Phosphorylation of adaptor protein containing pleckstrin homology domain, phosphotyrosinebinding domain, and leucine zipper motif 1 (APPL1) at Ser ⁴³⁰ mediates endoplasmic reticulum (ER) stress-induced insulin resistance in hepatocytes

APPL1 is an adaptor protein that plays a critical role in regulating adiponectin and insulin signaling. However, how APPL1 is regulated under normal and pathological conditions remains largely unknown. In this study, we show that APPL1 undergoes phosphorylation at Ser⁴³⁰ and that this phosphorylation is enhanced in the liver of obese mice displaying insulin resistance. In cultured mouse hepatocytes, APPL1 phosphorylation at Ser ⁴³⁰ is stimulated by phorbol 12-myristate 13-acetate, an activator of classicPKCisoforms, and by the endoplasmic reticulum (ER) stress inducer, thapsigargin. Overexpression of wild-type but not dominant negative PKCα increases APPL1 phosphorylation at Ser⁴³⁰ in mouse hepatocytes. In addition, suppressing PKCα expression by shRNA in hepatocytes reduces ER stressinduced APPL1 phosphorylation at Ser⁴³⁰ as well as the inhibitory effect of ER stress on insulin-stimulated Akt phosphorylation. Consistent with a negative regulatory role of APPL1 phosphorylation at Ser ⁴³⁰ in insulin signaling, overexpression of APPL1^S430D but not APPL1^S430A impairs the potentiating effect of APPL1 on insulin-stimulated Akt phosphorylation at Thr³⁰⁸. Taken together, our results identify APPL1 as a novel target in ER stress-induced insulin resistance and PKCα as the kinase mediating ER stress-induced phosphorylation of APPL1 at Ser⁴³⁰.