Figure 5: ROCK1 phosphorylation of Beclin1 at T119 promotes Bcl-2 dissociation.

(a) Inhibiting ROCK1 activity increases the association between Beclin1 and Bcl-2. HeLa cells, untreated or treated with 10 μM Y27632 (top panel), were cultured in control or glucose-free (4 h) medium, lysed and cell extracts prepared. Endogenous Bcl-2 was immunoprecipitated with cross-linked Bcl-2 agarose and resulting eluted immune complexes were resolved by SDS–PAGE and blotted against indicated antibodies. EJ shCont. and shROCK1#1 cells (bottom panel) were grown in control or starvation media for 4 h and Bcl-2 IP was performed as above. Inputs were rerun to confirm ROCK1 knockdown. (b) HeLa cells were transfected with full-length constructs of Flag-Beclin WT, Flag-Beclin T119A or Flag-Beclin T119E. Flag-Beclin WT cells were then treated with Y27632 for 8 h. Post transfection and treatment with Y27632, cells were starved in HBSS medium for 4 h, after which cells were collected and whole cell lysates prepared. IP with Flag-agarose was performed for 2 h and proteins bound to beads were eluted and used for western blotting. (c) Flag-Beclin1 Wt or T119A transfected HeLa cells were incubated in glucose-rich or nutrient-free (HBSS) media, in the presence or absence of ROCK1 inhibitor Y27632. Total cell extracts were used for IP using Flag agarose. Eluted protein was analysed by western blotting against Bcl-2 and Beclin1. Inputs and IP were run on separate gels and same exposure is shown. (d) HeLa cells with a transient knockdown using siCont. and siBeclin1–3′-untranslated region for 30 h were further left untreated or treated with 10 μM Y27632. Cells were then incubated in control or HBSS media for 8 h. Whole cell extracts were prepared, resolved by SDS–PAGE and were analysed for Beclin1, LC3 and β-actin. (e) Endogenous LC3 staining in WT- or T119A-Beclin1-transfected cells. HeLa cells transfected with Flag-Beclin WT (±Y27632) and T119A were cultured in HBSS media for 6 h, fixed with cold acetone and endogenous LC3 immunofluorescence was performed. Representative images are shown; scale bar, 20 μm. Arrows indicate punctate LC3 staining. Graph represents mean±s.d., n=25, LC3 puncta/cell.