A crucial role in cell spreading for the interaction of Abl PxxP motifs with Crk and Nck adaptors

Abstract

The dynamic reorganization of actin structures helps to mediate the interaction of cells with their environment. The Abl non-receptor tyrosine kinase can modulate actin rearrangement during cell attachment. Here we report that the Abl PxxP motifs, which bind Src homology 3 (SH3) domains, are indispensable for the coordinated regulation of filopodium and focal adhesion formation and cell-spreading dynamics during attachment. Candidate Abl PxxP-motif-binding partners were identified by screening a comprehensive SH3-domain phage-display library. A combination of protein overexpression, silencing, pharmacological manipulation and mutational analysis demonstrated that the PxxP motifs of Abl exert their effects on actin organization by two distinct mechanisms, involving the inhibition of Crk signaling and the engagement of Nck. These results uncover a previously unappreciated role for Abl PxxP motifs in the regulation of cell spreading.

Fig. 1

Involvement of Abl PxxP motifs in regulating filopodium formation during attachment. A. Expression of c-Abl and variants in Abl DKO cells detected by western blotting with anti-Abl antibody (K-12, recognizing a.a. 521-531 in kinase domain (Woodring et al., 2001)) which binds equally to wt and mutated forms of Abl used in this study (data not shown). Numbers on the left side of blots indicate relative molecular mass in kDa. B. Serum-starved Abl 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 c-Abl (green) with Hoechst 33342, phalloidin/Texas-red, and anti-Abl (8E9)/Alexa-fluora 488, respectively. White bar = 10 μm. C. The number of filopodia was counted from the fixed Abl DKO cells. Graph represents mean and standard error of mean (S.E.M.) of filopodia per cell. The numbers over the bars indicate the number of cells examined. ‡ indicates P<0.05 when compared with Abl DKO cells re-expressing c-Abl.

Fig. 2

c-Abl regulates filopodium formation during attachment through the interaction of PxxP motifs with Crk and Nck family proteins. A, B, and C. NIH3T3 cells were infected with control retrovirus (derived from pSUPER vector) or the one carrying shRNA sequence targeting CrkI/II/L or Nck1/2. These knockdown cells were super-infected with control retrovirus (derived from MIGR-1 vector) or virus expressing shRNA-resistant (human) CrkII or Nck2. D, E, and F. NIH3T3 cells overexpressing c-Abl, Nck2, or CrkII were treated with or without STI-571. A and D. Protein expression was examined by western blotting. B and 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. White bars = 20 μm. C and E. The number of filopodia was counted from the fixed cells. Graph represents mean and/or S.E.M. of filopodia per cell or percentage of cells showing filopodia per total cells. ‡ indicates P<0.05, and ¶ indicates P=0.143.

Fig. 3

c-Abl and its PxxP motif interaction partners modulate Rac1 activity during attachment. NIH3T3 cells overexpressing CrkII, Nck2, c-Abl or 1234 (A), STI-571 treated, or Crk or Nck family knockdown NIH3T3 cells (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.

Fig. 4

c-Abl and its PxxP motif interaction partners regulate the spreading speed of cells. NIH3T3 cells overexpressing Grb2, Nck2, CrkII, or c-Abl (A and B), Abl DKO cells re-expressing various forms of Abl (C and D), and Crk family knockdown NIH3T3 cells (G and 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 and F). A, C, E, and G. Spreading cells were stained for F-actin and DNA. Infected cells express EGFP (green) as marker (A). White bar = 50 μm. B, D, F, and H. Cell areas for the stained cells were measured. Each bar represents mean and S.E.M. of the cell area. ‡ indicates P<0.05.

Fig. 5

c-Abl and its PxxP motif interaction partners regulate focal adhesion formation during attachment. NIH3T3 cells overexpressing CrkII, Nck2, c-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 or 6 μg/ml fibronectin-coated dishes. WCL were subjected to western blotting to detect pY397-Fak or pY118-Paxillin.

Fig. 6

Model for roles of Abl, Crk, and Nck in cell spreading. Integrin engagement increases catalytic activity of c-Abl. Due to interaction of Abl PxxP motifs 1, 2, and 4 with CrkII, active c-Abl phosphorylates and inactivates CrkII (colored in grey), 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 c-Abl catalytic activity is low, CrkII remains active and promotes focal adhesion formation and Rac1 activation, resulting in increased formation of lamellipodia. c-Abl and Nck no longer productively interact and transduce signals (Nck under this situation is colored in grey). As a result, low c-Abl activity decreases filopodium formation and accelerates spreading speed of cells.