Phosphorylation Of Irs4 By Ck1 Gamma 2 Promotes Its Degradation By Chip Through The Ubiquitin/Lysosome Pathway

IRS4, a member of the insulin receptor substrate protein family, can induce constitutive PI3K/AKT hyperactivation and cell proliferation even in the absence of insulin or growth factors and promote tumorigenesis, but its regulation has only been explored at the transcriptional level.Methods: Scansite was used to predict the potential protein kinases that may regulate the functions of IRS4, and mass spectrometry was used to identify the E3 ligase for IRS4. The protein interaction was carried out by immunoprecipitation, and protein stability was measured by cycloheximide treatment. In vitro kinase assay was used to determine the phosphorylation of IRS4 by casein kinase I gamma 2 (CKl gamma 2). Colony formation assay and xenograft-bearing mice were employed to assess the cancer cell growth in vitro and in vivo, respectively. Immunohistochemistry was performed to examine protein levels of both IRS4 and CKl gamma 2 in osteosarcoma specimens and their relationship was evaluated by X-2 test. Two-tailed Student's t-test or the Mann-Whitney U test were used to compare the differences between subgroups.Results: IRS4 was phosphorylated at Ser859 by CKl gamma 2 in vitro and in vivo, which promoted the polyubiquitination and degradation of IRS4 through the ubiquitin/lysosome pathway by the carboxyl terminus of Hsc70-interacting protein(CHIP). Using osteosarcoma cell lines, the ectopic nonphosphorylated mutant of IRS4 by CKl gamma 2 triggered higher level of p-Akt and displayed faster cell proliferation and cancer growth in vitro and in nude mice. In addition, a negative correlation in protein levels between CKl gamma 2 and IRS4 was observed in osteosarcoma cell lines and tissue samples.Conclusions: IRS4, as a new substrate of CHIP, is negatively regulated by CKl gamma 2 at the posttranslational level, and specific CKl gamma 2 agonists may be a potentially effective strategy for treating patients with osteosarcoma.