Bim inhibits autophagy by recruiting Beclin 1 to microtubules. (2012)
Figure 6

LC8 Promotes the Bim-Beclin 1 Interaction

(A) Bim-147 does not bind to Beclin 1. Beclin 1-Flag/vector (IP negative control), Beclin 1-Flag/Myc-BimEL-1-147aa (Myc-147), or Beclin 1-Flag/Myc-BimEL-ΔBH3 (Myc-ΔBH3) were transfected into HeLa cells. Anti-Myc (Rabbit) was used for immunoprecipitation.

(B) Bim-147 is competent for LC8 binding. LC8/vector (IP negative control), LC8/Myc-BimEL-1-147aa (Myc-147), or LC8/Myc-BimEL-ΔBH3 (Myc-ΔBH3) were transfected into HeLa cells. Anti-Myc (Rabbit) was used for immunoprecipitation.

(C) Mutating LC8 binding consensus sites within Bim disrupts the Bim-LC8 interaction. Bim(EL)EE/vector (IP negative control), Bim(EL)EE/Myc-LC8, Bim(EL)EE-S109A, S113A, T114A (AAA)/ Myc-LC8, or Bim(EL)EE-T116E/ Myc-LC8 were transfected into HeLa cells. Anti-Myc (Rabbit) was used for immunoprecipitation. Data are shown as mean ± SD. ∗∗∗p < 0.001.

(D) Mutating LC8 binding consensus sites within Bim disrupts the Bim-Beclin 1 interaction. Bim(EL)EE/vector (IP negative control), Bim(EL)EE/Beclin 1-Flag, Bim(EL)EE-S109A, S113A, T114A (AAA)/Beclin 1-Flag, or Bim(EL)EE-T116E/Beclin 1-Flag were transfected into HeLa cells. Anti-Flag (M2) was used for immunoprecipitation. Data are shown as mean ± SD. ∗∗∗p < 0.001.

(E) Bim-S does not bind to LC8. BimEL-EE (EL)/vector (IP negative control), BimEL-EE (EL)/Myc-LC8, BimL-EE (L)/Myc-LC8, or BimS-EE (S)/Myc-LC8 were transfected into HeLa cells. Anti-Myc (Rabbit) was used for immunoprecipitation.

(F) Model of LC8 promoting Beclin 1-Bim interaction. In nutrients, LC8 binds the Bim consensus sequence (DKSTQTP). This enables a transition of the Bim C terminus from a disordered state to α helices and a β sheet (Barbar, 2008), which favors the Bim-Beclin 1 interaction. In starvation conditions, Bim is phosphorylated at T116 within the consensus sequence (DKSTQpTP) leading to its dissociation from LC8 (Lei and Davis, 2003). Bim returns to the disordered state, which binds to Beclin 1 less effectively. Grey boxes, higher degree of disorder; white box, lower degree of disorder (Barbar, 2008).

(G) LC8 enhances Bim-Beclin 1 interaction in vitro. His-Bim, His-LC8 and GST-Beclin 1 were expressed in BL21(DE3) E. coli and purified. Two micrograms of His-Bim was combined with 2 μg GST without or with 1 μg His-LC8 (as controls); 2 μg His-Bim was combined with 2 μg GST-Beclin 1 without or with 1 μg His-LC8. The mixtures were incubated in buffer A for 3 hr. Glutathione beads were then used to pull down GST or GST-Beclin 1. The pulldown products were detected with anti-Bim and anti-Beclin 1.

(H) LC8 siRNA knockdown reduces the Bim-Beclin 1 interaction. Control siRNA or LC8 siRNA were transfected into HeLa cells. After 48 hr, HA-Bim(EL)EE/vector (IP negative control) or HA-Bim(EL)EE/Beclin 1-Flag was transfected into control siRNA transfected HeLa cells; HA-Bim(EL)EE/Beclin 1-Flag was transfected into LC8 siRNA-transfected HeLa cells. Anti-Flag (M2) was used for immunoprecipitation. Note that in the presence of BimEE, endogenous LC8 was pulled down by Beclin 1 when cells were treated with control siRNA. Data are shown as mean ± SD. ∗∗∗p < 0.001.

See also Figure S6.