Ras Regulates Rb via NORE1A

Abstract

Mutations in the Ras oncogene are one of the most frequent events in human cancer. Although Ras regulates numerous growth-promoting pathways to drive transformation, it can paradoxically promote an irreversible cell cycle arrest known as oncogene-induced senescence. Although senescence has clearly been implicated as a major defense mechanism against tumorigenesis, the mechanisms by which Ras can promote such a senescent phenotype remain poorly defined. We have shown recently that the Ras death effector NORE1A plays a critical role in promoting Ras-induced senescence and connects Ras to the regulation of the p53 tumor suppressor. We now show that NORE1A also connects Ras to the regulation of a second major prosenescent tumor suppressor, the retinoblastoma (Rb) protein. We show that Ras induces the formation of a complex between NORE1A and the phosphatase PP1A, promoting the activation of the Rb tumor suppressor by dephosphorylation. Furthermore, suppression of Rb reduces NORE1A senescence activity. These results, together with our previous findings, suggest that NORE1A acts as a critical tumor suppressor node, linking Ras to both the p53 and the Rb pathways to drive senescence.

Keywords: NORE1A; RASSF; Ras protein; cancer; cellular senescence; protein phosphorylation; retinoblastoma protein (pRb, RB).

FIGURE 1.

NORE1A forms an endogenous, Ras-regulated complex with PP1A. A, exogenously expressed NORE1A and PP1A co-localize in the nucleus. COS-7 cells were transfected with GFP-PP1A in the presence or absence of KATE-NORE1A. A pool of GFP-PP1A co-localized with RFP-NORE1A within the nuclei. B, activated Ras enhances the interaction between NORE1A and PP1A. HEK-293T cells were co-transfected with expression constructs for PP1A, NORE1A, and activated H- or K-Ras for 24 h and lysed, and equal amounts of protein were immunoprecipitated (IP) with anti-GFP. The immunoprecipitates were analyzed by Western blotting with anti-HA and anti-GFP antibodies. IB, immunoblot. C, NORE1A and PP1A are found in an endogenous complex. HepG2 cells were immunoprecipitated for NORE1A and immunoblotted for PP1A. IgG incubated with HepG2 lysate and Ig/NORE1A antibody incubated with lysis buffer served as negative controls. Asterisk, nonspecific IgG band.

FIGURE 2.

Ras/NORE1A stabilizes PP1A. A, HEK-293 cells were transfected with PP1A, NORE1A, and activated H-Ras expression constructs for 24 h. The cells were treated with cycloheximide (20 μg/ml) and lysed at the indicated times after addition of cycloheximide. Levels of PP1A protein were measured by Western blotting analysis. Shown is a representative blot of three independent experiments. IB, immunoblot. B, the density of the bands was quantitated using ImageJ software, and relative PP1A expression was calculated after normalizing to β-Actin expression. No significant difference in PP1A expression levels was found in cells transfected with NORE1A or Ras individually. However, in cells transfected with both NORE1A and Ras, there was a significant increase in the levels of PP1A. C, HBEC-3KT cells stably knocked down for NORE1A were induced to express activated Ras. 3 days after induction, cell lysates were prepared and analyzed for PP1A expression by Western blotting analysis.

FIGURE 3.

NORE1A complexes with Rb and cooperates with Ras to scaffold PP1A to Rb. A and B, NORE1A forms a Ras-regulated complex with Rb. HEK-293T cells were co-transfected with expression constructs for Rb, NORE1A, and activated H-Ras for 24 h, lysed, and immunoprecipitated (IP) with anti-GFP. The immunoprecipitates were analyzed by Western blotting with anti-HA and anti-GFP antibodies. The blots were quantitated densitometrically using ImageJ software, and the amount of NORE1A bound to the immunoprecipitated Rb was determined after normalization to the NORE1A input levels. *, p < 0.05 compared with NORE1A alone. IB, immunoblot. C, NORE1A forms an endogenous complex with Rb. The HepG2 immunoprecipitates described in Fig. 1C were immunoblotted for the presence of Rb. D and E, Ras regulates the interaction between PP1A and Rb via NORE1A. HEK-293T cells were co-transfected with expression constructs for PP1A, Rb, NORE1A, and activated H-Ras. Cells were lysed and immunoprecipitated for GFP-PP1A, and the immunoprecipitates were analyzed by Western blotting with anti-HA, GFP, and FLAG antibodies. A representative blot is shown in D. E, the blots were quantified densitometrically to calculate the relative amount of Rb found in complex with PP1A after normalizing to the amount of Rb in the input. *, p ≤ 0.05 compared with cells transfected with empty vector. **, p ≤ 0.05 compared with cells transfected with either NORE1A or activated Ras alone.

FIGURE 4.

NORE1A promotes Rb-dephosphorylation. A and B, NORE1A suppresses Rb phosphorylation. A549 cells, which do not express NORE1A, were transiently transfected with GFP-NORE1A for 24 h. Cells were lysed and immunoblotted (IB) for Rb phosphorylated at serine 795 using a Ser-795-specific antibody (A). NCI-H1299 cells stably expressing NORE1A or an empty vector were lysed and immunoblotted for phospho-Rb at serine 795 (B). Shown are representative blots of two independent experiments. C, loss of NORE1A enhances Rb phosphorylation. HEK-293 cells were transiently knocked down for NORE1A expression using two different shRNA constructs to NORE1A. Cells were lysed and immunoblotted for phospho-Rb at serine 795.

FIGURE 5.

Rb is required for NORE1A-induced senescence. A, loss of Rb significantly impairs NORE1A-mediated senescence in MEFs. Wild-type and Rb^−/− MEFs were transfected with 1 μg of pcDNA-HA-vector or NORE1A. Cells were incubated for 72 h before assaying for β-galactosidase activity. *, p ≤ 0.05 compared with wild-type-transfected MEFs. Representative images are shown in B. C, Rb is a downstream effector of NORE1A-induced senescence in human cells. Stable scrambled or sh-Rb transduced A549 human lung cancer cells were transfected with 1 μg of GFP-NORE1A. After 72 h, cells were assayed for β-galactosidase activity. *, p ≤ 0.05 compared with scrambled control-transfected cells. Inset, Rb protein levels in A549 cells stably transfected with the two Rb shRNA constructs and scrambled control. D, representative images of NORE1A expression (top panels) and β-galactosidase-stained cells (bottom panels). E, Rb is an effector of NORE1A-induced IL-6 expression. Control and A549 cells stably knocked down for Rb as described above were co-transfected with GFP-NORE1A and an IL-6 promoter luciferase reporter construct. 48 h after transfection, luciferase activity was measured using a LightSwitch luciferase assay system. *, p ≤ 0.05. Representative images of NORE1A expression are shown in the right panels.

FIGURE 6.

NORE1A-induced Rb dephosphorylation and senescence require PP1A. A, loss of PP1A impairs NORE1A-induced dephosphorylation of Rb. A549 cells were transiently knocked down for PP1A expression using a validated pool of PP1A siRNA. The cells were transfected with GFP-NORE1A and, 24 h later, lysed and immunoblotted (IB) for phospho-Rb at serine 795, Rb, PP1A, and NORE1A. B, PP1A is required for NORE1A-induced senescence. A549 cells were transiently knocked down for PP1A as described above and transfected with GFP-NORE1A. 72 h later, cells were assayed for β-galactosidase activity. *, p ≤ 0.05 compared with control cells transfected with vector. **, p ≤ 0.05 compared with si-Scrm cells transfected with NORE1A. Representative images of NORE1A expression and β-galactosidase staining are shown in the bottom panels.