The identification of phosphorylation sites of IBTK by shotgun proteomics

Translation is a tightly regulated process, and the mTORC1-S6K signaling axis plays a critical role in this control. Binding of eIF4F to the cap is hindered by eIF4E binding proteins (4EBPs), which, when hypophosphorylated, sequester eIF4E and prevent its association with eIF4G. However, in response to positive stimuli such as growth factors, mitogens, and amino acids, mTORC1 phosphorylates 4EBPs and relieves this inhibition, allowing the formation of eIF4F and subsequent initiation of translation. We are interested to know whether IBTK-mediated eIF4A1 ubiquitination is regulated by mTORC1/S6K signaling. Indeed, quantitative phosphoproteomics studies revealed that several IBTK phosphorylation sites are markedly downregulated by treatment of mTOR inhibitor, Rapamycin or Torin 1. Thus, probable phosphorylation sites of IBTK were identified by MS analysis.