Disruption of protein phosphatase 2A subunit interaction in human ...

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Oncogene (2001) 20, 10 ± 15 2001 Nature Publishing Group All rights reserved 0950 ± 9232/01 $15.00 www.nature.com/onc

Disruption of protein phosphatase 2A subunit interaction in human cancers with mutations in the Aa subunit gene Ralf Ruediger1, Huong T Pham1 and Gernot Walter*,1 1

Department of Pathology, University of California at San Diego, La Jolla, California, CA 92093, USA

The A subunit of protein phosphatase 2A (PP2A) consists of 15 nonidentical repeats. The catalytic C subunit binds to C-terminal repeats 11 ± 15 and regulatory B subunits bind to N-terminal repeats 1 ± 10. Recently, four cancer-associated mutants of the Aa subunit have been described: Glu64?Asp in lung carcinoma, Glu64?Gly in breast carcinoma, Arg418?Trp in melanoma, and D171 ± 589 in breast carcinoma. Based on our model of PP2A, we predicted that Glu64?Asp and Glu64?Gly might be defective in B subunit binding, whereas Arg418?Trp and D171 ± 589 might bind neither B nor C subunits. We generated these mutants by site-directed mutagenesis and assayed their ability to associate with di€erent forms of B subunits (B, B', B'') or with the catalytic C subunit. The results demonstrate that all mutants are defective in binding either B or B and C subunits. Speci®cally, the Nterminal mutants, Glu64?Asp and Glu64?Gly, are defective in B' but normal in B, B'', and C subunit binding, whereas the C-terminal mutants Arg418?Trp and D171 ± 589 bind none of the B subunits nor the C subunit. The implications of these ®ndings with regard to the potential role of PP2A as a tumor suppressor are discussed. Oncogene (2001) 20, 10 ± 15. Keywords: PP2A; Aa subunit; mutation; human cancer; tumor suppressor Introduction Based on the observations that okadaic acid is both a strong inhibitor of the serine/threonine-speci®c protein phosphatase 2A (PP2A) (Bialojan and Takai, 1988) and a potent tumor promoter (Suganuma et al., 1988), it has been suggested that PP2A might be a tumor suppressor (Cohen and Cohen, 1989). Direct evidence for this suggestion has been lacking until recently, when Wang et al. (1998) discovered that the gene encoding the b isoform of the A subunit of PP2A was altered in 15% of primary lung tumors (small-cell- and non-small-cell lung carcinomas), in 6% of lung tumorderived cell lines and in 15% of primary colon tumors. Then, Calin et al. (2000) reported that both the Aa and

*Correspondence: G Walter Received 1 September 2000; revised 23 October 2000; accepted 26 October 2000

Ab subunit isoforms of PP2A are genetically altered in a variety of primary human cancers and Takagi et al. (2000) recently described additional mutations in the Ab subunit gene in four colon cancer cases. On the other hand, Campbell and Manolitsas (1999) detected no mutations in the Ab subunit gene from ovarian cancers. PP2A is composed of three subunits, A, B and C. The core enzyme consists of a 36 kDa catalytic C subunit and a 65 kDa regulatory A subunit. One of several regulatory B subunits can bind to the core enzyme, and the heterotrimer composed of the A, B and C subunits is called holoenzyme (Mumby and Walter, 1993, for review). The core and holoenzymes are expressed at similar levels in cells, and they are in equilibrium with each other (Kremmer et al., 1997). The A and C subunits each exist as two isoforms (a and b) (da Cruz e Silva et al., 1987; Green et al., 1987; Hemmings et al., 1990; Kitagawa et al., 1988; Stone et al., 1987; Walter et al., 1989), whereas the B subunits fall into three families designated B, B' (also called B56) and B''. The B family has three members, Ba,Bb and Bg, each with a molecular mass of around 55 kDa (Healy et al., 1991; Mayer et al., 1991; Pallas et al., 1992; Zolnierowicz et al., 1994). The B' family consists of numerous isoforms and splice variants whose molecular masses range from 54 ± 68 kDa (Csortos et al., 1996; McCright et al., 1996; McCright and Virshup, 1995; Tanabe et al., 1996; Tehrani et al., 1996). The B'' family has at least four members, designated PR48, PR59, PR72 and PR130 (Hendrix et al., 1993; Nagase et al., 1997; Voorhoeve et al., 1999; Yan et al., 2000). PR72 and PR130 are splice variants of the same gene. The combination of all subunits could give rise to over 50 di€erent forms of PP2A. However, it is unknown how many of the possible forms actually exist in cells. Regulatory subunits, in particular the B subunits, determine substrate speci®city and subcellular localization (Cegielska et al., 1994; McCright et al., 1996; Tehrani et al., 1996). Furthermore, some B subunits are expressed in a tissuesspeci®c fashion and at distinct developmental stages (Csortos et al., 1996; McCright et al., 1996; McCright and Virshup, 1995; Tehrani et al., 1996). In addition to B, B' and B'' subunits, certain tumor (T) antigens encoded by polyoma viruses represent a fourth class of proteins able to associate with the PP2A core enzyme (Walter and Mumby, 1993, for review). T antigens play key roles in neoplastic transformation by

Defective PP2A subunit interaction in human cancer R Ruediger et al

polyoma viruses. Interestingly, the B, B' and B'' subunits are unrelated by protein sequence and the T antigens are unrelated to the B subunits, although B subunits and T antigens bind to overlapping regions on the A subunit. The structural basis for the interaction between the A, B and C subunits in the core and holoenzymes and between the core enzyme and T antigens has been studied in our laboratory by site-directed mutagenesis (Ruediger et al., 1992, 1994). The resulting model of the A subunit structure and of the interactions between the A, B, and C subunits as well as T antigens are shown in Figure 1. The A subunit polypeptide consists of 15 nonidentical repeats (Hemmings et al., 1990; Walter et al., 1989). Each repeat is composed of two a helices connected by a loop (intra-repeat loop). Adjacent repeats are connected by inter-repeat loops. Collectively, the repeats form an extended molecule that is largely stabilized by hydrophobic interactions (Chen et al., 1989; Ruediger et al., 1994). We have shown that the intra-repeat loops are involved in binding B and C subunits as well as T antigens. The B subunits from all three families (B, B', B'') bind to repeats 1 ± 10, and the C subunit binds to repeats 11 ± 15 of the A subunit. Importantly, B subunits bind to the A subunit only when the C subunit is bound (Kamibayashi et al., 1992, 1994; Ruediger et al., 1992, 1994). To develop this model of PP2A, we have generated approximately 70 mutants of the A subunit, including N- and C-terminal deletions, deletions of individual repeats, intra- and inter-repeat loop replacement mutants, and point mutants at various positions (Ruediger et al., 1992, 1994, 1999). A remarkable ®nding was that certain point mutations in N-terminal repeats render the A subunit defective in binding speci®c B subunits (Ruediger et al., 1999). The recently published X-ray structure of the Aa subunit generally con®rmed and extended our model in regard to the a helical repeat structure and the location of intra- and inter-repeat loops (Groves et al., 1999). A key question is how the mutations in the Aa and Ab subunits, which were found in human cancers, alter the properties and functions of PP2A, and how the alterations relate to the presumed tumor suppressor function of PP2A. In the present study, we analysed

Figure 1 Model of the PP2A holoenzyme. The extended A subunit molecule consists of 15 repeats. Each repeat is composed of two a helices connected by an intra-repeat loop. Adjacent repeats are connected by inter-repeat loops. The B subunits bind to repeats 1 ± 10 and the C subunit to repeats 11 ± 15. T antigens bind within repeats 2 ± 8. The primary sites of subunit interaction are the intra-repeat loops. Repeat 5, which is deleted in mutant D5, is shaded

cancer-associated mutations in the Aa gene. The results demonstrate that all mutants are defective in binding either B, or B and C subunits.

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Results Calin et al. (2000) described four cancer-associated mutations in the Aa gene: Glu64?Asp in lung carcinoma, Glu64?Gly in breast carcinoma, Arg418?Trp in melanoma, and D171 ± 589 in breast carcinoma. Based on our model (Figure 1), both Glu64 mutations are located in the intra-repeat loop of the second repeat and have the potential of rendering the Aa subunit defective in binding B subunits. Both mutations are not expected to a€ect C subunit binding. The Arg418?Trp mutation, located in intra-repeat loop 11 may cause a defect in C subunit and, indirectly, in B subunit binding. The mutation D171 ± 589 is predicted to cause reduced B and C subunit binding since a large portion of the B subunit binding region and the entire C subunit binding region are deleted. These four mutations were generated by sitedirected mutagenesis of the Aa cDNA, and epitopetagged at either the N or C terminus with the EE-tag (EEEEYMPME). The ability of the mutant Aa subunits to form complexes with di€erent B subunits (Ba (Kamibayashi et al., 1994), B'a1 (Tehrani et al., 1996), B''/PR72 (Hendrix et al., 1993)) was assayed by incubating the in vitro synthesized, radiolabeled Aa subunits with in vitro synthesized, radiolabeled B subunits. The C subunit required for complex formation was supplied in unlabeled form as an endogenous component of the reticulocyte lysate. The complexes were immunoprecipitated with antibodies against the EE-tag and analysed by PAGE. The results obtained by this in vitro binding assay faithfully re¯ect in vivo binding, as demonstrated for several mutants in di€erent cell systems (Brewis et al., 2000; McCright and Virshup, 1995; Ruediger et al., 1997). As shown in Figure 2a,c, lanes 3 and 4, the mutants Glu64?Asp and Glu64?Gly bound the Ba and B''/PR72 subunits almost as eciently as the wild type Aa subunit (lane 1). The mutant D5, previously shown to be defective in binding all forms of B subunits but normal in C subunit binding (Ruediger et al., 1992, 1994), was used as a negative control (lane 2). As a further control, in vitro synthesized, radiolabeled and untagged luciferase instead of Aa subunit was incubated with radiolabeled B subunit and immunoprecipitated with antibodies against the EE-tag (lane 6). This illustrates the background level of B subunit obtained in the absence of any tagged Aa subunit. Note that this background has the same intensity as that obtained with the D5 mutant. Therefore, the B subunit signal obtained with D5 was used as a base line for the purpose of quanti®cation. It is also important to realize that since the A and B subunits are the only radioactive products in the immunoprecipitation reaction, some background at the position of these proteins is unavoidable. The most important and unexpected ®nding was that both Oncogene

Defective PP2A subunit interaction in human cancer R Ruediger et al

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mutants, Glu64?Asp and Glu64?Gly, were speci®cally defective in B'a1 subunit binding (Figure 2b, lanes 3 and 4). The ability of A subunit mutants to form complexes with the C subunit was assayed by incubating the radiolabeled, in vitro synthesized A subunits in the reticulocyte lysate in order to allow them to bind to endogenous (unlabeled) C subunit. The complexes were then immunoprecipitated with antibodies against the KL peptide (KVTRRTPDYFL), corresponding to the C terminus of the C subunit, and analysed for radiolabeled A subunit. As shown in Figure 3a, lanes 5 and 7, the mutants Glu64?Asp and Glu64?Gly bound the C subunit as eciently as the wild type or the mutant D5, which bind C subunit normally. As a control, the immunoprecipitations were carried out in the presence of KL peptide which inhibited precipitation. Importantly, the mutants Arg418?Trp and D171 ± 589 bound neither the C subunit, as shown in Figure 3a, lanes 9 and 11, nor any of the B subunits (Figure 2a,b,c, lanes 5 and 7). Quanti®cation of the

Figure 2 Complex formation of cancer-associated mutants of the Aa subunit of PP2A with regulatory subunits Ba (a), B'a1 (b) and B''/PR72 (c). Translation mixtures of [35S]methionine-labeled and EE-tagged A subunit constructs, named at the top, were mixed with [35S]methionine-labeled Ba, B'a1, or B''/PR72. After incubation at 308C for 4 h in the presence of unlabeled C subunit present in the reticulocyte lysate, complexes were immunoprecipitated with anti-EE antibodies and analysed by SDS ± PAGE and phosphorimaging. The eciency of immunoprecipitation of tagged Aa subunits was 90 ± 100%. The eciency of coimmunoprecipitation of Ba, B'a1, and B''/PR72 was 10, 3 and 15%, respectively, of the total amount synthesized. Considering the amounts of B subunits coimmunoprecipitated with AaEE, we calculated that 15, 15 and 60% of AaEE bound Ba, B'a1 and B''/ PR72, respectively. The Aa subunits in lanes 1 ± 5 were synthesized with 1/10 of the amount of radioactive methionine than the B subunits to avoid interference with the detection of B'a1 and B''/PR72, which run near the Aa subunits. The Aa subunits in lanes 7 ± 9 are more intense then those in lanes 1 ± 5 because they were synthesized with the full amount of radioactive methionine in order to make mutant D171 ± 589 better visible, since it contains only one methionine compared to 14 in wild type Aa. Therefore, although D171 ± 589 in lane 7 appears weaker than Aa in lanes 9, equal amounts of D171 ± 589 were synthesized and precipitated as determined by phosphorimgaing. A 10% polyacrylamide gel was used for lanes 1 ± 6, a 12% gel was used for lanes 7 ± 9. Arrow heads indicate the position of A subunits. Molecular mass markers are shown in the space between lanes 6 and 7 Oncogene

Figure 3 Complex formation of cancer-associated mutants of the Aa subunit of PP2A with the catalytic C subunit. Translation mixtures of 35S-cysteine-labeled Aa subunit constructs, named at the top, were incubated with anti-KL peptide antibodies for immunoprecipitation of unlabeled endogenous C subunit and coimmunoprecipitation of radioactive Aa subunits (a). (b) shows the in vitro synthesized products, demonstrating that the synthesis rate was similar for all constructs. D171 ± 589 is weaker because it has only three cysteines compared to 14 in wild type Aa. The molar amount of D171 ± 589 is the same as that of the wild type Aa subunit, as determined by phosphorimaging. While Aa subunits that co-immunoprecipitate with the C subunit increase in intensity (compare a vs b, lanes 1, 3, 5, 7 and 13) constructs not co-immunoprecipitating decrease in intensity (compare a vs b, lanes 9 and 11). Peptide KL was used as an inhibitor of immunoprecipitation. A 15% polyacrylamide gel was used

Defective PP2A subunit interaction in human cancer R Ruediger et al

binding experiments by phosphorimaging is shown in Figure 4. Discussion The present ®nding that four out of four cancerassociated mutants of the Aa subunit are defective in binding B, or C and B subunits suggests that loss or alteration of PP2A activity is an essential step in tumor development and supports the idea that PP2A acts as a tumor suppressor. We have previously generated numerous mutations in repeats 1 ± 10, the general region for binding B subunits, and tested the binding properties of the mutant proteins. They fell into four categories: (i) Defective in binding all types of B subunits (B- B'- B''-); (ii) defective in binding B and B'', normal in binding B' (B- B'+ B''-); (iii) defective in binding B' and B'', normal in binding B (B+ B'B''7); (iv) defective in binding B', normal in binding B and B'' (B+ B'- B''+). We concluded that the binding sites on the Aa subunit for the di€erent types of B subunits are composed of both distinct and common amino acids (Ruediger et al., 1999). The cancerassociated mutants E64D and E64G belong to the fourth category, which is the most speci®c in that binding of only one type of B subunit is a€ected. Whether the loss of B' subunit binding might cause loss of tumor suppressor activity, and whether B' itself or a B'-containing holoenzyme is in fact the tumor suppressor are open questions. Since B' subunits form a large family of gene products, a further question is whether a speci®c form of B' is involved in tumor suppression. It is likely, although not proven, that all B' family members bind with their highly conserved common region to the same site on the Aa subunit. This would imply that E64D and E64G are defective in binding all variants of B'.

We were surprised to ®nd that the small change of glutamic acid to aspartic acid in the E64D mutant has such a strong e€ect on B' subunit binding. According to the X-ray structure (Groves et al., 1999), E64 is located very close to the intra-repeat loop, and at the N terminus of helix B of repeat 2 (Figure 4). Its side chain is highly exposed and does not interact with other side chains of the A subunit. Therefore, E64 is likely to interact with the B' subunit directly. Apparently, the side chain of aspartic acid is too short to reach out to the B' subunit. Thus, the ®nding that the E64G mutant does not bind the B' subunit is not surprising. Previously, we found that mutation of proline 179 to alanine also produced a protein that is speci®cally defective in B' binding (Ruediger et al., 1999). This proline, like E64, is located C-terminally to the intra-repeat loop (of repeat 5) and at the beginning of helix B (Figure 4). It is also fully exposed at the surface, and it is reasonable to assume that P179 will be found mutated in some cancers. Furthermore, it is likely that still other amino acids are involved in B' subunit binding, all of which are potential targets for mutation in cancer. The mutants R418W and D171 ± 589 are likely to have a wider range of e€ects than E64D and E64G. Being unable to bind the C subunit and all forms of B subunits, they are expected to cause a general decrease in the levels of both core and holoenzyme and a corresponding increase in free C subunit, which normally is not present in cells. What e€ect the uncontrolled C subunit might have on cell growth (assuming it is not degraded) is an open question. As demonstrated previously, the substrate speci®city of the C subunit di€ers markedly from that of the core and holoenzymes (Cegielska et al., 1994; Kamibayashi et al., 1991). It is possible that homozygous expression of the mutants R418W and D171 ± 589 might be lethal since all control of PP2A function by regulatory

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Figure 4 Location of cancer-associated mutations within the Aa subunit of PP2A. Binding of the mutants to subunits Ba, B'a1, B''/PR72 and C. Shown are repeats 2, 5 and 11 of the Aa subunit, the a helices A and B, and the intra-repeat loops (underlined), as suggested by the X-ray structure (Groves et al., 1999). At the bottom, the amino acid positions within repeats are indicated. Binding of the mutants is expressed as a percentage of wild type Aa subunit binding. The values from 2 ± 3 independent experiments are shown. *The values for the proline to alanine mutation (P179A) are taken from a previous publication (Ruediger et al., 1999); n.d., not done Oncogene

Defective PP2A subunit interaction in human cancer R Ruediger et al

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Oncogene

subunits is lost. On the other hand, heterozygous expression of these mutants may stimulate cell growth without causing death. R418 assumes an equivalent position in repeat 11 as E64 and P179 in repeats 2 and 5, respectively (Figure 4). In addition, R418 is fully exposed and makes no interactions with other residues of the A subunit. Therefore, we reason that R418 interacts directly with the catalytic subunit. Mutation of the nearby Lys416 to Glu also causes a defect in C subunit binding (Turowski et al., 1997). Together, our ®ndings further con®rm our model of the A subunit structure and underline the importance of amino acids located in intra-repeat loop regions for binding B and C subunits. We propose that the Aa as well as the Ab subunits will be frequent targets for mutation not only in cancer but in other genetic diseases as well. In support of the latter idea, we have shown recently that transgenic expression of the D5 mutant in the heart of mice causes dilated cardiomyopathy and premature death (Brewis et al., 2000). The list of proteins that associate with PP2A has been growing rapidly in recent years (Virshup, 2000, for review). Some of these proteins bind to regulatory B subunits in the holoenzyme, others to the A or C subunit in the core enzyme. Therefore, loss of binding of B subunits to the core enzyme due to A subunit mutation will abolish associations that depend on B subunits. A speci®c example is the association of PP2A with the APC tumor suppressor, a component of the bcatenin-destabilizing complex that also contains axin and glycogen synthase kinase 3 (GSK3) (Seeling et al., 1999). The function of GSK3 is to phosphorylate bcatenin and marking it for degradation by the ubiquitin/proteasome pathway. Stimulation of the Wnt signaling pathway causes inhibition of GSK3 and accumulation of unphosphorylated b-catenin, which translocates to the nucleus and forms a complex with the T cell factor/lymphocyte enhancer binding factor (TCF/LEF). This complex stimulates growth by inducing the expression of genes such as c-myc, c-jun and cyclin D. The function of PP2A in the Wnt signaling pathway may be to downregulate b-catenin by dephosphorylating and activating GSK3. Stabilization of b-catenin plays an important role in cancer development. Thus, in over 80% of sporadic colon cancers the APC gene is mutated, resulting in a protein unable to form a b-catenin-destabilizing complex. In other cancers, including colon, pancreatic, hepatic and skin cancers, in which APC is normal, mutations of the b-catenin gene alter the GSK3 phosphorylation sites and render b-catenin degradation-resistant (Bienz, 1999; Peifer and Polakis, 2000, for review). A third mechanism for b-catenin stabilization may involve inactivation of PP2A, as proposed by Seeling et al. (1999). These authors found that the PP2A holoenzyme forms a speci®c complex with APC through its regulatory B' (B56) subunit. Furthermore, overexpression of the B' subunit causes proteasomal degradation of b-catenin as well as a reduction in b-catenindependent transcription. Our ®nding that two cancer-

associated point mutants (E64D and E64G) are speci®cally defective in B' subunit binding suggests that these mutants may contribute to cancer development by increasing b-catenin levels through decreased activation of GSK3 by the AB'C holoenzyme.

Materials and methods Mutants of PP2A-Aa Site-directed mutagenesis was performed with the Gene Editor kit from Promega following the manufacturer's instructions. To create the C-terminally tagged point mutants, plasmid pcDNA3-AaEE (Ruediger et al., 1999) was used together with mutagenic oligonucleotides, 36 ± 45 nucleotides in length, which, in addition to the desired mutation, also encoded a silent mutation introducing a new or eliminating a naturally occurring restriction site in order to identify the mutants. All mutations were con®rmed by sequencing (MacConnell Research Corporation, San Diego and UCSD Cancer Center Sequencing Facility). To create the N-terminally tagged deletion mutant D171 ± 589, the plasmid pRc/CMV-EEAa was constructed as follows: A piece of DNA was synthesized that encodes from N to C terminus an EagI site, an NheI site, a G/C rich region, a Shine ± Dalgarno sequence, the start codon, sequence coding for the tag EEEEYMPME, and sequence coding for three alanines and a naturally occurring EagI site (amino acids 2 ± 4 of Aa). This DNA was inserted into clone p16 (Walter et al., 1989), which has an EagI site in its polylinker upstream of the Aa subunit insert. The tagged Aa insert was then removed with NheI and EcoRI, blunt-ended, and inserted into HindIII-linearized and blunt-ended pRc/CMV (Invitrogen) to yield pRc/CMV-EEAa. The mutagenic oligonucleotide, a 62-mer, was designed to imitate the actual mutation occurring in cancer, which introduced one additional T after the second nucleotide of the codon for F170. Although without consequence for F170, this insertion of an extra T creates a frame shift resulting in a truncated protein, whose sequence after F170 is PEPVLRstop. For simplicity, rather than using the more precise name R171PEPVLR-stop, we call this mutant D171 ± 589. In vitro translation, complex formation, immunoprecipitation and quantitation To analyse for B subunit binding to Aa subunit mutants, Aa and B subunits were synthesized separately using Promega's TNT T7 Quick system and L-[35S]methionine (437 TBq/ mmol) (Amersham), followed by complex formation and immunoprecipitation with anti-EE antibodies as described previously (Ruediger et al., 1999). To analyse for C subunit binding to Aa subunit mutants, Aa subunits were synthesized using Promega's TNT coupled reticulocyte lysate system and L-[35S]cysteine (437 TBq/mmol) (Amersham), followed by complex formation and immunoprecipitation with antibody against the C subunit C-terminal peptide KL (KVTRRTPDYFL), as described previously (Ruediger et al., 1992). The immunoprecipitates were analysed by SDS ± PAGE and phosphorimaging as described previously (Ruediger et al., 1999). Acknowledgments We thank Marc Mumby for the Ba and B'a1 cDNA plasmids, and Brian Hemmings for the B''/PR72 cDNA

Defective PP2A subunit interaction in human cancer R Ruediger et al

plasmid. We also acknowledge the UCSD Cancer Center sequencing facility. This work was supported by the

Tobacco-Related Diseases Research Program, grant 8RT0037 and Public Health Service grant CA-36111.

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