Key words: protein phosphatase; PP2A; cancer; tumorigenesis; tumor suppressor

Abstract
The serine/threonine protein phosphatase 2A (PP2A) appears to be critically involved in cellular growth control and potentially in the development of cancer. A few studies indicated that this enzyme might actually exert tumor suppressive function. However, other findings demonstrated the requirement for PP2A in cell growth and survival, which is not a characteristic of a typical tumor suppressor. This apparent discrepancy might be due to the fact that PP2A is a multitask enzyme system, rather than a single enzyme. Its individual subunits are encoded by a heterogeneous group of genes which give rise to a multitude of different PP2A holoenzyme complexes. Thus, the puzzling observation that PP2A exerts inhibitory, as well as stimulatory, effects on cell growth could be due to the activity of different PP2A complexes with distinct subcellular location and divers substrate specificity. At the same time, this abundance of PP2A components provides a large target for mutations that might derail proper enzyme function and could contribute to the process of tumorigenesis. So far however, it has not been unequivocally established whether such mutations, examples of which have indeed been found in human cancer cells, result in the activation of an oncogenic function or rather in the inactivation of the presumed tumor suppressive role of PP2A. Therefore, the general opinion of PP2A as being a tumor suppressor needs to be viewed with caution.

INTRODUCTION
The adequate control of cellular growth and differentiation is a prerequisite for the proper development and functioning of higher eukaryotic organisms. Extracellular molecules, such as hormones and growth factors, are important agents in determining this control. The genetic response of cells to these molecules requires signal receivers (receptors), signal transducers (second and third messengers), and signal converters, i.e. transcription factors that subsequently stimulate or repress the transcription of target genes. As a consequence, the altered pattern of gene expression will generate the respective phenotypic response, such as cell proliferation, differentiation, or apoptosis. The importance of appropriate regulation of these signal transduction pathways has been emphasized by the finding that many components of these networks are products of protooncogenes. If mutated or inappropriately expressed, they become oncoproteins that are able to constitutively activate these pathways in the absence of external stimuli, and thus are able to promote unrestricted cellular proliferation, which eventually may lead to tumorigenesis and cancer (1-3).

A hallmark of these growth-regulatory signal transduction pathways is the reversible phosphorylation of many of their components, which results in the activation or inhibition of the respective substrate's function. Therefore, the opposing and spatiotemporarily well-balanced function of protein kinases and phosphatases is critical for proper cell growth behavior (and most likely for almost every other cellular function as well). Indeed, it has been demonstrated that many (proto)oncogene products are kinases, and their inappropriately increased levels of enzymatic activity can decidedly contribute to the process of tumorigenic transformation (4, 5).

On the other hand, the exact role that protein phosphatases are playing in these events is much less clear. However, the finding that some of these enzymes are crucial components of pathways that regulate cell growth and proliferation has brought them to the forefront of cancer research (6, 7). So far, only very few phosphatases have been directly implicated in the etiology of tumors. For example, the dual-specificity protein phosphatases CDC25A and CDC25B, which are important regulators of cell cycle progression, are able to transform cells in culture and therefore appear to harbor oncogenic potential (8). In contrast, the dual specificity protein phosphatase PTEN exhibits many characteristics of a typical tumor suppressor and is frequently found mutated or deleted in various types of advanced cancers (9). It should be mentioned, however, that in addition to its protein phosphatase activity, PTEN also displays lipid phosphatase activity (10). This latter function appears to determine its tumor suppressive function, although more recent results indicate that the importance of its protein versus lipid phosphatase activity may be cell type specific (11, 12). Another significant regulator of growth-regulatory signal transduction pathways and proliferation is the protein phosphatase 2A (PP2A) (13-16). Several lines of evidence have suggested that PP2A is a negative regulator of cell growth and even might function as a tumor suppressor. However, a couple of observations contradicting this view have emerged and have complicated the assessment of PP2A's role in cell growth regulation and tumorigenesis. It appears that PP2A exerts positive as well as negative functions, and some of these will be presented in detail below.

PP2A AS A HETEROGENEOUS TARGET FOR POTENTIALLY HARMFUL ASSAULT
PP2A is a member of the family of serine/threonine specific protein phosphatases which also contains PP1, PP2B (also called calcineurin), PP2C, PP3, PP4, PP5, PP6, PP7, and possible many more yet to be discovered members (17, 18). The activity of several of these enzymes has been linked to the mechanisms that control the cell cycle, and therefore, potentially, some of these members might be involved in aberrant cell growth and tumorigenesis. The role of PP1 in these processes has been reviewed in detail elsewhere (19), and a few other publications have indicated a correlation of PP2B, PP2C, PP5, and PP6 activity with cell growth (20-24). Among all these phosphatases, PP2A clearly is the best studied and several lines of evidence have established its involvement in cell growth regulation and tumorigenesis.

PP2A is a trimeric complex that is composed of a catalytic C subunit, a structural A subunit, and a regulatory B subunit (15, 25, 26). Each subunit provides a different function: C is the enzymatically active component; B acts as a targeting module that directs the enzyme to various intracellular locations and also provides distinct substrate specificity; and A appears to function primarily as a scaffolding protein that serves to assemble the different subunits into one holoenzyme complex. All of these subunits come in various different isoforms, so that the ABC holoenzyme is a structurally diverse enzyme in which a single catalytic C subunit can associate with a wide array of regulatory subunits (see Figure). The A and C subunits each exist as two isoforms, a and b, whereas the B subunits are made up of four unrelated families named B, B', B" and B'''. Each of these B families is composed of several different members, all of which are able to bind to the A subunit in a mutually exclusive manner to form a distinct ABC holoenzyme complex (see Figure legend for references). Overall, the combination of all subunits could produce more than 75 different trimeric ABC holoenzymes, although it is unknown how many of the possible combinations actually exist in cells. While the A and C subunits are present in all cells, some of the B subunits are expressed in a tissue-specific fashion and at distinct development stages (27).

The different isoforms of the various subunits clearly do not perform redundant functions. For example, knock-out mice in which the a isoform of the catalytic C subunit has been deleted are not viable and die in utero; i.e., the b subunit of the catalytic C subunit cannot make up for the missing a subunit - despite 97% identity in their primary protein sequence (28). Furthermore, complementation data from yeast have confirmed that the different B subunits perform non-redundant functions (29). Considering this complexity of PP2A composition, it is not surprising that this enzyme has been implicated in the regulation of a multitude of cellular functions, such as metabolism, transcription and translation, RNA splicing and DNA replication, development and morphogenesis, as well as cell cycle progression and transformation. Similarly, in view of the abundance of PP2A's different subunits and their ability to form a large repertoire of heteromultimeric holoenzymes, one would expect that this enzyme provides a large target for deleterious mutations or other detrimental impacts that may derail its proper cellular function. Indeed, a few of such harmful events have been discovered and will be described further below.

Besides the plenteousness of subunit composition, there are other covalent and noncovalent mechanisms that contribute to the regulation of PP2A's enzymatic activity, its substrate specificity, subunit assembly, or its subcellular localization. For instance, reversible phosphorylation (30) and methylation (31-33) of the catalytic C subunit has been shown to strongly affect PP2A activity, indicating that kinase, methyltransferase, and methylesterase enzymes are part of the PP2A-regulatory network. Furthermore, heat stable inhibitors (34), several other proteins (14, 15), as well as certain lipid second messengers such as ceramide (35), have been implicated in the management of PP2A function. Little is known, however, how these various regulatory forces are coordinated and integrated to direct the appropriate function of PP2A during its multifaceted tasks. Nonetheless, it is to be expected that these additional components of control may provide yet another group of targets whose malfunctioning could impinge on proper PP2A function - and thus might contribute to aberrant cell growth and potentially tumorigenesis.

PP2A AS A COMPONENT OF CELLULAR GROWTH CONTROL
Two major avenues of research have provided important clues as to the potential contribution of PP2A to cellular growth control. One was the discovery that okadaic acid, a tumor promoter, is a potent inhibitor of PP2A activity, implying a function of PP2A in the process of tumor promotion. The second piece of evidence was the finding that some proteins of DNA tumor viruses are able to form stable complexes with PP2A and thereby manipulate its enzymatic activity. This latter observation indicated that PP2A might be involved in the transformation process effected by these viruses. Both of these advances and their implications will be presented below.

Okadaic acid is a complex polyether derivative of a 38-carbon fatty acid that is synthesized by marine dinoflagellates (36, 37). Via its accumulation in filter feeding organisms such as mussels, it can gain entry into the human food chain and cause diarrheic shellfish poisoning. The discovery in 1988 (38) that this compound is able to bind to the catalytic subunit of PP2A and efficiently block its enzymatic activity provided a major impetus to the field of phosphatase research (39). Since okadaic acid had also been demonstrated to be a potent tumor promoter, it was inferred that inhibition of phosphatase activity contributed to tumor promotion - and thus, that PP2A might exert tumor-suppressing function. It was hypothesized (40) that the inhibition of phosphatase activity (by okadaic acid) would generate the same effect on growth-regulatory pathways as the activation of kinase cascades by phorbol ester-type tumor promoters which bind to and activate protein kinase C. In both cases, the balance of reversible phosphorylations would be shifted towards the increased net phosphorylation of cellular proteins. Since then, it has indeed been confirmed that treatment of cells with okadaic acid (or phorbol esters) caused the increased phosphorylation of a large number of cellular proteins (41).

Several growth-regulatory genes that were known to be activated by phorbol esters were also found to be stimulated by okadaic acid, indicating that the two tumor promoters might activate the same signal transduction pathway(s). Most prominent among those genes are the proto-oncogenes c-fos and c-jun, whose protein products are able to interact and form the heterodimeric transcription factor AP-1 (42) (for a detailed list of growth-regulatory genes that are controlled by okadaic acid, and the respective references, see (43)). The c-fos and c-jun genes belong to the group of immediate-early genes that are stimulated in response to treatment of cells with serum or growth factors, i.e. agents that stimulate cell proliferation. If appropriately mutated, both genes can be converted to oncogenes and contribute to the process of tumorigenic transformation (44). As PP2A is one of the major receptors for okadaic acid -and is inactivated by this compound (38)- it was concluded that PP2A might be responsible for the suppression of elevated c-fos and c-jun expression, and thereby might antagonize the activity of growth-stimulatory signals (see detailed references in (43)). This idea was supported further by experiments where microinjected PP2A was shown to inhibit the stimulation of c-fos gene expression in response to serum stimulation of cells (45).

The above studies were expanded to characterize the potential role of PP2A in the respective signal transduction pathways that governed the expression of those growth-regulatory genes. The mitogen activated protein (MAP) kinase pathway plays a central role in mediating transcriptional regulation in response to the stimulation of cell surface growth factor receptors (46). This pathway consists of the (proto)oncogene products Ras and Raf, MAP kinase kinase (MKK), and MAP kinase. It usually is only transiently activated, and its constitutive activation is sufficient to cause the tumorigenic transformation of susceptible cells (47). Treatment of cells with okadaic acid leads to the activation of this pathway (48), and numerous experiments have established that PP2A is a major negative regulator that acts at multiple points in this cascade (14, 49). From these observations, it has been concluded that PP2A might be important for the reversal of the activated MAP kinase pathway back to its pre-stimulatory, inactive state. Thereby, PP2A would not only act as a negative regulator of cell growth, but would also contribute to the transient nature of this activation and thus help avoid refractoriness, i.e., it would ensure the availability of the pathway for the next stimulus (14). It should be noted that in addition to PP2A, several other protein phosphatases, the MAP kinase phosphatases (MKPs), have been discovered that also contribute to the dephosphorylation and regulation of MAP kinases (50). The relative contributions of PP2A and MKPs to the regulation of MAP kinase signaling appear to vary, depending on the cell type and agonist under investigation. The reasons for this are currently unclear, but conceivably could be related to the presence of different forms of PP2A holoenzyme in different cells.

In addition to its effects on MAP kinases, PP2A has been found to form stable complexes with several other kinases, and numerous kinases have been identified as substrates of PP2A (see detailed references in (14)). In most instances, the targeted kinase is inactivated by PP2A, indicating a negative influence of PP2A on the respective signal transduction pathways. However, there are a few exceptions in which the action of PP2A results in the activation of the kinase substrate or the respective signal transduction pathway (see below).

The above mentioned similarities in cellular responses to the two tumor promoters, okadaic acid and phorbol esters, contributed in a major way to the view that PP2A might act as a negative regulator of cell growth and potentially even as a tumor suppressor (40, 51). However, there are also numerous observations that complicate this picture. For instance, even though okadaic acid treatment of cells leads to the increased phosphorylation of a large group of proteins, this group is not identical to the group of proteins that is phosphorylated in response to treatment of cells with phorbol esters (41, 52). In addition, there are several physiological differences between okadaic acid-generated and phorbol ester-generated transformed cells (53, 54). Similarly, the ability of okadaic acid to induce apoptosis has been well documented (55-58), and several cancer cell lines have been described where phorbol esters oppose this effect (59, 60). Moreover, independent groups reported on the puzzling observation that okadaic acid could act as an inhibitor of transformation in some in vitro transformation assays (61, 62), which is in stark contrast to those reports mentioned above that established this compound as a potent tumor promoter. Whereas the details of these apparent discrepancies remain to be investigated, the above data indicate that phorbol esters and okadaic acid, despite some similarities, also generate quite different consequences for the normal and transformed cellular phenotype.

The use of okadaic acid for the study of PP2A function became severely restricted with the discovery that the activity of other, newly discovered phosphatases could also be blocked at low concentrations of this drug. While PP1 was one hundred-fold less sensitive than PP2A, novel phosphatases such as PP4, PP5, and PP6 were shown to be efficiently inhibited by okadaic acid in the nanomolar range (for detailed references, see (16, 18)). Therefore, those experiments that exclusively rely on the use of okadaic acid need to be interpreted with caution as other serine/threonine-specific phosphatase(s) besides PP2A may be involved. The potential inhibition of phosphatases other than PP2A during okadaic acid-induced processes may also explain some of the apparent discrepancies that have been obtained with the use of this drug. More unequivocal data are obtained through a combination of different phosphatase inhibitors with distinct affinities for the various enzymes (63, 64), or, alternatively, through the application of diverse biochemical and immunological approaches (65, 66). In this regard, the microinjection of PP2A subunits or the respective antibodies has yielded valuable new insight into PP2A function (45, 66).

A further major finding to implicate PP2A in the processes of cell growth regulation and transformation was the discovery that certain proteins of DNA tumor viruses were able to bind to PP2A and affect its enzymatic activity (67, 68). These proteins include the small and medium T antigens of polyoma virus, and the small T antigen of simian virus 40 (SV40). Each of these proteins is able to associate with the AC core and displace the B subunit in the PP2A holoenzyme (for detailed references, see (69). In contrast to okadaic acid, which binds to the catalytic C subunit and effects complete inhibition of PP2A, these antigens bind to the structural A subunit and differentially reduce PP2A's enzymatic activity (70). Similar to the effects of okadaic acid, expression of these DNA tumor virus antigens lead to the activation of the MAP kinase pathway in the absence of growth factor-initiated signaling (49). It is thought that the reduction of PP2A activity is necessary in order to execute the transforming function of the respective DNA tumor virus (71). Similarly, a role for PP2A in the expression of human papilloma virus has been discovered which led to the suggestion that PP2A might play a role in the generation of human genital cancers (72). A few other examples have supported the view of PP2A as a potential inhibitor of transformation. For instance, PP2A can be inactivated in response to cell growth stimulation, i.e. by receptor tyrosine kinases or the (proto)oncogene product c-src, which is a non-receptor tyrosine kinase (73). PP2A has also been reported to inhibit the activity of telomerase (74), an enzyme that is mechanistically implicated and frequently found elevated in cancer cells (75). Furthermore, certain oncogene products, for example, the Hox11 and SET proteins, are able to interact with PP2A and impair its function (76, 77). Another intriguing, yet not well understood aspect of PP2A function is its association with the APC tumor suppressor (78), a protein that is mutated in over 80% or sporadic colon cancers; it is thought that the PP2A-APC complex plays a role in the turn-over of the ß-catenin protein, whose stabilization plays an important role in the development of cancer (79). In other studies, experimentally increased levels of PP2A catalytic subunit reduced the rate of transformation of mouse fibroblasts by the Ha-ras and the polyoma virus medium T oncogenes in vitro (80). This latter example provided the first demonstration that PP2A directly interfered with oncogenic transformation.

PP2A As A Positive Component of Cellular Functions
Despite numerous instances where PP2A has been found to negatively impinge on cell growth and proliferation, there are, surprisingly, a couple of findings that clearly indicate that PP2A is able to exert positive effects as well. In general terms, PP2A is an essential gene, as mice lacking the a isoform of its catalytic subunit are not viable and die in utero (28). While the primary germ layers ectoderm and endoderm are formed in these embryos, mesoderm is not formed, indicating that PP2A is required for the proper development of the embryo. Thus, despite of ample evidence (see above) that the impaired function of PP2A might contribute to tumorigenesis, it appears that the complete lack of this activity is quite detrimental to cell growth and survival. This view is further supported by the observation that long-term treatment of cells in culture with okadaic acid at concentrations that completely inhibit PP2A results in severe cytotoxicity (see detailed references in (39).

One particular function of PP2A that has been shown to directly impact the ability of cells to proliferate is its role in DNA replication. In this regard, it has been demonstrated that PP2A was required for the initiation of DNA replication in yeast, viral, and vertebrate systems (81-83). While neither the assembly of the prereplicative complexes nor the elongation of the replication forks is dependent on PP2A, the firing of the replication origins is suppressed in the absence of the phosphatase (83). Further recent studies revealed that one of the B" subunits of PP2A, PR48, is able to interact with Cdc6, which is a protein that is required for the formation of the prereplicative complexes and is rate limiting for initiation of DNA replication (84). Together, these results indicate that the initiation of eukaryotic DNA replication depends on dephosphorylation, and that PP2A is an essential component of this process.

In certain signal transduction pathways, PP2A has been found to have stimulatory effects as well. For example, PP2A has been identified as a positive regulator of Raf-1 function (85). Raf-1, a (proto)oncogene product, is an upstream component of the MAP kinase pathway and functions as a MAP kinase kinase kinase (86). Its activity is controlled, among other factors, by an inhibitory phosphorylation of a serine at position 259. PP2A is able to form stable complexes with Raf-1 and thereby appears to facilitate kinase activation by maintaining serine 259 in a dephosphorylated state (85). This finding was quite surprising, as PP2A had been shown before to inhibit other, further downstream components of the MAP kinase pathway (see above). It is not clear at this point what the exact implications of these seemingly contradictory events are. However, previous data showing impairment of Raf-dependent transformation by the tumor promoter okadaic acid are consistent with a positive role of PP2A in the activation of the Raf kinase (87). Other recent studies have indicated stimulatory functions of PP2A in signal transduction pathways initiated by the Wnt family of secreted glycoproteins which regulate biological processes such as cell growth, cell polarity, and tissue specification - and thus further added to the positive tasks of this phosphatase in cellular regulation (88).

Another area of cell growth regulation where positive -as well as negative- roles of PP2A were confirmed is the cell cycle. Progression of cells through the various phases of the cell cycle is governed by the sequential activity of different cyclin-dependent kinases (CDKs) which are required at specific points throughout the cycle (89). PP2A appears to have stimulatory effects during the early phases of the cell cycle by affecting the activity of G1-specific CDK complexes. In particular, the inhibition of PP2A activity results in the decreased expression of cyclin D2, cyclin E, and cyclin A, which are essential regulatory subunits of the respective CDK complexes. As a consequence, the unavailability of cyclins results in severely impaired CDK activity, and the cells become arrested in the G1 phase of the cell cycle (90, 91). Thus, combined with the results presented further above, these studies clearly indicate that PP2A activity is required for cells to progress through the early phases of the cell cycle and to enter S phase. This positive function of PP2A obviously contrasts with its well-established negative role during the G2/M transition. In this case, both genetic and biochemical studies have revealed that PP2A inhibits the activation of the M phase-specific CDK, the cdk1/cyclin B complex, which is essential for entry and progression through this phase of the cell cycle (77, 92-95). The molecular basis for these opposing functions of PP2A is currently unclear, but could be due to the actions of distinct PP2A holoenzymes that are targeted at different G1 versus G2/M phase substrates.

Considering the various known roles of PP2A, it becomes clear that there are multitudes of cellular tasks that are regulated by this phosphatase. Intriguingly, as outlined above, positive as well as negative functions of PP2A appear to be quite prominent, and some of these opposing activities may impinge even onto the same cellular process, such as, for example, stimulation of Raf-1 but inhibition of MAP kinase, both of which are components of the same signal transduction pathway. In light of the diversity of PP2A subunits and their ability to form a large repertoire of different holoenzymes, PP2A has been more appropriately described as a multitask enzyme system rather than a single enzyme (15, 96-98). The characterization of the relevant combinations and the assignment of specific roles to each of them will be an important and challenging task for the future.

PP2A As A Potential Tumor Suppressor
Based on the observations that okadaic acid is a strong inhibitor of PP2A, yet at the same time a potent tumor promoter, it was suggested that PP2A might be a tumor suppressor (40). However, considering the above-presented evidence of growth-stimulatory, as well as growth-inhibitory, functions of PP2A, it is difficult to certify PP2A as an obvious tumor suppressor. Clearly, PP2A differs from classical tumor suppressor genes, such as p53 or retinoblastoma (RB), in that the latter genes are not essential for cell growth and development, i.e., mice that are homozygously deficient in these genes are fully viable, but are prone to the development of tumors during their life (99). On the other hand, similar to PP2A, the above mentioned tumor suppressor phosphatase PTEN has been shown to be an essential gene as well, as knock-out mice die during embryonic development (100, 101). However, in contrast to PP2A-negative embryos, which are deficient in the development of a germ layer, PTEN-negative embryos harbor regions of increased proliferation, which is consistent with the absence of a tumor-suppressing function. At the cellular level, the deletion or inactivation of classical tumor suppressor genes, such as p53 or Rb, appears to promote, rather than restrict proliferation. In the case of the PTEN phosphatase, deletion of its gene confers onto cells the ability to proliferate in suspension, i.e. under anchorage-independent conditions, which is a characteristic of many tumor cells (102, 103). In contrast, no proliferation-stimulatory aspects are observed under conditions where PP2A activity is completely inhibited. As mentioned above, concentrations of okadaic acid that are sufficiently high to fully block all of PP2A's enzymatic activity are very toxic to cells. However, on occasion, cells are able to escape this inhibition either by amplifying genes encoding membrane pumps and thereby developing a multi-drug resistant phenotype, or by experiencing mutations in the PP2A catalytic subunit that reduce its sensitivity to inhibition by the drug (see detailed references in (16)). In yeast, deletion of the PP2A catalytic subunit gene is lethal (92, 104), and no cell lines that are derived from those early PP2A knock-out mouse embryos have been published (even if these cell lines existed, it could be argued that the presence of the ß-isoform of the catalytic subunit might substitute for at least some of the functions of the deleted a-subunit). Thus, in view of the fact that in addition to its negative roles in cell proliferation, PP2A clearly exhibits positive, even essential, functions as well, it may not be surprising after all that cells depend on PP2A in order to grow and proliferate. This may also be the reason why -in obvious contrast to conventional tumor suppressors- inactivating mutations or deletions of the PP2A catalytic subunit have never been found in any human tumors. From this, it should be concluded that PP2A does not easily qualify as a tumor suppressor.

However, while the considerations above do not agree with the notion that PP2A might be a tumor suppressor, it needs to be kept in mind that this enzyme is a multimeric complex composed of a variety of different subunits. Thus, although the catalytic subunit itself does not appear to qualify as a tumor suppressor, it is still possible that the complex as a whole, or rather its activity as such, might function in a tumor suppressive manner. In this regard, instead of being a matter of an all-or-nothing function (i.e., phosphatase activity 'on' or 'off'), it might be a matter of varying levels of quantity and quality (i.e., different complexes exhibiting distinct degrees of phosphatase activity, selective substrate specificity, and specific intracellular localization). It is conceivable, therefore, that tumor suppressive function might be achieved through the activity of a small subset of PP2A complexes that consist of particular combinations of subunits. By the same token, many PP2A aggregates that are not involved in cellular growth regulation are quite likely to exist and involved in some of the many other cellular processes that are affected by this phosphatase.

As mentioned above, inactivating mutations or deletions of the catalytic C subunit of PP2A have never been detected in human tumors, which probably is a reflection of this gene's indispensability for adequate cellular function and survival. Intriguingly, however, several studies were published that reported on the discovery of cancer-associated mutations in its structural A subunit, which suggested that the gene for this subunit could be the actual tumor suppressor (see below). In general, all PP2A complexes present in cells contain one molecule of A subunit, which appears to act as a scaffold, i.e., it is required for the assembly of the ABC holoenzyme and the recruitment of B-type subunits into the complex (see Figure). Studies using extensive site-directed mutagenesis have demonstrated that the catalytic subunit binds to the C-terminal part of the A subunit, whereas any one of the B-type subunits (B, B', B") binds to the N-terminal region of the A subunit (70). The finding of mutations in the A subunit in human tumor tissue was highly intriguing in view of the postulated role of PP2A as a tumor suppressor. The first of these studies (105) described that the gene encoding the ß isoform of the A subunit (Aß) was altered in 15% of primary lung tumors, in 6% of lung tumor-derived cell lines, and in 15% of colorectal carcinomas. Further studies reported that both the a as well as the ß isoforms of the A subunit were genetically altered at low frequency in a variety of primary human tumors (106, 107). The types of alterations included deletions, frameshifts, point mutations, and splicing abnormalities. It remains unclear, however, whether these mutations are causal or incidental, because it has not yet been investigated whether the introduction of the wild type A subunit gene into any of these tumor cell lines would restore normal cell growth. Furthermore, it was pointed out that several of the patients' samples had mutations in only one allele, which suggests that in some cases the mutant A subunit actually might act as a dominant oncogene (15).

So far, the various studies of PP2A subunit mutations in human cancers, although intriguing, have not unequivocally supported a tumor suppressive role of this enzyme. A recent study by Campbell and Manolitsas (108) concluded that the designation of the A subunit as a tumor suppressor should be regarded with caution. These authors found that the mutations in the Aß gene that they detected in primary ovarian cancers were not statistically different from the mutations present in non-cancer random controls. Moreover, at least one of the mutations appears to represent a non-pathological polymorphism, which raises the possibility that some of the other reported mutations of the A subunit may also be of no pathological relevance.

If any of the above discussed alterations in the A subunit gene were actually relevant for the process of tumorigenesis, one would expect that these mutations result in a protein that could modify the properties and functions of PP2A. For some of these cancer-associated mutations, this has indeed been demonstrated - at least in vitro, so far. In a study by Ruediger et al. (109), the authors investigated four different mutants of the Aa subunit that were previously described in various human tumors, and discovered that all four were defective in their binding of other PP2A subunits. Two of the mutants specifically could not bind the B' subunit, whereas the other two mutants were deficient in binding to the C subunit as well as all forms of B subunits. Although it remains to be established whether and how these altered subunit interactions affect cellular growth control, it is reasonable to assume that such changes would result in specific imbalances of cellular control. In this regard, it has been shown that transgenic expression of an A subunit that is incapable of binding to B subunits causes dilated cardiomyopathy and premature death in mice (110).

The observation that certain cancer-associated mutations of the A subunit specifically interfere with binding to certain B-type subunits further suggests that the third component of the ABC holoenzyme complex, the B subunit, could decidedly contribute to PP2A's cell growth-regulatory function. In this regard, PP2A's potential tumor suppressing function could be provided by distinct B subunits. This idea is appealing in light of the above presented findings that the small and medium T antigens of certain DNA tumor viruses are able to replace the B subunits in the holoenzyme and thereby manipulate PP2A activity during the virus-induced transformation process (68, 111).

In the B16 mouse melanoma model, a member of the B' family of subunits, B56g, was found to be highly expressed in metastatic cells, but not in the corresponding non-metastatic tumor cells or normal melanocytes (112, 113). Additionally, this gene was also found overexpressed in a number of human tumor cell lines and clinical samples from cancer patients, demonstrating a correlation of increased expression of B56g with tumor progression (113). In independent studies, Ito et al. (112) discovered the insertion of a retrotransposon into the B56g gene that resulted in an N-terminally truncated splice version (Dg1). These authors could demonstrate that the Dg1 isoform of B56g, when introduced into weakly metastatic B16 melanoma cells, resulted in highly metastatic versions of these cells. At the molecular level, it appeared that the B56g subunit was necessary for the recruitment of PP2A into focal adhesion complexes, and that the Dg1 isoform of this protein compromised the ability of PP2A to properly dephosphorylate certain components of these complexes. Considering the physiological roles of focal adhesion complexes, it was concluded that insufficient activity of the Dg1-containing PP2A heterotrimer could cause adhesion, spreading, migration, and invasion, which are characteristic features of malignant cells. In this regard, these studies provided a compelling model for earlier suggestions that PP2A might limit tumor motility (114-118) and suggested that this particular PP2A holoenzyme complex might function as a suppressor of the metastatic phenotype.

Summary
PP2A is an enzyme system that consists of a large repertoire of distinct heteromultimeric holoenzyme complexes. Considering this diversity, it is not surprising that multiple, sometimes even apparently opposing, functions of these enzymes have been described. Although it remains to be established, it is likely that the different roles can be ascribed to particular representatives of this complex enzyme system. Similarly, while it is evident that PP2A is a major component of signal transduction and cell growth regulation, it is not clear which specific holoenzyme regulates which aspect of these processes. Obviously, PP2A exerts positive as well as negative control on cell proliferation, and this might be a reflection of the activity of different holoenzymes during these processes. Because of its complexity, PP2A cannot easily be classified as a tumor suppressor - although certain members of this enzyme system indeed might have tumor suppressive roles. Complete loss of PP2A function does not contribute to tumor formation, as this enzymatic activity is essential for cell growth and survival. Thus, its potential tumor suppressive capacity might reside in the ability of particular regulatory B subunits to target the holoenzyme to specific locations within the cell and to modulate its activity at the respective subcellular area. The finding of cancer-associated mutations in the A subunit that prevent binding of specific B subunits might be indicative of such a scenario. In this case, the presumed tumor suppressive function that is provided by certain B subunits could be inactivated through mutations in either the A or the B subunits. Thus, in conclusion, the question as to whether PP2A is a tumor suppressor can be unequivocally answered with: "probably yes and no."

Acknowledgements
Research in the author's laboratory is supported in part by Public Health Service grant CA74278 from the National Cancer Institute and by the Margaret E. Early Medical Research Trust. Michael R. Stallcup (University of Southern California) is appreciated for reading of this manuscript.

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Figure 1
Each PP2A holoenzyme is composed of three subunits as shown: a catalytic subunit (C), a regulatory scaffolding subunit (A), and a highly variable regulatory targeting subunit (B). The A and C subunits each exist as two isoforms, a and b, whereas the B subunit is derived from one of four structurally unrelated families named B, B', B" and B'''. Each of the different B families contains several members as indicated. For detailed references on these subunits, see review by Janssens and Goris [97].

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