Figure 3
MCP1-induced p21Cip1 degradation requires cortactin phosphorylation at Y446.
(a) Cells transfected with the indicated cortactin expression vectors were growth-arrested, treated with vehicle or MCP1 for 6 hrs and equal amount of protein from control and each treatment were immunoprecipitated with anti-pTyr antibodies and the immunocomplexes were analyzed by immunoblotting for GFP-CTTN. Same cell extracts were also analyzed by immunoblotting for GFP-CTTN and p21Cip1 levels and normalized to β-tubulin. (b) Cells were transfected with Myc-tagged WT or mutant (Y421F or Y446F) cortactin expression vectors, growth-arrested, treated with vehicle or MCP1 for 6 hrs and co-immunostained for p21Cip1 and proteasome 20Sα/β using their specific antibodies. (c) All the conditions were same as in panel b except that cells were subjected to MCP1-induced DNA synthesis. (d) The cytoplasmic and nuclear fractions (CF and NF, respectively) of control and the indicated time periods of MCP1-treated HASMCs were analyzed by Western blotting for p21Cip1 levels and reprobed for MEK1 and p53 levels to show the relative purity of the cytoplasmic and nuclear fractions, respectively. (e) Cells were transfected with GFP-tagged WT or Y446F mutant cortactin expression vector, growth-arrested, treated with vehicle or MCP1 for 4 hrs, cell extracts prepared and an equal amount of protein form the cytoplasmic and nuclear fractions was analyzed by Western blotting for p21Cip1, MEK1 and p53 levels as described in panel d. The same blot was reprobed for GFP to show the overexpression of WT or mutant cortactin levels. (f) Cells were transfected with GFP-tagged WT or mutant (Y421F or Y446F) cortactin expression vectors, growth-arrested, treated with vehicle or MCP1 for 6 hrs and equal amounts of protein from each condition were immunoprecipitated with anti-GFP antibodies and the immunocomplexes were analyzed by immunoblotting using anti-pTyr, anti-pSer/Thr or anti-Ac-Lys antibodies and normalized to GFP-CTTN. The bar graphs in panels a and c–f represent Mean ± SD values of three independent experiments. *p < 0.05 vs vehicle control or WT-CTTN; **p < 0.05 vs MCP1 or WT-CTTN + MCP1.
