Fig. 2: TAB1 interacts with GFAT1 in a Ser438 phosphorylation-dependent manner.

a TAB1 could interact with GFAT1. A549 cells with stable expression of Flag-GFAT1 were cultured for 8 h under glucose deprivation. Immunoprecipitation and immunoblotting analyses were performed using the indicated antibodies. Silver staining analysis of the immunoprecipitates was performed. b TAB1–GFAT1 interaction was phosphorylation-dependent. A549 cells were cultured for 8 h under glucose deprivation. Immunoprecipitation and immunoblotting analyses were performed using the indicated antibodies. c TAB1 phosphorylation mediated TAB1–GFAT1 interaction. The indicated purified GST-GFAT1 or His-TAB1 protein was mixed with cell lysates from A549 cells cultured for 8 h under glucose deprivation. Pull-down and immunoblotting analyses were performed using the indicated antibodies. d JNK/p38 mediated TAB1–GFAT1 interaction. A549 cells were pretreated with Compound C (10 μM), SP600125 (20 μM), and SB203580 (10 μM) for 1 h before being cultured for 8 h under glucose deprivation. Immunoprecipitation and immunoblotting analyses were performed using the indicated antibodies. e Ser438 of TAB1 is evolutionarily conserved in the indicated species (upper panel). TAB1 Ser438 phosphorylation mediated TAB1–GFAT1 interaction. A549 cells with transient expression of WT TAB1, TAB1-S423A, TAB1-T431A or TAB1-S438A were cultured for 8 h under glucose deprivation. Immunoprecipitation and immunoblotting analyses were performed using the indicated antibodies (lower panel). f His-TAB1 Ser438 could be phosphorylated by p38α or JNK1. Purified His-TAB1 proteins were subjected to in vitro kinase assay. g, h In vitro binding of His-TAB1 and GST-GFAT1. Purified His-TAB1 WT and His-TAB1-S438A protein with phosphorylation by p38α (g) or JNK1 (h) was mixed with purified GST-GFAT1. Pull-down and immunoblotting analyses were performed using the indicated antibodies. i Expression of rTAB1-S438A inhibited p38 activation at late time points. A549 cells with depleted TAB1 and reconstituted expression of WT rTAB1 or rTAB1-S438A were cultured for indicated time under glucose deprivation. Immunoblotting analyses were performed using the indicated antibodies. In i, the values are presented as means ± SEM, n = 3; *P < 0.05, ***P < 0.001, Student’s t-test, two-sided.