The Mechanism Of IRTKS Regulates Insulin Signaling Pathway

BACKGROUNDS AND AIMS:Insulin signaling is mediated by a highly integrated network that controls glucose metabolism,protein synthesis,cell growth,and differentiation.Our previous work indicates that the insulin receptor tyrosine kinase substrate?IRTKS?,also known as BAI1-associated protein 2-like 1?BAIAP2L1?,is a novel regulator of insulin network,but the detailed mechanism has not been fully studied.We identified one potential partner of IRTKS,Src homology?SH2?containing inositol polyphosphate5-phosphatase-2?SHIP2?,which specifically hydrolyzes the 5-phosphate of phosphatidylinositol 3,4,5-triphosphate?PI?3,4,5?P₃,PIP3?to produce PI?3,4?P₂.PIP3 is one of the corner-stone of insulin signaling pathway,it is interesting to investigate the relationship between IRTKS and SHIP2,which is important to understand the detailed mechanism of IRTKS regulates insulin signaling transduction.METHODS:Firstly,we evaluated the interaction between IRTKS and SHIP2 in vitro and in vivo through co-localization and co-immunopricipitation experiments.We further constructed series of mutant IRTKS and SHIP2 plasmids to investigate the binding domain by GST pull-down assay.The enzyme activity of SHIP2 also was determined by malachite green phosphatase assay under IRTKS overexpression or knock-down.The influence of interaction between IRTKS and SHIP2 in insulin signaling transduction was evaluated by western-blot,cell proliferation curve and colony formation assays.RESULTS:In this work we reveal that IRTKS co-localizes with Src homology?SH2?containing inositol polyphosphate 5-phosphatase-2?SHIP2?,and the SH3 domain of IRTKS directly binds to SHIP2's catalytic domain INPP5c.IRTKS suppresses SHIP2 phosphatase to convert phosphatidylinositol 3,4,5-triphosphate?PI?3,4,5?P₃,PIP3?to phosphatidylinositol?3,4?bisphosphate?PI?3,4?P₂?.IRTKS-knockout significantly increases PI?3,4?P₂ level and decreases cellular PI?3,4,5?P₃content.Interestingly,the interaction between IRTKS and SHIP2 is dynamically regulated by insulin,which feeds back and affects the tyrosine phosphorylation of IRTKS.Furthermore,IRTKS overexpression elevates PIP3,activates the Akt–mTOR signaling pathway,and increases cell proliferation.Thereby,IRTKS not only associates with insulin receptors to activate PI3K but also interacts with SHIP2 to suppress its activity,leading to PIP3 accumulation and the activation of the Akt–mTOR signaling pathway to modulate cell proliferation.