Fig 2: mTORC2 phosphorylates RRM1 at Ser 631. (A) H1299 cells were transfected with Flag-RRM1, followed by flag immunoprecipitated and analysis of LC-MS/MS peptide spectra of RRM1 phosphorylation. (B) Sequence alignment of S631 phosphorylation site in RRM1 from different species. (C) H1299 cells were transfected with indicated flag tagged RRM1 variants, followed by analysis of RRM1 phosphorylation through IP with flag and western blot with anti-pRXXS/T antibody. Analysis of RRM1 phosphorylation in H1299 Rictor KO cells (E) or H1299 cells treated with 1 µM PP242 or 1 µM rapamycin for 4 hrs (F) through RRM1 IP and western blot with anti-pRXXS/T antibody. (G) Active mTORC2 was immunoprecipitated from HEK293T cells with purified GST-tagged WT or S631A mutant RRM1 protein in kinase buffer containing [?-32P] ATP. RRM1 phosphorylation was analyzed by autoradiography.

Fig 3: mTORC2 interacts RRM1 and regulates RNR activity. (A-B) Co-immunoprecipitation (Co-IP) analysis of interaction between Rictor and RRM1 using anti-Rictor antibody (A) or anti-RRM1 antibody (B) in H1299 and H460 cells. (C) RNR activities in H1299 parental or Rictor KO cells were assessed as described in “Materials and methods”. (D) Intracellular dNTPs pool size in H1299 parental or Rictor KO cells were measured by High Performance Liquid Chromatography (HPLC). (E) H1299 parental or Rictor KO cells were labeled sequentially with CldU and IdU for 20 min each, the DNA replication progressions were visualized by DNA fiber assay using specific antibodies against CIdU (red) or IdU (green). P value was calculated using two-tailed t-test (C, D) and Mann–Whitney U test (E). * P < 0.05, **P < 0.01 when compared with parental.