The interaction of Akt with APPL1 is required for insulin-stimulated Glut4 translocation.
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.
Saito
Tsugumichi
T
Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts 02118, USA.
Jones
Christine C
CC
Huang
Shaohui
S
Czech
Michael P
MP
Pilch
Paul F
PF
D048868:Adaptor Proteins, Signal Transducing; D017667:Adipocytes / Q000378:metabolism*; D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D051275:Glucose Transporter Type 4 / Q000378:metabolism*; D006801:Humans; D007328:Insulin / Q000378:metabolism*; D008297:Male; D051379:Mice; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley
C464801:APPL1 protein, human; D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D051275:Glucose Transporter Type 4; D007328:Insulin; D051057:Proto-Oncogene Proteins c-akt