The interaction of AKT with APPL1 is required for insulin-stimulated Glut4 translocation
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.
- SourceAvailable from: etd.library.vanderbilt.edu
- [Show abstract] [Hide abstract]
ABSTRACT: We propose a new approach for incorporating RE ions into Al-bearing III-V compound semiconductor systems such as AlGaAs by converting the RE-doped III-V crystal to its native oxide through the wet-thermal oxidation process. In such an approach, the RE-doped native oxide regions could ultimately be optically excited by a monolithically integrated semiconductor pump laser. We present the results of preliminary exploratory studies on the suitability of AlGaAs native oxides as a host for optically-active Er<sup>3+</sup> ions. Room temperature, CW photoluminescence (PL) has been performed on various Al <sub>x</sub>Ga<sub>1-x</sub>As films doped with Er during crystal growth by molecular beam epitaxy (MBE) and subsequently thermally oxidizedLasers and Electro-Optics Society Annual Meeting, 1997. LEOS '97 10th Annual Meeting. Conference Proceedings., IEEE; 12/1997
- [Show abstract] [Hide abstract]
ABSTRACT: Human adaptor protein, phosphotyrosine interaction, PH domain and leucine zipper containing 1 (APPL1) and adaptor protein, phosphotyrosine interaction, PH domain and leucine zipper containing 2 (APPL2) are homologous effectors of the small guanosine triphosphatase RAB5 that interact with a diverse set of receptors and signaling proteins and are proposed to function in endosome-mediated signaling. Herein, we investigated the membrane-targeting properties of the APPL1 and APPL2 Bin/Amphiphysin/Rvs (BAR), pleckstrin homology (PH) and phosphotyrosine binding (PTB) domains. Coimmunoprecipitation and yeast two-hybrid studies demonstrated that full-length APPL proteins formed homooligomers and heterooligomers and that the APPL minimal BAR domains were necessary and sufficient for mediating APPL-APPL interactions. When fused to a fluorescent protein and overexpressed, all three domains (minimal BAR, PH and PTB) were targeted to cell membranes. Furthermore, full-length APPL proteins bound to phosphoinositides, and the APPL isolated PH or PTB domains were sufficient for in vitro phosphoinositide binding. Live cell imaging showed that full-length APPL-yellow fluorescent protein (YFP) fusion proteins associated with cytosolic membrane structures that underwent movement, fusion and fission events. Overexpression of full-length APPL-YFP fusion proteins was sufficient to recruit endogenous RAB5 to enlarged APPL-associated membrane structures, although APPL1 was not necessary for RAB5 membrane targeting. Taken together, our findings suggest a role for APPL proteins as dynamic scaffolds that modulate RAB5-associated signaling endosomal membranes by their ability to undergo domain-mediated oligomerization, membrane targeting and phosphoinositide binding.Traffic 03/2008; 9(2):215-29. DOI:10.1111/j.1600-0854.2007.00680.x · 4.71 Impact Factor