Fig. 2: α4 regulates IR Tyr phosphorylation via YBX1 and PTP1B.

a Schematic diagram showing the regulation of insulin signaling in brown preadipocytes via α4. Specifically, α4 binds to the catalytic subunit of protein phosphatase 2A (PP2Ac) and is known to regulate Ser/Thr phosphorylation. For the mass spectroscopic proteomic analysis. we compared the Control (n = 1) and α4 overexpressing (n = 2, biological replicates) samples. b α4-complex containing YBX1. Flag-tagged α4 immunoprecipitation showed the interaction between YBX1 with α4 as well as that between PP2Ac and α4 in brown preadipocytes (n = 3 biologically independent cell clones/group). c Western blotting of phosphorylation of insulin signaling molecules in Control and α4KO brown preadipocytes following indicated concentrations of insulin stimulation for 10 min. d Effect of α4 on insulin signaling-based densitometric immunoblotting analysis with antibodies to α4 and phosphorylated IR, Akt, YBX1, as well as PTP1B in lysates of α4 Knockout brown preadipocytes (α4 KO) before and 10 min after insulin stimulation (see c). Data are presented as mean ± SEM (one-way ANOVA post hoc Bonferroni test, n = 3, each cell was generated from biologically independent animals from three independent experiments, Statistical significance is shown as p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***)). e Luciferase reporter assay for PTP1B transcription was performed as described by Fukada and Tong⁴⁵. Cells were transfected with the expression vectors of α4 and/or YBX1 in the presence of Ptp1b-Luc reporter or mock plasmid DNA in HEK293cells. Data are presented as mean ± SEM (One-way ANOVA post hoc Bonferroni test, n = 8 technical replicates per group, ***p < 0.0001). f Model of the regulation of α4 in insulin-activated IR phosphorylation. Insulin-bound IR phosphorylates itself and IRS1/2, and activates the PI3K-AKT and S6K pathways. α4 stabilizes PP2A and dephosphorylates mTORC2 with Rictor. α4 also binds to Y-box protein 1 (YBX1), which functions as a transcription factor for Tyr phosphatase PTP1B. α4 blocks YBX1 nuclear translocation and inhibits PTP1B expression, thus in feedback regulation, promoting the enhanced signaling through the insulin receptor. Source data are provided as a Source Data file.