Pim Serine/Threonine Kinases Substrate Identification And Investigation

The Pim family of Ser/Thr kinas,which is consisted of three members:Pim-1, Pim-2, and Pim-3. Pim-1was originally identified as proviral integration sites in Moloney murine leukemia virus (MoMuLV)-induced lymphoma. Insertion in this region stabilised the mRNA for Pim-1thus up-regulating the protein expression. Pim-1and Pim-2are highly expressed in hematopoietic tissues and are subject to regulation by a variety of cytokines and growth factors.Up-regulation of Pim-1and Pim-2plays an important role in cellular proliferation, survival and differentiation. The third isoform, Pim-3, which is72%identical to Pim-1, also exists in mammalian cells although the oncogenic properties of this protein are less well studied.Several substrates have been identified for Pim-1and Pim-2. eIF4B (eukaryotic initiation factor4B) as Pim-2kinase substrate had been identified to be directly phosphorylatyed on Ser406by Pim-2kinase. The translation initiation factor eIF4B stimulates the RNA helicase activity of eIF4A in unwinding secondary structures in the5’untranslated region (5’UTR) of the mRNA. Eukaryotic initiation factor4B (eIF4B) is a multidomain protein with a critical role in the initiation of protein synthesis, and has long been known as a hyperphosphorylated protein.In this study, we first tested eIF4B as Pim-1kinase substrate. We found that Pim-1, Pim-2but not Pim-3can upregulate phosphorylation levels of eIF4B on Ser406and Ser422using293T cell system, while kinase-dead mutants of Pim-1or Pim-2significantly inhibit phosphorylation of eIF4B. Furthermore, in vitro kinase assay revealed that Pim-1kinase can directly phosphorylate eIF4B on Ser422. As expected, we found that Pim-1kinase directly bound to eIF4B by using GST-pull down assay experiments. Interestingly, levels of eIF4B protein in v-Abl transformants treated with the Ab1kinase inhibitor imatinib decreased markedly, indicating that expression of eIF4B is Ab1kinase dependant. Our results suggest that Pim kinases regulate cellular processes via phosphorylating downstream eIF4B.