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.
From: The gluconeogenic enzyme PCK1 phosphorylates INSIG1/2 for lipogenesis
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). q–s, 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)). t–v, 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.
