Figure 2. ROCK1 is activated upon metabolic stress.

(a) Inhibition of ROCK1 activity ablates the interaction between Beclin1 and ROCK1. HeLa cells were untreated (H₂O) or treated with 10 µM ROCK1 inhibitor, Y27632, for 8 h and then cultured in control media (+) or HBSS (-) for 4 h. Cells were harvested, lysed and immunoprecipitated with Beclin1 (left panel) or ROCK1 (right panel) antibody. Left panel- Immune-complexes were divided into two (ROCK1-MOPS buffer and Beclin1-MES buffer) and probed for ROCK1 and Beclin1. Relative values of ROCK1 versus Beclin1 are shown. (b) An increase in ROCK1-mediated phosphorylation of MYPT1 upon starvation. HeLa and EJ cells were starved in HBSS for indicated time-points and endogenous ROCK1 was immunoprecipitated. An in vitro kinase assay was performed using MYPT1 as substrate and immune-complexed ROCK1. Proteins were resolved and visualized with Coomassie (top), western blot (middle) and autoradiography (bottom). Inputs were examined for ROCK1 and β-actin. (c) HeLa cells incubated in HBSS were harvested and endogenous phosphorylation of MYPT1 was determined by western blotting. The same blot was then washed in TBST for 30 min, blocked in 5% milk and analyzed for total MYPT1 as a control. Extracts from this experiment were re-run on 7.5% gel and analyzed for ROCK1 and β-actin. (d) ROCK activity is increased upon starvation. Cell lines (HeLa and EJ) grown in control (+Glu) or starved with HBSS for 1 and 3 h were lysed, extracts normalized and ROCK kinase activity was measured by ELISA. Graphs represent ROCK activity in starved cells plotted as percent of ROCK1 activity in control (glucose-rich) cells. Data was obtained from triplicates performed at the same time (mean±s.d.). p-value was calculated using Student’s t-test. (e) ROCK1 activity is not dependent on Beclin1. siControl (siCont.) and siBeclin1 knockdown cells were incubated in starvation media (HBSS) for indicated time. Resulting cell extracts were used to confirm Beclin1 knockdown and ROCK1 expression (left panel). Cell extracts from the same experiment were used for ELISA measuring ROCK activity (right panel). Graph represents mean±s.d. of duplicate samples read at the same time (Student t-test: NS=not significant).