IndraLab

"Here we show that blocking phosphorylation of the AKT substrate, endothelial nitric oxide synthase (eNOS or NOS3), inhibits tumour initiation and maintenance. Moreover, eNOS enhances the nitrosylation and activation of endogenous wild-type Ras proteins,"

"Collectively, we speculated that AKT activation of eNOS maintains tumour growth in the absence of oncogenic Ras by activating wildtype Ras through S-nitrosylation of C118. To test this, activated AKT in PI3K-TtH was shown to foster HRas nitrosylation through eNOS. Specifically, most HRas nitrosylation was lost by treatment with the PI3K inhibitor wortmannin (Fig. 2a), by mutating C118 in HRas to serine (a minor change that exchanges the sulphur atom for oxygen but nevertheless blocks nitrosylation14) (Fig. 2b), or by knocking down eNOS (Fig. 2c). Conversly, HRas nitrosylation was elevated upon activation of AKT by p110-CAAX (Fig. 2c). Reduction of HRas nitrosylation by eNOS shRNA also reduced levels of active GTP-bound HRas (Fig. 2c). Because TtH cells express HRas and NRas, but not KRas (not shown), and C118 is conserved among all Ras proteins, we tested and confirmed that activated AKT in PI3KTtH cells also led to elevated levels of nitrosylated and GTP-bound endogenous NRas, which were reduced upon knockdown of eNOS (Fig. 2c). Thus, AKT activation of eNOS promotes nitrosylation and activation of wild-type Ras proteins"