Abstract
Akt2 and its downstream effectors mediate insulin-stimulated GLUT4-storage vesicle (GSV) translocation and fusion with the plasma membrane (PM). Using mass spectrometry, we identify actin-capping protein Tropomodulin 3 (Tmod3) as an Akt2-interacting partner in 3T3-L1 adipocytes. We demonstrate that Tmod3 is phosphorylated at Ser71 on insulin-stimulated Akt2 activation, and Ser71 phosphorylation is required for insulin-stimulated GLUT4 PM insertion and glucose uptake. Phosphorylated Tmod3 regulates insulin-induced actin remodelling, an essential step for GSV fusion with the PM. Furthermore, the interaction of Tmod3 with its cognate tropomyosin partner, Tm5NM1 is necessary for GSV exocytosis and glucose uptake. Together these results establish Tmod3 as a novel Akt2 effector that mediates insulin-induced cortical actin remodelling and subsequent GLUT4 membrane insertion. Our findings suggest that defects in cytoskeletal remodelling may contribute to impaired GLUT4 exocytosis and glucose uptake.
Highlights
Akt[2] and its downstream effectors mediate insulin-stimulated GLUT4-storage vesicle (GSV) translocation and fusion with the plasma membrane (PM)
The identification of Akt substrates that are involved in regulating the GSV exocytosis and understanding the cellular and molecular mechanisms of their actions in glucose transport will enable us to pinpoint the major nodes of molecular pathways underlying this process, as defects in this process are responsible for impaired glucose uptake in insulin resistance and diabetes
The mechanisms that regulate membrane fusion of GLUT4 are not completely understood, it has become clear that cortical actin remodelling plays a pivotal role[18,19,20,21,27,41,42], in coordination with the membrane fusion machinery and its regulators, including SNARE proteins, small GTPases and Sec-1/Munc[18] proteins, to bridge and eventually merge the two lipid bilayers between GSVs and the PM, allowing cell surface exposure of GLUT4 proteins and the ensuing glucose uptake[1]
Summary
Tmod[3] is phosphorylated by Akt[2] on insulin stimulation. To search for novel Akt substrates involved in ISGT, we performed a proteomic screening in 3T3-L1 adipocytes expressing FLAGtagged constitutively active form of Akt[2] (myristoylated human Akt[2], FLAG-Akt2-CA). Mass spectrometry (MS) analysis of Tmod[3] purified from HEK293T cells co-expressing Tmod[3] and active Akt[2] showed that Akt[2] phosphorylated Tmod[3] at Ser[71], but not other residues (Supplementary Fig. 1g). These findings support that Tmod[3] is directly phosphorylated at S71 by Akt[2] both in vitro and in vivo
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