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The gluconeogenic enzyme PCK1 phosphorylates INSIG1/2 for lipogenesis

Abstract

Cancer cells increase lipogenesis for their proliferation and the activation of sterol regulatory element-binding proteins (SREBPs) has a central role in this process. SREBPs are inhibited by a complex composed of INSIG proteins, SREBP cleavage-activating protein (SCAP) and sterols in the endoplasmic reticulum. Regulation of the interaction between INSIG proteins and SCAP by sterol levels is critical for the dissociation of the SCAP–SREBP complex from the endoplasmic reticulum and the activation of SREBPs1,2. However, whether this protein interaction is regulated by a mechanism other than the abundance of sterol—and in particular, whether oncogenic signalling has a role—is unclear. Here we show that activated AKT in human hepatocellular carcinoma (HCC) cells phosphorylates cytosolic phosphoenolpyruvate carboxykinase 1 (PCK1), the rate-limiting enzyme in gluconeogenesis, at Ser90. Phosphorylated PCK1 translocates to the endoplasmic reticulum, where it uses GTP as a phosphate donor to phosphorylate INSIG1 at Ser207 and INSIG2 at Ser151. This phosphorylation reduces the binding of sterols to INSIG1 and INSIG2 and disrupts the interaction between INSIG proteins and SCAP, leading to the translocation of the SCAP–SREBP complex to the Golgi apparatus, the activation of SREBP proteins (SREBP1 or SREBP2) and the transcription of downstream lipogenesis-related genes, proliferation of tumour cells, and tumorigenesis in mice. In addition, phosphorylation of PCK1 at Ser90, INSIG1 at Ser207 and INSIG2 at Ser151 is not only positively correlated with the nuclear accumulation of SREBP1 in samples from patients with HCC, but also associated with poor HCC prognosis. Our findings highlight the importance of the protein kinase activity of PCK1 in the activation of SREBPs, lipogenesis and the development of HCC.

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Fig. 1: IGF1-induced and AKT-phosphorylated PCK1 translocates to the ER and binds to INSIG1/2.
Fig. 2: PCK1 phosphorylates INSIG1 Ser207 and INSIG2 Ser151, thereby reducing the binding of oxysterols to INSIG1/2.
Fig. 3: PCK1-mediated phosphorylation of INSIG1/2 releases SCAP from the ER and promotes SREBP1 activation for lipogenesis.
Fig. 4: PCK1-mediated phosphorylation of INSIG1/2 promotes liver tumour growth and correlates with poor prognosis for HCC.

Data availability

All source data for immunoblotting are shown in Supplementary Fig. 1. All the other source data for Figs. 24 and Extended Data Figs. 110, containing the raw data for all experiments, are provided with the paper. All images and data were created and analysed by the authors and will be available from the lead corresponding author (Z.L.) on reasonable request.

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Acknowledgements

We thank L. Li for technical assistance. The mass spectrometry was supported in part by the Clinical and Translational Proteomics Service Center at the University of Texas Health Science Center. This work was supported by Zhejiang University Research Fund (188020*194221901/029) (Z.L.); the Leading Innovative and Entrepreneur Team Introduction Program of Zhejiang (2019R01001) (Z.L.); MOHW109-TDU-B-212-010001 (M.-C.H.); the Drug Development Center, China Medical University from the Ministry of Education in Taiwan (M.-C.H.); and The Odyssey Fellowship from The University of Texas MD Anderson Cancer Center (D.X.). Z.L. is Kuancheng Wang Distinguished Chair.

Author information

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Authors

Contributions

Z.L. and D. Xu conceived and designed the study and wrote the manuscript; Z.L. and M.-C.H. acquired the funding support and supervised the study; D. Xu and Z.W. performed most experiments; Y.X., F.S., X.L. and X.Q. provided support for generating different knock-in mutation cell lines and protein purification; W.X., Y.W. and G.L. provided support for IHC staining; F.S., J.-H.L. and L.D. were involved in the studies with mice; M.-C.H., D. Xing and T.L. reviewed and edited the manuscript; H.W. provided the HCC samples and technical support; Y.Z., J.-s.L., D. Xing and T.L. performed statistical analysis and interpretation of the data.

Corresponding authors

Correspondence to Daqian Xu, Mien-Chie Hung or Zhimin Lu.

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Competing interests

Z.L. owns shares in Signalway Biotechnology (Pearland, TX), which supplied rabbit antibodies that recognize PCK1(pS90), INSIG1(pS207) and INSIG2(pS151). The interest of Z.L. in this company had no bearing on its being chosen to supply these reagents.

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Extended data figures and tables

Extended Data Fig. 1 IGF1-induced AKT activation induces the translocation of PCK1 to the ER and the binding of PCK1 to INSIG1/2.

a, Huh7 cells with or without expression of Flag–INSIG1 (left) or Flag–INSIG2 (right) were treated with or without IGF1 (100 ng ml−1) for 1 h. An immunoprecipitation assay was performed using anti-Flag antibody, and immunoprecipitates of Flag–INSIG1 or Flag–INSIG2 were eluted with Flag peptide, separated using SDS–PAGE and stained with Coomassie Brilliant Blue. Selected peptide hits of proteins associated with Flag–INSIG1 or Flag–INSIG2, identified through mass spectrometry, are shown. b, Hep3B cells were treated with or without IGF1 (100 ng ml−1) for 1 h. c, Huh7 cells expressing Flag–PCK1 or Flag–PCK2 were stimulated with or without IGF1 (100 ng ml−1) for 1 h. d, Huh7 cells expressing Flag–PCK1 or Flag–PCK2 were stimulated with or without IGF1 (100 ng ml−1) for 1 h. Immunofluorescence analyses were performed with the indicated antibodies (top). The colocalization coefficients between the indicated proteins in the presence or absence of IGF1 are shown (bottom). At least n = 50 cells from each independent experiment were analysed and representative data are shown. Data are mean ± s.d. **P < 0.001 (two-tailed t-test). The regions in white boxes are shown at higher magnification on the right. e, Whole cell lysate (WCL), cytosolic and ER fractions were prepared from Huh7 and Hep3B cells stimulated with or without IGF1 (100 ng ml−1) for 1 h. Cellular fractions from equal numbers of cells were analysed using immunoblotting with the indicated antibodies. f, Huh7 cells expressing Flag–PCK1 or Flag–PCK2 were stimulated with or without IGF1 (100 ng ml−1) for 1 h. ER fractions and total lysates were prepared for immunoblotting analyses with the indicated antibodies. g, Total lysates were prepared from Huh7 cells pretreated with or without U0126 (20 μM), SP600125 (25 μM), SU6656 (4 μM) or MK-2206 (10 μM) for 30 min before treatment with or without IGF1 (100 ng ml−1) for 1 h. h, Huh7 cells expressing wild-type HA–AKT1 or HA–AKT1-DN (K179A, T308A, S473A were treated with or without IGF1 (100 ng ml−1) for 1 h. i, Huh7 cells were pretreated with or without U0126 (20 μM), SU6656 (4 μM), SP600125 (25 μM) or MK-2206 (10 μM) for 30 min before treatment with or without IGF1 (100 ng ml−1) for 1 h. ER fractions and total lysates were isolated for immunoblotting analyses with the indicated antibodies. j, Huh7 cells were stably transfected with a control vector or a vector expressing HA–AKT1-DN (K179A, T308A, S473A). The cells were treated with or without IGF1 (100 ng ml−1) for 1 h. ER fractions and total lysates were isolated for immunoblotting analyses with the indicated antibodies. k, Huh7 cells were pretreated with or without MK-2206 (10 μM) for 30 min and treated with or without IGF1 (100 ng ml−1) for 1 h. Immunofluorescence analyses were performed with the indicated antibodies (left). The colocalization coefficients between the indicated proteins are shown (right). At least n = 50 cells from each independent experiment were analysed and representative data are shown. Data are mean ± s.d. **P < 0.001 (two-tailed t-test). l, Huh7 cells expressing Flag–PCK1 or Flag–PCK2 were transfected with or without HA–myr-AKT1. m, Huh7 and Hep3B cells were treated with or without IGF1 (100 ng ml−1) for 1 h. In b, c, ej, l, m, immunoprecipitation and immunoblotting analyses were performed with the indicated antibodies, and the experiments were repeated three times independently with similar results.

Source Data

Extended Data Fig. 2 AKT-mediated phosphorylation of PCK1 Ser90 is necessary and sufficient for the translocation of PCK1 to the ER and the binding of PCK1 to INSIG1/2, and inhibits the canonical role of PCK1 in gluconeogenesis.

a, A GST pull-down assay was performed by mixing purified His–PCK1 with purified GST or GST–AKT1. Immunoblotting analyses were performed as indicated. b, Left, schematic of AKT1 full-length and deletion mutants. Right, 293T cells were transfected with the indicated constructs, and GST pull-down assays and immunoblotting analyses with the indicated antibodies were performed. c, In vitro kinase assays were performed by mixing purified GST–PCK1 with purified active or inactive His–AKT1 in the presence of [γ-32P]ATP. Autoradiography and immunoblotting analyses were performed as indicated. d, In vitro kinase assays were performed by mixing purified His–PCK1 with or without purified GST–AKT1 in the presence of ATP. Mass spectrometric analysis of a tryptic fragment at m/z 805.91107 Da (−0.06 mmu/−0.08 ppm), which was matched with the +2 charged peptide 88-IESKTVIVTQEQR-100, suggested that PGK1 Ser90 was phosphorylated. The Mascot score was 31, and the expectation value was 0.25. e, Alignment of protein sequences spanning PCK1 Ser90 from different species. f, Alignment of PCK1 Ser90 to the non-canonical AKT-phosphorylated substrate motif (RXXS/T). The reported AKT substrates (TFEB, YAP1, CREB, CDK2, β-catenin, FAM129A, CDC25B and NPM) are shown. Red, phosphoacceptor residue; blue, basic residue (R). g, In vitro kinase assays were performed by mixing purified GST–PCK1 or GST–PCK1(S90A) and active His–AKT1 in the presence or absence of MK-2206 (10 μM) for 1 h. Immunoblotting analyses were performed as indicated. h, Left, IHC analyses of human HCC samples were performed with the indicated antibodies in the presence or absence of a blocking peptide for PCK1(pS90). Right, Huh7 cells expressing Flag–PCK1 were treated with or without IGF1 (100 ng ml−1) for 1 h. Immunoprecipitation and immunoblotting analyses were performed with the indicated antibodies in the presence or absence of a blocking peptide for PCK1(pS90). i, Huh7 cells expressing wild-type Flag–rPCK1 or Flag–rPCK1(S90A) were pretreated with or without MK-2206 (10 μM) for 30 min. The cells were treated with or without IGF1 (100 ng ml−1) for 1 h. Immunoprecipitation and immunoblotting analyses were performed with the indicated antibodies. j, PCK1-depleted Huh7 cells were reconstituted with the indicated shRNA-resistant PCK1 proteins. After transfection with HA–myr-AKT1, ER fractions and total cell lysate were prepared for immunoblotting analyses as indicated. k, Huh7 and Hep3B cells expressing the indicated Flag–rPCK1 proteins were treated with or without IGF1 (100 ng ml−1) for 1 h. ER fractions and total cell lysate were prepared for immunoblotting analyses as indicated. l, m, Genomic DNA was extracted from two individual clones of parental Huh7 or Hep3B cells with knock-in expression of PCK1(S90A). PCR products were amplified from the indicated DNA fragment (l) and separated on an agarose gel (m). n, Sequencing of parental Huh7 and Hep3B cells and two individual clones of parental cells with knock-in expression of PCK1(S90A). The red line indicates the sgRNA-targeting sequence. The black line indicates the protospacer adjacent motif (PAM). Blue arrows indicate mutated nucleotides. A mutated amino acid and its wild-type counterpart are indicated by the solid red box. o, p, Parental Huh7 and Hep3B cells and the indicated clones of Huh7 and Hep3B cells with knock-in expression of PCK1(S90A) were stimulated with or without IGF1 (100 ng ml−1) for 1 h. ER fractions and total cell lysates were prepared and immunoblotting analyses were performed as indicated (o). Immunofluorescence staining of Huh7 cells was performed with the indicated antibodies (p, left). The colocalization coefficients between the indicated proteins are shown (p, right). At least n = 50 cells from each independent experiment were analysed and representative data are shown. Data are mean ± s.d. **P < 0.001 (two-tailed t-test). qs, Bacterially purified wild-type His–PCK1 or His–PCK1(S90A) on Ni-NTA agarose beads were incubated with or without purified active GST–AKT1 in the presence of ATP for an in vitro AKT kinase assay. Immunoblotting analyses were performed as indicated (q). After washing wild-type His–PCK1 or His–PCK1(S90A)-conjugated beads with PBS five times, the binding affinity of PCK1 to oxaloacetate (OAA) (r) and the relative PCK1 activity (s) were measured. Data are mean ± s.d. (n = 6 biological replicates). **P < 0.001 (two-tailed t-test); NS, not significant (P = 0.923 (r); P = 0.728 (s)). tv, Wild-type His–PCK1 and His–PCK1(S90E) were purified from bacteria and Coomassie Brilliant Blue staining analyses were performed (t). The binding affinity of the His–PCK1 proteins to oxaloacetate (u) and the relative PCK1 activity (v) were measured. Data are mean ± s.d. (n = 6 biological replicates). **P < 0.001 (two-tailed t-test). w, Bacterially purified wild-type GST–PCK1 or GST–PCK1(S90A) on glutathione agarose beads were incubated with or without active His–AKT1 in the presence of ATP for an in vitro kinase assay. The GST-tagged proteins were then incubated with or without CIP (10 U) for 30 min at 37 °C, followed by incubation with Flag/His-tagged INSIG1 or INSIG2 purified from Huh7 cells for a pull-down assay. x, SFB-tagged wild-type PCK1 or PCK1(S90A) were pulled down from Huh7 cells treated with or without IGF1 (100 ng ml−1) for 1 h. These proteins were incubated with or without CIP (10 U) for 30 min at 37 °C. Immunoblotting analyses were performed as indicated. y, Parental Hep3B cells and the indicated clones of cells with knock-in expression of PCK1(S90A) were stimulated with or without IGF1 for 1 h. Immunoprecipitation and immunoblotting analyses were performed as indicated. z, Huh7 cells expressing INSIG1 shRNA and INSIG2 shRNA were treated with or without IGF1 (100 ng ml−1) for 1 h (left) or transfected with HA–myr-AKT1 (right). ER fractions and total cell lysate were prepared for immunoblotting analyses as indicated. All experiments were repeated three times independently with similar results.

Source Data

Extended Data Fig. 3 INSIG1/2 is required for the translocation of PCK1 to the ER and PCK1 functions as a protein kinase to phosphorylate INSIG1 Ser207 and INSIG2 Ser151.

a, Top, the topological structures of INSIG1 and INSIG2, which have 69% amino acid sequence identity and contain six transmembrane-spanning regions. Bottom, different INSIG1 (left) or INSIG2 (right) truncation mutants were expressed in 293T cells. These cells were treated with or without IGF1 (100 ng ml−1) for 1 h. Immunoprecipitation and immunoblotting analyses were performed with the indicated antibodies. b, Bacterially purified GST–PCK1 was incubated with or without active His–AKT1 in the presence of ATP for 1 h. After the in vitro kinase assay, GST–PCK1-conjugated beads were washed with PBS five times and then incubated with purified Flag/His–INSIG1 or Flag/His–INSIG2 in the absence or presence of a PCK1(pS90) peptide for 2 h. Immunoblotting analyses were performed with the indicated antibodies. c, Endogenous PCK1-depleted Huh7 cells with reconstituted expression of shRNA-resistant wild-type rPCK1 or rPCK1(C228S) were transfected with Flag–INSIG1 (left) or Flag–INSIG2 (right), respectively. After being treated with or without IGF1 (100 ng ml−1) for 1 h, the cells were collected for immunoprecipitation and immunoblotting analyses as indicated. d, Bacterially purified wild-type His–PCK1, His–PCK1(S90A) or His–PCK1(C288S) on Ni-NTA agarose beads were incubated with or without active GST–AKT1 in the presence of ATP for an in vitro AKT kinase assay. The beads were washed with PBS five times and incubated with SFB–INSIG2 in the presence of [γ-32P]GTP. Autoradiography and immunoblotting analyses with the indicated antibodies were performed. e, In vitro kinase assays were performed by mixing purified His–PCK1 on Ni-NTA agarose beads with purified active GST–AKT1 in the presence of ATP for 1 h. His–PCK1-conjugated Ni-NTA agarose beads were washed with PBS five times and then incubated with SFB–INSIG1 or SFB–INSIG2 purified from Huh7 cells in the presence of [γ-32P]ATP or [γ-32P]GTP for 1 h. Autoradiography and immunoblotting analyses with the indicated antibodies were performed. f, An in vitro kinase assay was performed as in e, except that the His–PCK1-conjugated beads were incubated with SFB–INSIG1 purified from Huh7 cells in the presence of GTP for 1 h. Mass spectrometric analysis of a tryptic fragment at m/z 764.40387 Da (−1.96 mmu/−2.57 ppm), which was matched with the +2 charged peptide 205-RSRSGLGLGITIAF-218, suggested that INSIG1 Ser207 was phosphorylated. The Mascot score was 53, and the expectation value was 0.021. g, PCK1-mediated phosphorylation residues in the cytosolic loop 2 of both INSIG1 and INSIG2. h, Alignment of protein sequences spanning INSIG1 Ser207 and INSIG2 Ser151 from different species. i, Bacterially purified wild-type His–PCK1 on Ni-NTA agarose beads was incubated with or without purified active GST–AKT1 in the presence of ATP for 1 h for an in vitro AKT kinase assay. The beads were then washed with PBS five times and incubated with or without wild-type SFB–INSIG2 or SFB–INSIG2(S151A) in the presence of [γ-32P]GTP. Autoradiography and immunoblotting analyses with the indicated antibodies were performed. j, IHC analyses of human HCC samples were performed with the indicated antibodies in the presence or absence of a blocking peptide for INSIG1(pS207) and INSIG2(pS151). k, Huh7 cells expressing Flag–INSIG1 (top) or Flag–INSIG2 (bottom) were treated with or without IGF1 (100 ng ml−1) for 1 h. Immunoprecipitation and immunoblotting analyses were performed with the indicated antibodies in the presence or absence of a blocking peptide for INSIG1(pS207) and INSIG2(pS151). l, Enzyme kinetics plots of velocity relative to GTP concentration between purified wild-type His–PCK1 and His–PCK1(S90E). The Vmax and Km of PCK1 in phosphorylating an INSIG1 peptide at Ser207 were calculated (n = 6). Data are mean ±s.d. m, n, Bacterially purified wild-type His–PCK1, His–PCK1(S90E) or His–PCK1(S90D) on Ni-NTA agarose beads were incubated with wild-type SFB–INSIG1 or SFB–INSIG1(S207A) (m) or wild-type SFB–INSIG2 or SFB–INSIG2(S151A) (n) in the presence of [γ-32P]GTP. Autoradiography and immunoblotting assays with the indicated antibodies were performed. o, Wild-type Flag–INSIG1 or Flag–INSIG1(S207A) (left) or wild-type Flag–INSIG2 or Flag–INSIG2(S151A) (right) were transfected into Huh7 cells. Huh7 cells were stimulated with or without IGF1 (100 ng ml–1) for 1 h. Immunoprecipitation and immunoblotting analyses were performed with the indicated antibodies. p, Wild-type Flag–INSIG1 or Flag–INSIG1(S207A) (left) or wild-type Flag–INSIG2 or Flag–INSIG2(S151A) (right) were co-transfected with or without HA–myr-AKT1 into Huh7 cells. Immunoprecipitation and immunoblotting analyses were performed with the indicated antibodies. q, Parental Huh7 cells and the indicated clones of cells with knock-in expression of PCK1(S90A) were transfected with SFB–INSIG1 (left) or SFB–INSIG2 (right). These cells were then stimulated with or without IGF1 (100 ng ml–1) for 1 h. All experiments were repeated three times independently with similar results.

Source Data

Extended Data Fig. 4 Generation of Huh7 and Hep3B cells with knock-in expression of both INSIG1(S207A) and INSIG2(S151A), and PCK1-mediated phosphorylation of INSIG1/2 reduces the binding of oxysterols to INSIG1/2.

a, b, Genomic DNA was extracted from two individual clones of parental Huh7 and Hep3B cells with knock-in expression of INSIG1(S207A) (a) and INSIG2(S151A) (b). PCR products amplified from the indicated DNA fragments were separated on an agarose gel. c, d, Sequencing of parental and two individual clones of parental Huh7 and Hep3B cells with knock-in expression of INSIG1(S207A)/INSIG2(S151A) double mutants. The red line indicates the sgRNA-targeting sequence. The black line indicates the PAM. Blue arrows indicate mutated nucleotides. A mutated amino acid and its wild-type counterpart are indicated by the solid red box. e, Top, Flag/His-tagged INSIG2 immunoprecipitated and purified from Huh7 cells was incubated with the indicated GST–PCK1 proteins with or without active GST–AKT1 in the presence of ATP and GTP for 1 h. Immunoblotting analyses were performed with the indicated antibodies. Bottom, INSIG2-conjugated Ni-NTA agarose beads were washed and incubated with 400 nM [3H]25-hydroxycholesterol. Specifically bound [3H]25-hydroxycholesterol was measured (n = 6). **P < 0.001 (two-tailed t-test); NS, not significant (P=0.875, 0.846, 0.969, 0.924 (left to right)). f, g, Top, Flag/His-tagged wild-type INSIG1 or INSIG1(S207A) (f) or Flag/His-tagged wild-type INSIG2 or INSIG2(S151A) (g) purified from Huh7 cells were incubated with purified wild-type GST–PCK1, GST–PCK1(S90D) or GST–PCK1(S90E) in the presence of GTP for 1 h. Immunoblotting analyses were performed with the indicated antibodies. Bottom, the INSIG1 or INSIG2 proteins on Ni-NTA agarose beads were washed with PBS five times and incubated with 400 nM [3H]25-hydroxycholesterol. Specifically bound [3H]25-hydroxycholesterol was measured (n = 6). **P < 0.001 (two-tailed t-test); NS, not significant (P=0.823, 0.445, 0.185 (left to right) (f); P=0.320, 0.196, 0.735 (left to right) (g)). h, Top, Flag/His-tagged wild-type INSIG2 or INSIG2(S151A) purified from Huh7 cells were incubated with purified wild-type GST–PCK1 with or without purified active GST–AKT1 in the presence of ATP and GTP for 1 h. Immunoblotting analyses were performed with the indicated antibodies. Bottom, the INSIG2 protein on Ni-NTA agarose beads was washed with PBS five times and incubated with 400 nM [3H]25-hydroxycholesterol. Specifically bound [3H]25-hydroxycholesterol was measured (n = 6). **P < 0.001 (two-tailed t-test); NS, not significant (P = 0.682, 0.947 (left to right)). i, j, Flag/His-tagged wild-type INSIG1 or INSIG1(S207A) (i) or wild-type INSIG2 or INSIG2(S151A) (j) were purified from Huh7 treated with or without IGF1 (100 ng ml−1) for 12 h. Immunoblotting analyses were performed with the indicated antibodies (left). The INSIG1 or INSIG2 proteins on Ni-NTA agarose beads were incubated with the indicated concentration of [3H]25-hydroxycholesterol. Specifically bound [3H]25-hydroxycholesterol was measured (n = 6) (right). k, l, Top, Flag/His-tagged INSIG1 (k) or INSIG2 (l) was expressed in parental Huh7 cells and the Huh7 cells with knock-in expression of PCK1(S90A). After these cells were treated with or without IGF1 (100 ng ml−1) for 12 h, immunoblotting analyses were performed with the indicated antibodies. Bottom, the immunoprecipitated and purified INSIG1 or INSIG2 was incubated with the indicated concentration of [3H]25-hydroxycholesterol. Specifically bound [3H]25-hydroxycholesterol was measured (n = 6). m, n, Top, the immunoprecipitated and purified Flag/His-tagged wild-type INSIG1 or INSIG1(S207E) (m) or Flag/His-tagged wild-type INSIG2 or INSIG2(S151E) (n) from Huh7 cells was incubated with the indicated concentration of [3H]25-hydroxycholesterol. Specifically bound [3H]25-hydroxycholesterol was measured (n = 6). **P < 0.001 (two-tailed t-test). Data in en are mean ± s.d. All experiments were repeated three times independently with similar results.

Source Data

Extended Data Fig. 5 PCK1-mediated phosphorylation of INSIG1/2 promotes the translocation of SCAP from the ER to the Golgi apparatus.

a, Parental Hep3B cells and the indicated clones of Hep3B cells with knock-in expression of INSIG1(S207A)/INSIG2(S151A) double mutants were transfected with Myc–SCAP and stimulated with or without IGF1 (100 ng ml−1) for 16 h. Immunoprecipitation and immunoblotting analyses with the indicated antibodies were performed. b, Validation of the specificity of the SCAP antibody. SCAP shRNA was expressed in 293T cells. Immunoblotting analyses were performed with the indicated antibodies. c, Parental Huh7 or Hep3B cells and the indicated clones with knock-in expression of PCK1(S90A) were transfected with the indicated plasmids and stimulated with or without IGF1 (100 ng ml−1) for 16 h. Ni-NTA pull-down assays, immunoprecipitation and immunoblotting analyses with the indicated antibodies were performed. d, PCK1-depleted Huh7 (left) and Hep3B (right) cells were stably transfected with shRNA-resistant wild-type rPCK1 or rPCK1(S90A). Immunoblotting analyses were performed with the indicated antibodies. e, f, PCK1-depleted Huh7 (left) and Hep3B (right) cells with reconstituted expression of shRNA-resistant wild-type rPCK1 and rPCK1(S90A) (e) or rPCK1(C288S) (f) were transfected with vectors expressing Flag–INSIG1 and His–INSIG2. The cells were then stimulated with or without IGF1 (100 ng ml−1) for 16 h. Ni-NTA pull-down assays, immunoprecipitation and immunoblotting analyses with the indicated antibodies were performed. g, h, Parental Huh7 cells and the indicated clones of Huh7 cells with knock-in expression of INSIG1(S207A)/INSIG2(S151A) were stimulated with or without IGF1 (100 ng ml−1) for 16 h. The cells were then subjected to homogenization and cell fractionation using gradient centrifugation. Immunoblotting analyses were performed with the indicated antibodies (g). The relative distribution of each protein in different fractions was quantified by densitometric analysis of the blots (n = 3) (h). i, j, Parental Huh7 cells and the indicated clones of Huh7 cells with knock-in expression of PCK1(S90A) were stimulated with or without IGF1 (100 ng ml−1) for 16 h. The cells were then subjected to homogenization and cell fractionation using gradient centrifugation. Immunoblotting analyses were performed with the indicated antibodies (i). The relative distribution of each protein in different fractions was quantified by densitometric analysis of the blots (n = 3) (j). k, Colocalization coefficients between SCAP and calnexin in the indicated cells. At least n = 50 cells from each independent experiment were analysed and representative data are shown. **P < 0.001 (two-tailed t-test). l, Parental Huh7 cells and the indicated clones of Huh7 cells with knock-in expression of INSIG1(S207A)/INSIG2(S151A) were stimulated with or without IGF1 for 16 h. Immunofluorescence analyses were performed with the indicated antibodies. The white arrows indicate the Golgi-localized SCAP. Scale bar, 20 μm. m, Colocalization coefficients between SCAP and golgin-97. At least n = 50 cells from each independent experiment were analysed and representative data are shown. **P <0.001 (two-tailed t-test). n, o, Top, parental Huh7 cells and the indicated clones of Huh7 cells with knock-in expression of PCK1(S90A) were stimulated with or without IGF1 (100 ng ml−1) for 16 h. Immunofluorescence analyses were performed with the indicated antibodies. The white arrows indicate the Golgi-localized SCAP. Bottom, colocalization coefficients between SCAP and calnexin (n) or golgin-97 (o). At least n = 50 cells from each independent experiment were analysed and representative data are shown. **P < 0.001 (two-tailed t-test). Scale bars, 20 μm. p, q, Top, parental Huh7 cells and the indicated clones of Huh7 cells with knock-in expression of INSIG1(S207A)/INSIG2(S151A) double mutants were stably transfected with Myc–SCAP and stimulated with or without IGF1 (100 ng ml−1) for 16 h. Immunofluorescence analyses were performed with the indicated antibodies. The white arrows indicate the Golgi-localized SCAP. Bottom, colocalization coefficients between Myc–SCAP and calnexin (p) or golgin-97 (q). At least n = 50 cells from each independent experiment were analysed and representative data are shown. **P < 0.001 (two-tailed t-test). Scale bars, 20 μm. r, Parental Huh7 and Hep3B cells and the indicated clones of these cells with knock-in expression of INSIG1(S207A)/INSIG2(S151A) were stimulated with or without IGF1 (100 ng ml−1) for 16 h. N, N terminus of SREBP1; P, precursor of SREBP1. s, Parental Huh7 cells and the indicated clones of Huh7 cells with knock-in expression of INSIG1(S207A)/INSIG2(S151A) were transiently transfected with vectors expressing β-galactosidase and an SRE-driven luciferase reporter. Twenty-four hours later, the cells were stimulated with or without IGF1 (100 ng ml−1) for 16 h. The relative SRE luciferase activity after normalization to β-galactosidase activity is shown (n = 6). **P < 0.001 (two-tailed t-test). t, Parental Huh7 and Hep3B cells and the indicated clones of these cells with knock-in expression of INSIG1(S207A)/INSIG2(S151A) were stimulated with or without IGF1 (100 ng ml−1) for 16 h. Immunoblotting analyses were performed with the indicated antibodies. Data in h, j, k, mq, s are mean ± s.d. All experiments were repeated three times independently with similar results.

Source Data

Extended Data Fig. 6 PCK1-mediated INSIG1/2 phosphorylation is required for IGF1- induced SREBP activation for lipogenesis and does not affect the lipid depletion-induced translocation of SCAP from the ER to the Golgi apparatus.

a, Parental Huh7 (left) and Hep3B (right) cells and the indicated clones with knock-in expression of PCK1(S90A) were stimulated with or without IGF1 (100 ng ml−1) for 16 h. Immunoblotting analyses were performed as indicated. b, Parental Huh7 cells and the indicated clones of Huh7 cells with knock-in expression of PCK1(S90A) were stimulated with or without IGF1 (100 ng ml−1) for 16 h. Immunofluorescence analyses were performed as indicated. Scale bar, 20 μm. c, Parental Huh7 cells and the indicated clones with knock-in expression of PCK1(S90A) were transiently transfected with vectors expressing β-galactosidase and an SRE-driven luciferase reporter and stimulated with or without IGF1 (100 ng ml−1) for 16 h. The relative SRE luciferase activity after normalization to β-galactosidase activity is shown (n = 6). Data are mean ± s.d. **P < 0.001 (two-tailed t-test). d, Parental Huh7 cells and the indicated clones with knock-in expression of PCK1(S90A) were stimulated with or without IGF1 (100 ng ml−1) for 16 h. The mRNA expression levels for SREBP target genes were measured using quantitative PCR (n = 6). Data are mean ± s.d. **P < 0.001 (two-tailed t-test). e, Parental Huh7 (left) and Hep3B (right) cells and the indicated clones with knock-in expression of PCK1(S90A) were stimulated with or without IGF1 (100 ng ml−1) for 16 h. Immunoblotting analyses were performed as indicated. f, Parental Huh7 cells and the indicated clones with knock-in expression of INSIG1(S207A)/INSIG2(S151A) double mutants (left) or PCK1(S90A) (right) were stimulated with or without IGF1 (100 ng ml−1) for 16 h. The mRNA expression levels for SREBF1 were measured using quantitative PCR (n = 6). Data are mean ± s.d. *P = 0.002, **P < 0.001 (two-tailed t-test). g, Parental Huh7 cells and the indicated clones with knock-in expression of PCK1(S90A) were stimulated with or without IGF1 (100 ng ml−1) for 16 h. The incorporation of 14C-glucose into triglycerides (left) and fatty acids (right) was measured (n = 6). Data are mean ± s.d. and were compared between groups using a two-tailed t-test. *P = 0.014, 0.012 (left to right); **P = 0.004, 0.001 (left to right). h, Endogenous PCK1-depleted CHL-1 human melanoma cells, U87 human glioblastoma cells and H1993 human non-small-cell lung cancer cells with reconstituted expression of shRNA-resistant wild-type HA–rPCK1 or HA–rPCK1(S90A) were stimulated with or without IGF1 (100 ng ml−1) for 16 h. The incorporation of 14C-glucose into fatty acids was measured (n = 6). Data are mean ± s.d. and were compared between groups using a two-tailed t-test. *P = 0.011, **P < 0.001. i, j, Parental Huh7 or Hep3B cells and the indicated clones with expression of PCK1(S90D) (i) or INSIG1(S207D)/INSIG2(S151D) double mutants (2S/D) (j) were collected for immunoblotting analyses as indicated. k, l, Parental Huh7 cells and the indicated clones with knock-in expression of PCK1(S90A) (k) or INSIG1(S207A)/INSIG2(S151A) (l) were stimulated with or without IGF1 (100 ng ml−1) for 16 h. Immunoblotting analyses were performed as indicated. m, n, Parental Huh7 cells and the indicated clones with knock-in expression of PCK1(S90A) (m) or INSIG1(S207A)/INSIG2(S151A) (n) were stimulated with or without IGF1 (100 ng ml−1) for 16 h. The mRNA expression levels for SREBP2 target genes were measured using quantitative PCR (n = 6). Data are mean ± s.d. **P < 0.001 (two-tailed t-test). o, p, Parental Huh7 cells and the indicated clones with knock-in expression of INSIG1(S207A)/INSIG2(S151A) double mutants were incubated with a lipid-depleted medium and treated with lovastatin (an inhibitor of HMGCR) to inhibit cholesterol synthesis for 16 h. The cells were subjected to homogenization and cell fractionation using gradient centrifugation. Immunoblotting analyses were performed as indicated (o). The relative distribution of each protein in different fractions was quantified by densitometric analysis of the blots (n = 3) (p). Data are mean ± s.d. q, r, Parental Huh7 cells and the indicated clones with knock-in expression of PCK1(S90A) were incubated with or without lipid-depleted medium for 16 h. The cells were subjected to homogenization and cell fractionation using gradient centrifugation. Immunoblotting analyses were performed as indicated (q). The relative distribution of each protein in different fractions was quantified by densitometric analysis of the blots (n = 3) (r). Data are mean ± s.d. sx, Parental Huh7 cells and the indicated clones with knock-in expression of INSIG1(S207A)/INSIG2(S151A) double mutants (s, v) or PCK1(S90A) (t, w) were incubated with or without lipid-depleted medium for 16 h. Immunofluorescence analyses were performed as indicated. Scale bars, 20 μm. The white arrows indicate the Golgi-localized SCAP. The colocalization coefficients between SCAP and the ER marker calnexin (u) and the Golgi apparatus marker golgin-97 (x) are shown. At least n = 50 cells from each independent experiment were analysed and representative data are shown. Data are mean ± s.d. and were compared between groups using a two-tailed t-test. NS, not significant (P = 0.788, 0.514, 0.689, 0.693 (left to right) (u); P = 0.976, 0.606, 0.750, 0.940 (left to right) (x)). All experiments were repeated three times independently with similar results.

Source Data

Extended Data Fig. 7 PCK1-mediated phosphorylation of INSIG1/2, which does not affect lipid-depletion-induced activity of SREBP1, promotes rapid activation of SREBP1 and degradation of INSIG1.

a, Parental Huh7 cells and the indicated clones of Huh7 cells with knock-in expression of INSIG1(S207A)/INSIG2(S151A) double mutants (top) or knock-in expression of PCK1(S90A) (bottom) were incubated with or without lipid-depleted medium for 16 h. Immunoblotting analyses were performed with the indicated antibodies. b, Parental Huh7 cells and the indicated clones of Huh7 cells with knock-in expression of NSIG1(S207A)/INSIG2(S151A) double mutants (left) or knock-in expression of PCK1(S90A) (right) were incubated with or without lipid-depleted medium for 16 h. Immunofluorescence analyses were performed with the indicated antibodies. c, Parental Huh7 cells and the indicated clones of Huh7 cells with knock-in expression of INSIG1(S207A)/INSIG2(S151A) double mutants (left) or knock-in expression of PCK1(S90A) (right) were transiently transfected with vectors expressing β-galactosidase and an SRE-driven luciferase reporter. Luciferase activity was determined after the cells were incubated with or without lipid-depleted medium for 16 h. The relative SRE luciferase activity after normalization to β-galactosidase activity is shown (n = 6). NS, not significant (P = 0.965, 0.699, 0.967, 0.767 (left to right)). d, Huh7 cells expressing Flag–INSIG1 and His–INSIG2 were stimulated with or without IGF1 (100 ng ml−1) for 16 h in the presence of 40 nM or 120 nM cholesterol or 25-hydroxycholesterol. Ni-NTA pull-down assays, immunoprecipitation and immunoblotting analyses were performed with the indicated antibodies. 25-HC, 25-hydroxycholesterol. e, Parental Huh7 cells and the indicated clones of Huh7 cells with knock-in expression of both INSIG1(S207D) and INSIG2(S151D) (left) or knock-in expression of PCK1(S90D) (right) were incubated with 40 nM or 120 nM cholesterol or 25-hydroxycholesterol for 16 h. Immunoprecipitation and immunoblotting analyses were performed with the indicated antibodies. f, Huh7 cells were transiently transfected with vectors expressing β-galactosidase and an SRE-driven luciferase reporter. Twenty-four hours later, the cells were stimulated with or without IGF1 (100 ng ml−1) for 16 h in the presence of 40 nM or 120 nM cholesterol or 25-hydroxycholesterol. The relative SRE luciferase activity after normalization to β-galactosidase activity is shown (n = 6). *P = 0.001, **P < 0.001 (two-tailed t-test); NS, not significant (P = 0.921, 0.579 (left to right)). g, Parental Huh7 cells and the indicated clones of Huh7 cells with knock-in expression of INSIG1(S207D)/INSIG2(S151D) double mutants (left) or knock-in expression of PCK1(S90D) (right) were transiently transfected with vectors expressing β-galactosidase and an SRE-driven luciferase reporter. Luciferase activity was determined after the cells were incubated with 40 nM or 120 nM cholesterol or 25-hydroxycholesterol for 16 h. The relative SRE luciferase activity after normalization to β-galactosidase activity is shown (n = 6). *P = 0.002, **P < 0.001 (two-tailed t-test); NS, not significant (P = 0.642, 0.957, 0.842, 0.372 (left to right)). h, Huh7 cells were treated with IGF1 (100 ng ml−1) for the indicated time periods. Immunoprecipitation and immunoblotting analyses were performed with the indicated antibodies. i, Parental Huh7 cells and the indicated clones of Huh7 cells with knock-in expression of PCK1(S90A) (left) or INSIG1(S207A)/INSIG2(S151A) double mutants (right) were treated with or without IGF1 (100 ng ml−1) for 48 h. Immunoblotting analyses were performed with the indicated antibodies. j, k, Serum-starved parental Huh7 (j) and Hep3B (k) cells or the indicated clones with knock-in expression of INSIG1(S207A)/INSIG2(S151A) double mutants were stimulated with or without IGF1 (100 ng ml−1) in the presence or absence of CHX (100 μg ml−1) for the indicated time periods. Immunoblotting analyses were performed with the indicated antibodies (left). The relative abundance of remaining INSIG1 or INSIG2 protein was quantified (right) (n = 6). **P < 0.001 (two-tailed t test); NS, not significant (P = 0.721, 0.637 (left to right) (j); P = 0.310, 0.853 (left to right) (k)). l, Huh7 cells expressing Flag-tagged wild-type INSIG1 (top) or INSIG1(S207D) (bottom) were treated with CHX (100 μg ml−1) for the indicated time periods. Immunoblotting analyses were performed with the indicated antibodies (left). The relative abundance of the remaining INSIG1 protein was quantified (right) (n = 6). **P < 0.001 (two-tailed t test). m, Huh7 cells expressing Flag-tagged wild-type INSIG2 (top) or INSIG2(S151D) (bottom) were treated with CHX (100 μg ml−1) for the indicated time periods. Immunoblotting analyses were performed with the indicated antibodies (left). The relative abundance of the remaining INSIG2 protein was quantified (right) (n = 6). **P < 0.001 (two-tailed t-test); NS, not significant (P = 0.395, 0.973, 0.636, 0.882 (left to right)). n, Endogenous PCK1-depleted Huh7 cells with reconstituted expression of shRNA-resistant wild-type rPCK1 or rPCK1(S90A) were treated with IGF1 (100 ng ml−1) for the indicated time periods. Immunoprecipitation and immunoblotting analyses were performed with the indicated antibodies. Data in c, f, g, jm are mean ± s.d. All experiments were repeated three times independently with similar results.

Source Data

Extended Data Fig. 8 PCK1-mediated phosphorylation of INSIG1 Ser207 and INSIG2 Ser151 and SREBP1 activation are induced by oncogene- or growth-factor-mediated activation of AKT in several cancer types.

a, b, el, Cells were transfected with the indicated plasmids and immunoprecipitation or immunoblotting analyses were performed with the indicated antibodies. a, Endogenous PCK1-depleted HL7702 (left) and THLE-2 (right) cells with reconstituted expression of shRNA resistant wild-type HA–rPCK1 or HA–rPCK1(S90A) were treated with or without IGF1 (100 ng ml−1) for 16 h. b, Endogenous PCK1-depleted HL7702 (left) and THLE-2 (right) cells with reconstituted expression of shRNA resistant wild-type HA–rPCK1 or HA–rPCK1(S90D) were analysed. c, C57BL/6J male mice (eight-week-old) fasted for 24 h were intraperitoneally injected with or without glucose (1 g per kg body weight). After 6 h, the mouse livers were dissected for immunoprecipitation and immunoblotting analyses. d, HL7702, THLE-2, Huh7 and Hep3B cells were analysed by Ni-NTA pull-down, immunoprecipitation and immunoblotting assays. e, Endogenous PCK1-depleted HL7702, THLE-2, Huh7 and Hep3B cells with reconstituted expression of shRNA resistant wild-type HA–rPCK1 or HA–rPCK1(S90A) were analysed. f, Parental Huh7 cells and the indicated clones with knock-in expression of PCK1(S90A) (left) or INSIG1(S207A)/INSIG2(S151A) double mutants (right) were transfected with or without Flag–KRAS(G12V). g, Endogenous PCK1-depleted CHL-1, U87 and H1993 cells with reconstituted expression of shRNA resistant wild-type HA–rPCK1 or HA–rPCK1(S90A) were transfected with the indicated plasmids. h, Parental Huh7 cells and the indicated clones with knock-in expression of PCK1(S90A) (left) or INSIG1(S207A)/INSIG2(S151A) double mutants (right) were transfected with or without constitutively active Flag–IGF1R(V922E). i, Endogenous PCK1-depleted CHL-1, U87 and H1993 cells with reconstituted expression of shRNA resistant wild-type HA–rPCK1 or HA–rPCK1(S90A) were transfected with the indicated plasmids. j, Endogenous PCK1-depleted CHL-1, U87 and H1993 cells with reconstituted expression of shRNA resistant wild-type HA–rPCK1 or HA–rPCK1(S90A) were transfected with the indicated plasmids and treated with or without PDGF (30 ng ml−1) for 16 h. k, m, Endogenous PCK1-depleted Huh7, Hep3B, SNU-398 and SNU-475 cells with reconstituted expression of shRNA resistant wild-type HA–rPCK1 or HA–rPCK1(S90A) were analysed by immunoprecipitation and immunoblotting analyses with the indicated antibodies (k). The cells were plated and then collected and counted for 3 days (n = 6) (m). Data are mean ± s.d. **P < 0.001 (two-tailed t-test). l, n, Endogenous PCK1-depleted Huh7, Hep3B, SNU-398 and SNU-475 cells with reconstituted expression of shRNA resistant wild-type HA–rPCK1 or HA–rPCK1(S90A) were transfected with Flag–INSIG1 and His–INSIG2. After incubation with or without lipid-depleted medium for 16 h, the cells were collected for Ni-NTA pull-down, immunoprecipitation and immunoblotting analyses as indicated (l). Viable cells were counted for 3 days after lipid depletion (n=6) (n). Data are mean ± s.d. NS, not significant (P = 0.708, 0.619, 0.888, 0.901, 0.633, 0.788, 0.902, 0.764 (left to right)). All experiments were repeated three times independently with similar results.

Source Data

Extended Data Fig. 9 PCK1-mediated phosphorylation of INSIG1/2 promotes the proliferation of liver cancer cells and the growth of tumours in mice.

a, Parental Hep3B cells (2 × 105) and the indicated clones of Hep3B cells (2 × 105) with knock-in expression of PCK1(S90A) (left) or INSIG1(S207A)/INSIG2(S151A) double mutants (right) were plated for 3 days. The cells were then collected and counted. Data are mean ± s.d. (n = 6). **P < 0.001 (two-tailed t-test). b, c, Parental Huh7 and Hep3B cells (2 × 105) and the indicated clones of Huh7 and Hep3B cells with knock-in expression of PCK1(S90D) (b) or INSIG1(S207D)/INSIG2(S151D) double mutants (c) were plated for 3 days. The cells were then collected and counted. Data are mean ± s.d. (n = 6). **P < 0.001 (two-tailed t-test). dh, Parental Huh7 cells (1 × 106) or the clones with knock-in expression of PCK1(S90A) or INSIG1(S207A)/INSIG2(S151A) double mutants were subcutaneously injected into the left or right flanks of athymic nude mice, respectively (n= 7 per group) (d, left). The resulting tumours were resected 28 days after injection (d, right). The growth of xenografted tumours in the mice was measured (e) and the tumours were weighed (f). Data are mean ± s.d. (n = 7). *P = 0.002, **P < 0.001 compared with the wild-type group (two-tailed t-test). g, IHC analyses of tumour samples were performed with an anti-Ki67 antibody. Ki67-positive cells were quantified (right). h, TUNEL analyses of the indicated tumour samples were performed. Apoptotic cells were stained brown and quantified in n = 10 microscopic fields (right). Data are mean ± s.d. **P < 0.001 compared with the wild-type group (two-tailed t-test). i, j, Huh7 cells (1 × 106) with or without knock-in expression of PCK1(S90A) or INSIG1(S207A)/INSIG2(S151A) double mutants were intrahepatically injected into athymic nude mice (n = 7 per group). At 28 days after injection, the mice were euthanized and the liver tumours were dissected for immunoprecipitation, immunoblotting (i) and immunofluorescence (j) analyses with the indicated antibodies. k, Huh7 cells (1 × 106) with or without knock-in expression of PCK1(S90D) (top) or INSIG1(S207D)/INSIG2(S151D) double mutants (bottom) were intrahepatically injected into athymic nude mice (n = 7 per group). The mice were euthanized and examined for tumour growth 22 days after injection. The arrows indicate tumours. Tumour volumes were calculated (right). Data are mean ± s.d. (n = 7). **P < 0.001 compared with the wild-type group (two-tailed t-test). l, m, Huh7 cells (1 × 106) and Huh7 cells with knock-in expression of PCK1(S90D) (l) or INSIG1(S207D)/INSIG2(S151D) double mutants (m) were subcutaneously injected into the left or right flanks of athymic nude mice, respectively (n = 7 per group). The resulting tumors were resected 22 days after injection (left). The growth of xenografted tumours in the mice was measured (middle) and the tumours were weighed (right). Data are mean ± s.d. (n = 7). **P < 0.001 compared with the wild-type group (two-tailed t-test). All experiments were repeated three times independently with similar results.

Source Data

Extended Data Fig. 10 Activation of the IGF1R–AKT–PCK1–INSIG1/2 signalling cascade is required for the growth of liver tumours and correlates with poor prognosis for HCC.

a, b, Huh7 cells (1 × 106) with or without knock-in expression of PCK1(S90A) (a) or INSIG1(S207A)/INSIG2(S151A) double mutants (b) were stably transfected with or without Flag–IGF1R(V922E) and intrahepatically injected into athymic nude mice (n = 7 per group). The mice were euthanized and examined for tumour growth 28 days after injection. The arrows indicate tumours. Tumour volumes were calculated (right). Data are mean ± s.d. (n = 7). *P =0.011 (a), 0.013 (b), **P <0.001 (two-tailed t-test). c, IHC analyses of xenografted tumours from nude mice were performed with the indicated antibodies. The regions in white boxes are shown at higher magnification below. d, e, Huh7 cells (1 × 106) with or without knock-in expression of PCK1(S90A) (d) or INSIG1(S207A)/INSIG2(S151A) double mutants (e) were stably transfected with or without Flag–myr-AKT1 and intrahepatically injected into athymic nude mice (n = 7 per group). The mice were euthanized and examined for tumour growth 28 days after injection. The arrows indicate tumours. Tumour volumes were calculated (right). Data are mean ± s.d. (n = 7). *P = 0.015 (d), 0.010 (e), **P < 0.001 (two-tailed t-test). f, IHC analyses of xenografted tumours from nude mice were performed with the indicated antibodies. The regions in white boxes are shown at higher magnification below. g, h, Huh7 cells (1 × 106) with or without knock-in expression of PCK1(S90D) (g) or INSIG1(S207D)/INSIG2(S151D) double mutants (h) were stably transfected with Flag–IGF1R(L1003R) and intrahepatically injected into athymic nude mice (n = 7 per group). The mice were euthanized and examined for tumour growth 28 days after injection. The arrows indicate tumours. Tumour volumes were calculated (right). Data are mean ± s.d. (n = 7). *P = 0.003 (g), 0.005 (h), **P < 0.001 (two-tailed t-test). in, Huh7 cells (1 × 106) with or without knock-in expression of PCK1(S90D) (ik) or INSIG1(S207D)/INSIG2(S151D) double mutants (ln) were stably transfected with or without Flag–IGF1R(L1003R) and subcutaneously injected into the left flanks of athymic nude mice (n = 7 per group). The resulting tumours were resected 28 days after injection (i, l). The growth of xenografted tumours in the mice was measured (j, m). The tumours were weighed (k, n). Data are mean ± s.d. (n = 7). *P= 0.002 (j), 0.011 (k), 0.009 (m), 0.016 (n), **P < 0.001 compared with the PCK1 wild-type + IGF1R(L1003R) group (jk) or compared with the INSIG1/2 wild-type + IGF1R(L1003R) group (mn) (two-tailed t-test). oq, Huh7 cells (1 × 106) with or without knock-in expression of PCK1(S90D) or INSIG1(S207D)/INSIG2(S151D) double mutants were stably transfected with or without Flag–IGF1R(L1003R) and intrahepatically injected into athymic nude mice (n = 7 per group). The mice were euthanized 28 days after injection. IHC analyses of xenografted tumours from nude mice were performed with the indicated antibodies. The regions in white boxes are shown at higher magnification below. rt, Wild-type pT3-EF1α-Flag-PCK1 (or pT3-EF1α-Flag-PCK1(S90A)), pT3-EF1α-HA-myr-AKT1 and pT3-EF1α-V5-c-Met, along with the sleeping beauty transposase (SB), were stably expressed in the mouse liver using hydrodynamic transfection into FVB/N mice (n = 7 per group). After 14 weeks, the mice were euthanized and representative liver tumours are shown (r, left). The average tumour volumes were measured (r, right) (n = 7 per group). **P < 0.001 (two-tailed t-test). s, IHC analyses of tumour samples were performed with an anti-Ki67 antibody (left). Ki67-positive cells were quantified in 10 microscopic fields (right). **P < 0.001 (two-tailed t-test). t, IHC analyses of liver tumours from nude mice were performed with the indicated antibodies. The regions in white boxes are shown at higher magnification below. u, IHC staining of 30 human HCC and matched non-tumour tissue samples was performed with the indicated antibodies. Representative images of two cases are shown. The regions in white boxes are shown at higher magnification below. v, Representative immunoblotting analyses of two cases of human HCC and matched non-tumour tissue samples was performed with the indicated antibodies. w, The indicated staining scores for PCK1(pS90), INSIG1(pS207)/INSIG2(pS151) and SREBP1 expression levels in HCC and matched non-tumour liver samples (n = 30) were compared using a paired t-test (two-tailed). Data are mean ± s.d. **P < 0.001 compared with the non-tumour adjacent tissue. x, Representative immunoblotting analyses of four different human HCC samples were performed with the indicated antibodies. y, IHC staining of human HCC samples with the indicated antibodies was scored, and correlation analyses were performed. A Pearson correlation test was used (two-tailed) (n = 40). Note that the scores for some samples overlap. z, Mechanism for PCK1-promoted activation of SREBP1 and lipogenesis. PEP, phosphoenolpyruvate; OAA, oxaloacetate; HC, hydroxycholesterol; bHLH, basic helix-loop-helix protein; Reg, regulatory domain of SREBP. All experiments were repeated three times independently with similar results.

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This file contains a Supplementary Discussion and associated references, legends to two Supplementary Tables, and Supplementary Figure 1 (raw gel data from Figures 1-3 and Extended Data Figures 1-10).

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Supplementary Table 1 | Full list of Insig1 associated proteins upon IGF1 treatment for 1h.

Supplementary Table 2 | Full list of Insig2 associated proteins upon IGF1 treatment for 1h.

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Xu, D., Wang, Z., Xia, Y. et al. The gluconeogenic enzyme PCK1 phosphorylates INSIG1/2 for lipogenesis. Nature 580, 530–535 (2020). https://doi.org/10.1038/s41586-020-2183-2

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