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. 2021 Jun 11;23(7):643–652. doi: 10.1016/j.neo.2021.05.007

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© 2021 The Authors. Published by Elsevier Inc.

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

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Fig 4 — 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 [γ-32P] ATP. RRM1 phosphorylation was analyzed by autoradiography.