Phosphorylation of tyrosine residues 31 and 118 on paxillin regulates cell migration through an association with CRK in NBT-II cells

Abstract

Identification of signaling molecules that regulate cell migration is important for understanding fundamental processes in development and the origin of various pathological conditions. The migration of Nara Bladder Tumor II (NBT-II) cells was used to determine which signaling molecules are specifically involved in the collagen-mediated locomotion. We show here that paxillin is tyrosine phosphorylated after induction of motility on collagen. Overexpression of paxillin mutants in which tyrosine 31 and/or tyrosine 118 were replaced by phenylalanine effectively impaired cell motility. Moreover, stimulation of motility by collagen preferentially enhanced the association of paxillin with the SH2 domain of the adaptor protein CrkII. Mutations in both tyrosine 31 and 118 diminished the phosphotyrosine content of paxillin and prevented the formation of the paxillin-Crk complex, suggesting that this association is necessary for collagen-mediated NBT-II cell migration. Other responses to collagen, such as cell adhesion and spreading, were not affected by these mutations. Overexpression of wild-type paxillin or Crk could bypass the migration-deficient phenotype. Both the SH2 and the SH3 domains of CrkII are shown to play a critical role in this collagen-mediated migration. These results demonstrate the important role of the paxillin-Crk complex in the collagen-induced cell motility.

Figure 3

Adhesion and spreading of clones expressing wild-type and mutant forms of paxillin are similar on collagen. (A) Mock-transfected (clone C1) and cells stably transfected with wild-type (clone P1), or mutant forms of paxillin F31 (clone F31-1), F118 (clone F118-2), or F31/118 (clone Fx1) were allowed to adhere on plates coated with the indicated concentrations of collagen for 1 h at 37°C. Absorbance values corresponding to crystal violet uptake were determined for each point and expressed as percent adhesion. (B) Cell spreading was quantified by allowing cells to adhere on plates coated with 20 μg/ml collagen for the indicated times. Cell spreading was determined by calculating the percentage of spread cells at each time point. Values represent the mean of three independent experiments carried out in duplicate ± SD. Selected clones are representative of all the tested clones.

Figure 5

Adhesion on collagen enhances the association of CrkII with paxillin in NBT-II cells. (A) Total cell lysates prepared from cells plated on PL, collagen, FN, or LN for 2 h were immunoprecipitated with anti–Crk antibodies, subjected to SDS-PAGE and revealed with antipaxillin and anti–Crk antibodies. (B) Total cell lysates prepared from cells plated on either PL or collagen for 30, 60, 90, 120 min at 37°C were immunoprecipitated with anti–Crk antibodies. Total lysates (left panels) and immunoprecipitates (right panels) were subjected to SDS-PAGE and immunoblotted with antipaxillin, anti–p130Cas and anti–Crk antibodies. Note the presence of the CrkII protein doublet.

Figure 6

Plating on collagen increases the association of tyrosine-phosphorylated paxillin with GST-CrkSH2 in NBT-II cells. NBT-II cells were plated on either PL or collagen for 90 min at 37°C. Cell lysates were incubated for 2 h at 4°C with GST-CrkSH2, GST-Grb2SH2 fusion proteins or GST alone that were previously immobilized on glutathione-Sepharose 4B beads. Total cell lysates (left panel) and the bound proteins (right panel) were separated by SDS-PAGE and immunoblotted with antiphosphotyrosine, antipaxillin and anti–p130Cas antibodies. The arrows indicate the position of p130Cas (upper) and paxillin (lower).

Figure 7

CrkII binding to paxillin is impaired in F31/118-transfected cells. (A) NBT-II cells were transiently transfected for 48 h with constructs encoding the different mutant forms of paxillin fused to GFP. Cells were stimulated on collagen for 90 min at 37°C, and cell lysates were incubated for 2 h at 4°C with GST-CrkSH2 fusion protein, or with GST alone, immobilized on glutathione-Sepharose 4B beads. Total cell lysates (left panel) and bound proteins (right panel) were separated by SDS-PAGE and immunoblotted with antipaxillin antibodies. (B) Cells transfected for 48 h with wild-type paxillin (pax-GFP) or the double mutant form (F31/118-GFP) were collagen stimulated, and the total cell lysates were incubated for 2 h with anti–GFP antibodies. After SDS-PAGE separation, immunoblots were revealed with antipaxillin and anti–Crk antibodies.

Figure 9

CrkII can rescue the migration deficiency of F31/118-transfected cells. Two stable transfected cell lines (clones Fx1 and Fx2) expressing the F31/118 mutations were transiently transfected for 48 h with wild-type CrkII or paxillin (Pax), together with a vector encoding GFP (10:1 ratio). (A) Cells were allowed to attach to collagen for 2 h at 37°C and cell motility determined as described in Fig. 4. Values obtained from the quantification of track paths are represented in micrometers per hour. Each bar represents the mean of at least three independent experiments ± SD. (B) Lysates obtained from cells transfected with the different constructs were analyzed for expression of paxillin and Crk with specific antibodies.