Bcl-xL and UVRAG cause a monomer-dimer switch in Beclin1

Abstract

Beclin1 has a key regulatory role in the initiation of autophagy and is a tumor suppressor. We have examined the interplay between viral or human Bcl-2-like proteins and UVRAG and their opposite effects on Beclin1. We show that Beclin1 forms a dimer in solution via its coiled-coil domain both in vivo and in vitro. Viral Bcl-2 binds independently to two sites on the Beclin1 dimer, one with high affinity and one with lower affinity, whereas human Bcl-x(L) binds both sites equally with relatively low affinity. UVRAG disrupts the Beclin1-dimer interface, forming a heterodimer with Beclin1, suggesting that this is how UVRAG causes its effects on Beclin1 to activate autophagy. Both Bcl-2-like proteins reduce the affinity of UVRAG for Beclin1 approximately 4-fold, suggesting that they stabilize the Beclin1 dimer. Moreover, coimmunoprecipitation assays show that UVRAG substantially reduces Beclin1 dimerization in vivo. These data explain the concentration-dependent interplay between Bcl-2, UVRAG, and Beclin1, as both tumor suppressors, UVRAG and Beclin1, have single-copy mutations in human cancers. Furthermore, our data suggest that an alternative strategy for developing anti-cancer compounds would be to disrupt the Beclin1-dimer interface.

FIGURE 1.

Beclin1 CCD dimerizes in solution. Beclin1 CCD (A) and Beclin1 BH3-CCD (B) were analyzed by sedimentation equilibrium and fitted to an ideal single-species model. Representative fits for each protein are shown. C, Beclin1 CCD and Beclin1 BH3-CCD were also analyzed by sedimentation velocity and fitted to the c(S) and c(M) size-distribution functions. The c(M) distribution is shown for both proteins.

FIGURE 2.

Two vBcl-2 molecules bind the Beclin1 dimer; one with high and one lower affinity. A, binding isotherm using ITC for vBcl-2 in the injection syringe titrated into Beclin1 BH3-CCD in the cell. The data were fitted to a two-site binding model (lower panel). B, a complex of vBcl-2-Beclin1 BH3-CCD was co-purified and analyzed by sedimentation equilibrium. The data were fitted to a heterodimer model in which vBcl-2 bound by two separate events to the Beclin1 dimer. A representative fit is shown. C, ITC titration for vBcl-2 in the syringe and Beclin1 BH3 peptide in the cell, and the data were fitted to a single-site model.

FIGURE 3.

Bcl-x_L binds to Beclin1 by a different mechanism from vBcl-2. ITC titrations with Bcl-x_L in the syringe and Beclin1 BH3-CCD (A) or Beclin1 BH3 (B) peptide in the cell. The data were fitted to single-site models.

FIGURE 4.

UVRAG disrupts the Beclin1 dimer interface. A, ITC titration for UVRAG CCD in the injection syringe and Beclin1 BH3-CCD in the cell. B, analytical gel-filtration profiles, measured at 210 nm for Beclin1 CCD, UVRAG CCD, or a mixture of the two. C, ITC titration for vBcl-2 in the injection syringe and Beclin1 BH3-CCD in the cell in the presence of a 2-fold excess of UVRAG CCD. The ITC data were fitted to a single-site model.

FIGURE 5.

Bcl-x_L and Bcl-2 have a similar effect on the affinity of UVRAG for Beclin1. ITC titrations for UVRAG CCD in the syringe and Beclin1 BH3-CCD in the cell in the presence of a 2-fold excess of vBcl-2 (A) or Bcl-x_L (B). The data were fitted to a single-site model and in the presence of Bcl-x_L. n was held constant at 1.

FIGURE 6.

Beclin1 co-localization with Bcl-x_L in mitochondria is dependent on its BH3 domain. A, HeLa cells were transfected with YPet-Beclin1 (a-c), YPet-Beclin1 and mCherry-Bcl-x_L (d-f), or YPet-Bcl-x_L (h-i) and fixed after 6 h. Mitochondrial co-localization was assessed using MitoTracker (b and g). Panels c, f, and i are merged images of a and b, d and e, and g and h, respectively. The insets show an enlarged area (×3.4). B, representative images of COS-7 cells expressing YPet-Beclin1-(95-266) and mCherry-Bcl-x_L (a-c) or YPet-Beclin1-(133-266) and mCherry-Bcl-x_L (d-f). Panels c and f are merged images of a and b and or d and e, respectively.

FIGURE 7.

Beclin1 dimerizes via its CCD domain and this interaction is inhibited by UVRAG in vivo. A, COS7 total cell lysate (TCL, 400 μg) co-expressing FLAG-SBP-Beclin1 and YPet-fusion proteins as indicated were collected on 25 μl of streptavidin-agarose beads, and after washing, 10% of material was assessed by Western blotting for bound YPet proteins. Total cell lysate (5%) was used for comparison. GFP, green fluorescent protein. B, COS7 cells expressing hemagglutinin (HA)-Beclin1 BH3-CCD, YPet-Beclin1 BH3-CCD, and FLAG constructs as indicated were collected on 25 μl of streptavidin-agarose beads and processed as for A.