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.
From: The gluconeogenic enzyme PCK1 phosphorylates INSIG1/2 for lipogenesis
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, j–m are mean ± s.d. All experiments were repeated three times independently with similar results.
