Onnockx Sheela, De Schutter Julie, Blockmans Marianne, Xie Jingwei, Jacobs Christine, Vanderwinden Jean-Marie, Erneux Christophe, Pirson Isabelle
Institute of Interdisciplinary Research (IRIBHM), Brussels, Belgium.
J Cell Physiol. 2008 Jan;214(1):260-72. doi: 10.1002/jcp.21193.
SHIP2 (SH2-containing inositol polyphosphate 5-phosphatase 2) is a phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P(3)) 5-phosphatase containing various motifs susceptible to mediate protein-protein interaction. In cell models, SHIP2 negatively regulates insulin signalling through its catalytic PtdIns(3,4,5)P(3) 5-phosphatase activity. We have previously reported that SHIP2 interacts with the c-Cbl associated protein (CAP) and c-Cbl, proteins implicated in the insulin cellular response regulating the small G protein TC10. The first steps of the TC10 pathway are the recruitment and tyrosine phosphorylation by the insulin receptor of the adaptor protein with Pleckstrin Homology and Src Homology 2 domains (APS). Herein, we show that SHIP2 can directly interact with APS in 3T3-L1 adipocytes and in transfected CHO-IR cells (Chinese hamster ovary cells stably transfected with the insulin receptor). Upon insulin stimulation, APS and SHIP2 are recruited to cell membranes as seen by immunofluorescence studies, which is consistent with their interaction. We also observed that SHIP2 negatively regulates APS insulin-induced tyrosine phosphorylation and consequently inhibits APS association with c-Cbl. APS, which specifically interacts with SHIP2, but not PTEN, in turn, increases the PtdIns(3,4,5)P(3) 5-phosphatase activity of SHIP2 in an inositol phosphatase assay. Co-transfection of SHIP2 and APS in CHO-IR cells further increases the inhibitory effect of SHIP2 on Akt insulin-induced phosphorylation. Therefore, the interaction between APS and SHIP2 provides to both proteins potential negative regulatory mechanisms to act on the insulin cascade.
SHIP2（含SH2结构域的肌醇多磷酸5-磷酸酶2）是一种磷脂酰肌醇(3,4,5)-三磷酸（PtdIns(3,4,5)P(3)）5-磷酸酶，含有多种易于介导蛋白质-蛋白质相互作用的基序。在细胞模型中，SHIP2通过其催化性的PtdIns(3,4,5)P(3) 5-磷酸酶活性对胰岛素信号传导进行负调控。我们之前报道过SHIP2与c-Cbl相关蛋白（CAP）和c-Cbl相互作用，这些蛋白参与胰岛素细胞反应，调节小G蛋白TC10。TC10信号通路的第一步是具有普列克底物蛋白同源结构域和Src同源结构域2的衔接蛋白（APS）被胰岛素受体招募并发生酪氨酸磷酸化。在此，我们表明SHIP2可在3T3-L1脂肪细胞和转染的CHO-IR细胞（稳定转染胰岛素受体的中国仓鼠卵巢细胞）中直接与APS相互作用。免疫荧光研究显示，胰岛素刺激后，APS和SHIP2被招募到细胞膜上，这与它们之间的相互作用一致。我们还观察到SHIP2对APS胰岛素诱导的酪氨酸磷酸化起负调控作用，从而抑制APS与c-Cbl的结合。反过来，特异性与SHIP2而非PTEN相互作用的APS，在肌醇磷酸酶测定中增加了SHIP2的PtdIns(3,4,5)P(3) 5-磷酸酶活性。在CHO-IR细胞中共转染SHIP2和APS进一步增强了SHIP2对Akt胰岛素诱导的磷酸化的抑制作用。因此，APS与SHIP2之间的相互作用为这两种蛋白提供了作用于胰岛素信号级联反应的潜在负调控机制。
