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Creatine kinase B suppresses ferroptosis by phosphorylating GPX4 through a moonlighting function

Nature Cell Biology volume 25, pages 714–725 (2023)Cite this article

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Abstract

Activation of receptor protein kinases is prevalent in various cancers with unknown impact on ferroptosis. Here we demonstrated that AKT activated by insulin-like growth factor 1 receptor signalling phosphorylates creatine kinase B (CKB) T133, reduces metabolic activity of CKB and increases CKB binding to glutathione peroxidase 4 (GPX4). Importantly, CKB acts as a protein kinase and phosphorylates GPX4 S104. This phosphorylation prevents HSC70 binding to GPX4, thereby abrogating the GPX4 degradation regulated by chaperone-mediated autophagy, alleviating ferroptosis and promoting tumour growth in mice. In addition, the levels of GPX4 are positively correlated with the phosphorylation levels of CKB T133 and GPX4 S104 in human hepatocellular carcinoma specimens and associated with poor prognosis of patients with hepatocellular carcinoma. These findings reveal a critical mechanism by which tumour cells counteract ferroptosis by non-metabolic function of CKB-enhanced GPX4 stability and underscore the potential to target the protein kinase activity of CKB for cancer treatment.

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Fig. 1: CKB T133 phosphorylation increases GPX4 stability.
Fig. 2: CKB-mediated GPX4 S104 phosphorylation stabilizes GPX4.
Fig. 3: GPX4 phosphorylation blocks CMA-mediated GPX4 degradation.
Fig. 4: GPX4 S104 phosphorylation suppresses ferroptosis.
Fig. 5: GPX4 S104 phosphorylation promotes tumour growth.
Fig. 6: GPX4 pS104 predicts clinical aggressiveness of HCC.

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Data availability

The human HCC data were derived from the TCGA Research Network. The dataset derived from this resource that supports the findings of this study is available at the following link: https://tcga.xenahubs.net/download/TCGA.LIHC.sampleMap/HiSeqV2.gz. Mass spectrometry data have been deposited in ProteomeXchange with the accession code PXD040322. UniProt protein database (EMBL-EBI) was used for protein identification. Source data are provided with this paper.

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Acknowledgements

This study was supported by grants from the Ministry of Science and Technology of the People’s Republic of China (2021YFA0805600, D.X.; 2020YFA0803300, Z.L.), the National Natural Science Foundation of China (82188102 and 82030074, Z.L.; 92157113 and 82072630, D.X.; 82173114, Z.W.; 82072903 and 82272872, T.L.; 82002811, M.Y.; 82030065 and 81873932, Q.Z.), the Zhejiang Natural Science Foundation Key Project (LD22H160002, D.X.; LD21H160003, Z.L.), Zhejiang Natural Science Foundation Discovery Project (LQ22H160023, Z.W.), Chinese Academy of Sciences Intramural Research Program (QTJC20220010-A, Q.X.) and the Leading Innovative and Entrepreneur Team Introduction Program of Zhejiang (2019R01001, Z.L.). Z.L. is the Kuancheng Wang Distinguished Chair. The authors received no specific funding for this work.

Author information

Author notes

  1. These authors contributed equally: Ke Wu, Meisi Yan, Tong Liu, Zheng Wang.

Authors and Affiliations

  1. Zhejiang Provincial Key Laboratory of Pancreatic Disease, The First Affiliated Hospital, Institute of Translational Medicine, Zhejiang University School of Medicine, Zhejiang University, Hangzhou, China

    Ke Wu, Zheng Wang, Yuran Duan, Guimei Ji, Yuli Shen, Lei Wang, Lin Li, Peixiang Zheng, Bofei Dong, Qingang Wu, Liwei Xiao, Haochen Shen, Ting Wen, Jingjing Zhang, Zhimin Lu & Daqian Xu

  2. School of Basic Medical Sciences, Harbin Medical University, Harbin, China

    Meisi Yan, Jinfeng Yi & Qin Zhou

  3. Department of Breast Surgery, Harbin Medical University Cancer Hospital, Harbin, China

    Tong Liu & Yuhan Deng

  4. Cancer Center, Zhejiang University, Hangzhou, China

    Zheng Wang, Qingang Wu, Liwei Xiao, Xueying Yang, Zhimin Lu & Daqian Xu

  5. Department of Cancer Biology, Department of Neuro-Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA

    Yan Xia

  6. Department of Hematology, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China

    Xueying Yang

  7. Department of Clinical Laboratory, Cancer Hospital of the University of Chinese Academy of Sciences (Zhejiang Cancer Hospital), Institute of Basic Medicine and Cancer (IBMC), Chinese Academy of Sciences, Hangzhou, China

    Xu Qian

  8. The Affiliated Hospital of Qingdao University and Qingdao Cancer Institute, Qingdao, China

    Leina Ma & Jing Fang

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  1. Ke Wu
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Contributions

D.X., Z.L. and Q.Z. conceived and designed the study and wrote the paper. K.W., M.Y., T.L., Z.W., Y.D., G.J., Y.S., L.W., L.L., P.Z., B.D., X.Y., H.S., T.W., J.Z., J.Y. and Y.D. performed the experiments and analysed the data. Y.X., Q.W. and L.X. reviewed and edited the paper. X.Q., L.M. and J.F. provided the technical support in gene editing and interpretation of the data.

Corresponding authors

Correspondence to Qin Zhou, Zhimin Lu or Daqian Xu.

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Competing interests

Z.L. owns shares in Signalway Biotechnology (Pearland, TX), which supplied rabbit antibodies that recognize CKB pT133 and GPX4 pS104. Z.L.’s interest in this company had no bearing on its being chosen to supply these reagents. The other authors declare no competing interests.

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Extended data

Extended Data Fig. 1 CKB T133 phosphorylation upregulates GPX4 stability.

(b, d-h, l,m, r) Immunoprecipitation and immunoblotting analyses were performed with the indicated antibodies. (a, b, q-s) Data are the mean ± SD (n = 6). N.S., not significant (a, q, s); **P < 0.001 (r); ***P < 0.0001(b) by two-tailed Student’s t-test. (a, m, q-s) Huh7 and HCCLM3 were treated with IGF1 for the indicated time (a). The indicated cells expressing CKB shRNA with reconstituted expression of the indicated CKB proteins were harvested (m, s), or treated with IGF1 for 12 h (q), or treated with CHX for the indicated time (r). The mRNA expression levels of Gpx4 gene were measured using qPCR (a, q, s). (b) Huh7 and HCCLM3 cells were treated with CHX for the indicated time in the presence of IGF1. The quantification of GPX4 protein levels relative to initial protein levels is shown. (c) Selected mass spectrometry identified peptide hits are shown. (d) Huh7 cells were pretreated with or without indicated inhibitors for 30 min before treatment with or without IGF1 for 1 h. (e) Huh7 cells transfected with or without the indicated plasmid were treated with or without Tris DBA (10 μM) for 12 h. (f) Huh7 cells were treated with or without IGF1 (100 ng/ml) for 1 h. (g) A GST pull-down assay was performed as indicated. (h) In vitro kinase assays were performed in the presence of [γ-32P] ATP. Autoradiography were performed. (i) Purified GST-CKB proteins were incubated with His-AKT1 for an in vitro kinase assay. Mass-spectrometric analysis was performed. (j) Alignment of protein sequences spanning CKB T133 from different species. (k, l) Huh7 cells expressing Flag-CKB were treated with or without IGF1 for 1 h (l). IHC analyses of human HCC samples (k) or immunoblotting (l) were performed with the indicated antibodies and a CKB pT133-blocking peptide. (n-p) Genomic DNA was extracted from two individual clones of indicated cells with knock-in expression of CKB T133A. PCR products amplified from the indicated DNA fragments were shown (n, o) and sequenced (p).

Source data

Extended Data Fig. 2 GPX4 S104 phosphorylation stabilizes GPX4.

(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.

Source data

Extended Data Fig. 3 CKB-mediated GPX4 S104 phosphorylation stabilizes GPX4.

(a, b) Alignment of protein sequences spanning GPX4 S104 and the adjacent CMA-target motif from different species. The structure of GPX4 exhibiting CMA-target motif and S104. (c) Purified His-CKB WT or T133D proteins on Ni-NTA beads were incubated with the indicated GST-GPX4 proteins for an in vitro kinase assay. Then the beads were removed and the remaining GST-GPX4 proteins were incubated with His-HSC70 followed by a GST pulldown assay as indicated. (d, e) Huh7 and HCCLM3 cells expressing GPX4 shRNA with reconstituted expression of the indicated GPX4 proteins were harvested for immunoblotting and mRNA detection using quantitative PCR as indicated. Data are the mean ± SD (n = 6). N.S., not significant (two-tailed Student’s t-test).

Source data

Extended Data Fig. 4 GPX4 S104 phosphorylation suppresses Lipid peroxidation.

(a-d) Parental Huh7 and HCCLM3 cells and the indicated clones with knock-in expression of CKB T133A mutants (upper) or GPX4 S104A (lower) were treated with or without cystine deprivation (a, c) or 20 μM Erastin (b, d) and IGF1 for 24 h. Reduced GSH (a, b) and GSSG (c, d) were measured respectively. Data are the mean ± SD (n = 6), ^P < 0.05; *P < 0.01; **P < 0.001; ***P < 0.0001 by two-tailed Student’s t-test. (e) Parental Huh7 and HCCLM3 cells and the indicated clones with knock-in expression of CKB T133A mutants (left) or GPX4 S104A (right) were treated with or without cystine deprivation or 20 μM Erastin in the presence or absence of IGF1 (100 ng/ml) for 24 h. The cell lysates were harvested for immunoblotting analyses as indicated. (f-k) Parental HCCLM3 (f, g, j,k) or Huh7 (h, i) cells and the indicated clones with knock-in expression of CKB T133A or GPX4 S104A mutants were treated with or without cystine deprivation (f, h, j) or 20 μM Erastin (g, i, k) combined with Fer-1 (2 μM) in the absence or presence of IGF1 for 24 h. Lipid ROS-positive cells were measured. Data are the mean ± SD (n = 3), *P < 0.01 by two-tailed Student’s t-test (f, g). Lipid peroxidation was assessed using BODIPY 581/591 C11 staining followed by FACS analysis.

Source data

Extended Data Fig. 5 GPX4 S104 phosphorylation suppresses ferroptosis.

(a, b, d-k) Data are the mean ± SD, *P < 0.01; **P < 0.001;***P < 0.0001 by two-tailed Student’s t-test. (a, b, d-g) Parental Huh7 cells and the indicated clones with knock-in expression of CKB T133A or GPX4 S104A mutants were treated with or without cystine deprivation (a, d, f) or 20 μM Erastin (b, e, g) in the presence or absence of IGF1 (100 ng/ml) for 24 h. The indicated mRNA expression levels were measured using quantitative PCR. GSSG (d, e) and reduced GSH (f, g) were measured respectively (n = 6). (c) Huh7 cells stably transfected with GLRX and GSTA1 shRNA were harvested for immunoblotting analyses as indicated. (h, i) Parental HCCLM3 cells and the indicated clones with knock-in expression of CKB T133A or GPX4 S104A mutants were treated with or without cystine deprivation (h) or 20 μM Erastin (i) combined with Fer-1 (2 μM) in the absence or presence of IGF1 for 24 h. Cell death were measured respectively (n = 3). (j, k) Parental HCCLM3 cells and the indicated clones with knock-in expression of CKB T133A mutants or GPX4 S104A were treated with different doses of sulfasalazine (SAS) (j) or Erastin (k) for 24 h, cell viability were measured (n = 3). (l) Parental Huh7 and HCCLM3 cells and the indicated clones with knock-in expression of CKB T133A mutants were treated with or without IGF1 for 12 h. Immunoblotting analyses were performed with the indicated antibodies.

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Extended Data Fig. 6 CKB suppresses ferroptosis in a GPX4 pS104 dependent manner.

(a-l) Data are the mean ± SD (n = 3), *P < 0.01; **P < 0.001; N.S., not significant by two-tailed Student’s t-test. (a-d) Huh7 and HCCLM3 cells expressing GPX4 shRNA with reconstituted expression of the indicated GPX4 proteins were treated with or without cystine deprivation (a, c) or 20 μM Erastin (b, d) for 24 h. Lipid ROS-positive cells (a, b) and cell death (c, d) were measured respectively. (e, f) Huh7 and HCCLM3 cells expressing GPX4 shRNA with reconstituted expression of the indicated GPX4 proteins were treated with different doses of sulfasalazine (SAS) (e) or Erastin (f) for 24 h, cell viability were measured. (g-l) Parental Huh7 cells and the indicated clones with knock-in expression of GPX4 S104A mutants were stably transfected with CKB shRNA and reconstituted with indicated CKB proteins. The cells were treated with or without cystine deprivation (g, i) or 20 μM Erastin (h, j) for 24 h. Lipid ROS-positive cells (g, h) and cell death (i, j) were measured respectively. The cells were treated with different doses of sulfasalazine (SAS) (k) or Erastin (l) for 24 h, cell viability were measured.

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Extended Data Fig. 7 CKB suppresses ferroptosis through its protein kinase activity.

(a-l) Data are the mean ± SD (n = 3), ^ P < 0.05; *P < 0.01; **P < 0.001; N.S., not significant by two-tailed Student’s t-test. (a-d) Huh7 and HCCLM3 cells expressing IGF1R-CA and CKB shRNA with reconstituted expression of the indicated CKB proteins were treated with or without cystine deprivation (a, c) or 20 μM Erastin (b, d) for 24 h. Lipid ROS-positive cells (a, b) and cell death (c, d) were measured respectively. (e, f) Huh7 and HCCLM3 cells expressing IGF1R-CA and CKB shRNA with reconstituted expression of the indicated CKB proteins were treated with different doses of sulfasalazine (SAS) (e) or Erastin (f) for 24 h, cell viability were measured. (g-j) Parental Huh7 and HCCLM3 cells and the indicated clones with knock-in expression of CKB T133A (g, i) or GPX4 S104A mutants (h, j) were treated with or without 2 μM RSL3 for 8 h. Lipid ROS-positive cells (g, h) and cell death (i, j) were measured respectively. (k, l) Parental Huh7 and HCCLM3 cells and the indicated clones with knock-in expression of CKB T133A (k) and GPX4 S104A mutants (l) were treated with different doses of RSL3 for 8 h, cell viability were measured.

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Extended Data Fig. 8 CKB phosphorylates GPX4 to inhibit ferroptosis.

(a, b) MDA-MB-231, A549 and HCT116 cells expressing CKB shRNA with reconstituted expression of the indicated CKB proteins were treated with or without IGF1 for 12 h. Immunoblotting analyses were performed with the indicated antibodies. (c, d) MDA-MB-231, A549 and HCT116 cells expressing CKB shRNA with reconstituted expression of the indicated CKB proteins were treated with or without 20 μM Erastin in the absence or presence of IGF1 for 24 h. Lipid ROS-positive cells (c) and cell death (d) were measured respectively. Data are the mean ± SD (n = 3), ^ P < 0.05; *P < 0.01; **P < 0.001 by two-tailed Student’s t-test. (e-g) Genomic DNA was extracted from parental MDA-MB-231, A549 and HCT116 cells and the indicated clones with knock-in expression of GPX4 S104A. PCR products amplified from the indicated DNA fragments were shown (e, f) and sequenced (g). (h) Parental MDA-MB-231, A549 and HCT116 cells and the indicated clones with knock-in expression of GPX4 S104A mutants were treated with or without IGF1 for 12 h. Immunoblotting analyses were performed with the indicated antibodies. (i, j) Parental MDA-MB-231, A549 and HCT116 cells and the indicated clones with knock-in expression of GPX4 S104A mutants were treated with or without 20 μM Erastin for 24 h. Lipid ROS-positive cells (i) and cell death (j) were measured respectively. Data are the mean ± SD (n = 3), *P < 0.01; **P < 0.001 by two-tailed Student’s t-test.

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Extended Data Fig. 9 AKT-CKB-GPX4 axis suppresses ferroptosis.

(a) The whole cell lysates of LN18, LN229, U87 and U251 were harvested for immunoblotting analyses as indicated. (b, c) LN18, LN229, U87 and U251 cells were treated with or without 20 μM Erastin combined with Fer-1 (2 μM) for 24 h. Lipid ROS-positive cells (b) and cell death (c) were measured respectively. Data are the mean ± SD (n = 3), *P < 0.01; **P < 0.001; *** P < 0.0001 by two-tailed Student’s t-test. (d) U251 and U87 cells were treated with or without MK-2206 (5 μM) for 12 h. Immunoblotting analyses were performed with the indicated antibodies. (e, f) Parental U251 and U87 cells were treated with or without 20 μM Erastin in the absence or presence of MK-2206 (5 μM) for 24 h. Lipid ROS-positive cells (e) and cell death (f) were measured respectively. Data are the mean ± SD (n = 3), *P < 0.01; **P < 0.001 by two-tailed Student’s t-test.

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Extended Data Fig. 10 CKB-mediated GPX4 phosphorylation promotes tumour growth.

(a, b, e-i, k, l, n, o, q) Data are the mean ± SD, ^P < 0.05; *P < 0.01; **P < 0.001; ***P < 0.0001; N.S., not significant by two-tailed Student’s t-test (a, e, g, h, i, l, o) and by two-tailed Mann-Whitney U test (b, f, k, n, q). (b, f, j, m, p) IHC analyses of the indicated xenograft tumors from nude mice were performed with the indicated antibodies. Representative staining images and staining scores are shown. (a, b) Huh7 cells were subcutaneously injected into 6-week-old male athymic nude mice. When the tumor reached 50 mm3, the mice were assigned randomly into different treatment groups. Sulfasalazine (SAS) and Liproxstatin-1 (Lip-1) were intraperitoneally injected daily at a dose of 100 mg/kg and 10 mg/kg respectively until the endpoint at Day 28. Tumor volume and weight were analyzed (n = 7) (a). (c, r) The whole cell lysates of WT and GPX4 knockout Huh7 cells (c) and L02, THLE-2, Huh7, HCCLM3 (r) were harvested for immunoblotting analyses as indicated. (d-f) WT and GPX4 knockout Huh7 cells were subcutaneously injected into the left or right flanks of 6-week-old male athymic nude mice with and without daily Lip-1 intraperitoneal injection from the 4th day. The resulting tumors were resected 28 days after injection (d). Tumor volume and weight were analyzed (n = 6) (e). (g-q) IGF1R CA-expressing parental Huh7 cells and the indicated clones with knock-in expression of CKB T133A (g) and GPX4 S104A (h) or Huh7 cells expressing CKB shRNA with reconstituted expression of the indicated CKB proteins (i-k) or Parental Huh7 cells and the indicated clones with knock-in expression of GPX4 S104D mutants stably transfected with CKB shRNA and reconstituted with indicated CKB proteins (l-q) were intrahepatically (g, h) or subcutaneously (i-q) injected into 6-week-old male athymic nude mice (n = 7). When the tumor reached 50 mm3, the mice were assigned randomly into different treatment groups. SAS was intraperitoneally injected daily at a dose of 100 mg/kg until the endpoint at Day 28. Tumor volume and weight were analyzed.

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Wu, K., Yan, M., Liu, T. et al. Creatine kinase B suppresses ferroptosis by phosphorylating GPX4 through a moonlighting function. Nat Cell Biol 25, 714–725 (2023). https://doi.org/10.1038/s41556-023-01133-9

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