Interaction between Her2 and Beclin-1 proteins underlies a new mechanism of reciprocal regulation

Abstract

Beclin-1 is a key regulator of autophagy that functions in the context of two phase-specific complexes in the initiation and maturation of autophagosomes. Its known interacting proteins include autophagy effectors, Bcl-2 family members, and organelle membrane anchor proteins. Here we report a newly identified interaction between Beclin-1 and the protein tyrosine kinase receptor Her2. We demonstrate that in Her2-expressing breast carcinoma cells that do not succumb to lapatinib, this Her1/2 inhibitor disrupts the cell surface interaction between Her2 and Beclin-1. The data suggest that the ensuing autophagic response is correlatively associated with the release of Beclin-1 from its complex with Her2 and with the subsequent increase in cytosolic Beclin-1. Upon its interaction with Her2, Beclin-1 up-regulates the phosphorylation levels of Her2 and Akt. The Beclin-1 evolutionarily conserved domain is required both for the interaction of Beclin-1 with Her2 and for the increased Her2 and Akt phosphorylation. These findings shed new light on mechanisms involved in lapatinib-mediated autophagy in Her2-expressing breast carcinoma cell lines and in Beclin-1 signaling in these cells.

Keywords: Apoptosis; Autophagy; Beclin-1; Breast Cancer; Epidermal Growth Factor Receptor (EGFR); Her2; Lapatinib; Receptor Tyrosine Kinase.

FIGURE 1.

Transmission electron microscopy evidence for LP-mediated autophagy in LP-resistant BT474 cells. A, increase in clonogenic potential of LP-selected BT474 cells as compared with their WT counterpart and assessed by a long term culture (35 days). The results are means ± S.E. of five plates for each group in one of three experiments with equivalent results. LP-R, LP-resistant. B and C, LP-resistant BT474 cells that were freshly treated with LP (2 μm, 16 h) were assessed by transmission electron microscopy for the presence of autophagosomes and autophagolysosomes. Scale bars: left and middle panels, 2 μm; right, 500 nm; the right panel is an increased magnification of the square area indicated in the middle panel. The arrows indicate autophagosomes. E/P, E64D/PepA. D and E, LP induces autophagic flux in LP-resistant BT474, as indicated by the significantly increased number of AVds in the presence of lysosomal inhibitors. Of note, active and inhibited AVds demonstrate a distinct EM appearance; in panel a, the square shows an active AVd, and in panel b, the square shows an inhibited AVd. The quantification in B and D was performed on at least 30 cells per treatment in three independent experiments. *, p < 0.05; ***, p < 0.0001 (Mann-Whitney U). Results shown in B and D and their corresponding quantification (C and E, respectively) represent two independent experiments.

FIGURE 2.

Altered expression of endogenous LC3 puncta in LP-treated BrCA cells. LP-selected BT474 cells (A–C), SKBR3 cells (D–F), and cisplatin-selected MCF7 cells (G–J) were treated with LP in the absence or presence of E64D/PepA (E/P). Expression patterns for LC3 and LAMP2 were assessed by confocal microscopy (A, D, and G) and quantified by MetaMorph. Scale bars: A and D, 30 μm; G, 50 μm. The LC3 dot number per cell was determined by manual counting (B, E, and H), whereas the cumulative LC3 dot area × 10⁻⁴ per cell area was determined by MetaMorph (C, F, and I). The percentage of LC3-LAMP2 co-localization (LC3 over LAMP2) was quantified by MetaMorph. All quantifications were performed on at least 50 cells per treatment in each of three independent experiments with equivalent results; ***, p < 0.0001 (Mann-Whitney U).

FIGURE 3.

Induction of autophagic flux as assessed by LC3-II level in LP-treated BrCA cells. A–H, accumulation of LC3-II was tested in the absence or presence of saturating concentrations of E64D/PepA (E/P) (10 μg/ml each). The immune-probed protein bands (A, C, E, and G) were quantified as described under “Experimental Procedures,” and the ratio to control treatment is presented in the corresponding charts (B, D, F, and H). A and B, LP-selected BT474 re-treated with LP (2 μm, 16 h). C and D, LP-treated SKBR3 cells (2 μm, 6 h). E and F, LP-treated cisplatin-resistant MCF7 cells (5 μm, 16 h). G and H, LP-treated 4T1 and NT5.1, Her2-positive murine BrCA cell lines were obtained from Her2 transgenic mouse (2 μm, 16 h). Please note that in certain cell lines, only LC3-II, and not its precursor, LC3-I, is detected.

FIGURE 4.

Beclin-1 knockdown shifts the response of LP-resistant cells from autophagy to apoptosis. A–F, Beclin-1 knockdown by distinct siRNAs induces LP apoptosis susceptibility in LP-selected BT474 cells (A and B); SKBR3 cells (C and D); and cisplatin-selected MCF7 cells (E and F). B, D, and F, evidence for Beclin-1 RNAi in cells utilized in A, C, and E, respectively. Different Beclin-1 siRNA targeting sequences 1 and 2 are indicated.

FIGURE 5.

LP disrupts the constitutive co-localization of cell surface Her2 and Beclin-1. A and B, LP-selected BT474 cells (A) and SKBR3 cells (B) were treated with LP (2 μm, 15 min) and assessed by confocal microscopy for the expression of Her2, Beclin-1, and DAPI. Scale bars: 10 μm. C–H, cellular accumulation of Beclin-1 dots in LP-treated BT474 cells (C–E) and in LP-treated SKBR3 cells (F–H). Quantification was performed by MetaMorph, and at least 50 cells were individually assessed for each group in three independent experiments with similar results. ***, p < 0.0001 (Mann-Whitney U).

FIGURE 6.

A Her2-Beclin-1 complex demonstrates two-way co-immunoprecipitation (IP), which is disrupted by LP treatment. A, SKBR3 cells were transfected with Beclin-1 or a matching vector and subjected to IP by control IgG, anti-Beclin-1, or anti-Her2 antibodies. A slight shift detected in the gel migration of pelleted Beclin-1 relative to the input may relate to unknown post-translational modifications of the Her2-interacting Beclin-1. B and C, BT474 cells were transfected with Beclin-1 or a matching vector and subjected to Beclin-1 IP (B) or Her2 IP (C). The ratio between the pellet and the input loading is 3:1. In C, the input and the pellets were run on the same gel, but different exposure times of the chemiluminescence reaction are shown. D and E, LP treatment disrupts the complex between Her2 and Beclin-1 in BT474 cells (D) and in NT5.1 cells (E). Following treatments by LP (2 μm, 15 min) or vehicle control, the cells were subjected to Her2 IP. F, control IP with non-immune rabbit IgG precipitates neither Her2 nor Beclin-1 in lysates of the indicated cell lines. The asterisks indicate unidentified protein bands.

FIGURE 7.

The ECD of Beclin-1 is required for its interaction with Her2. BT474 or SKBR3 cells were transfected (Tx.) with 3×FLAG WT Beclin-1 or with the indicated deletion mutants of Beclin-1 (ΔCCD, ΔBBD, and ΔECD), each with an N-terminal 3×FLAG tag. A–C, the cells were then subjected to IP of endogenous Her2 (A and C) or of an exogenous 3×FLAG Beclin-1 variant (B). The asterisks indicate unidentified protein bands.

FIGURE 8.

Increased expression of Beclin-1, but not Beclin-1ΔECD, enhances the phosphorylation levels of Her2 and Akt and reduces the access of LP to Her2. A–F, MDA-MB-361 cells (A and B), SKBR3 cells (C and D), and WT BT474 cells (E and F) were transfected with vector control or WT Beclin-1 and treated with LP (6 h, 1 or 2.5 μm (A–D) and 0.25 or 0.5 μm) (E and F). The cell lysates were assessed by immunoblotting for the expression of the indicated proteins. Results of one representative experiment of three performed for each cell line are shown. The expression levels of p-Her2 and p-Akt were quantified, and the percentages of increases in Beclin-1 transfected cells versus vector-transfected cells are presented in the corresponding charts (B, D, and F). G and H, the Beclin-1 ECD domain is required for the observed Her2 and Akt phosphorylation changes. MDA-MB-361 cells (G), SKBR3 cells (G), and WT BT474 cells (H) were transfected with vector control or Beclin-1ΔECD and treated with LP, as described above for the corresponding cell line. The asterisks indicate unidentified protein bands.