N terminus of ASPP2 binds to Ras and enhances Ras/Raf/MEK/ERK activation to promote oncogene-induced senescence

Abstract

The ASPP2 (also known as 53BP2L) tumor suppressor is a proapoptotic member of a family of p53 binding proteins that functions in part by enhancing p53-dependent apoptosis via its C-terminal p53-binding domain. Mounting evidence also suggests that ASPP2 harbors important nonapoptotic p53-independent functions. Structural studies identify a small G protein Ras-association domain in the ASPP2 N terminus. Because Ras-induced senescence is a barrier to tumor formation in normal cells, we investigated whether ASPP2 could bind Ras and stimulate the protein kinase Raf/MEK/ERK signaling cascade. We now show that ASPP2 binds to Ras–GTP at the plasma membrane and stimulates Ras-induced signaling and pERK1/2 levels via promoting Ras–GTP loading, B-Raf/C-Raf dimerization, and C-Raf phosphorylation. These functions require the ASPP2 N terminus because BBP (also known as 53BP2S), an alternatively spliced ASPP2 isoform lacking the N terminus, was defective in binding Ras–GTP and stimulating Raf/MEK/ERK signaling. Decreased ASPP2 levels attenuated H-RasV12–induced senescence in normal human fibroblasts and neonatal human epidermal keratinocytes. Together, our results reveal a mechanism for ASPP2 tumor suppressor function via direct interaction with Ras–GTP to stimulate Ras-induced senescence in nontransformed human cells.

ASPP2, also known as 53BP2L, is a tumor suppressor whose expression is altered in human cancers (1). Importantly, targeting of the ASPP2 allele in two different mouse models reveals that ASPP2 heterozygous mice are prone to spontaneous and γ-irradiation–induced tumors, which rigorously demonstrates the role of ASPP2 as a tumor suppressor (2, 3). ASPP2 binds p53 via the C-terminal ankyrin-repeat and SH3 domain (4–6), is damage-inducible, and can enhance damage-induced apoptosis in part through a p53-mediated pathway (1, 2, 7–10). However, it remains unclear what biologic pathways and mechanisms mediate ASPP2 tumor suppressor function (1). Indeed, accumulating evidence demonstrates that ASPP2 also mediates nonapoptotic p53-independent pathways (1, 3, 11–15).

The induction of cellular senescence forms an important barrier to tumorigenesis in vivo (16–21). It is well known that oncogenic Ras signaling induces senescence in normal nontransformed cells to prevent tumor initiation and maintain complex growth arrest pathways (16, 18, 21–24). The level of oncogenic Ras activation influences its capacity to activate senescence; high levels of oncogenic H-RasV12 signaling leads to low grade tumors with senescence markers, which progress to invasive cancers upon senescence inactivation (25). Thus, tight control of Ras signaling is critical to ensure the proper biologic outcome in the correct cellular context (26–28).

The ASPP2 C terminus is important for promoting p53-dependent apoptosis (7). The ASPP2 N terminus may also suppress cell growth (1, 7, 29–33). Alternative splicing can generate the ASPP2 N-terminal truncated protein BBP (also known as 53BP2S) that is less potent in suppressing cell growth (7, 34, 35). Although the ASPP2 C terminus mediates nuclear localization, full-length ASPP2 also localizes to the cytoplasm and plasma membrane to mediate extranuclear functions (7, 11, 12, 36). Structural studies of the ASPP2 N terminus reveal a β–Grasp ubiquitin-like fold as well as a potential Ras-binding (RB)/Ras-association (RA) domain (32). Moreover, ASPP2 can promote H-RasV12–induced senescence (13, 15). However, the molecular mechanism(s) of how ASPP2 directly promotes Ras signaling are complex and remain to be completely elucidated.

Here, we explore the molecular mechanisms of how Ras-signaling is enhanced by ASPP2. We demonstrate that ASPP2: (i) binds Ras-GTP and stimulates Ras-induced ERK signaling via its N-terminal domain at the plasma membrane; (ii) enhances Ras-GTP loading and B-Raf/C-Raf dimerization and forms a ASPP2/Raf complex; (iii) stimulates Ras-induced C-Raf phosphorylation and activation; and (iv) potentiates H-RasV12–induced senescence in both primary human fibroblasts and neonatal human epidermal keratinocytes. These data provide mechanistic insight into ASPP2 function(s) and opens important avenues for investigation into its role as a tumor suppressor in human cancer.

Results

ASPP2 Interacts with Activated Ras-GTP.

Because structural data suggest that ASPP2 contains a potential Ras interacting domain (32), we determined whether ASPP2 colocalized with Ras at the plasma membrane. We coexpressed green fluorescent protein (GFP)–ASPP2 with mCherry (mc)-tagged–H-RasV12 (a constitutively active GTP-loaded Ras mutant) in 293T cells and analyzed transfected cells by confocal fluorescence microscopy (Fig. 1A). As expected, we found mc–H-RasV12 localized at the membrane (Fig. 1A Center). Interestingly, GFP-ASPP2 (Fig. 1A Left) was colocalized with mc–H-RasV12 at the plasma membrane (Fig. 1A Right). Cytoplasmic and plasma membrane localization of ASPP2 was not due to enforced coexpression of H-RasV12 because ASPP2 had a similar intracellular localization pattern in the absence of serum (Ras inactive state) or when a dominant negative H-RasN17 mutant was expressed (Fig. S1). To determine whether Ras and ASPP2 could be coimmunoprecipitated from cells, Flag–H-Ras, Flag–H-RasV12, or Flag–H-RasN17 expression vectors were cotransfected with an ASPP2 expression vector in 293T cells (Fig. 1B). We found that Flag–H-RasV12, but not Flag–H-RasN17, coimmunoprecipitated with ASPP2 (Fig. 1B, lane 2 vs. lane 1). To determine whether mitogen-induced Ras activation enhanced association with ASPP2, we performed coimmunoprecipitations from lysates prepared from cells transfected with Flag–H-Ras and ASPP2 expression vectors that were grown in media with or without epidermal growth factor (EGF) or serum stimulation. We found the interaction between Flag–H-Ras and ASPP2 was strongly stimulated by serum (Fig. 1B, lane 4 vs. lane 5). Furthermore, the addition of EGF to cells grown in the absence of serum enhanced the interaction of Flag–H-Ras with ASPP2 (Fig. 1B, lane 6 vs. lane 5). To determine whether endogenous ASPP2 could interact with mitogen-activated Ras, we performed coimmunoprecipitations from cells transfected with a Flag–H-Ras expression vector, and found enhanced binding of Flag–H-Ras with endogenous ASPP2 upon EGF stimulation (Fig. 1C). Because ASPP2 also binds p53 to promote apoptosis (7), we explored whether the endogenous Ras–ASPP2 interaction was affected by p53 using the matched p53 isogenic HCT116 cell lines (37). We performed coimmunoprecipitations using an anti-Ras antibody (Fig. 1D, lanes 2 and 4) or control IgG (Fig. 1D, lanes 1 and 3). Compared with HCT116p53^+/+ cells (Fig. 1D, lane 1 and 2), we did not find differences in association between endogenous activated Ras and endogenous ASPP2 in HCT116p53^−/− cells (Fig. 1D, lane 2 vs. lane 4). Collectively, these results demonstrate that ASPP2 interacts with activated GTP-bound Ras independent of p53.

Fig. 1.

ASPP2 interacts with activated Ras. (A) Confocal fluorescence microscopy on 293T cells cotransfected with GFP-ASPP2 (green) and mc-H-RasV12 (red). (B) Western blot of anti-Flag immunoprecipitations (IP) probed with anti-ASPP2 antibody. Indicated expression vectors transfected into 293T cells followed by serum starvation and/or 20 ng/mL EGF treatment for 10 min. (Lower) Total input. (C) Western blot of anti-Flag IP probed with anti-ASPP2 antibody on lysates prepared from 293T cells after transfection with Flag-Ras expression vector, serum starvation and then treatment with/without EGF for 10 min. (Lower) Total input. (D) Western blot of indicated IPs probed with anti-ASPP2 antibody on lysates prepared from p53 isogenic HCT116 cells grown in complete media. (Lower) Total input.

ASPP2 N Terminus Mediates the ASPP2–Ras Interaction.

To determine whether disruption of the ASPP2 N terminus would attenuate Ras association, we used the naturally occurring N-terminal truncated ASPP2 splice isoform BBP (Fig. 2A) (7, 34, 35). As expected, ASPP2 coimmunoprecipitated with HA–H-RasV12 (Fig. 2B, lane 2). In contrast, BBP did not coimmunoprecipitate with HA–H-RasV12 (Fig. 2B, lane 3). Although ASPP2 localization to the plasma membrane was not dependent on Ras activation (Fig. S1), we wondered whether the interaction between ASPP2 and Ras-GTP required Ras to localize to the plasma membrane. Ras plasma membrane localization is mediated by the C-terminal CAAX motif via cysteine farnesylation (28). In contrast to Flag–H-RasV12, ASPP2 did not coimmunoprecipitate with Flag–H-RasV12–SAAX, a mutant that is constitutively GTP-loaded but mislocalized to the cytosol (Fig. 2C, lane 3 vs. lane 2). Likewise, GFP–ASPP2 did not colocalize with mc–H-RasV12–SAAX (Fig. 2D). These data demonstrate that ASPP2 associates with activated Ras at the plasma membrane, and that the ASPP2 N terminus mediates this interaction.

Fig. 2.

The ASPP2 N terminus mediates the interaction with Ras. (A) Schematic of ASPP2 and BBP proteins. (B) Western blot of anti-HA IP probed with anti-ASPP2 antibody, on lysates prepared from 293T cells transfected with indicated expression vectors. (Lower) Total input. (C) Western blot of anti-Flag IP probed with anti-ASPP2 antibody on lysates prepared from 293T cells transfected with indicated expression vectors. (Lower) Total input. (D) Confocal fluorescence microscopy on 293T cells cotransfected with GFP-ASPP2 (green) and mc-H-RasV12SAAX (red). Merged image shows no colocalization.

ASPP2 N Terminus Stimulates Ras-Induced ERK Signaling.

Growth factors and oncogenes activate Ras, which in turn activates complex signaling cascades such as RAF/MEK/ERK (28). To test whether Ras downstream signaling pathways would be modulated by ASPP2, we coexpressed H-RasV12 with ASPP2 in 293T cells, and found that ASPP2 enhanced H-RasV12–induced ERK phosphorylation (Fig. 3A, lane 3). In contrast, when H-RasV12 and BBP were coexpressed, ERK phosphorylation was diminished compared with H-RasV12 alone (Fig. 3A, lane 2). To test whether EGF signaling through the MEK/ERK pathway could be enhanced by ASPP2, we transfected ASPP2 into 293T cells, stimulated with EGF, and found enhanced pERK1/2, but not pAKT, levels (Fig. 3B, lanes 4–6 vs. lanes 1–3). Consistent with N terminus involvement, BBP did not enhance EGF stimulation of pERK1/2 levels (Fig. 3C, lanes 4–6 vs. lanes 1–3). When ASPP2 expression was silenced using siRNA and cells stimulated with EGF, we found reduced pERK1/2 levels compared with control siRNA (Fig. 3D, lanes 4–6 vs. lanes 1–3). Together, these data suggest that the ASPP2 N terminus potentiates Ras-mediated signaling through the MEK/ERK pathway.

Fig. 3.

The ASPP2 N terminus stimulates Ras-induced ERK signaling. (A) Western blot using indicated antibodies on equivalent amounts of cell lysates prepared from 293T cells transfected with indicated expression vectors and then serum starved until harvested (B) Western blot with indicated antibodies on equivalent amounts of cell lysates prepared from 293T cells transfected with ASPP2 expression vector, or empty vector, followed by serum starvation for 18 h, and then treated with 20 ng/mL EGF for the indicated times. (C) Western blot with indicated antibodies on equivalent amounts of cell lysates prepared from 293T cells transfected with BBP expression vector, or empty vector, followed by serum starvation for 18 h, and then treated with 20 ng/mL EGF for the indicated times. (D) Western blot with indicated antibodies on equivalent amounts of cell lysates prepared from 293T cells transfected with ASPP2, or scrambled, siRNA followed by serum starvation for 18 h and then treatment with 20 ng/mL EGF for the indicated times.

ASPP2 Enhances Ras Activation and B-Raf/C-Raf Dimerization.

Because the ASPP2 N terminus binds Ras–GTP to stimulate pERK1/2 levels (Figs. 1–3), we determined whether the ASPP2 interaction with Ras stimulated Ras activation. Enforced ASPP2 expression enhanced EGF-induced Ras–GTP activation (Fig. S2A, lanes 4–6 vs. lanes 1–3); in contrast, ASPP2 siRNA knockdown attenuated EGF-induced Ras–GTP activation (Fig. S2B, lanes 4–6 vs. lanes 1–3). Because active Ras-GTP induces B-Raf/C-Raf dimerization (26, 27, 38), we wondered whether ASPP2 could promote B-Raf/C-Raf dimerization. As expected, endogenous B-Raf and C-Raf dimerization was enhanced in 293T cells transfected with Flag–H-RasV12 but not Flag–H-RasN17 (Fig. 4A, lane 2 vs. lane 3). However, cotransfection of ASPP2 with Flag–H-RasV12 significantly increased endogenous B-Raf/C-Raf association (Fig. 4B, lane 4 vs. lane 2). Unlike ASPP2, cotransfection of BBP and Flag–H-RasV12 did not increase endogenous B-Raf/C-Raf dimerization (Fig. 4B, lane 8 vs. lane 2). We also coexpressed Flag–C-Raf and HA–H-RasV12 with ASPP2 and similarly found increased B-Raf/C-Raf association (Fig. 4C, lane 3 vs. lane 2); in contrast, BBP did not stimulate B-Raf/C-Raf association (Fig. 4C, lane 4 vs. lane 2). Transfection with ASPP2 siRNA modestly reduced Flag–H-RasV12–induced B-Raf/C-Raf association (Fig. 4B, lane 6 vs. lane 2). Together, these results demonstrate that the ASPP2 N terminus can enhance Ras-induced B-Raf/C-Raf dimerization.

Fig. 4.

ASPP2 enhances B-Raf/C-Raf dimerization. (A) Western blot of anti–B-Raf IP probed with anti–C-Raf antibody, on lysates prepared from 293T cells transfected with Flag-H-RasV12, Flag-H-RasN17, or empty expression vectors. (Lower) Total input probed with indicated antibodies. (B) Western blot of anti–B-Raf IP probed with anti–C-Raf antibody, on lysates prepared from 293T cells transfected with ASPP2 or control siRNAs, or ASPP2 or BBP expression vectors. (Lower) Total input probed with indicated antibodies. (C) Western blot of anti-Flag IP probed with anti–B-Raf antibody, on lysates prepared from 293T cells transfected with indicated expression or empty vectors. (Lower) Total input probed with indicated antibodies.

Ras Association with the ASPP2 N Terminus Enhances ASPP2/Raf Complex Formation.

Because Ras recruits Raf to the plasma membrane to initiate signaling (26, 27, 38) and ASPP2 binds Ras–GTP (Figs. 1–3), we explored whether ASPP2 existed in a complex with Raf. After HA–H-RasV12 expression in 293T cells, we found enhanced association between endogenous ASPP2 and endogenous C-Raf (Fig. 5A). To test whether the ASPP2 N terminus could mediate ASPP2/Raf complex formation, we transfected 293T cells with HA–H-RasV12 and Flag–C-Raf along with increasing amounts of ASPP2 or BBP (Fig. 5B). We found that BBP was deficient, relative to ASPP2, in complex formation with Flag–C-Raf (Fig. 5B, lanes 3 and 4 vs. lanes 1 and 2). Likewise, ASPP2 associated with Flag–B-Raf, in contrast to BBP (Fig. 5C, lanes 1 and 2 vs. lanes 3 and 4). These findings suggest that ASPP2 can complex with C-Raf and B-Raf, and that this requires the ASPP2 N terminus.

Fig. 5.

Ras association with ASPP2 N terminus enhances ASPP2/Raf complex formation. (A) Western blot of anti-ASPP2 IP probed with anti–C-Raf antibody, on lysates prepared from 293T cells transfected with Flag-H-RasV12 or empty expression vectors. (Lower) Total input probed with indicated antibodies. (B) Western blot of anti-Flag IP probed with anti-ASPP2 antibody, on lysates prepared from 293T cells cotransfected with equivalent amounts of Flag-C-Raf and HA-H-RasV12, and increasing amounts (0.5–1.5 μg) of ASPP2 or BBP expression vectors. (Lower) Total input probed with indicated antibodies. (C) Similar to B except used Flag-B-Raf.

ASPP2 Enhances Ras-Induced C-Raf Phosphorylation and Activation.

Because Raf phosphorylation and activation can mediate Ras signaling (26, 27), we determined whether ASPP2 could activate C-Raf by promoting phosphorylation at Ser-338 (39–41). Cells were cotransfected with Flag–H-RasV12 and increasing amounts of ASPP2, lysates immunoprecipitated with an anti-Flag antibody, and then probed with an anti–C-Raf–p338 antibody (Fig. 6). Although ASPP2 did not appreciably increase the association between Flag–H-RasV12 and endogenous C-Raf (Fig. 6A Top, lanes 3 and 4 vs. lane 2), ASPP2 significantly increased the association of endogenous C-Raf-p338 with Flag-H-RasV12 (Fig. 6A Middle, lanes 3 and 4 vs. lane 2). Consistent with ASPP2 stimulation of pERK1/2 (Fig. 3), we found increased pMEK levels after ASPP2 expression (Fig. 6A, lanes 3 and 4 vs. lane 2). To determine the importance of the ASPP2 N terminus in stimulating C-Raf–p338, we coexpressed BBP and Flag–H-RasV12, but found no increase in C-Raf–p338 levels (Fig. 6B, lane 5 vs. lane 3). We also consistently observed a modest decrease in C-Raf–p338 levels with ASPP2 siRNA compared with control siRNA (Fig. 6B, lane 4 vs. lane 2). We found similar results using a different ASPP2 siRNA (Fig. S3). These data suggest that ASPP2 stimulation of Ras/Raf/MEK/ERK signaling is mediated at least in part through C-Raf activation.

Fig. 6.

ASPP2 enhances Ras-induced C-Raf phosphorylation and activation. (A) Western blot of anti-Flag IP probed with anti–C-Raf or anti–C-Raf-p338 antibody, on lysates prepared from 293T cells cotransfected with Flag-H-RasV12 and increasing amounts (2.0–4.0 μg) of ASPP2 expression vector. (Lower) Total input probed with indicated antibodies. (B) Western blot of anti-Flag IP probed with anti–C-Raf–p338 antibody, on lysates prepared from 293T cells transfected with ASPP2 or control siRNAs, or ASPP2 or BBP expression vectors. (Lower) Total input probed with indicated antibodies.

ASPP2 Potentiates H-RasV12–Induced Senescence.

We investigated whether Ras-induced senescence and pERK1/2 activation could be enhanced by ASPP2 in nontransformed primary human cells (Fig. 7). Using a well-established senescence assay, we expressed H-RasV12 in IMR90 primary human fibroblasts by infecting them with a recombinant H-RasV12 lentivirus, and then quantified senescent cells with β-galactosidase staining 7 d after infection. As expected, H-RasV12 induced senescence compared with control vector. However, when ASPP2 expression was attenuated by siRNA, we found a reduction in H-RasV12–induced senescence (Fig. 7 A and B). Additionally, H-RasV12–induced senescence was blocked by the MEK inhibitor PD98059, which is consistent with studies demonstrating the MEK/ERK pathway mediates Ras-induced senescence (16, 22, 23, 42). As expected, we found that H-RasV12 increased pERK1/2 levels (Fig. 7C Middle, lane 2 vs. lane 1). Likewise, we also found that H-RasV12 stimulated ASPP2 levels (Fig. 7C Top, lanes 2–4 vs. lane 1), which is consistent with our prior findings that ASPP2 expression is regulated by E2F or mitogenic stimulation (9). When we used ASPP2 siRNA to inhibit H-RasV12–induced stimulation of ASPP2 levels, we found reduced pERK1/2 levels (Fig. 7C, lane 5 vs. lanes 2 and 3), similar to that achieved by PD98059 (Fig. 7C, lane 4 vs. lanes 2 and 3). As an additional control for reduction in ASPP2 levels, we used ASPP2^{+/−(exon10-17);}p53^+/− freshly isolated MEFs (3) and found a reduced ability to undergo normal senescence in culture compared with ASPP2^+/+;p53^+/− freshly isolated MEFs (Fig. S4). To test whether reduced ASPP2 expression would similarly attenuate H-RasV12–induced senescence and promote transformation in epithelial cells, we used normal neonatal human epidermal keratinocytes (HEKn) and found that ASPP2 siRNA knockdown significantly attenuated H-RasV12–induced senescence compared with scrambled siRNA (Fig. S5). Taken together, these findings reveal a role for ASPP2 in enhancing Ras/Raf/MEK/ERK signaling to induce cellular senescence as a barrier to transformation in nontransformed primary cells.

Fig. 7.

ASPP2 potentiates H-RasV12–induced senescence and pERK1/2 activation in primary human fibroblasts. (A) Light microscopy (20×) of β-gal stained IMR90 cells that were infected for 24 h with recombinant lentiviral H-RasV12, or control vector, before transfection with either ASPP2 or scrambled siRNA, or treated with 25 μM PD98095. Cells were stained for β-gal activity 7 d after infection. (B) Quantification of SA-β-gal positive cells from A. Error bar represents SD from duplicate experiments. (C) Western blot on equivalent lysates prepared from IMR90 cells treated as above, using the indicated antibodies. ASPP2 and pERK1/2 levels were analyzed 3 d after infection.

Discussion

The mechanism(s) of ASPP2 tumor suppressor function (1–3) have remained unclear (1, 43, 44). Our observation that ASPP2 and Ras bind and colocalize at the plasma membrane (Fig. 1) suggests that ASPP2 modulates Ras function. Indeed, ASPP2-Ras-GTP binding stimulates Ras/Raf/MEK/ERK signaling to promote oncogene-induced senescence (Figs. 3, 6, and 7). This ASPP2 cytoplasmic function is independent of p53-binding (7) because Ras-GTP binds ASPP2 in HCT116p53^+/+ or HCT116p53^−/− cells (Fig. 1). ASPP2 was originally described to promote p53-dependent apoptosis (7). Although our findings that ASPP2 can stimulate Ras-signaling seem paradoxical to these original observations, our current results are actually consistent with a role for ASPP2 as a tumor suppressor because Ras-induced senescence is a known barrier to tumor initiation in nontransformed cells (16–21, 24, 25). These different ASPP2 tumor suppressor mechanisms are highly cell context specific, which allows us to reconcile our data that, in addition to promoting p53-dependent apoptosis (7), ASPP2 stimulates Ras activation independent of p53-binding. Our findings provide significant mechanistic insight into a p53-independent mechanism for ASPP2 tumor suppressor function (Fig. S6), and add to the mounting evidence that ASPP2 mediates nonapoptotic pathways including stimulation of Ras-induced senescence (1, 3, 11–15).

We found the ASPP2 splice isoform BBP (34, 35) could not bind Ras (Fig. 2), nor enhance Ras-induced stimulation of pERK1/2 and C-Raf-p338 (Figs. 3 and 6). This would be predicted because BBP is missing the N-terminal 123 amino acids that contain the Ras-binding domain (32). BBP has an intact C-terminal p53-interacting domain yet can still, albeit less efficiently, promote p53-induced apoptosis compared with full-length ASPP2 (7). These data suggest that the inability of BBP to stimulate Ras signaling is not affected by p53 binding. Relative to ASPP2, we found that BBP had a reduced ability to inhibit cell growth as measured by MTS assay in Ras mutant HCT116p53^−/− tumor cells (Fig. S7). This finding reinforces the notion that the ASPP2 N terminus is important for additional p53-independent nonapoptotic tumor suppression functions (1, 3, 11–15). BBP inhibition of pERK1/2 activation appeared more pronounced compared with inhibition of Raf dimerization (Figs. 3 and 4). Because pERK1/2 is a downstream consequence of Ras activation, this may reflect amplification of signals as the signal progresses along the cascade. It is also possible that BBP may interfere with Ras signaling in other ways as well. Structural models predict an ASPP2/BBP intermolecular interaction via the C-terminal domain (31, 45). Thus, it is tempting to speculate that expressed BBP binds endogenous ASPP2 to inhibit its Ras interaction, although this remains to be tested. In this regard, the ratio of ASPP2 and BBP isoforms could influence the ability of ASPP2 to modulate Ras-activation, which may be important for signaling in the appropriate cellular context. Moreover, alterations in the ASPP2/BBP ratio could contribute to cancer development and we are investigating this hypothesis.

Although ASPP2 promoted Ras-induced MEK/ERK signaling, we did not observe enhanced pAKT levels (Fig. 3). This observation suggests that ASPP2 stimulates specific Ras effector pathways rather than globally enhancing Ras signaling, although this remains to be proven. Given the complexity of Ras signaling (28), it is not unexpected that ASPP2 siRNA cannot completely block EGF-induced or H-RasV12–induced pERK1/2 levels (Figs. 3 and 7). Other mechanisms could also influence ASPP2-mediated MEK/ERK activation via Ras-independent pathways (27, 46). ASPP2 also binds protein phosphatase 1 (PP1) (14, 47), which in turn could modulate Ras-induced senescence (48). Intriguingly, a recent report found that the ASPP2 N terminus can mediate Ras-induced senescence and inhibit autophagy by competing with ATG16 to bind ATG5/ATG12 (15). How direct ASPP2-Ras binding/activation modulates these pathways remains to be explored.

Our findings that ASPP2, but not BBP, promotes B-Raf/C-Raf dimerization (Fig. 4) suggest the ASPP2 N terminus stimulates Ras-signaling. Attenuation of ASPP2 expression inhibited EGF-stimulation of Ras–GTP formation, suggesting at least one upstream mechanism for ASPP2-mediated Raf-activation (Fig. S2). ASPP2 might function as a scaffolding protein to facilitate Ras signaling as suggested by detection of ASPP2 in complex with Raf proteins (Fig. 5). It remains unknown whether ASPP2 binds Raf proteins directly, or indirectly via Ras–GTP. The latter possibility is supported by data showing ASPP2-Raf complex formation requires Ras–GTP and the ASPP2 N terminus. Ras-signaling occurs through a complex series of events mediated in part through C-Raf phosphorylation (26, 27, 39–41). Importantly, we found that ASPP2 stimulated Ras-induced C-Raf phosphorylation at p338 (Fig. 6). This suggests that ASPP2 functions upstream of pERK1/2 activation. ASPP2 did not increase Ras–Raf binding, suggesting that ASPP2 might stimulate kinase activity (or inhibit phosphatase-activity) leading to activation of C-Raf–p338. Stimulation of C-Raf phosphorylation requires the ASPP2 N terminus because BBP fails to do so (Fig. 6). ASPP2 siRNA knockdown demonstrated only a partial inhibition of C-Raf–p388, which is not surprising given the multiple mechanisms that activate C-Raf (26, 27, 49). How ASPP2 can fine-tune these pathways remains to be determined.

We found that attenuation of ASPP2 expression inhibited H-RasV12–induced senescence in nontransformed human fibroblasts (Fig. 7) and HEKn epithelial cells (Fig. S5). Because Ras-activating mutations contribute to tumorigenesis in epithelial cells, the demonstration that ASPP2 can enhance Ras-induced senescence in HEKn cells provides a clinically relevant observation implying that the loss of ASPP2 could be an important early event in the initiation of human epithelial cancers. Recent studies found that ASPP2 potentiated H-RasV12–induced cellular senescence in MEFs, although the direct mechanism(s) that activated Ras were unclear (13, 15). Our data demonstrating that the ASPP2 N terminus binds Ras-GTP and stimulates Ras/Raf/MEK/ERK activation reveal a molecular mechanism by which ASPP2 can directly enhance Ras signaling. The importance of the ASPP2 N terminus was also shown in the recent report demonstrating that BBP could not stimulate Ras-induced senescence (15). Stimulation of ASPP2 expression by H-RasV12 (Fig. 7C) could be envisioned as a feedback mechanism to enhance senescence as a protective barrier against inappropriate proliferative signals in normal cells (16, 21–23, 42, 50). Together, our findings (i) mechanistically establish ASPP2 as an important component of Ras-induced senescence, (ii) expand on the complexity of ASPP2 tumor suppressor function and, (iii) open new avenues for investigation that may eventually be translated into the oncology clinic.

Methods

HCT116 isogenic cell lines were a gift from Dr. Bert Vogelstein (Johns Hopkins University, Baltimore, MD). The Ras-GTP activation assay was performed using a GST-tagged Ras binding domain (RBD) of RalGDS (gift from Dr. Johannes Bos, Utrecht University, Utrecht, The Netherlands). Details of these methods as well as lentiviral infections, senescence assays, plasmids, antibodies, siRNAs, and fluorescence microscopy are described in SI Methods.