Role of PIM1 kinase in the regulation of translation elongation

The ribosome is one of the most complex and regulated molecular machines in the cell. It is a large ribonucleoprotein complex that translates mRNA into protein, thus synthesizing all the proteins within the cell. Defects in the synthesis, structure or function of ribosomes trigger a cell response defined as "ribosomal stress". This phenomenon and the signaling pathways activated downstream are considered at the base of a heterogeneous class of rare diseases called ribosomopathies such as Diamond-Blackfan Anemia (DBA), Dyskeratosis Congenita (DC) and the acquired 5q-syndrome. Although the molecular mechanisms underlying these diseases are still unclear, a well characterized aspect is that ribosomopathy patients show an increased predisposition to malignancies. Recent studies have shown that ribosomal stress induced by depletion of a ribosomal protein can lead to a decrease in the elongation rate but does not affect initiation. This response is dependent on eukaryotic Elongation Factor 2 kinase (eEF2K) activation that phosphorylates its target, eEF2, on Thr56 compromising its binding to the active ribosome. However, the consequence of eEF2K activation is not limited to reducing total protein synthesis. Several studies reported the evidence of a role of eEF2K in specific mRNA translation. In addition, translation elongation appears to play a role in the adaptive stress response in cancer cells. Besides the classical regulation of eEF2K by mTORC1, AMPK, ERK and cAMP kinase (PKA), an unconventional role of PIM1 kinase is emerging. PIM1 belongs to the PIM kinase family (together with PIM2 and PIM3), a family of short-lived, constitutively active serine/threonine kinases involved in translation, survival, cell cycle, and MYC-dependent transcription. The lack of regulatory domain implies that the regulation of these proteins occurs at the transcriptional and translational levels, as well as by proteasomal degradation. It has been reported that: I) the proto-oncogene PIM1 has an important role in the regulation of cell cycle and proliferation in hematopoietic cells with induced RPS19 deficiency; II) whereas elevated levels of PIM1 and PIM2 were mostly found in hematologic malignancies and prostate cancer, an increased PIM3 expression was observed in different solid tumors. Starting from the evidence that PIM1 could represent a potential sensor of ribosomal stress and, as a consequence, could cause the activation of eEF2K, we focused on the possible mechanism through which this regulation occurs. In this thesis work we applied in silico and in vitro approaches to explore the involvement of PIM1 in the elongation phase of translation. Through a kinase affinity prediction analysis we highlighted a possible direct phosphorylation of eEF2K by PIM1 kinase. We chose different cell lines, cancerous and non, to widely investigate the molecular mechanism of PIM1 involvement in the elongation process. We, therefore, demonstrated in vitro that PIM1 can interact and phosphorylate eEF2K on the inhibitory site Ser366. Then we assessed the role of PIM1 in the modulation of EF2 activity and, as a consequence, in the modulation of translation elongation. Indeed, we demonstrated that this modulation does not involve the canonical mTOR pathway showing that eEF2K phosphorylation is not affected by S6K in a S6K double-knockout cellular system. We further investigated how variations of PIM1 level or activity can influence global protein synthesis in cultured cells. We modulated PIM1 expression in vitro and, measuring isotopically-labelled methionine and puromycin incorporation, we demonstrated that protein synthesis is influenced by PIM1 activity. Our data uncover a novel role of PIM1 and support the model of a PIM1- eEF2K-eEF2 pathway involved in the regulation of translation elongation. As PIM1 is known as a pro-survival kinase, the proposed characterization opens a new scenario that can be exploited in future anti-cancer therapeutic approaches.