Novel Endogenous, Insulin-Stimulated Akt2 Protein Interaction Partners in L6 Myoblasts.

Michael Caruso,Zhao Yang,Zhengping Yi,Yue Qi,Danjun Ma,Xiangmin Zhang

doi:10.1371/journal.pone.0140255

Michael Caruso, Zhao Yang + Show 4 more

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https://doi.org/10.1371/journal.pone.0140255

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Abstract

Insulin resistance and Type 2 diabetes are marked by an aberrant response in the insulin signaling network. The phosphoinositide-dependent serine/threonine kinase, Akt2, plays a key role in insulin signaling and glucose uptake, most notably within skeletal muscle. Protein-protein interaction regulates the functional consequence of Akt2 and in turn, Akt2’s role in glucose uptake. However, only few insulin-responsive Akt2 interaction partners have been identified in skeletal muscle cells. In the present work, rat L6 myoblasts, a widely used insulin sensitive skeletal muscle cell line, were used to examine endogenous, insulin-stimulated Akt2 protein interaction partners. Akt2 co-immunoprecipitation was coupled with 1D-SDS-PAGE and fractions were analyzed by HPLC-ESI-MS/MS to reveal Akt2 protein-protein interactions. The pull-down assay displayed specificity for the Akt2 isoform; Akt1 and Akt3 unique peptides were not detected. A total of 49 were detected with a significantly increased (47) or decreased (2) association with Akt2 following insulin administration (n = 4; p<0.05). Multiple pathways were identified for the novel Akt2 interaction partners, such as the EIF2 and ubiquitination pathways. These data suggest that multiple new endogenous proteins may associate with Akt2 under basal as well as insulin-stimulated conditions, providing further insight into the insulin signaling network. Data are available via ProteomeXchange with identifier PXD002557.

Highlights

Insulin-stimulated glucose uptake and metabolism in target tissues is regulated through intracellular protein-protein interactions, as well as by protein post-translational modifications, notably phosphorylation [1,2,3]
Novel Akt2 interactions may further elucidate insulin signaling and provide insight into abnormal Akt2 protein interaction that contributes to the development of insulin resistance and/or type II diabetes
L6 myoblasts have been shown to be a suitable model for insulin-stimulated GLUT4 translocation as well as glucose uptake, of which Akt2 plays a defining role [31]

Summary

Introduction

Insulin-stimulated glucose uptake and metabolism in target tissues is regulated through intracellular protein-protein interactions, as well as by protein post-translational modifications, notably phosphorylation [1,2,3]. Dysregulation of insulin signaling may lead to several debilitating disorders such as insulin resistance, metabolic syndrome, type 2 diabetes (T2D), cardiovascular disease, and/or cancer [4,5,6]. Two canonical insulin-stimulated signaling pathways have emerged: the phosphatidylinositide 3-kinase (PI3K) and the mitogen-activated protein kinase (MAPK) signaling pathways [7]. The PI3K insulin-stimulated pathway carries out the primary metabolic functions while MAPK regulates cell survival and mitogenesis [7]. The serine/threonine kinase, Akt, is a keystone mediator in the PI3K pathway, associating with numerous downstream proteins that affect metabolism, growth, and cell survival [8].

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