Extended Data Fig. 8: Phosphorylation of FABP4 on T126 by PAK4.
From: p21-activated kinase 4 counteracts PKA-dependent lipolysis by phosphorylating FABP4 and HSL
a. Recombinant FABP4 was incubated with recombinant PAK4 and [32P]ATP for 30 min, and proteins in the mixture were resolved by SDS-PAGE. The band was visualized by autoradiography of 32P-labeled protein. Protein loading was confirmed by Coomassie blue staining. b. 3T3-L1 adipocytes were transfected with PAK4 and then treated with 1 μM isoproterenol for 1 h. FABP4 binding with CGI-58 was analyzed by PLA (n = 26). c. 3T3-L1 adipocytes were transfected as indicated. PAK4 phosphorylation of FABP4 was analyzed by Western blotting. d. A predicted model of the interaction between PAK4 and FABP4, represented by green and yellow, respectively, in their ternary structure. The green ball-and-stick model depicts ATP bound to PAK4, while the yellow stick model represents palmitate binding to FABP4. Additionally, the sphere located in the ATP binding pocket of PAK4 indicates the presence of Mg2+ ion. Notably, the model suggests that the phosphorylation of FABP4 on the T126 residue by PAK4 is structurally feasible. e. 3T3-L1 adipocytes were transfected as indicated and then treated with 1 μM isoproterenol for 1 h. The size of lipid droplets was then analyzed (n = 52). Data are representative of at least three independent experiments. Values are mean ± SD. Two-way ANOVA followed by Bonferroni’s post hoc analysis (b, e) was conducted for statistical analyses. Source data are provided as a Source Data file.
