Extended Data Fig. 2: GPX4 S104 phosphorylation stabilizes GPX4.
From: Creatine kinase B suppresses ferroptosis by phosphorylating GPX4 through a moonlighting function
(a,b, h, j, k, p, t-x) Immunoprecipitation and immunoblotting analyses were performed with the indicated antibodies. (a-e, o, q-s, v-x) Data are the mean ± SD (n = 6). **P < 0.001 (v-x); ***P < 0.0001 (a-e, o, q-s, v-x); N.S., not significant (b, c, r, s) by two-tailed Student’s t-test. (a) The indicated cells treated with IGF1 for 1 h were harvested. (b-f) The indicated purified His-CKB on Ni-NTA agarose beads were incubated with active GST-AKT1 for an in vitro kinase assay. The binding affinity of CKB to creatine (b, d) and the relative CKB activity (c, e) were measured. AKT-phosphorylated CKB conjugated on Ni-NTA beads were washed and incubated with His-GPX4 proteins for an in vitro kinase assay. Mass-spectrometric analysis was performed (f). (g) Alignment of protein sequences spanning GPX4 S104. (h) Purified His-CKB were incubated with GST-GPX4 in the presence of [γ-32P] ATP for in vitro kinase assay. Autoradiography was performed. (i, j) Huh7 cells expressing Flag-GPX4 were treated with IGF1 for 1 h (j). IHC analyses of human HCC samples (i) or immunoblotting (j) were performed with the indicated antibodies and GPX4 pS104-blocking peptide. (k) Huh7 cells expressing Flag-GPX4 were transfected with HA-myr-AKT1. (l-n) Genomic DNA was extracted from the indicated cells. Amplified PCR products were shown (l, m) and sequenced (n). (o, p) Huh7 cells were pretreated with NAC (5 mM) for 30 min before IGF1 incubation for 12 h. The intercellular ROS levels were measured (o). (q-s) Relative CKB activity (q), binding affinity of the His-CKB proteins to creatine (r) and ATP (s) were measured. (t, u) Huh7 or HCCLM3 cells expressing CKB shRNA with reconstituted expression of the indicated CKB proteins were treated with or without IGF1 for 1 h . (v-x) The indicated cells expressing CKB shRNA with reconstituted expression of the indicated CKB proteins (v) or with knock-in expression of GPX4 mutants (w, x) were treated with CHX for the indicated time. The quantification of GPX4 protein levels relative to initial protein levels is shown.
