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First published online September 3, 2008
doi: 10.1242/10.1242/jcs.031575

Journal of Cell Science 121, 3071-3082 (2008)
Published by The Company of Biologists 2008
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A crucial role in cell spreading for the interaction of Abl PxxP motifs with Crk and Nck adaptors

Susumu Antoku1, Kalle Saksela2, Gonzalo M. Rivera1 and Bruce J. Mayer1,*

1 Raymond and Beverly Sackler Laboratory of Genetics and Molecular Medicine, Department of Genetics and Developmental Biology, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, CT 06030-3301 USA
2 Department of Virology, Haartman Institute, University of Helsinki and Helsinki University Central Hospital, Helsinki, FIN-00014, Finland


Figure 1
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  		Fig. 1. Involvement of Abl PxxP motifs in regulating filopodium formation during attachment. (A) Expression of Abl and variants in Abl-double-knockout cells detected by western blotting with anti-Abl antibody [K-12, recognizing residues 521-531 in kinase domain (Woodring et al., 2001Go)], which binds equally to wild-type and mutated forms of Abl used in this study (data not shown). Numbers to the left of blots indicate relative molecular mass in kDa. (B) Serum-starved Abl-double-knockout (DKO) cells stably expressing various forms of Abl were plated on coverslips coated with 10 µg/ml fibronectin and fixed at 20 minutes. The fixed cells were stained for DNA (blue), F-actin (red), and Abl (green) with Hoechst 33342, Texas-Red-labelled phalloidin and Alexa Fluor 488-labeled anti-Abl (8E9), respectively. Scale bar: 10 µm. (C) The number of filopodia was counted on fixed Abl-double-knockout cells. Graph represents mean ± s.e.m. number of filopodia per cell. The number of cells examined is shown above the bars. {ddagger}P<0.05 when compared with Abl-double-knockout cells re-expressing Abl.

 

Figure 2
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  		Fig. 2. Abl regulates filopodium formation during attachment through the interaction of PxxP motifs with Crk and Nck family proteins. (A-C) NIH3T3 cells were infected with control retrovirus (derived from pSUPER vector) or ones carrying shRNA sequence targeting CrkI, CrkII and CrkL, or Nck1 or Nck2. These knockdown cells were superinfected with control retrovirus (derived from MIGR-1 vector) or virus expressing shRNA-resistant (human) CrkII or Nck2. (D-F) NIH3T3 cells overexpressing Abl, Nck2 or CrkII were treated with or without STI-571. (A,D) Protein expression was examined by western blotting. (B,F) Serum-starved cells were plated on coverslips coated with 10 µg/ml fibronectin, fixed at 20 minutes after plating, and stained for F-actin and DNA. Scale bars: 20 µm. (C,E) The number of filopodia was counted on fixed cells. Graph represents mean ± s.e.m. number of filopodia per cell or percentage of cells showing filopodia. {ddagger}P<0.05; P=0.143.

 

Figure 3
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  		Fig. 3. Abl and its PxxP motif interaction partners modulate Rac1 activity during attachment. NIH3T3 cells overexpressing CrkII, Nck2, Abl or 1234 (A), STI-571 treated or NIH3T3 cells with knockdown of Crk or Nck (B) were harvested in suspension or 5 minutes after attachment on 4 µg/ml fibronectin-coated dishes. Whole cell lysates (WCL) were subjected to GTPase assay, and the amounts of Rac1 and Cdc42 were detected by western blotting.

 

Figure 4
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  		Fig. 4. Abl and its PxxP motif interaction partners regulate the spreading speed of cells. NIH3T3 cells overexpressing Grb2, Nck2, CrkII or Abl (A,B), Abl double-knockout cells re-expressing various forms of Abl (C,D) and Crk family knockdown NIH3T3 cells (G,H), were plated on fibronectin-coated surface and fixed at 10 minutes when cells were actively spreading. Nck family knockdown or STI-571-treated NIH3T3 cells were fixed at 9 minutes (E,F). (A,C,E,G) Spreading cells were stained for F-actin and DNA. Infected cells express EGFP (green) as marker (A). Scale bars: 50 µm. (B,D,F,H). Cell areas for the stained cells. Each bar represents mean ± s.e.m. cell area. {ddagger}P<0.05.

 

Figure 5
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  		Fig. 5. Abl and its PxxP motif interaction partners regulate focal adhesion formation during attachment. NIH3T3 cells overexpressing CrkII, Nck2, Abl or 1234 (A), STI-571 treated or Crk or Nck family knockdown NIH3T3 cells (B) were harvested in suspension or 10 minutes after attachment on 2 µg/ml or 6 µg/ml fibronectin-coated dishes. Whole cell lysates were subjected to western blotting to detect pY397-Fak or pY118-Paxillin.

 

Figure 6
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  		Fig. 6. Model for roles of Abl, Crk and Nck in cell spreading. Integrin engagement increases catalytic activity of Abl. Owing to interaction of Abl PxxP motifs 1, 2 and 4 with CrkII, active Abl phosphorylates and inactivates CrkII (gray), together with interaction between PxxP motif 3 and Nck, inhibiting focal adhesion formation and Rac1 activation and thus decreasing lamellipodium formation. These actions also cause increased filopodium formation and eventually slow cell spreading. When Abl catalytic activity is low, CrkII remains active and promotes focal adhesion formation and Rac1 activation, resulting in increased formation of lamellipodia. Abl and Nck no longer productively interact and transduce signals (gray). As a result, low Abl activity decreases filopodium formation and accelerates spreading speed of cells.

 

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