CHO/IRF960/T962 cells express a mutant human insulin receptor in which Tyr960 and Ser962 in the juxtamembrane region of the receptor's β-subunit are replaced by Phe and Thr, respectively. The mutant insulin receptor undergoes autophosphorylation normally in response to insulin; however, insulin fails to stimulate thymidine incorporation into DNA, glycogen synthesis, and tyrosyl phosphorylation of an endogenous substrate pp185 in these cells. Another putative substrate of the insulin receptor tyrosine kinase is phosphatidylinositol 3-kinase (Ptdlns 3-kinase). We have previously shown that Ptdlns 3-kinase activity in Chinese hamster ovary cells expressing the wild-type human insulin receptor (CHO/IR) increases in both antiphosphotyrosine [anti-Tyr(P)] immunoprecipitates and intact cells in response to insulin. In the present study a new technique (detection of the 85-kDa subunit of Ptdlns 3-kinase using [32P]phosphorylated polyoma virus middle T-antigen as probe) is used to monitor the Ptdlns 3-kinase protein. The 85-kDa subunit of Ptdlns 3-kinase is precipitated by anti-Tyr(P) antibodies from insulin-stimulated CHO/IR cells, but markedly less protein is precipitated from CHO/IRF96O/T962 cells. The amount of Ptdlns 3-kinase activity in the immunoprecipitates was also reduced in the CHO/IRF960/T962 cells compared to CHO/IR cells. In intact CHO/IRF96O/T962 cells, insulin failed to stimulate phosphate incorporation into one of the products of activated Ptdlns 3-kinase, phosphatidylinositol-3, 4-bisphosphate[Ptdlns(3, 4)P2], whereas it caused a 12-fold increase in CHO/IR cells. In contrast, phosphate incorporation into another product, phosphatidylinositol trisphosphate [PtdlnsP3], was only partially depressed in the CHO/IRF960/T962 cells. The data indicate that disruption of the juxtamembrane region of the insulin receptor impairs its ability to modulate Ptdlns 3-kinase activity and suggest that Ptdlns 3-kinase may play an important role in insulin signaling. The results further suggest that the levels of Ptdlns(3, 4)P2 and PtdlnsP3 can be differentially regulated in the intact cell, and that production of the former may be important for some of the biological actions of insulin.
ASJC Scopus subject areas
- Molecular Biology