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Cell Death and Survival

B56γ Tumor-Associated Mutations Provide New Mechanisms for B56γ-PP2A Tumor Suppressor Activity

Yumiko Nobumori, Geoffrey P. Shouse, Yong Wu, Kyu Joon Lee, Binghui Shen and Xuan Liu
DOI: 10.1158/1541-7786.MCR-12-0633 Published September 2013

Abstract

The hetero-trimeric PP2A serine/threonine phosphatases containing the regulatory subunit B56, and in particular B56γ, can function as tumor suppressors. In response to DNA damage, the B56γ subunit complexes with the PP2A AC core (B56γ-PP2A) and binds p53. This event promotes PP2A-mediated dephosphorylation of p53 at Thr55, which induces expression of p21, and the subsequent inhibition of cell proliferation and transformation. In addition to dephosphorylation of p53, B56γ-PP2A also inhibits cell proliferation and transformation by a second, as yet unknown, p53-independent mechanism. Here, we interrogated a panel of B56γ mutations found in human cancer samples and cell lines and showed that these mutations lost B56γ tumor-suppressive activity by two distinct mechanisms: one is by disrupting interactions with the PP2A AC core and the other with B56γ-PP2A substrates (p53 and unknown proteins). For the first mechanism, due to the absence of the C catalytic subunit in the complex, the mutants are unable to mediate dephosphorylation of any substrate and thus failed to promote both the p53-dependent and -independent tumor-suppressive functions of B56γ-PP2A. For the second mechanism, the mutants lacked specific substrate interactions and thus partially lost tumor-suppressive function, i.e., either the p53-dependent or p53-independent contingent upon which substrate binding was affected. Overall, these data provide new insight into the mechanisms of tumor suppression by B56γ.

Implications: This study further indicates the importance of B56γ-PP2A in tumorigenesis. Mol Cancer Res; 11(9); 995–1003. ©2013 AACR.

This article is featured in Highlights of This Issue, p. 965

Introduction

The protein phosphatase 2A (PP2A) is a family of serine/threonine phosphatases that is involved in a multitude of cell-signaling pathways. PP2A exists either in the cell as a heterodimer of scaffolding A subunit and catalytic C subunit (the AC core), or as a heterotrimeric complex where the AC core additionally associates with one of the variable B subunits. The B subunits have 4 gene families based on sequence homology: the B (B55 or PR55), B′ (B56 or PR61), B″ (PR48/59/72/130), and B″(PR93/110). Each B subunit family contains 2 to 5 isoforms and many contain alternatively spliced variants. Binding of a specific B subunit determines diverse cellular localization and substrate specificities, allowing PP2A holoenzyme to have a diverse enzymatic activity in the cell (1, 2).

Recent evidence suggested that a subset of PP2A holoenzymes that contain B56 (B56-PP2A), in particular B56γ (PPP2R5C), functions as tumor suppressor (3, 4). Although the underlying mechanism is not fully understood, B56γ–PP2A is known to dephosphorylate and regulate specific substrates involved in cellular functions. For example, dephosphorylation of tumor suppressor p53 (TP53) at Thr55 activates p53, resulting in the induction of the CDK inhibitor p21 (CDKN1A), and inhibition of cell growth. However, in the absence of p53, B56γ–PP2A can still reduce cell growth, suggesting that it dephosphorylates other unknown substrates that also play roles in tumor suppression (4). Although the mechanism of the p53-independent function is unknown, additional proteins have been shown to interact with B56γ and potentially could be dephosphorylated by B56γ–PP2A. These proteins include the mitogen-activated kinase ERK (MAPK; 5), transcription cofactor p300 (EP300; 6), and centromeric cohesion recruited by Sgo1 (SGOL1; 7, 8). Overall, these studies suggest that B56γ–PP2A acts as a tumor suppressor by dephosphorylating specific target substrates to regulate their effects on cellular functions. In support of this view, some viral oncoproteins function by displacing the B56 subunits from AC core (3, 9). In addition, mutations in PP2A Aα gene (PPP2R1A) and Aβ gene (PPP2R1B) identified in cancers are known to lose interaction with either the C subunit or the B56 subunits (10–13). Despite its importance in tumorigenesis, mechanisms for inactivation of B56γ–PP2A tumor-suppression by B56γ mutations are not well studied.

In this study, we characterized a panel of B56γ mutations previously identified in human tumor samples and cancer cell lines. Our results revealed three classes of mutations in the B56γ gene. The class I mutants, which includes A61V, A212T, S251R, E266R, P274T, H287Q, and P289S, could bind to both the AC core and p53, and displayed a tumor suppressive activity similar to wild-type (WT) B56γ, suggesting single mutations within this class have no effect on the B56γ tumor suppressor function. The class II mutants (C39R, E164K, Q256R, and L257R) lost all B56γ–PP2A tumor suppressor activity, although still able to bind p53, they could not complex with the AC core. In contrast, the class III mutants (S220N, A383G, and F395C) had a partial tumor suppressor activity compared with WT B56γ and could complex with the AC core. Two mutations, A383G and F395C, fail to bind to p53, thus explaining their loss of p53-dependent function (14). In contrast, S220N still bound and dephosphorylated p53 but reduced B56γ–PP2A tumor-suppressive activity, indicating that it disrupts p53-independent mechanism, although the involved substrate is unknown. These results provide mechanistic insight into the inactivation of tumor-suppressive function of B56γ and further support the notion that multiple pathways are involved in B56γ–PP2A–mediated tumor suppression.

Materials and Methods

Cell culture and plasmids

U2OS and HCT116 cells were cultured in McCoy's 5A medium supplemented with 10% fetal calf serum. The B56γ point mutants were generated using the QuikChange Site-Directed Mutagenesis Kit (Stratagene).

Western blot and immunoprecipitation

Whole-cell extract was prepared by lysing the cells in a buffer containing 50 mmol/L Tris-HCl (pH 8.0), 120 mmol/L NaCl, 0.5% NP-40, 1 mmol/L dithiothreitol, 2 μg/mL aprotinin, and 2 μg/mL leupeptin. Cell lysates were subjected to SDS-PAGE, then analyzed by Western blot analysis using anti-p53 (DO1, Santa Cruz Biotechnology), anti-PP2A A subunit (Upstate), anti-PP2A C subunit (1D6, Upstate), anti-p21 (Santa Cruz Biotechnology), anti-PP2A B56γ (14), anti-HA (12CA5), anti-ERK (Santa Cruz Biotechnology), anti-SGOL1 (ABNOVA), anti-cyclin G (Santa Cruz Biotechnology), or anti-vinculin (VIN-11-5, Sigma) antibodies. For Thr55 dephosphorylation experiments, the cell lysate was immunoprecipitated with a phospho-specific antibody for phos-Thr55 (Ab202) and then immunoblotted with anti-p53 antibody (4). For interaction of endogenous proteins with transfected B56γ proteins, U2OS cells were transfected with various B56γ plasmids using FuGene (Roche) or BioT (Bioland Scientific) and lysed 28 hours after transfection. Immunoprecipitation was conducted using anti-HA monoclonal antibody. The amounts of coprecipitated proteins were determined by immunoblotting.

RT-PCR

Total RNA was extracted using TRIzol reagent (Ambion), and RT-PCR was conducted using a SuperScript One-Step RT-PCR kit (Invitrogen) according to manufacturer's protocol. RT-PCR for p21 mRNA was conducted with (F) 5′-CGACTGTGATGCGCTAATGG–3′ and (R) 5′-GGCGTTTGGAGTGGTAGAAATC-3′, and for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA was conducted with (F) 5′-AGGTGAAGGTCGGAGTCAAC-3′ and (R) 5′- GACAAGCTTCCCGTTCTCAG-3′.

Identification of cancer-derived mutation

The NCBI AceView program (http://www.ncbi.nlm.nih.gov/IEB/Research/Acembly/index.html) provides a comprehensive sequence of the human transcriptome and genes of all quality-filtered human complementary DNA data from GenBank, RefSeq, dbEST, and Trace in a strictly complementary DNA-supported manner. Using this program, we looked for B56γ mutations in the annotated sequences of tumor samples and cancer cell lines.

Cell proliferation and anchorage-independent growth assays

To generate proliferation curves for HCT116 cells, cells were transfected with WT, mutant B56γ, or a control cytomegalovirus (CMV) empty vector using BioT. Transfected cells were seeded in triplicate, and then counted at 120-hour postseeding. The presence of overexpressed B56γ protein in the cell was verified by immunoblotting. For anchorage-independent growth assays, HCT116 cells were transfected with WT, mutant B56γ or a control empty vector seeded in triplicate in 0.35% Noble Agar (Fisher) and colony numbers were counted 4 weeks post seeding.

Results

Identification of potential tumor-derived mutations in B56γ gene

To better understand the role of B56γ in human cancers, we conducted an AceView search for all of the known, tumor-associated mutations in the B56γ gene. AceView regularly downloads the whole set of cDNA sequences from the public databases, aligns them on the current genome available at NCBI, and clusters them into reference transcripts. Because all of the identified alternative transcripts were originally cloned from cancer samples or cancer cell lines, they represent potential tumor-inducing mutations. We identified 24 point mutations scattered throughout the B56γ coding region (Table 1; Fig. 1), whereas a search of the NCBI single-nucleotide polymorphism database did not yield any of these mutations. The fact that B56γ mutations are found in several different types of tumors (Table 1) suggests that B56γ may play a broad role in tumor suppression. The B56γ protein consists of 8 pseudo Huntington-elongation-A subunit-TOR (HEAT) repeats. Interestingly, although the mutations are spread across the entire B56γ sequence, they cluster more frequently toward the center of the gene, notably on the HEAT-repeat 4, 5, and 6 and in a very small domain (aa 383–410) that contains the p53-binding domain (Fig. 1), suggesting there are potential cancer mutation hot spots in the gene. The human B56γ transcript has at least 3 long splice variants known as γ1, γ2, and γ3 (15, 16) and most of the mutations we identified are common to all 3 variants. To begin investigating their function in tumor suppression, we used site-directed mutagenesis to generate 11 of the new mutations (A61V, E164K, A212T, S220N, S251R, Q256R, L257R, E266R, P274T, H287Q, and P289S; Fig. 1). All of these mutations, plus 3 previously reported C39R, A383G, and F395C mutations, are shared by the 3 spliced isoforms and represent different clusters located in the B56γ gene (Fig. 1).

Figure 1.
Figure 1.

Distribution of B56γ mutations identified by AceView. Distribution of 24 mutations on three splice-variants known as γ1, γ2, and γ3, of B56γ. Each shaded rectangle represents a HEAT-repeat and each box represents a α-helix. Mutants in bold were chosen for characterization.

View this table:
Table 1.

Identification of tumor-derived mutations in B56γ

Effect of identified mutations on the tumor-suppressive functions of B56γ

Because the mutations were identified in tumor samples, we assessed their effect on B56γ tumor suppressor activity. Previously, we showed that overexpression of WT B56γ inhibits cell proliferation and anchorage-independent cell growth in both p53-dependent and p53-independent manner (4). To evaluate the effect of the mutants, we first tested whether mutations affect the ability of B56γ to inhibit cell proliferation. Human colon cancer cells, HCT116 cells with either a p53−/− or p53+/+ background, were transfected with WT B56γ or each of the mutants. As shown in Fig. 2A, overexpression of WT B56γ in the presence of p53 (HCT116 p53+/+ cells) led to approximately 45% decrease in cell number compared with the vector control after 120 hours of cell growth. Similar level of decrease was also observed in a tetracycline-inducible B56γ overexpression U2OS cell line with p53+/+ background (Supplementary Fig. S1), suggesting it represented both p53-dependent and p53-independent inhibition. In contrast, in the p53−/− cells, overexpression of WT B56γ had a reduced effect on cell proliferation, with a 20% decrease in cell number, which represented the level of p53-independent inhibition.

Figure 2.
Figure 2.

Effect of B56γ mutants on tumor suppressor function. HCT116 human colon cancer cells with p53+/+ or p53−/− background were transfected with HA-tagged WT or B56γ mutants. For the control (EV), cells were transfected with an empty CMV vector. A, representatives of cell proliferation assay where transfected cells were seeded, harvested and counted after 120 hours of growth. Numbers of cells were normalized against the representative empty vector controls and plotted in a bar graph. Error bars show average ± SD from triplicate plates in one representative experiment. Cells harvested were lysed and protein expression for endogenous B56γ (lower), HA-B56γ (upper), p53, and vinculin (vinc) were analyzed by Western blot analysis. B, representatives of anchorage-independent growth assay where transfected cells were seeded in soft agar and number of colonies were counted. Error bars show average ± SD from triplicate plates in one representative experiment. Cells at initial seeding were lysed and analyzed for B56γ protein expression.

In comparison, overexpression of B56γ mutants led to cell growth inhibition ranged from similar to WT, to partial reduction, to no inhibition at all. On the basis of their growth inhibition property, we classified all 14 mutants tested into one of 3 classes (Table 2). Class I, including A61V, A212T, S251R, E266R, P274T, H287Q, and P289S, had little or no effect on B56γ-mediated growth inhibition in both HCT116 p53+/+ cells and p53−/− cells, suggesting those individual single mutations have no effect on the B56γ tumor suppressor function. In contrast, class II, including C39R, E164K, Q256R, and L257R, were unable to inhibit cell growth in both p53−/− and p53+/+ HCT116 cells, suggesting these mutants lost their ability to block cell proliferation regardless of p53 status. Class III, including S220N, A383G, and F395C, only partially inhibited cell proliferation compared with WT B56γ. As previously described, A383G and F395C show reduced inhibitory effect in p53+/+ cells but not in p53−/− cells. This can be explained by their inability to bind and dephosphorylate p53 (14). Interestingly, S220N showed a partial inhibitory effect in p53+/+ cells, but not in p53−/− cells, suggesting that this mutant specifically lost the p53-independent tumor suppressor activity of B56γ–PP2A.

View this table:
Table 2.

Characterization of 14 tumor-derived B56γ mutations

To provide further evidence, we tested the effect of the mutations on anchorage-independent cell growth. On the basis of the result from cell proliferation assay (Fig. 2A), we assayed A212T and P274T from class I, E164K, Q256R, L257R from class II, and S220N from class III. HCT116 p53+/+ cells and p53−/− cells were transfected with WT B56γ or the mutants, and seeded in soft agar. As shown in Fig. 2B, overexpression of WT B56γ in p53+/+ cells led to 65% reduction in the number of colonies compared with empty vector control, which represents both p53-dependent and p53-independent inhibition. In contrast, overexpression of WT B56γ in p53−/− cells only led to 18% decrease in colony numbers, which represents the p53-independent inhibition.

When class I A212T and P274T mutants were overexpressed, no significant changes in the number of colonies were observed compared with WT, suggesting those mutants have no effect on anchorage-independent growth suppression of B56γ. Class II E164K, Q256R, and L257R mutants, however, completely abolished WT B56γ-mediated anchorage-independent growth suppression in both p53−/− and p53+/+ cells, indicating that those mutations lost their ability to suppress anchorage-independent cell growth in both p53-dependent and -independent manner. Compared with WT, class III S220N mutant partially lost its ability to inhibit anchorage-independent growth in p53+/+ cells and completely lost its ability in p53−/− cells, suggesting that it specifically disrupts p53-independent function of B56γ. A383G and F395C from class III were previously shown to specifically block p53-dependent function of B56γ (14). Taken together, our results show that class II and III cancer-associated B56γ mutations disrupt, either completely or partially, tumor suppressive activity of B56γ.

B56γ mutations interfere with interaction with either the AC core or substrates

To understand the mechanisms for inactivation of B56γ tumor-suppressive function, we next assayed the ability of the mutants to interact with the AC core and with p53. WT or mutant B56γ was expressed in U2OS cells, and their interaction with the AC core and p53 was assayed by immunoprecipitation. As shown in Fig. 3A and summarized in Table 2, none of the 11 new mutations tested affect interaction of B56γ with p53. This is perhaps not surprising because most of the mutations are not located near the mapped p53-binding domain (aa 391–401). Furthermore, all class I mutants (A61V, A212T, S251R, E266R, P274T, H287Q, and P289S) showed little or no effect on the AC interaction (Fig. 3), supporting the notion that these individual mutations do not affect the B56γ tumor suppressor function (Fig. 2 and Table 2). To further prove this, we examined the effect of these mutations on p53 Thr55 dephosphorylation and function. Results of 4 representatives (A61V, A212T, E266R, and P274T) are shown in Fig. 3B. Overexpression of these mutants led to efficient dephosphorylation of p53 at Thr55 and activation of the p53 transcription target p21 at levels similar to WT B56γ3 (summarized in Table 2). Because p53 Thr55 is the only known residue that is directly dephosphorylated by B56γ–PP2A, we were unable to assess the effect of the mutations on other potential dephosphorylation by B56γ–PP2A. However, given their ability to fully support p53-independent tumor-suppressive function (Fig. 2), it is likely that class I mutants fully support B56γ–PP2A dephosphorylation.

Figure 3.
Figure 3.

Interaction of B56γ mutants with PP2A A and C and p53. U2OS cells were transfected with empty vector control (EV), HA-tagged WT, or mutant B56γ. A, WT and mutant B56γ were immunoprecipitated and interacting proteins were analyzed by Western blot using antibodies listed. B, p53 Thr55 dephosphorylation, p21 protein levels, p53, and vinculin (vinc) were analyzed by Western blot. p53 Thr55 phosphorylation levels were analyzed by phospho-specific antibody for Thr55 in the presence of MG132. The p21 protein levels were tested in the absence of MG132. C, the p21 mRNA levels were analyzed by RT-PCR. D, class II mutations are shown on the crystal structure of B56γ–PP2A holoenzyme (adapted from Protein Data Bank, accession code 2NYM) and prepared by PyMOL. The PP2A holoenzyme is displayed with A, C, and B56γ. Mutation residues are indicated for E164, Q256, and L257. Dashed lines indicate hydrogen bonding.

In contrast, class II mutants (E164K, Q256R, and L257R) remained bound to p53, but lost their ability to interact with the AC core (Fig. 3A). In addition, C39R was also unable to bind to AC core (17). We note that all residues in this class, C39, E164, Q256, and L257, are not making any direct contact to A or C subunits according to B56γ–PP2A crystal structure (18, 19). However, they are located in close proximity from the interaction interface (Fig. 3D). E164 residue is located within intraloop of HEAT-repeat 3 and its negatively charged side chain is important to form hydrogen bond to E118 and R167. Mutation of glutamic acid to lysine in this position would abolish these hydrogen bonds and thus destabilize intraloop of HEAT-repeat 2 that mediates interaction with A and C subunits. Q256 and L257 residues are located within the second helix of HEAT-repeat 5. The polar side chain of Q256 points toward a helix of HEAT-repeat 4 and interacts with E216. In Q256R mutation, arginine has a positively charged side chain that is larger than glutamine and additionally contacts E213. Alternatively, the hydrophobic side chain of L257 points toward the first helix of HEAT-repeat 5. In L257R mutation, a larger side chain of arginine would protrude into the adjacent helix and a positive charge of arginine would induce further alterations in the environment. Both cases would result in displacement of helices, leading to rearrange the location of intraloops that mediate the interaction between B56γ and AC core. Interestingly, all 4 residues are conserved among the B56 family isoforms, indicating the importance of these residues for maintaining the interaction between B56 and AC core.

Because the C subunit is required for PP2A catalytic activity, our data explain why the class II mutants completely abolished all tumor-suppressive function of B56γ–PP2A (Fig. 2 and Table 2). To support this view, we assayed their effect on p53 Thr55 dephosphorylation and induction of p21 (Fig. 3B and C and Table 2). The assays showed that, unlike WT B56γ, overexpression of class II mutants fail to induce dephosphorylation of p53 at Thr55 and activation of p53 transcription target p21. These results show that the interaction of B56γ with the AC core is required for B56γ–PP2A to dephosphorylate and activate p53. Consequently, class II mutants have lost their tumor-suppressive function through disruption of the AC core interaction and loss of catalytic activity.

Previous study has shown that 2 mutants in class III, A383G and F395C, remain bound to AC core, but have lost their ability to bind and dephosphorylate p53, leading to disruption of the p53-dependent tumor suppressor activity of B56γ–PP2A (14). Interestingly, unlike A383G and F395C, S220N still interacted with p53 at a level comparable with WT B56γ. Importantly, the protein also promoted dephosphorylation of p53 at Thr55 and induced p21 expression to levels similar to WT B56γ (Fig. 3B and C, and Table 2). These results suggest that p53 is unlikely responsible for partial loss of tumor suppressor activity of the S220N protein (Fig. 2). Together, our results show that class III mutations inactivate the tumor suppressor activity of B56γ–PP2A by preventing B56γ from binding to its substrates, thereby indicating the importance of B56γ in recruiting the AC core to substrate, so that B56γ–PP2A can function correctly.

S220N binds to B56γ-interacting proteins

Because S220N specifically abolished the p53-independent tumor suppressor activity of B56γ–PP2A, we hypothesize that this may be due to its lack of interaction with another unknown substrate. Interestingly, S220 is located on a large-concave surface of B56γ that is unoccupied by A and C subunits and leans toward the catalytic pocket of the C subunit (Fig. 4A). It has been previously suggested that this open area may be important for recruiting substrates (18). To identify potential substrate that may bind to WT B56γ but not S220N, we assayed the ability of the S220N mutant protein to interact with several known B56γ interacting proteins including ERK, cyclin G2 (CCNG2), Shugoshin 1, and p300 (Fig. 4B and data not shown). The assay showed that the interaction of S220N with all of these proteins was similarly to WT B56γ (Fig. 4B), suggesting that interactions with these proteins are unlikely to be responsible for loss of p53-independent tumor suppressor activity of S220N. Nevertheless, our results indicate that class III mutations specifically abolished individual substrate interaction, thus partially disrupting B56γ tumor suppressor activity. Furthermore, our results also indicate that B56γ contains multiple substrate-binding domains, implying the role of a specific B subunit in multiple pathways. Further study will provide insight into these pathways and their role in PP2A-dependent tumor suppression

Figure 4.
Figure 4.

S220N interacts with AC core and p53. A, S220 is indicated on the crystal structure of B56γ–PP2A holoenzyme. B, lysates of U2OS cells that were transfected with empty vector control (EV), hemagglutinin (HA)-tagged WT, or S220N B56γ were immunoprecipitated with anti-HA antibody, then analyzed by Western blot against PP2A A and C, ERK, p53, Sgo, cyclin G, HA, and vinculin (vinc).

Discussion

In this study, we characterized a panel of B56γ mutations that were previously identified in human cancers and defined the molecular mechanisms behind loss of B56γ–PP2A tumor suppression. Since B56γ has been suggested to be a tumor suppressor gene and function as a B56γ–PP2A complex to dephosphorylate proteins involved in cancer, mutants that lost their interaction with either AC core or substrates could potentially affect its tumor suppressive function. Indeed, we have shown, the mutations could be categorized into 3 groups: “no effect” (class I); “loss of AC core interaction” (class II); and “loss of substrate interaction” (class III; Fig. 5). The “loss of AC core interaction” group (C39R, E164K, Q256R, and L257R) failed to bind to AC core and thus disrupted the B56γ–PP2A complex in the cells. This leads to the loss of all B56γ–PP2A tumor suppressor-related functions. As a result, overexpression of these mutants failed to promote p53-dependent and p53-independent tumor suppression. Interestingly, the “loss of AC core interaction” mechanism has been observed with several previously described A subunit tumor-associated mutations and SV40 ST antigen, emphasizing the importance of the B56γ–PP2A complex in cancer suppression. In contrast, the “loss of substrate interaction” group (S220N, A383G, and F395C) remains bound to the AC core, but failed to bind B56γ–PP2A substrates. The A383G and F395C mutations are located at the p53-binding domain, and thus caused the loss of p53 interaction, which specifically abolished p53-dependent B56γ tumor suppressor function. In contrast, the S220N mutation is located at another potential substrate binding domain. Although we have not identified the substrate(s) involved in this loss of B56γ–PP2A function, we hypothesize that an unknown protein(s) plays a role in p53-independent tumor suppression. Taken together, our data defined detailed mechanisms for the inactivation of B56γ–PP2A in cancer. Given the fact that increased B56γ protein level is required for its function (4), those inactivation mechanisms may also apply for mutations that are heterozygous.

Figure 5.
Figure 5.

Inactivation of B56γ–PP2A tumor-suppressive function by B56γ mutations. The class II mutations (C39R, E164K, Q256R, and L257R) failed to bind to AC core and thus disrupted all B56γ–PP2A tumor suppressor-related functions. The class III mutations (S220N, A383G, and F395C) failed to bind B56γ–PP2A substrates and thus partially lost tumor-suppressive function of B56γ–PP2A.

Although the B56γ mutations identified so far spread across the entire B56γ sequence, they appear more frequently in 2 regions: the center of the B56γ gene (HEAT-repeat 4, 5, and 6) and a small region toward the C-terminus (Fig. 1). These clusters suggest possible existence of hot spots that are more susceptible to tumor-associated mutations. The existence of hot spots for somatic mutations often indicates that the region is essential for the function of protein. Indeed, the small region at the C-terminus (aa 383–401) contains a domain crucial for p53 binding (14), arguing that p53 is an important substrate for B56γ–PP2A function. Interestingly, although 11 mutations are located on HEAT-repeat 4, 5, and 6 alone, the majority of them did not affect the B56γ ability to inhibit cell growth. Aside from sequencing errors, it is possible that more than one-point mutation is needed to change the protein function supported by those repeats. In fact, multiple interacting interfaces have been suggested for the complex formed between B56γ and the A and C subunits (18, 19). Further study of this region may lead to a better understanding of those interfaces and their roles in PP2A function.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Authors' Contributions

Conception and design: Y. Nobumori, G.P. Shouse, B. Shen, X. Liu

Development of methodology: Y. Nobumori, G.P. Shouse, B. Shen, X. Liu

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): Y. Nobumori, G.P. Shouse, Y. Wu, K.J. Lee, B. Shen, X. Liu

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): Y. Nobumori, B. Shen, X. Liu

Writing, review, and/or revision of the manuscript: Y. Nobumori, G.P. Shouse, B. Shen, X. Liu

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): Y. Wu, X. Liu

Study supervision: X. Liu

Grant Support

This work was supported by the NIH grant R01CA075180 (to X. Liu) and by the American Heart Association Postdoctoral Fellowship POST3530033 (to Y. Wu)

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Acknowledgments

The authors thank Dr. Margaret Morgan for careful editing of this manuscript.

Footnotes

  • Note: Supplementary data for this article are available at Molecular Cancer Research Online (http://mcr.aacrjournals.org/).

  • Received November 5, 2012.
  • Revision received April 22, 2013.
  • Accepted May 6, 2013.

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Molecular Cancer Research: 11 (9)
September 2013
Volume 11, Issue 9

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B56γ Tumor-Associated Mutations Provide New Mechanisms for B56γ-PP2A Tumor Suppressor Activity
Yumiko Nobumori, Geoffrey P. Shouse, Yong Wu, Kyu Joon Lee, Binghui Shen and Xuan Liu
Mol Cancer Res September 1 2013 (11) (9) 995-1003; DOI: 10.1158/1541-7786.MCR-12-0633
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