LYRIC/AEG-1 overexpression modulates BCCIPalpha protein levels in prostate tumor cells

Abstract

LYRIC/AEG-1 is a unique protein that has been shown to promote tumor cell migration and invasion through activation of the transcription factor NF-kappaB. We performed yeast two-hybrid screening to detect LYRIC/AEG-1 associated proteins, and identified BCCIP, a CDKN1A and BRCA2-associated protein involved in cell cycle regulation and DNA repair. Here, we demonstrate association between LYRIC/AEG-1 and BCCIP in mammalian cells, and define the region of interaction. Co-expression of the two proteins resulted in decreased levels of BCCIPalpha, an effect partially abrogated by proteasome inhibition. A truncated LYRIC/AEG-1 construct lacking the interaction region did not alter BCCIPalpha protein levels. Coincidentally, it was observed that overexpression of BCCIPalpha in DU145 prostate tumor cells induced an apparent neuroendocrine differentiation. In summary, our data suggest LYRIC/AEG-1 is a negative regulator of BCCIPalpha, promoting proteasomal degradation either through direct interaction, or potentially through an indirect mechanism involving downstream effects of the NF-kappaB signaling pathway.

Figure 1. LYRIC/AEG-1 associates with BCCIP

(a) 293T cells were co-transfected with plasmids encoding full-length FLAG-tagged LYRIC/AEG-1 with empty HA-vector (lanes1&4), HA-BCCIPc (lanes 2&5) or HA-BCCIPα (lanes 3&6). Expression was verified by Western blotting of cell lysates with anti-FLAG and anti-HA (lanes 1–3). FLAG-LYRIC was immunoprecipitated using anti-FLAG agarose (lanes 4–6, upper), and both isoforms of BCCIP co-precipitated (lanes 4–6, lower). (b) HA-BCCIPα (+) or empty vector (−) were transfected into 293T and endogenous LYRIC was IP’d using PAb5393. IP with pre-immune rabbit serum (IgG) was the negative control. BCCIPα co-precipitated with LYRIC (upper), and precipitation of LYRIC was confirmed by blotting with PAb5393 (lower). (c) HA-BCCIPα was transfected into 293T and IP’d with anti-HA agarose. Endogenous LYRIC co-precipitated and was detected with PAb5393.

Figure 2. NH₂-terminal region of LYRIC is required for association with BCCIPα

(a) FLAG-tagged deletion constructs, indicating the region expressed, drawn to scale with full-length LYRIC (RNF). (b) 293T cells were transfected with BCCIPα and each of the LYRIC constructs. Protein expression was verified by Western blotting of total cell lysates (left). BCCIPα was precipitated with anti-HA agarose, and only constructs Δ238 and Δ463, and full length LYRIC co-precipitated.

Figure 3. LYRIC overexpression reduces BCCIPα protein levels

(a) DU145 cells were co-transfected with plasmids encoding HA-BCCIPα and LYRIC or β-galactosidase. Pairs of lanes represent two independent transfections. LYRIC overexpression resulted in decreased levels of BCCIPα protein (lanes 3&4), relative to negative control lacZ plasmid (lanes 5&6). Endogenous BCCIPβ did not appear to be affected. (b) DU145 cells were transfected with HA-BCCIPα and LYRIC or lacZ plasmids then treated with MG-132 (+) or DMSO (−)overnight. Western blot shows LYRIC overexpressed approximately 4-fold, resulting in a two-fold decrease in BCCIPα protein levels. Proteasome inhibition by MG-132 abrogated the effect. p21 was detected only with proteasome inhibition. β-actin was a loading control. (c) DU145 cells were transfected with plasmids encoding BCCIPα and full length LYRIC, deletion mutant hsNΔ169, or β-galactosidase and analyzed by Western blot. Decreased levels of HA-BCCIP were seen only with full-length LYRIC, the deletion mutant had no effect. (d) HA-BCCIP levels were determined by densitometry of the bands from Western blots as shown in panel (c). Graph depicts average and standard deviation of HA-BCCIP expression from three independent transfection experiments. The difference in HA-BCCIP protein levels in cells co-transfected with LYRIC and hsNΔ169 was statistically significant (p<0.01).

Figure 4. Overexpression of BCCIPα induces formation of neurite-like extensions

DU145 cells on chamber slides were transfected with HA-BCCIPα or HA-CLASP1, incubated overnight, fixed and stained with anti-HA and a fluorescent-tagged secondary antibody. (a) CLASP-1 transfected cells displayed a normal polygonal morphology, while a sub-population of cells overexpressing BCCIPα were rounded with elaborate branching extensions, suggestive of neuroendocrine (NE) differentiation. Two patterns were commonly seen, cells with a single long extension (upper panel) and cells with multiple convoluted processes (lower panel). Size bar represents 25µ. (b) To quantitate cells putatively undergoing NE differentiation, the number of rounded cells with extensions in a given microscope field was counted and expressed as a percent of the total number of transfected cells visible in the field. Graph depicts the average and standard deviation from 10 random microscope fields (approximately 200 cells for each transfection). Ten percent of BCCIPα expressing cells developed this phenotype, while less than 1% of these cells were observed in the control. The difference was statistically significant as determined by Student’s t-test (p<0.001).