IRTKS Phosphorylation And Degradation

IRTKS (insulin receptor tyrosine kinase substrate) is a member of IRSp53 family. IRTKS has been reported to be associated with F-actin dynamics and membrane protrusion. In order to deeply understand the function of IRTKS, we analyzed tyrosine phosphorylation of IRTKS, and we also studied the ubiquitination and degradation of IRTKS.We discovered that IRTKS tyrosine phosphorylation status is variant in different cell lines, and may depend on the activity of proto-oncogene c-Src. Activated Src was able to stimulate tyrosine phosphorylation of IRTKS in vivo, and when Src was inhibited by its specific inhibitor PP2 or knocked down by small interference RNA, phosphorylation of IRTKS was decreased or even disappeared. Co-immunoprecipitation experiment showed that Src could interact with IRTKS, suggesting that Src might phosphorylate IRTKS directly. Using IRTKS truncations, we discovered that IRTKS contains multiple sites which could be phosphorylated by Src. A site directed mutagenesis technology was used to identify tyrosine phosphorylation site of IRTKS. We found that six tyrosine residues of IRTKS could be phosphorylated by Src, including Y37, Y156, Y163, Y274, Y293, Y439. Moreover, IRTKS could promote cell migration, and this ability disappears when Src is knocked down. This result shows that IRTKS enhances cell migration depending on Src.On the other hand, IRTKS could interact with MDM2. Overexpression of MDM2 accelerated IRTKS degradation and induced its polyubiquitination. This data indicated that MDM2 mediated IRTKS polyubiquitination and targeted its degradation by 26s proteasomes. Intetestingly, Src could inhibit the degradation and ubiquitination of IRTKS, suggesting phosphorylated IRTKS may be more stable in cells.