Figure 1

Activation of Gα _q stimulates Fhit Tyr¹¹⁴phosphorylation in a Src-dependent mannar while activated Gα _q can associate with Fhit independent of Src. A, HEK293 cells were co-transfected with either pcDNA3 (Vector) or pcDNA3-Fhit in combination with pcDNA3 or pcDNA3-Src. Phosphorylated Src at Tyr⁴¹⁶ and phosphorylated Fhit at Tyr¹¹⁴ were determined by Western blotting. B, HEK293 cells stably expressing type 2 bradykinin receptor were transiently transfected with pFlag-CMV2-Fhit and then seeded onto 6-well plates. One day later, cells were serum starved, pretreated with DMSO, 10 μM PP1 or 25 μM PP2 for 30 min and then treated with or without 100 nM bradykinin for 24 h. The levels of phosphorylated Fhit were quantified relative to the controls without bradykinin treatment (set as 1). * Significantly different from the indicated controls (Dunnett t test, P < 0.05). C, Serum starved HEK293 cells were pretreated with 10 μM PP1 for 30 min and then treated with or without 100 μM carbachol (CCH) in the absence or presence of 100 μM Na₃VO₄ for 24 h. * Carbachol treatment significantly increased the phosphorylation of endogenous Fhit, while inhibition of Src significantly suppressed this effect (Dunnett t test, n = 4, P < 0.05). D, HEK293 cells were co-transfected with pFlag-CMV2-Fhit or pFlag-CMV2-Fhit Y114F in combination with wild-type or constitutively active mutant of Gα_q or Gα₁₄. The cDNA amount of Fhit for transfection was adjusted to achieve comparable expression levels. E, HEK293 cells were co-transfected with different combinations of pFLAG-CMV2-Fhit (F), pFLAG-CMV2 expression vector (V), Gα₁₆, Gα₁₆QL, pcDNA3 (Vector) or Src constructs. After 24 h, cells were collected and immunoprecipitated with anti-Flag affinity gel. * The upper bands represent Src while the lower bands were heavy chains of immunoglobulin G. Immunoblots shown represent one of three sets; two other sets yielded similar results.