Article

The interaction of Akt with APPL1 is required for insulin-stimulated Glut4 translocation.

Department of Biochemistry , Boston University, Boston, Massachusetts, United States
Journal of Biological Chemistry (Impact Factor: 4.6). 12/2007; 282(44):32280-7. DOI: 10.1074/jbc.M704150200
Source: PubMed

ABSTRACT APPL1 (adaptor protein containing PH domain, PTB domain, and leucine zipper motif 1) is an Akt/protein kinase B-binding protein involved in signal transduction and membrane trafficking pathways for various receptors, including receptor tyrosine kinases. Here, we establish a role for APPL1 in insulin signaling in which we demonstrate its interaction with Akt2 by co-immunoprecipitation and pulldown assays. In primary rat adipocytes and skeletal muscle, APPL1 and Akt2 formed a complex that was dissociated upon insulin stimulation in both tissues. To investigate possible APPL1 function in adipocytes, we analyzed Akt phosphorylation, 2-deoxyglucose uptake, and Glut4 translocation by immunofluorescence following APPL1 knockdown by small interfering and short hairpin RNAs. We show that APPL1 knockdown suppressed Akt phosphorylation, glucose uptake, and Glut4 translocation. We also tested the effect in 3T3-L1 adipocytes of expressing full-length APPL1 or an N- or a C-terminal APPL1 construct. Interestingly, expression of full-length APPL1 and its N terminus suppressed insulin-stimulated 2-deoxyglucose uptake and Glut4 translocation to roughly the same extent (40-60%). We confirmed by cellular fractionation that Glut4 translocation was substantially blocked in 3T3-L1 adipocytes transfected with full-length APPL1. By cellular fractionation, APPL1 was localized mainly in the cytosol, and it showed a small degree of re-localization to the light microsomes and nucleus in response to insulin. By immunofluorescence, we also show that APPL1 partially co-localized with Glut4. These data suggest that APPL1 plays an important role in insulin-stimulated Glut4 translocation in muscle and adipose tissues and that its N-terminal portion may be critical for APPL1 function.

0 Followers
 · 
99 Views
  • [Show abstract] [Hide abstract]
    ABSTRACT: The protein kinase AKT is a central kinase in the heart and has major impact on growth/hypertrophy, survival/apoptosis and metabolism. To gain more insight into AKT isoform specific signaling at the molecular level we investigated the phosphoproteome of HL-1 cardiomyocytes carrying AKT1 or AKT2 isoform specific knock down, respectively. We combined stable isotope labeling with high resolution mass spectrometry and identified 377 regulated phosphopeptides. Although AKT1 is expressed at four-fold higher levels, insulin stimulation mainly activated AKT2, which might in part rely on a preferred interaction of AKT2 with mammalian target of rapamycin complex 2. In line with this result, the highest number of regulated phosphopeptides was identified in the AKT2 knock down cells. Isoform specific regulation of AKT targets not previously described could be observed and specific regulation of indirect target sites allows a deeper insight into affected biological processes. In the myocardial context we identified many phosphosites supporting a connection of AKT to excitation-contraction coupling. Phosphoproteins identified included L-type calcium channel, ryanodine receptor, junctophilin, histidine-rich calcium binding protein, phospholamban, heat shock protein beta-6, and Ca2+/calmodulin-dependent kinase II. In conclusion, AKT isoform specific knock down combined with quantitative phosphoproteomics provided a powerful strategy to unravel AKT isoform specific signaling.
    Journal of Proteome Research 08/2014; 13(10). DOI:10.1021/pr500131g · 5.00 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Abstract Cell migration is fundamental to a variety of physiological processes, including tissue development, homeostasis, and regeneration. Migration has been extensively studied with cells on two-dimensional (2D) substrates, but much less is known about cell migration in 3D environments. Tissues and organs are 3D, which is the native environment of cells in vivo, pointing to a need to understand migration and the mechanisms that regulate it in 3D environments. To investigate cell migration in 3D environments, we developed microfluidic devices that afford a controlled, reproducible platform for generating 3D matrices. Using these devices, we show that the Rho family guanine nucleotide exchange factor (GEF) Asef2 inhibits cell migration in 3D type I collagen (collagen I) matrices. Treatment of cells with the myosin II (MyoII) inhibitor blebbistatin abolished the decrease in migration by Asef2. Moreover, Asef2 enhanced MyoII activity as shown by increased phosphorylation of serine 19 (S19). Furthermore, Asef2 increased activation of Rac, which is a Rho family small GTPase, in 3D collagen I matrices. Inhibition of Rac activity by treatment with the Rac-specific inhibitor NSC23766 abrogated the Asef2-promoted increase in S19 MyoII phosphorylation. Thus, our results indicate that Asef2 regulates cell migration in 3D collagen I matrices through a Rac-MyoII-dependent mechanism.
    Cell adhesion & migration 12/2014; 8(5). DOI:10.4161/19336918.2014.983778 · 3.40 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Phosphoinositides are key players in many trafficking and signaling pathways. Recent advances regarding the synthesis, location and functions of these lipids have dramatically improved our understanding of how and when these lipids are generated and what their roles are in animal physiology. In particular, phosphoinositides play a central role in insulin signaling, and manipulation of PtdIns(3,4,5)P3 levels in particular, may be an important potential therapeutic target for the alleviation of insulin resistance associated with obesity and the metabolic syndrome. In this article we review the metabolism, regulation and functional roles of phosphoinositides in insulin signaling and the regulation of energy metabolism. This article is part of a Special Issue entitled Phosphoinositides.
    Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids 11/2014; 1851(6). DOI:10.1016/j.bbalip.2014.11.008 · 4.50 Impact Factor