Insulin-stimulated Interaction between insulin receptor substrate 1 and p85alpha and activation of protein kinase B/Akt require Rab5.
Binding of insulin to the insulin receptor initiates a cascade of protein phosphorylation and effector recruitment events leading to the activation of multiple distinct signaling pathways. Previous studies suggested that the diversity and specificity of insulin signal transduction are accomplished by both subcellular localization of receptor and the selective activation of downstream signaling molecules. The small GTPase Rab5 is a key regulator of endocytosis. Three Rab5 isoforms (Rab5a, -5b, and -5c) have been identified. Here we exploited the RNA interference technique to specifically knock down individual Rab5 isoforms to determine the cellular function of Rab5 in distinct insulin signaling pathways. Small interference RNA against a single Rab5 isoform had no effect on protein kinase B (PKB)/Akt or MAPK activation by insulin in NIH3T3 cells overexpressing human insulin receptor. However, simultaneous knockdown of all three Rab5 isoforms dramatically attenuated PKB/Akt activation by insulin without affecting MAPK activation. This inhibition of PKB/Akt activation was because of the impaired interaction between insulin receptor substrate 1 and the p85alpha subunit of phosphatidylinositol 3-kinase. These results indicate a requirement of Rab5 in presenting p85 to insulin receptor substrate 1. Additional evidence supporting a role for Rab5 was suggested by studies with GAPex-5, a vps9 domain containing exchange factor. Down-regulation of GAPex-5 impaired insulin-stimulated PKB/Akt activation. Collectively, this study indicates the involvement of Rab5 in insulin signaling.
Su
Xiong
X
Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Lodhi
Irfan J
IJ
Saltiel
Alan R
AR
Stahl
Philip D
PD
D000818:Animals; D034285:Dynamin II / Q000502:physiology; D004705:Endocytosis; D004789:Enzyme Activation; D005260:Female; D020662:Guanine Nucleotide Exchange Factors / Q000502:physiology; D007328:Insulin / Q000494:pharmacology*; D055504:Insulin Receptor Substrate Proteins; D051379:Mice; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D041681:NIH 3T3 Cells; D019869:Phosphatidylinositol 3-Kinases / Q000502:physiology*; D010750:Phosphoproteins / Q000502:physiology*; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000494:pharmacology; D051381:Rats; D011924:Rats, Zucker; D011972:Receptor, Insulin / Q000378:metabolism; D015398:Signal Transduction; D020696:rab5 GTP-Binding Proteins / Q000502:physiology*
D020662:Guanine Nucleotide Exchange Factors; C526292:IRS1 protein, human; D007328:Insulin; D055504:Insulin Receptor Substrate Proteins; C526295:Irs1 protein, mouse; C526296:Irs1 protein, rat; D010750:Phosphoproteins; D034741:RNA, Small Interfering; D019869:Phosphatidylinositol 3-Kinases; D011972:Receptor, Insulin; D051057:Proto-Oncogene Proteins c-akt; D020928:Mitogen-Activated Protein Kinases; D020696:rab5 GTP-Binding Proteins; D034285:Dynamin II