Figure 5.

RASA5 Functions as a RasGAP and Antagonizes RAS Signaling

(A) Western blot of phosphor-MEK (pMEK), phosphor-ERK (pERK), and phosphor-Akt (pAKT) following RASA5 expression in RAS-wild-type (wt) KYSE150 and HNE1 cells, as well as KRAS-mutant (mut) MB231 cells. Ectopic expression of RASA5 downregulated phosphorylation levels of RAS signaling effectors, whereas active RAS mutant form (HRAS Q61L) counteracted this suppression. GAPDH used as a control. Quantification of the phosphor-immunoblots normalized to corresponding total protein levels (right). Data were presented as mean ± SEM of three independent experiments via Student's t test and representative data were shown. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

(B) Active GTP-bound form of Ras was pulled down by Raf-RBD and subjected to Western blot analysis. RASA5 downregulated Ras-GTP level, but overexpression of oncogenic HRAS Q61L reversed this suppression. Graph represents quantification of Western blots with fold change compared with controls (right). Data were presented as mean ± SEM of three independent experiments via Student's t test and representative data were shown. ***, p < 0.001.

(C) Semi-quantitative RT-PCR analyses of RASA5 inhibitory effect on Ras target oncogenes MCL1 and c-MYC at mRNA levels. Ectopic expression of HRAS Q61L mutant abrogated this inhibition. wt, wild-type; mut, mutant.