PMID: 23947369 eFIP 3 Check other iTextMine results Issue Report
Results-10
| 1. | Activation of Gαstimulates Fhit Tyrphosphorylation in a Src-dependent mannar while activated Gαcan 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. |
| 2. | Phosphorylated Src at Tyr416 and phosphorylated Fhit at Tyr114 were determined by Western blotting. |
| 3. | B, HEK293 cells stably expressing type 2 bradykinin receptor were transiently transfected with pFlag-CMV2-Fhit and then seeded onto 6-well plates. |
| 4. | 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. |
| 5. | The levels of phosphorylated Fhit were quantified relative to the controls without bradykinin treatment (set as 1). |
| 6. | * Significantly different from the indicated controls (Dunnett t test, P < 0.05). |
| 7. | 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). |
| 8. | 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 Gαq or Gα14. |
| 9. | The cDNA amount of Fhit for transfection was adjusted to achieve comparable expression levels. |
| 10. | 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. |
| 11. | After 24 h, cells were collected and immunoprecipitated with anti-Flag affinity gel. |
| 12. | * 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. |
Results-23
| 1. | In an attempt to unveil the biological function of the Gαq/Fhit interaction, we asked if such association is affected by Fhit phosphorylation at Tyr114 or Fhit’s ability to bind Ap3A. |
| 2. | Previous studies have shown that Fhit undergoes degradation upon phosphorylation by Src kinase at Tyr114[13] and activated Gαq can stimulate tyrosine kinases [25]. |
| 3. | Many signaling molecules regulate their binding to protein partners through tyrosine phosphorylation. |
| 4. | To test if this holds true for Fhit, we employed the Fhit Y114F mutant in co-immunoprecipitation assays. |
| 5. | Since Flag-Fhit Y114F appeared to interact with constitutively active GαqRC to an extent similar to Flag-Fhit (Figure 5A), it suggests that phosphorylation of Fhit Tyr114 is not a prerequisite for the formation of Gαq/Fhit complexes. |
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