Anales RANF

S7-02 GUANOSINE/GUANYLATE-DEPENDENT REGULATION OF SIGNAL TRANSDUCTION IN CANCER CELLS Mikhail A. Nikiforov 1* and Anna Bianchi-Smiraglia 2 1 Wake Forest University Comprehensive Cancer Center, NC, USA; 2 Roswell Park Comprehensive Cancer Center, NC, USA. Recently, we have reported a connection between activity of RHO-GTPase and GTP metabolism enzymes ( GMEs ) including GMPR, GMPS, and IMPDH-1 and 2 (Fig. 1). In particular, we demonstrated that in cancer cells overexpression of GMPR or partial inhibition of IMPDH led to a moderate ~25% depletion of intracellular GTP pools and a dramatic drop in the amounts of GTP-bound i.e. active RHO-GTPases. As a result, cancer cell invasion was significantly downregulated. Restoration of GTP levels or expression of RAC1 G12V mutant insensitive to changes in GTP, reverted these effects. Additionally, GMPR overexpression or inhibition of GMPS , a functional antagonist of GMPR, suppressed melanoma cell tumorigenicity. Historically, changes in GTP levels have not been considered as a regulatory step in activation of RAC1 (or other G- proteins) in live cells. This is because average GTP concentration in the cell measured by HPLC (~500 μ M) is much higher than the GTP dissociation constant of RAC1, which even in presence of GEF, is ~15 μ M GTP. However, HPLC cannot account for free vs bound GTP or for sub- cellular variations in GTP levels. Up until now no methods existed to detect such variations. We recently reported genetically encoded intracellular sensors of free GTP ( Bianchi-Smiraglia et al, Nature Methods, 2017 ). These sensors for the first time made possible visualization of GTP changes in live cells and identified regions with low (~30 μ M) and high local GTP concentration. We will present data demonstrating that distribution of intracellular GTP levels is previously unrecognized major mechanism controlling activity of RHO-GTPases and possibly other G-proteins, and that physiological modulation of GME activity regulates GTP loading on RHO-GTP and therefore can be exploited therapeutically.