation. On the other hand, an Abba mutant deficient in Rac1 binding failed to promote membrane ruffling and Rac1 activation in response to PDGF. However, the cells overexpressing a truncated mutant carrying the I-BAR domain alone displayed numerous filopodia-like microspikes in a manner independent of growth factors. Also, the Rac-binding activity of the mutant was not affected by PDGF treatment. Our data indicates that the interaction between full-length Abba and Rac1 is implicated in membrane deformation and subjected to a growth factor-mediated regulation through the C-terminal sequence.

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Figures

Figure 1
Figure 1. Overexpression of Abba promotes the PDGF-mediated membrane ruffling
(A) NIH3T3 cells infected with retroviruses carrying GFP (a–d) and GFP-Abba (e–h) were starved in 0.2% serum-containing medium. After 24 h, cells were exposed to PDGF at 30 ng/ml for 10 min (c, d, g and h) followed by staining with GFP antibody (a, c, e and g) and phalloidin (b, d, f and h). The scale bar: 20 μm. The inset of d is an enlarged box as indicated to show typical filopodia-like microspikes. (B) Quantification of PDGF-treated cells with prominent dorsal membrane ruffles. (C) Quantification of the formation of microspikes. The data presented were mean ± SEM of three independent experiments, and p values (student's t-test) refer to the difference between GFP and GFP-Abba cells as indicated by *.
Figure 2
Figure 2. Abba interacts with and activates Rac1
(A) Abba interacts with Rac1. 3T3 cells were co-transfected with Myc-Rac1 and Flag-Abba (lane 1), Myc-Rac1 alone (lane 2) and Flag-Abba alone (lane 3). After transfection, the cell lysates were immunoprecipitated with Myc antibody, and Rac1-associated Flag-Abba proteins in the pellets were measured by Western blot using Flag antibody. The same blot was re-probed with Myc antibody to show equal immunoprecipitation. The expression level of Flag-Abba in the transfected cells was determined by immunoblotting of whole cell lysates (WCL) with Flag antibody as shown in the bottom panel. (B–C) Analysis of the effect of overexpression of GFP-Abba on Rac activation in response to PDGF. Cells were starved at 0.2% serum-containing medium for 24 h and stimulated with PDGF for 10 to 60 min. GTP-bound Rac (GTP-Rac) in cell lysates was determined by pull-down with GST-PAK-CRIB followed by Western blot with Rac1 antibody. Aliquots of the cell lysates were also immunoblotted with Rac antibody as the input control. The chart represents the normalized levels of GTP-Rac to the input Rac1, and error bars were standard derivations of three independent experiments. (C) Cells co-expressing GFP-Abba with Myc-Rac1 or with Myc-Rac1G12Vcells were incubated in 0.2% serum-containing medium for 24 h and stimulated with PDGF for 30 min. The lysates derived from treated cells were immunoprecipitated with Myc antibody, and the pellets were immunoblotted with GFP and Myc antibody, respectively. Aliquots of cell lysates were also blotted with α-tubulin antibody for the loading control. (D) Cells stably expressing Myc-Rac1G12V (a and b) or Myc-Rac1 (c and d) were transiently transfected with GFP-Abba, and stained with phalloidin (a and c) and GFP antibody (b and d). A cell co-expressing GFP-Abba and Myc-Rac1G12V developed prominent ruffles. Scale bar: 10 μm.
Figure 3
Figure 3. Mutation at the basic batch abolished the interaction of Abba with Rac1 and Rac1 activation
(A) Comparison of the basic patch of human Abba with that of MIM. The Lys residues in Abba that had been mutated to Glu are bolded. (B) Cells were transiently co-transfected with constructs as indicated. After 24 h of transfection, cells were analyzed for Rac1 binding as described above. The same membrane was also re-blotted with Myc antibody for equal immunoprecipitation. Also, aliquots of the lysates were immunoblotted with GFP antibody to determine expression of GFP-tagged proteins. (C) Cells were transiently transfected with GFP-Abba and GFP-AbbaK/D, respectively. The level of GTP-Rac in the transfected cells was analyzed as described in the legend of Fig. 3. (D) GFP-AbbaK/D cells (a–d) and GFP-Abba cells (e–h) were plated on fibronectin-coated coverslips and arrested in 0.2% serum-containing medium for 24 h. The arrested cells were treated without (a, b, e and f) or with PDGF (c, d, g and h) for 10 min, and co-stained with GFP antibody (a, c, e and g) and phalloidin (b, d, f and h). Bar: 10 μm.
Figure 4
Figure 4. Interaction between Abba-IMD alone and Rac1 was not regulated by PDGF
Cells transfected with GFP-Abba-IMD were grown in 10% serum-containing medium (a and b), 0.1% serum-containing medium (c and d), and 0.1% serum medium plus PDGF for 10 min (e and f). The morphology of the transfected cells was examined by immunofluorescent microscopy after staining with GFP antibody (a, c and e) and phalloidin (b, d and f). Bar: 10 μm. (B) Serum-starved 3T3 cells transiently transfected with GFP-Abba and GFP-Abba-IMD were stimulated with PDGF for 30 min. Rac activation was measured by the pull-down assay. Aliquots of lysates of the samples were also analyzed for Rac input. (C) 3T3 cells stably expressing Myc-RacG12V were transiently transfected with GFP-Abba-IMD and treated with PDGF for 30 min. The interaction between GFP-Abba-IMD and Myc-RacG12V in the treated cells was analyzed as described in the legend of Fig. 4.
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