Cross-talk between the two divergent insulin signaling pathways is revealed by the protein kinase B (Akt)-mediated phosphorylation of adapter protein APS on serine 588.
The APS adapter protein is recruited to the autophosphorylated kinase domain of the insulin receptor and initiates the phosphatidylinositol 3-kinase (PI3K)-independent pathway of insulin-stimulated glucose transport by recruiting CAP and c-Cbl. In this study, we have identified APS as a novel substrate for protein kinase B/Akt using an antibody that exhibits insulin-dependent immunoreactivity with a phosphospecific antibody raised against the protein kinase B substrate consensus sequence RXRXX(pS/pT) and a phosphospecific antibody that recognizes serine 21/9 of glycogen synthase kinase-3alpha/beta. This phosphorylation of APS is observed in both 3T3-L1 adipocytes and transfected cells. The insulin-stimulated serine phosphorylation of APS was inhibited by a PI3-kinase inhibitor, LY290004, a specific protein kinase B (PKB) inhibitor, deguelin, and knockdown of Akt. Serine 588 of APS is contained in a protein kinase B consensus sequence for phosphorylation conserved in APS across multiple species but not found in other members of this family, including SH2-B and Lnk. Mutation of serine 588 to alanine abolished the insulin-stimulated serine phosphorylation of APS and prevented the localization of APS to membrane ruffles. A glutathione S-transferase fusion protein containing amino acids 534-621 of APS was phosphorylated by purified PKB in vitro, and mutation of serine 588 abolished the PKB-mediated phosphorylation of APS in vitro. Taken together, this study identifies APS as a novel physiological substrate for PKB and the first serine phosphorylation site on APS. These data therefore reveal the molecular cross-talk between the insulin-activated PI3-kinase-dependent and -independent pathways previously thought to be distinct and divergent.
Katsanakis
Kostas D
KD
Institute of Cell Signaling and School of Biomedical Sciences, University of Nottingham Medical School, UK.
Pillay
Tahir S
TS
D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017667:Adipocytes / Q000166:cytology / Q000378:metabolism; D020816:Amino Acid Motifs; D000818:Animals; D002460:Cell Line; D017124:Conserved Sequence; D007328:Insulin / Q000378:metabolism*; D051379:Mice; D009154:Mutation; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D051381:Rats; D015398:Signal Transduction; D013379:Substrate Specificity
D048868:Adaptor Proteins, Signal Transducing; D007328:Insulin; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010768:Phosphoserine; D051057:Proto-Oncogene Proteins c-akt; C488162:Sh2b2 protein, rat