Figure 6: Phosphorylation of ATF4 S245 by RSK2 enhances its binding to USP8 and protects ATF4 from ubiquitin-mediated degradation.

(a) Knockdown of RSK2 in a HCT116 PIK3CA mutant clone reduces pS245 ATF4 protein levels. HCT116 PIK3CA mutant cells were transfected with either control siRNA or siRNA against RSK2. Cell lysates were made 48 h post transfection, immunoprecipitated with anti-ATF4 antibodies and blotted with antibodies against either pS245 ATF4 or total ATF4. (b) Levels of pS245 ATF4 are higher in the HCT116 PIK3CA mutant clone than the WT clone. ATF4 proteins were immunoprecipitated and blotted with an anti-pS245 ATF4 antibody. (c) The ATF4 S245A mutant is less stable than the WT protein. The indicated constructs were expressed in the HCT116 PIK3CA mutant clone and cell lysates were blotted with the indicated antibodies. A Myc-paxillin construct was co-transfected as an internal control. (d) Ubiquitination levels of ATF4 S245A mutant are higher than that of WT protein. Constructs expression FLAG-tagged WT or S245A ATF4 were co-transfected with HA-ubiquitin into HCT116 PIK3CA mutant cells. Seventy-two hours post transfection, FLAG-tagged ATF4 proteins were immunoprecipitated and blotted with anti-human influenza hemagglutinin (HA) antibodies to detect ubiquitin. (e) The ATF4 S245A mutant binds to less USP8 than the WT protein. The indicated ATF4 constructs were transfected into HCT116 PIK3CA mutant cells. ATF4 immunocomplexes were pulled down by anti-FLAG antibodies and blotted with an anti-USP8 antibody. (f) Knockdown of USP8 with two different siRNAs in the HCT116 PIK3CA mutant clone reduces ATF4 protein levels. (g) Knockdown of USP8 increases the levels of ATF4 ubiquitination. Control siRNA or siRNA against USP8 was transfected into HCT116 PIK3CA mutant cells. Seventy-two hours post transfection, cells were treated with 5 μM MG132 for 6 h. Cell lysates were immunoprecipitated with anti-ATF4 antibodies and blotted with an anti-ubiquitin antibody.