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RET downstrean effectors: The function of Rai as a neuronal survival factor and its mechanism of action through PI3K-Akt

Mechanisms of stress-induced activation of Rai. 1.1 We have analyzed the effects of H2O2 (an inducer of oxidative stress) and CoCl2 (an inducer of cellular hypoxia) on Rai protein expression using primary neuronal mouse cortical cultures. Notably, H2O2 and CoCl2 treatments consistently induced an acceleration of the mobility (shift-down) of endogenous Rai proteins in polyacrilamide gels. The shift down of Rai polypeptides appeared at late time points after treatment how 1 hr and 2 hr for H2O2 and CoCl2 respectively and returned to the basal levels within 24 hours. The same modification was also detected in a transformed cell line (PFSK1) and in vivo in different areas of the brain mouse following 15 minutes of ischemia and 24 hours of reperfusion. In vitro 32P labeling experiments and usage of dual specificity or tyrosine-specific phosphatases indicated that the stress-induced gel-shift of Rai proteins is due to dephosphorylation of serine-threonine residues. Rai contains 40 conserved serine and threonine residues in the CH1 region. Using site-directed mutagenesis we have restricted the putative phosphorylation-sites to two serine residues. These two serines are predicted to be phosphorylation sites for the CaMKII (Ca2+/calmodulin-dependent protein kinase).

CaMKII is enriched in neuronal tissue (up to 2% of total protein) and highly concentrated in the post-synaptic densities and mediates a variety of different cellular response to Ca2+ influx. It is involved in synaptic plasticity and has a role in learning and memory. So far we have demonstrated that the kinase is able to phosphorylate in vitro Rai proteins. 2.2 Identification of Rai interactors. To characterize the Rai-signalling potential (and upstream effectors), we attempted purification of Rai-complexes. To this end we expressed N-terminally or C-terminally Flag-HA-epitope-tagged Rai at stable and low levels in the neuroectoderma cell line PFSK-1 endogenously expressing Rai.

We isolated protein complexes by affinity purification and analyzed them by mass spectrometry. The experiments have been performed in growing cells and after stress stimuli (H2O2 treatment and hypoxia). From the mass-spectrometry analysis we identified the protein PP2C. PP2C is a Mg2+ dependent serine/threonine phosphatase present in the brain. It is activated by cellular stress and is known to dephosphorylate CaMKII. By in vitro coimmunoprecipitation studies using specific antibodies we validated the Rai-PP2C association. Rai is constitutively bound to PP2C and the binding increases after 5 minutes of H2O2 treatment. We are investigating the protein domain of interaction between the two proteins. To demonstrate the role of Rai dephosphorylation on activation of its biological activity we are performing PP2C RNA interference in Rai expressing cells and we are evaluating the cellular resistance to stress stimuli.

The study of the molecular mechanisms involved in Rai-dependent induction of anti-apoptotic activity, the identification of the main phosphorylation sites and of the kinase/ phosphatase involved can identify Rai as a new potential diagnostic/prognostic marker and, eventually, a target of therapeutic intervention for neurodegenerative diseases. Moreover, the identification of other proteins that interact with Rai will improve our knowledge on which pathways are activated in different stress conditions, such as neuronal injury or neurodegenerative diseases.

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Istituto Europeo di Oncologia
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