Phosphorylation of Adaptor Protein Containing Pleckstrin Homology Domain, Phosphotyrosine Binding Domain, and Leucine Zipper Motif 1 (APPL1) at Ser430 Mediates Endoplasmic Reticulum (ER) Stress-induced Insulin Resistance in Hepatocytes

Meilian Liu,Lijun Zhou,Li Wei,Ricardo Villarreal,Xin Yang,Derong Hu,Ramon A Riojas,Bekke M Holmes,Paul R Langlais,Hakjoo Lee,Lily Q Dong

doi:10.1074/jbc.m112.372292

Abstract

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 Ser(430) 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 classic PKC isoforms, and by the endoplasmic reticulum (ER) stress inducer, thapsigargin. Overexpression of wild-type but not dominant negative PKCα increases APPL1 phosphorylation at Ser(430) in mouse hepatocytes. In addition, suppressing PKCα expression by shRNA in hepatocytes reduces ER stress-induced APPL1 phosphorylation at Ser(430) 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 Thr(308). 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 Ser(430).

Highlights

Adaptor protein APPL1 plays a critical role in regulating both adiponectin and insulin signaling pathways
A recent mass spectrometry study revealed that APPL1 is phosphorylated at several sites including Ser151, Ser401 Ser427, Ser430, and Ser691/693/696 and that the protein could be a potential target for multiple important cellular kinases including PKC, AMPK, and MAPK family members Erk, p38 MAPK, and c-Jun N-terminal kinase (JNK) [10]
To determine whether these kinases were involved in the phosphorylation of APPL1, we performed in vivo labeling experiments with C2C12 cells transiently expressing Myc-APPL1

Summary

Introduction

Adaptor protein APPL1 plays a critical role in regulating both adiponectin and insulin signaling pathways. Results: ER stress-induced APPL1 phosphorylation at Ser430 blocks the insulin-sensitizing effect of APPL1 in a PKC␣-dependent manner. Conclusion: APPL1 phosphorylation at Ser430 mediates ER stress-induced insulin resistance. APPL1 is an adaptor protein that plays a critical role in regulating adiponectin and insulin signaling. Suppressing PKC␣ 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 Ser430 in insulin signaling, overexpression of APPL1S430D but not APPL1S430A impairs the potentiating effect of APPL1 on insulin-stimulated Akt phosphorylation at Thr308. Our results identify APPL1 as a novel target in ER stress-induced insulin resistance and PKC␣ as the kinase mediating ER stress-induced phosphorylation of APPL1 at Ser430

Methods

Results

Conclusion