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 V_max and K_m 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.

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