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. Author manuscript; available in PMC: 2011 Dec 18.
Published in final edited form as: Life Sci. 2010 Oct 8;87(23-26):659–666. doi: 10.1016/j.lfs.2010.10.003

Functions of B56-Containing PP2As in Major Developmental and Cancer Signaling Pathways

Jing Yang 1,1, Christopher Phiel 1
PMCID: PMC2993835  NIHMSID: NIHMS243865  PMID: 20934435

Abstract

Members of the B’/B56/PR61 family regulatory subunits of PP2A determine the subcellular localization, substrate specificity, and catalytic activity of PP2A in a wide range of biological processes. Here, we summarize the structure and intracellular localization of B56-containing PP2As, and review functions of B56-containing PP2As in several major developmental/cancer signaling pathways.

Introduction

The reversible phosphorylation of proteins, regulated by opposing functions of protein kinases and protein phosphatases, is one of the major mechanisms that controls the stability, localization, and function of numerous proteins and is essential for all aspects of biology. During phosphorylation, protein kinases transfer phosphate groups from ATP to the hydroxyl side chain of three amino acid residues: serine (Ser), threonine (Thr), and tyrosine (Tyr). In humans, the majority of phosphorylation occurs on Ser and Thr residues and is catalyzed by more than 400 Ser/Thr protein kinases (Manning, et al. 2002). These kinases often recognize specific peptide sequences and exhibit distinct substrate specificities. In contrast to the large number of protein kinases, there are only a dozen of genes that encode catalytic subunits of phosphoprotein phosphatases (PPPs). Five of them encode catalytic subunits of PP1 (Sasaki, et al. 1990) and PP2A (Stone, et al. 1987), the two most abundant Ser/Thr protein phosphatases. Catalytic subunits of PP1 and PP2A have fairly broad substrate specificity and are capable of dephosphorylating many phophoproteins in vitro. In the cell, however, PP1 and PP2A catalytic subunits are always associated with other subunits to form multimeric enzymes. These subunits often modify the substrate specificity and catalytic activity of the enzyme and localize catalytic subunits to specific intracellular compartments or protein complexes.

In the case of PP2A, PP2A exists predominantly as a heterotrimer. The holoenzyme consists of a catalytic subunit (C), scaffold subunit (A), and variable regulatory subunit (B). C subunits are encoded by two distinct genes, Cα and Cß (Stone, et al. 1987). These two proteins are abundantly expressed and make up to 0.1% of total cellular proteins (Ruediger, et al. 1991). A subunits, also known as PR65, are encoded by two distinct genes as well (Hemmings, et al. 1990). While Aα and Aß share 87% identity, Aα is found in ~90% of PP2A holoenzymes; only 10% of PP2A holoenzymes contain the Aß subunit. The diversity of PP2A holoenzymes derives largely from B subunits. The human genome contains at least 15 regulatory subunits of PP2A, falling into four regulatory subunit families. These include B/B55/PR55, B’/B56/PR61, B”/PR72, and B’”/PR93/PR110. In addition, many PP2A regulatory subunits are alternatively spliced/translated. This ensures assembly of a large number of distinct PP2A heterotrimeric holoenzymes. Interestingly, formation of the heterotrimeric complex not only affects the substrate specificity and subcellular localization of PP2A, but also regulates the stability of PP2A subunits. In Drosophila S2 cells, knockdown of B subunits accelerates turnover of A and C subunits, and vice versa (Li, et al. 2002,Silverstein, et al. 2002). Similarly, mammalian C and most B (B/B55/PR55 and B’/B56/PR61) subunits are stable only when they complex with the A subunit (Chen, et al. 2005,Li and Virshup 2002,Sablina, et al. 2007,Strack, et al. 2004,Strack, et al. 2002). Monomeric subunits are degraded rapidly through the ubiquitin/protesome protein degradation pathway (Strack, et al. 2004,Strack, et al. 2002). Interestingly, it has been noted that the stability of yeast PP2A subunits is not linked to heterotrimer formation (Gentry and Hallberg 2002,Wei, et al. 2001,Wu, et al. 2000).

The purpose of this review is to provide a focused discussion on the structure, intracellular localization, and functions of B’/B56/PR61-containing PP2A holoenzymes, with an emphasis on their functions in major developmental and cancer pathways. Due to the scope of the paper, post-translational modification of A or C subunits will not be discussed, although it plays essential roles in regulating PP2A holoenzymes. We must also omit some important functions of B56-containing PP2As, for example, regulation of circadian rhythms (Sathyanarayanan, et al. 2004), some important transcription factors (Firulli, et al. 2003), and nutrient signaling by B56-containing PP2As (Yan, et al. 2010). Due to the page limit, we have to apologize to some colleagues for not discussing their elegant studies on phosphorylation of B56 (Ahn, et al. 2007,Letourneux, et al. 2006,Margolis, et al. 2006,Ruvolo, et al. 2008,Usui, et al. 1998). Readers are referred to a number of recent reviews for information about other forms of PP2A and other protein phosphatases (Eichhorn, et al. 2009,Janssens, et al. 2008,Shi 2009,Virshup and Shenolikar 2009). From here on, B’/B56/PR61 will be referred to as B56.

Structure of B56 regulatory subunits

All five mammalian B56 family members, including B56α (PPP2R5A), B56β (PPP2R5B), B56δ (PPP2R5D), B56ε (PPP2R5E), and B56γ (PPP2R5C), were discovered in the mid 1990s (Csortos, et al. 1996,McCright, et al. 1996,McCright, et al. 1996,McCright and Virshup 1995,Tehrani, et al. 1996). Later, B56 subunits in other species were identified. Unlike vertebrates (from zebrafish to mammals), lower organisms such as fission yeast (Tanabe, et al. 2001,Tehrani, et al. 1996) and Drosophila (Berry and Gehring 2000) have only two B56 subunits, indicating that the B56 family is expanded during evolution. In vertebrates, B56δ (Tanabe, et al. 1997,Tanabe, et al. 1996) and B56γ (McCright, et al. 1996,Muneer, et al. 2002,Tehrani, et al. 1996) genes are alternatively spliced, with each giving rise to at least three splicing variants (Csortos, et al. 1996). B56ε is alternatively translated (Jin, et al. 2009) (Figure 1).

Figure 1. Schematic drawing to show the structure of B56 family members.

Figure 1

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Top panel shows the structure of the B56 core domain. All 18 a helices are numbed and HEAT-like repeats are coated with different colors. Amino acid residues making direct contact with A and C subunits of PP2A in the crystallized holoenzyme are indicated by green and red arrows, respectively. Positions of ASBD1 and ASBD2 were underlined with blue lines. Bottom panels are schematic representation of B56 family members. Of note, B56γ and B56δ have 3 alternative splicing isoforms, whereas B56ε has an alternative translation isoform.

B56 subunits share a highly conserved B56 core domain (~400 amino acid residues), which is responsible for binding to the A/C heterodimer. The B56 core domain contains 18 α helices, which are organized into 8 HEAT-like (huntingtin-elongation-A subunit-TOR-like) repeats (Figure 1). The crystal structure of the PP2A holoenzyme containing Aα, B56γ1, and Cα has been determined by two groups independently (Cho and Xu 2007,Xu, et al. 2006). In the crystallized holoenzyme, 18 α helices of B56 stack against each other and form a superhelical structure. B56 uses a number of highly conserved amino acid residues to interact with the A and C subunits. Positions of residues making direct contact with the A and C subunits are indicated in Figure 1. Residues which physically interact with the A subunit are located in HEAT2, HEAT5 and HEAT6, while residues interacting with the C subunit are mainly located in HEAT6, HEAT7 and HEAT8. In addition to the B56/C and B56/A interactions, the C-terminal tail of the C subunit docks at the interface of the A and C subunits. It has been proposed that the C-terminal tail of the C subunit plays a role in recruiting the B56 subunits to the A/C heterodimer and enhances the binding between the A subunit and B56. As the consequence of holoenzyme assembly, B56 is placed to a position close to the active site of the C subunit and modifies the conformation of the substrates docking site of the holoenzyme (Cho and Xu 2007). It is important to mention that some viral proteins function by disrupting the assembly of B56-containing PP2A holoenzymes. For example, simian virus 40 (SV40) small t antigen and polyoma small t and middle T compete with B56s for A subunit binding (Chen, et al. 2007,Cho, et al. 2007,Ruediger, et al. 1999,Ruediger, et al. 1994,Ruediger, et al. 1992). In addition, mutations, which affect the interaction between B56s and the A subunit, have been identified from breast cancer and lung cancer cells (Chen, et al. 2005).

Surprisingly, some disagreements between crystal structural analysis and other biochemical studies have been noticed. Earlier biochemical analysis identified two A subunit binding domains (ASBD) within the B56 core domain. ASBD1 is located between the 6th and the 10th α helices, whereas ASBD2 lies within a region containing the 12th-15th α helices (Li and Virshup 2002). The HEAT2, which directly interacts with the A subunit in the crystallized holoenzyme, does not interact with a GST-tagged A subunit in the in vitro GST pull-down assay. By contrast, the ASBD2, which binds strongly to the A subunit in vitro, does not make any direct contact with the A subunit in the crystal structure. Intriguingly, the HEAT1 of the core domain, which is located far away from the C subunit and does not interact with the A subunit, is required for the formation of the heterotrimeric holoenzyme. B56ß-Δ1-99, a trunvated B56ß lacking the N-terminal region and the first α helix of the HEAT1, dose not interact with A and C subunits in vivo (Saraf, et al. 2010). A similar observation has been made with B56ε-s, a naturally occurring alternative translation isoform of B56ε which lacks the N-terminal sequence and the first α helix of the core domain (Jin, et al. 2009). In this case, B56ε-s cannot be co-immunoprecipitated by the anti-PP2A C antibody or by microcystin beads (Jin, et al. 2010). An explanation of these results is that some sequences of the B56 core domain may affect holoenzyme assembly indirectly through the folding of B56 proteins. Alternatively, assembly of B56-containing PP2A holoenzyme may be a dynamic process. While only a few regions on the surface the B56 core domain interact with the A and C subunits in the holoenzyme, transient interactions between the B56 core domain and A-C heterodimer may be required for the assembly of B56-containing PP2A holoenzymes. In support of this hypothesis, Magnusdottir et al. observed several significant conformational changes of the B56γ/PP2A-C interaction surface during holoenzyme assembly (Magnusdottir, et al. 2009). Clearly, more experimental evidence will be needed to thoroughly understand assembly of B56-containing PP2A holoenzymes.

Intracellular localization of B56-containing PP2A

An important function of B56 subunits is to localize PP2A holoenzymes to specific intracellular compartments. Since monomeric B56s are degraded rapidly in vivo (Chen, et al. 2005,Li and Virshup 2002,Sablina, et al. 2007,Strack, et al. 2004,Strack, et al. 2002), localization of B56s reflects the intracellular distribution of B56-containing PP2A holoenzymes. During mitosis, B56 family members can be detected at centromeres from the prophase to metaphase (Kitajima, et al. 2006,Riedel, et al. 2006). During interphase, B56α, B56ß, and B56ε undergo nuclear-cytoplasmic shuttling (Flegg, et al. 2010,Jin, et al. 2009). Likely due to a faster nuclear export rate, they are mainly localized in the cytoplasm (McCright, et al. 1996). B56α, B56ß, and B56ε all contain a “LxxxLxxLxL” motif adjacent to the C-terminus of the B56 core domain. Replacing any leucine residues with alanine results in nuclear accumulation of B56α, demonstrating that this “LxxxLxxLxL” motif is a functional nuclear export signal (Flegg, et al. 2010). In fact, the nuclear export signal of B56ε can even overcome the activity of SV40 nuclear localization signal (Jin, et al. 2009). In addition to the C-terminal nuclear export signal, nuclear localization signals have also been identified experimentally from the N-termini of B56α and B56ε. B56α and B56ε accumulate in the nuclei when the nuclear export pathway is blocked (Flegg, et al. 2010,Jin, et al. 2009). Interestingly, B56α colocalized with pericentrin, a centrosomal marker. Based on the observation that both ASBD1 and ASBD2 are capable of mediating the centrosome accumulation of B56α, it has been proposed that B56α is recruited to centrosomes by the A/C herterodimer (Flegg, et al. 2010). This observation suggests that B56-containing PP2As may be involved in some centrosome-dependent cellular processes. Since the basal bodies of primary cilia are derived from centrioles, it would be of interest to determine whether B56-containing PP2A holoenzymes play a role during ciliogenesis. Sequences outside the B56 core domain are divergent among family members. Recent studies have identified an isoform specific subcellular targeting signal from the C-terminus of B56α. In cardiac myocytes, B56α is strongly localized to the M-lines. This M-line localization of B56α requires a physical interaction between ankyrin-B and an unique 13 amino acids C-terminal motif of B56α (Bhasin, et al. 2007). Since this 13 amino acids motif is not present in other B56s, it is likely that B56α has an isoform specific function in cardiac myocytes.

Unlike B56α/ß/ε, B56δ and B56γ play important roles in the nucleus (McCright, et al. 1996). In rat cardiomyocytes, B56γ1 accumulates into nuclear speckles. Nuclear speckles are irregular and punctate sub-nuclear structures that are enriched in pre-messenger RNA splicing factors. Overexpression of B56γ1 attenuates the dynamic structural reorganization of nuclear speckles (Gigena, et al. 2005). In addition, B56γ3-containing PP2A holoenzyme, which is ubiquitously distributed normally, is markedly accumulated in the nucleus at the G1/S border and in S phase and regulates the G1 to S transition (Lee, et al. 2010).

Functions of B56s in major developmental pathways

Currently, functions of B56s during mammalian embryonic development remain unclear. While all five B56s are expressed in embryonic day 7 mouse embryos (Martens, et al. 2004), their spatial expression patterns have not been investigated. In addition, none of B56 genes has been knocked out in mouse. Our knowledge about functions of B56s during development was derived from a few studies performed in lower organisms. These studies, together with studies in tissue culture cells, demonstrate that B56s are involved in a number of major developmental signaling pathways.

Canonical Wnt pathway

The canonical Wnt pathway is an evolutionarily conserved signaling pathway and is involved in numerous biological processes. Wnt ligands regulate the expression of their target genes by controlling the stability/function of ß-catenin destruction complex, a protein complex consisting of APC, Axin, GSK3, and other proteins. The Wnt pathway plays essential roles during embryonic development and tumorigenesis (He, et al. 2004,Logan and Nusse 2004,MacDonald, et al. 2009,Moon, et al. 2002). During axis specification, Wnt signaling is responsible for the development of dorsal tissues such as CNS, notochord, muscle et al., whereas inhibition of the Wnt pathway impairs axis formation (Heasman 2006,Sokol 1999,White and Heasman 2008).

In 1999, Seeling and colleagues reported that B56s interact with APC and promote proteosome-dependent turnover of ß-catenin (Seeling, et al. 1999). As expected, overexpression of B56 family members inhibits secondary axis induced by Wnt and impairs endogenous axis specification during Xenopus embryonic development. Based on epistasis analysis, B56s were placed downstream of Casein Kinase I, GSK3, and Axin, but upstream of ß-catenin in the Wnt pathway (Gao, et al. 2002,Li, et al. 2001). However, other evidence suggests that regulation of Wnt signaling by B56s is more complex. In addition to APC, B56s interact with other components of the Wnt pathway. Using co-immunoprecipitation assay, Li et al. showed that Axin interacts with B56-containing PP2As (Li, et al. 2001). In 2000, Sokol’s group reported that in the yeast-2-hybrid system Xenopus B56ε interacts with Dishevelled, a component of the Wnt pathway acting upstream of the ß-catenin destruction complex (Ratcliffe, et al. 2000). Surprisingly, overexpression of B56ε inhibits ectopic axis formation induced by a N-terminal truncated ß-catenin or by a constitutively active Tcf (Ratcliffe, et al. 2000,Yang, et al. 2003), raising the possibility that B56 family members may have additional functions downstream of the ß-catenin destruction complex as well. It is important to note that these studies all relied on overexpression approaches. While B56s are capable of inhibiting Wnt signaling in overexpression experiments, it remains unclear which B56 family member plays an inhibitory role in the Wnt pathway under physiological conditions. Given that B56 family members share a very high sequence homology, overexpression of a specific B56 may mimic the functions of other B56s, or displace endogenous regulatory subunits from the PP2A heterotrimer and thus act in a quasi dominant-negative fashion. This makes it essential to study roles of B56s in loss-of-function settings.

To date, only a few loss-of-function studies of B56 have been reported. In striking contrast to results from overexpression experiments, B56ε was found to play a positive role in the Wnt pathway. During Xenopus development, B56ε is maternally expressed. After gastrulation, B56ε is abundantly expressed in the developing neural ectoderm (Yang, et al. 2003). A similar expression pattern of B56ε has been observed during zebrafish development (Thisse and Thisse 2004). In Xenopus embryos, depletion of maternal B56ε phenocopies ß-catenin loss-of-function phenotypes and results in ventralization. This ventralization phenotype can be rescued by Dishevelled, ß-catenin, or a constitutively active Tcf, but cannot be rescued by Wnt ligand. This demonstrates that B56ε plays a positive role in the canonical Wnt pathway upstream or at the parallel level of Dishevelled. In addition to axis specification, knockdown of B56ε in the neural ectoderm impairs the formation of midbrain-hindbrain boundary, another well-studied developmental process under the control of Wnt signaling (Yang, et al. 2003). Interestingly, B56ε is alternatively translated (Jin, et al. 2009). The alternative translation isoform B56ε-s, which does not interact with the C subunit of PP2A (Jin, et al. 2010), is required for Wnt signaling (Jin, et al. 2009). It will be of interest to determine whether B56ε-s functions as a naturally occurring dominant negative form, which inhibits the activity of B56-containing PP2As in the Wnt pathway.

Planar cell polarity pathway

The planar cell polarity (PCP) pathway is also known as the non-canonical Wnt pathway. The PCP and canonical Wnt pathways share a number of components, for example, Frizzled and Dishevelled (Fanto and McNeill 2004). The PCP pathway controls the polarity of Drosophila wing hair. During Xenopus and zebrafish development, the PCP pathway coordinates gastrulation cell movement. It also regulates the polarity of stereociliary bundles within the sensory epithelium in the cochlea during mouse development (Barrow 2006,Dabdoub and Kelley 2005,Roszko, et al. 2009).

From a screen for genes regulating the polarity of Drosophila wing hair, Hannus and colleagues found that widerborst (wdb), Drosophila homologue of B56ε, is a component of the PCP pathway and controls the polarized membrane outgrowth of fly wing hair. As expected, knockdown of zebrafish wdb severely impairs convergent extension during gastrulation. WDB localizes to the distal side of a planar microtubule web and is required for localization of Dishevelled and other PCP pathway components in developing wing cells (Hannus, et al. 2002). Like B56ε, wdb is alternatively translated (Hannus, et al. 2002,Jin, et al. 2009). It seems that the full-length and alternative translation isoforms of wdb function redundantly in the PCP pathway (Hannus, et al. 2002). Currently, the detailed molecular mechanism by which WDB/B56ε-containing PP2A regulates the PCP pathway is not fully understood.

Hedgehog pathway

The Hedgehog (Hh) pathway is an evolutionarily conserved developmental pathway. Since the discovery of the Hh mutant about 30 years ago (Nusslein-Volhard and Wieschaus 1980), functions of Hh signaling during development, adult tissue homeostasis, and tumorigenesis have been extensively studied. It has been established that Hh signaling plays fundamental roles during neural tube and limb bud patterning. Disruption of Hh signaling induces cyclopia, impairs cell fate determination in the ventral neural tube, and causes abnormal digit formation (Ingham and McMahon 2001,McMahon, et al. 2003). Hh proteins act as morphogens during vertebrate development (Briscoe 2009,Fuccillo, et al. 2006,Jia and Jiang 2006,McGlinn and Tabin 2006). At the molecular level, Hh signaling regulates the expression of its target genes by controlling the stability/activity of Gli/Ci transcriptional factor (Jiang and Hui 2008,Osterlund and Kogerman 2006,Varjosalo and Taipale 2008). A number of studies demonstrate that WDB/B56ε-containing PP2A is essential for Hh signaling (Jia, et al. 2009,Nybakken, et al. 2005,Rorick, et al. 2007).

In 2005, Nybakken and colleagues reported a genome-wide RNAi screen for Drosophila components of the Hh signaling pathway. About 500 regulators of the Hh pathway were identified from 21,000 transcripts. Several PP2A subunits were found to regulate the Hh pathway positively. These include the C subunit, wdb, and B’ (the other B56 family member in the Drosophila genome). In contrast, knockdown of the B/B55 and B”/PR72 regulatory subunits had little if any effects (Nybakken, et al. 2005), demonstrating that the B56 family is the only regulatory subunit family involved in Hh signaling. Parallel to the unbiased RNAi screen in Drosophila, by characterizing functions of B56ε during eye development, Rorick et al. independently found that B56ε is required for Hh signaling in the Xenopus embryo. Knockdown of B56ε reduced the expression of Hh target genes and induced cyclopia. Based on epistasis analysis, it was proposed that that B56ε regulates Hh signaling at the level of Gli (Rorick, et al. 2007). This hypothesis was confirmed by studies from other groups. By analyzing functions of PP2A and PP4 in the Hh pathway, Jia et al. were able to show that PP2A is a Ci phosphatase in Drosophila. WDB-containing PP2A counteracts kinases to prevent Ci phosphorylation and, consequently, up-regulates the signaling activity of Ci by preventing its proteolytic cleavage (Jia, et al. 2009). It has also been observed that PP2A antagonizes the action of the mammalian target of rapamycin kinase complex 1 (mTORC1) on Gli3 and causes cytosolic retention of Gli3 (Krauss, et al. 2008). Thus, regulation of Ci/Gli by B56-containing PP2As is an important regulatory mechanism of the Hh pathway.

Receptor tyrosine kinase pathway

Receptor tyrosine kinase (RTK) pathways play fundamental roles during cell fate determination, cell proliferation, and cell survival. At the molecular level, RTKs are activated upon binding between growth factors and their receptors. Activation of RTKs triggers a series of intracellular events, which ultimately leads to the activation of the PI3K/Akt pathway and the MAPK pathway.

Regulation of the RTK by B56-containing PP2As has been documented. However, functions of B56-containing PP2As in the RTK pathways seem to be context-dependent. Elegant studies from Strack’s group have demonstrated that B56-containing PP2As is required for growth factor-induced activation of Akt, but not that of ERK, in PC6-3 cells.

Based on the observation that EGF induces Tyr phosphorylation of EGFR in the absence of B56-containing PP2As, it has been concluded that B56-containing PP2As regulate the pathway downstream of EGFR (Van Kanegan, et al. 2005). Interestingly, the same group also found that B56-containing PP2As can act at the receptor level in other contexts. In PC12 cells, B56ß and B56δ dephosphorylate Ser/Thr on NGF receptor and potentiate its Tyr kinase activity (Van Kanegan and Strack 2009). IGF signaling plays critical roles during Xenopus and zebrafish eye induction (Eivers, et al. 2004,Pera, et al. 2003,Pera, et al. 2001,Richard-Parpaillon, et al. 2002). B56ε mediates IGF signaling during Xenopus eye induction. Neural ectoderm-specific knockdown of B56ε completely blocks IGF-induced eye development and severely impairs the initiation of a number of eye field transcription factor in Xenopus embryos. Epistasis experiments reveal that B56ε functions upstream, or at the parallel level of PI3K in this context. In other experimental contexts, however, it has been observed that B56-containing PP2As directly dephosphorylate Akt (Rocher, et al. 2007,Vereshchagina, et al. 2008,Xu, et al. 2009). In fact, PPTR-1, a C. elegans B56 regulatory subunit, is responsible for dephosphorylation of Akt-1 at Thr350 and controls worm life span, dauer, stress resistance, and fat storage (Padmanabhan, et al. 2009). In addition to the PI3K/Akt branch, B56-containing PP2As have been implicated in the MAPK pathway as well. Effects of B56-containing PP2As on the MAKP pathway are, again, context-dependent. In Drosophila S2 cells, knockdown of B56s prevents ERK activation in response to oxidative stress (Liu, et al. 2007). In contrast, B56-containing PP2As dephosphorylate ERK in some mammalian culture cells (Letourneux, et al. 2006,Rocher, et al. 2007). It is likely that multiple components of the RTK pathways are substrates of B56-containing PP2As. Individual B56 family members may even regulate the RTK pathways through distinct mechanisms.

Functions of B56s in tumorigenesis

Tumorigenesis by definition is uncontrolled cell growth, which is often caused by genetic mutations or exposure to carcinogens that inactivate the DNA damage response and the mitotic checkpoint functions. During malignant transformation, many cancer cells are characteristic of chromosomal instability. Increasing data suggests that B56-containing PP2As are involved in DNA damage response and the mitotic checkpoint regulation.

The p53 network

Tumor suppressor p53 is an important player of DNA damage response and plays fundamental roles in apoptosis. p53 regulates the balance between the pro- and anti-apoptotic Bcl family members through transcription-independent (Green and Kroemer 2009,Vaseva and Moll 2009) or transcription-dependent (Shen and White 2001,Vousden and Lu 2002) mechanisms. In normal cells, mdm2 and other negative regulators maintain p53 protein at low levels. DNA damage and other stress signals, through post-translational modification of p53 protein, increase the stability and activity of p53 and activate pro-apoptotic Bcl family members. This results in mitochondrial membrane permeabilization, cytochrome c leakage, and ultimately activation of executioner caspases. Since SV40 small t antigen, a potent tumor inducer, disrupts assembly of B56-containing PP2A holoenzyme (Cho, et al. 2007,Cho and Xu 2007,Sablina and Hahn 2008,Xu, et al. 2006), it was speculated that B56-containing PP2As play important roles in the p53 network. This is indeed the case.

Earlier studies in Drosophila have demonstrated that B56 family members function upstream of p53 to inhibit apoptosis (Li, et al. 2002,Silverstein, et al. 2002). Similar to these observations, knockdown of all mammalian B56 family members elevates the level of p53 in PC12 cells (Stefan Strack, personal communication), suggesting an evolutionarily conserved inhibitory effect of total B56s on p53. However, accumulating evidence indicates that B56-containing PP2As regulate the p53 network at multiple levels. It was noted that B56γ-containing PP2A could act downstream of p53 to dephosphorylate Bcl2 and prevent proteasome-dependent degradation of Bcl2 (Lin, et al. 2006). Interestingly, B56γ-containing PP2A can also regulate DNA damage-induced p53 stabilization directly. In this case, DNA damage induces ATM-dependent phosphorylation of p53 at Ser15. This phosphorylation increases the binding between p53 and B56γ-containing PP2A and triggers Thr55 dephosphorylation of p53 by B56γ-containing PP2A. Unlike knockdown of all B56 family members, which stabilizes p53, B56γ specific knockdown abolishes Thr55 dephosphorylation and p53 stabilization in response to DNA damage (Li, et al. 2007,Shouse, et al. 2008). B56 family members can regulate p53 through controlling the interaction between p53 and mdm2 as well. Several B56s were found to interact with cyclin G (Bennin, et al. 2002,Okamoto, et al. 1996). Cyclin G can function as a scaffold protein and bring B56-containing PP2As and mdm2 together. Once being recruited to the mdm2 complex, B56-containing PP2As dephosphorylate mdm2 at Thr216 and Ser166. This increases the affinity of mdm2 to p53 and enhances degradation of p53 (Kimura and Nojima 2002,Okamoto, et al. 1996,Okamoto, et al. 2002). From a genetic study aiming to identify human single nucleotide polymorphisms (SNP) that affect p53 signaling, Grochola et al. recently found an intriguing link between B56ε and human soft tissue sarcoma. It was observed that e-SNP2, a SNP located in the third intron of B56ε, is associated with earlier onset/increased risk, but increased survival time of human soft tissue sarcoma (Grochola, et al. 2009). While it remains unclear how e-SNP2 alters the expression of B56ε, our studies indicate that B56ε plays a positive role in stabilizing p53 and triggering p53-dependent apoptosis in Xenopus embryos. Intriguingly, B56ε is a substrate of caspase-3. Caspase-dependent cleavage of B56ε occurs on the carboxyl side of an evolutionarily conserved N-terminal “DKxD” motif and results in a less stable form of B56ε (Jin, et al. 2010), suggesting a potential feedback loop between p53 and B56ε-containing PP2A.

Chromosome segregation

During each cell cycle, sister chromatids of replicated chromosomes are paired by cohesin, a ring-shaped multi-protein complex. Triggered by cohesin kinase(s)-dependent phosphorylation (Hauf, et al. 2005,Ishiguro, et al. 2010,Katis, et al. 2010,Tang, et al. 2006), the vast majority of cohesin on the chromosome arm is removed during the prophase, leaving sister chromatids being held together by the centromeric fraction of cohesin. Centromeric cohesin is cleaved byseparase at the metaphase–anaphase transition, allowing chromosomes being equally separated into two daughter cells. Mis-segregation of chromosome results in aneuploidy, the most common characteristic of human malignant tumors. It is widely believed that aneuploidy contributes to, or even drives, tumor development.

B56-containing PP2As are essential for protecting centromeric cohesin prior to the metaphase-anaphase transition. Interfering with B56-containing PP2As causes precocious separation of mitotic chromosomes and results in aneuploidy (Chen, et al. 2007,Kitajima, et al. 2006,Riedel, et al. 2006). It turns out that B56-containing PP2As is recruited to centrosome by shugoshin (Sgo1), a protein that protects centromeric cohesin from the prophase cleavage (Kitajima, et al. 2004). Homodimerization of Sgo1 appears to be a prerequisite for its interaction with B56-containing PP2As (Xu, et al. 2009). In the centromere, B56-containing PP2As counteract the action of cohesin kinase(s). In agreement with this view, chromosome mis-segregation caused by PP2A interference can be rescued by knockdown of cohesin kinase (Tang, et al. 2006). On the other hand, forcing cohesin kinase to centromeres promotes cleavage of centromeric cohesin, without affecting the centromeric localization of PP2A (Ishiguro, et al. 2010). These observations, together with the finding that B56-containing PP2As are important regulators of the p53 network, provide important clues to understand functions of PP2A as a tumor suppressor.

Remaining Questions

One and a half decades of B56 studies have led to many exciting discoveries. While many important functions of B56s have already been uncovered, we are still far from a thorough understanding of this important PP2A regulatory subunit family. Many questions remain, ranging from structural to functional aspects. For example, more structural analysis is needed to understand the dynamics of B56-containing PP2A holoenzyme assembly and to identify potential regulatory mechanisms. Existing data indicates that B56 family members function redundantly in some experimental settings, but seem to play distinct roles in others. Obviously, the sequence and structural similarities account for functional redundancy among B56 family members. The same reason, however, makes it technically challenging to understand specific functions of individual B56 family members, even with many available structural, proteomic, biochemical, cell biological, and functional analysis techniques. Since B56 often mimics functions of other family members in overexpression experiments, future investigation must be based on loss-of-function studies. This makes it critically important to generate a variety of B56 knockout animals that allow an investigation of B56 function under physiological conditions. Efforts are also needed to identify additional substrates of B56-containing PP2As and to investigate the relevance of B56-dependent dephosphorylation. Some recent studies begin to illustrate a common regulatory mechanism for B56-containing PP2As. In a number of cases, non-substrate binding partners of B56-containing PP2As recruit holoenzymes to specific intracellular compartments or protein complexes, allowing precisely regulated B56-dependent dephosphorylation. Thus, identification and characterization of non-substrate B56 binding partners will provide fundamental insights into mechanisms by which the activity of B56-containing PP2A is regulated. Finally, while it has been noticed for a long time that B56s are phosphoproteins (McCright, et al. 1996), significance of B56s phosphorylation just begins to emerge. Recent studies indicate that phosphorylation status of B56s has important impact on the formation of heterotrimeric holoenzyme (Letourneux, et al. 2006,Margolis, et al. 2006), activity of PP2A holoenzyme (Ahn, et al. 2007,Margolis, et al. 2006,Usui, et al. 1998), and the stability of B56s (Ruvolo, et al. 2008). It will be of interest to further investigate regulation of B56-containing PP2As by phosphorylaton and other post-translational modifications.

Acknowledgements

JY is supported by grant 1R01GM093217-01A1 from NIGMS. CP is supported by grant 1R01AG031833 from NIA.

Footnotes

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