Figure 7
Cortactin phosphorylation at Y446 residue is required for only GPCR agonist but not RTK agonist-induced p21Cip1 degradation and HASMC proliferation.
(a) HASMCs were transfected with GFP-tagged WT or Y446F mutant cortactin expression vector, growth-arrested, treated with vehicle, MCP1 (50 ng/ml), thrombin (0.5 U/ml) or PDGF-BB (20 ng/ml) for 6 hrs and cell extracts were prepared. Equal amounts of protein from control and each treatment were immunoprecipitated with anti-GFP antibodies and the immunocomplexes were analyzed by Western blotting with anti-pTyr antibodies and normalized for GFP. (b) All the conditions were the same as panel a except that cells were treated with vehicle or the indicated agonist for 8 hrs and equal amounts of protein from control and each treatment were analyzed by Western blotting for p21Cip1 levels and normalized to β-tubulin. (c) All the conditions were same as in panel a except that cells were subjected to the indicated agonist-induced DNA synthesis. (d) Cells were transfected with WT or Y5F mutant cortactin expression vector, growth-arrested, treated with vehicle or PDGF-BB for 6 hrs and cell extracts were prepared. Equal amount of protein from control and each treatment were immunoprecipitated with anti-GFP antibodies and the immunocomplexes were analyzed by Western blotting with anti-pTyr antibodies and normalized to GFP. (e) All the conditions were the same as in panel d except that cells were treated with vehicle or PDGF-BB for 8 hrs and equal amounts of protein from each condition were analyzed by Western blotting for p21Cip1 levels using its specific antibodies and normalized to β-tubulin. The same cell extracts were also analyzed by Western blotting for GFP to show the overexpression of WT or mutant cortactin. (f) All the conditions were the same as in panel d except that cells were subjected to PDGF-BB-induced DNA synthesis. The bar graphs in panels a–f represent Mean ± SD values of three independent experiments. *p < 0.05 vs WT-CTTN; **p < 0.05 vs WT-CTTN+ the indicated agonist.
