Serine/Threonine-Protein Phosphatase 2A
Since Krebs and Fischer (1956) discovered that the activity of an enzyme (glycogen phosphorylase) can be regulated by a reversible phosphorylation, much attention has been paid to the enzymes catalyzing these covalent modifications: the protein kinases and protein phosphatases. As opposed to protein kinases, it has taken much more time to understand the action of protein phosphatases, mainly because of their complex molecular structure and broad substrate specificities. Nevertheless, protein phosphatases are equally important in controlling biochemical pathways, adding reversibility and sensitivity to these processes.
While the first kinase (phosphorylase kinase) was isolated soon after the discovery of the concept of reversible protein phosphorylation, the biochemical isolation of the first phosphatase needed about 20 more years: ethanol denaturation (accidently applied!) yielded a “pure” and active 35-kDa phosphorylase phosphatase that exhibited broad catalytic specificity (Brandt et al. 1975). Later, this phosphatase turned out to be a mixture of the catalytic subunits of the most abundant serine/threonine phosphatases in most cells and tissues: protein phosphatase type 1 (PP1) and protein phosphatase type 2A (PP2A). For a long time, the classification of serine/threonine phosphatases into type 1 and type 2, and further into type 2A, 2B, and 2C, was based on biochemical purification schemes and in vitro enzymatic properties (Ingebritsen and Cohen 1983). Like that, type 2A phosphatases were characterized as preferentially dephosphorylating the α-subunit of phosphorylase kinase, being insensitive to the heat-stable proteins inhibitor-1 and inhibitor-2 (also called “modulator”) and being stimulated by polycations such as protamine or polylysine (hence their alternative name: polycation-stimulated phosphatases or PCS phosphatases). Extensive efforts to purify PP2A from animal tissues based on these properties soon revealed the existence of distinct enzyme complexes, founding the concept that PP2A phosphatases are actually holoenzymes, consisting of two or three subunits (Waelkens et al. 1987). With the coming of age of molecular biology and genetics techniques, several new phosphatases and related molecules were identified, and a better classification, based on evolutionary relationships and primary structure homologies, rather than biochemical properties, came into place. Thus, PP2A became part of the PPP (phosphoprotein phosphatase) family, together with its closest relatives PP4, PP6, PP1, PP2B (calcineurin), PP5, and PP7 (Shi 2009). The PP2A catalytic subunit is one of the most evolutionary conserved proteins, with orthologues in all eukaryotes (yeast, plants, animals), and its levels are under strict auto-regulatory control, in as much that it cannot be overexpressed in tissue cultured cells. Several pharmacologic inhibitors, including okadaic acid, fostriecin, cytostatin and norcantharadin, directly target the catalytic subunit and can be used to selectively inhibit PP2A in cells, but unfortunately, do not distinguish between different holoenzymes (Lambrecht et al. 2013).
Structure of PP2A
Canonical PP2A Holoenzymes
Besides their assembly into trimeric PP2A complexes, A and C subunits can form active A–C heterodimers, which represent about one-third of PP2A in a given cell. The combinatorial assembly of one C and one A or one C, one A, and one B-type subunit theoretically gives rise to four different heterodimers and at least 92 heterotrimers (Fig. 1), all exhibiting potentially different physiologic functions. Thus, the diversity in PP2A composition creates specificity and constitutes the basis for the multiple cellular and physiologic functions of these phosphatases. In addition, PP2A composition largely defines regulation by upstream factors, which is again determined by the nature of the specific B-type subunit in the complex.
Atypical PP2A Complexes
Besides the prototypical PP2A holoenzymes described above, several “atypical” PP2A complexes have been identified that occur within cells largely as catalytically inactive PP2A forms. For example, the interaction between the C subunit and the α4 protein (encoded by IGBP1) stabilizes the C subunit as a latent, inactive form, although there is also some evidence that this complex might be active in specific contexts (Sents et al. 2013). Another example is the catalytically inactive complex between the C subunit, the A subunit, and PME-1 (PP2A methyl esterase-1, encoded by PPME1) that has been estimated to represent up to 25% of the cellular PP2A pool (Sents et al. 2013). PME-1 also represents the enzyme catalyzing the carboxy-demethylation of the C subunit. It is postulated that these atypical PP2A complexes constitute stable, intermediate complexes during the process of PP2A holoenzyme biogenesis or holoenzyme disassembly.
PP2A Holoenzyme Biogenesis
The precise assembly mechanism of active PP2A holoenzymes is still incompletely understood (Sents et al. 2013). Intriguingly, the C subunit is translated as an inactive protein that is subsequently activated in a way that is strictly coupled to its incorporation into the complete holoenzyme. Like that, promiscuous and unregulated phosphatase activity of the free C subunit can be avoided (Hombauer et al. 2007). Several cellular PP2A regulators are involved in stabilizing the free C subunit during this process, in its subsequent catalytic activation, or its interaction with the A- and B-type subunits. These regulators include α4, PME-1, PTPA (protein phosphatase two A phosphatase activator, encoded by PPP2R4), and LCMT1 (leucine carboxyl methyl transferase 1, encoded by LCMT1) (Sents et al. 2013). PTPA is an ATP/Mg2+-dependent activation chaperone that modulates incorporation of catalytic metal ions into the PP2A-C active site and affects PP2A-C conformation through its ATP/Mg2+-dependent prolyl-peptidyl cis/trans isomerase activity. LCMT1 is an S-adenosylmethionine-dependent methyltransferase catalyzing the carboxy-methylation of the C subunit. PP2A-C carboxy-methylation requires an active PP2A-C conformation, is facilitated by the presence of the A subunit, and enhances the affinity of the PP2A-AC dimer for specific regulatory B-type subunits (Janssens et al. 2008). All regulators of PP2A holoenzyme biogenesis (α4, PME-1, PTPA, and LCMT1) are indispensable for mammalian survival, indicative for their physiologic importance (Sents et al. 2013).
Regulation of PP2A
No different from protein kinases, protein phosphatases serve as signaling enzymes in their own right and are responsive to extracellular stimuli or stresses. Not illogically, PP2A regulation is largely determined by its subunit composition.
Regulation of PP2A Composition
Regulation of PP2A holoenzyme composition is mainly driven by posttranslational modifications of the subunits involved. Some of these modifications serve a “housekeeping” function and are directly involved in PP2A holoenzyme assembly; others are subject to extracellular regulation.
All PP2A-C orthologues have a T 304 PDYFL 309 motif at their C-terminus, which is a focal point of posttranslational modification. The free carboxy-terminus of Leu309 is subject to reversible methylation (catalyzed by LCMT1 and PME-1), which is a determining factor for facilitating binding of specific B-type subunits (Janssens et al. 2008). Thr304 is specifically phosphorylated during mitosis and may contribute to mitotic inactivation of PP2A-B55 trimers or prevent their de novo formation (Schmitz et al. 2010). Tyr307 becomes phosphorylated in v-src-transformed fibroblasts (Chen et al. 1992) or upon serum, interleukin-1, tumor necrosis factor-α, or insulin stimulation (Janssens and Goris 2001) and in some pathologic conditions (e.g., cancer). This modification inactivates PP2A and prevents formation of PP2A-B56 trimers and, indirectly, of PP2A-B55 trimers through interference with PP2A-C methylation (Janssens et al. 2008).
Outside the C-tail, PP2A-C Tyr284 nitration results in its dissociation from the A subunit and is observed in endotoxin-conditioned cells as part of an adaptation mechanism which makes cells resistant to Toll-like receptor signaling (Ohama and Brautigan 2010). Nitration also occurs on a conserved tyrosine residue in the B56 subunits upon reactive oxygen species (ROS) production, impairing their interaction with A and C subunits (Low et al. 2014).
Most B56 subunits are phosphoproteins, and in some cases, this modification affects A and C interaction. For instance, extracellular regulated MAP kinase (ERK)-mediated phosphorylation of B56β/γ1 subunits induces A/C subunit dissociation (Letourneux et al. 2006), and in mitosis, Ser167 phosphorylation of B55α has a negative impact on A/C subunit binding (Schmitz et al. 2010). Conversely, ataxia telangiectasia mutated (ATM) kinase-dependent phosphorylation of B56γ2,3/δ upon DNA damage increases interaction with A/C subunits (Shouse et al. 2011).
Subunit Modifications Affecting PP2A Activity or PP2A–Substrate Interaction
B-type subunit phosphorylation can directly affect the catalytic activity of the PP2A holoenzyme toward specific physiologic substrates. This is, for instance, the case for protein kinase A (PKA)-mediated phosphorylation of B56δ, which increases PP2A-B56δ activity toward dopamine- and cAMP-regulated phosphoprotein of 32-kDa (DARPP-32) in dopaminergic neurons (Ahn et al. 2007a) and toward cAMP phosphodiesterase PDE4D3 in muscle (Dodge-Kafka et al. 2010). In B56α, double-stranded RNA-dependent protein kinase (PKR)-mediated phosphorylation activates PP2A-B56α toward eukaryotic translation initiation factor-2α (eIF2α) and B-cell lymphoma-2 (Bcl-2) (Ruvolo 2016). Conversely, protein kinase Cα (PKCα)-mediated phosphorylation of B56α decreases PP2A-B56α activity toward several cardiac myofilament substrates (Kirchhefer et al. 2014).
B-type subunit phosphorylation can also modulate PP2A interaction with its substrate(s). For instance, in fibroblast growth factor (FGF)-stimulated chondrocytes, B55α dephosphorylation increases both the affinity of B55α for the PP2A-AC dimer and for the PP2A-B55α substrate, p107 (Kolupaeva et al. 2013). Another example is seen upon DNA damage, where interaction of PP2A-B56γ3 with its substrate p53 significantly increases upon ATM-mediated phosphorylation of B56γ3 (Shouse et al. 2011).
Inhibition by Cellular Inhibitors
PP2A catalytic activity can be directly inhibited by a growing set of specific cellular inhibitory proteins, which play an increasingly important role in mediating PP2A dysfunction under pathologic conditions. The most important ones are acidic nuclear phosphoprotein-32a (ANP32a), suvar 3–9/enhancer-of-zeste/trithorax (SET), cancerous inhibitor of PP2A (CIP2A), members of the cAMP-regulated phosphoprotein/α-endosulfin (ARPP-16/19/ENSA) family, type 2A-interacting protein-1 (TIPRL1), and biorientation of chromosomes in cell division-1 (Bod1). Although often poorly biochemically characterized, these inhibitors either directly bind to the C subunit or target very specific PP2A holoenzymes (Haesen et al. 2012). Additionally, their inhibitory activities are frequently regulated through direct (de)phosphorylation, adding yet another level of regulatory complexity (Haesen et al. 2012).
Activation by Second Messengers
Although for several years calcineurin (PP2B) appeared the major Ca2+-responsive Ser/Thr phosphatase in most cells and tissues, two specific PP2A holoenzyme families are equally well regulated by Ca 2+ -ions. All B″/PR72 subunits harbor two canonical Ca2+-binding EF-hand motifs: a high-affinity one, directly involved in A subunit interaction, and a lower-affinity one, mediating stimulation of PP2A-B″ activity by rising intracellular Ca2+ (Janssens et al. 2003). Moreover, all B‴/striatin subunits contain a Ca2+/calmodulin binding domain that becomes occupied when Ca2+ levels rise. So far, it remains unclear how this affects PP2A-B‴ activity or function.
Sphingolipids, like ceramide, have been reported to activate PP2A, without any mechanistic information, until recently (Oaks and Ogretmen 2015). Ceramide directly binds the PP2A inhibitor SET, relieving PP2A from SET and increasing PP2A activity. These observations form the basis for the therapeutic use of synthetic sphingosine analogs, such as FTY720, as PP2A activators in SET overexpressing cancers (mainly hematologic malignancies) (Perrotti and Neviani 2013).
Signaling Functions and Substrate Specificity of PP2A
Given the ubiquitous expression, high abundance, and structural variety of PP2A holoenzymes, there is hardly not any phosphorylation-regulated cellular or physiologic process in which PP2A does not play a role. “PP2A” – i.e., the large family of distinct PP2A holoenzymes – may exert collaborating as well as opposing functions within a given signaling pathway by acting at different levels in the cascade. This is, for instance, the case in growth factor-induced ERK signaling, PI3K/Akt signaling, mTOR signaling, TGFβ signaling, or Wnt signaling (Eichhorn et al. 2009; Janssens and Rebollo 2012; Wlodarchak and Xing 2016). In addition, different PP2A complexes may dephosphorylate the same substrate, even on the same site, depending on the regulatory stimulus involved, the cell type, or the broader physiologic context (Ahn et al. 2007a, b; Janssens and Rebollo 2012). Recently, major insights were obtained in potential determinants governing PP2A substrate selection for both PP2A-B55 and PP2A-B56 trimers: apparently, these B subunits recognize specific “short linear interaction motifs” residing in intrinsically disordered protein domains that serve as direct or indirect substrate determinants (Hertz et al. 2016; Cundell et al. 2016). Many protein kinases are direct PP2A substrates (Millward et al. 1999), further underscoring PP2A’s pleiotropic functions in regulating cell growth and proliferation (Janssens and Goris 2001; Eichhorn et al. 2009), cell death and apoptosis (Janssens and Rebollo 2012), cell cycle progression and mitosis (Kolupaeva and Janssens 2013; Hunt 2013; Wlodarchak and Xing 2016), cell adhesion and migration (Basu 2011), transcription, and translation (Janssens and Goris 2001; Wlodarchak and Xing 2016). Nonetheless, the specific PP2A regulatory subunits controlling dephosphorylation of a given substrate in a given mammalian cell or tissue often remain poorly defined, particularly in the physiologic context of a whole organism. To further clarify this, in vivo mammalian studies through systematic characterization of PP2A knockout mice are eagerly being awaited.
PP2A Dysfunction in Human Disease
PP2A dysregulation substantially contributes to disease, although the full clinical potential of these insights for improved treatment, diagnostic, or prognostic purposes has so far not been properly exploited.
Together with pRb and p53, PP2A is recognized as a major human tumor suppressor, whose function needs to be abolished to fully transform human epithelial cells (Hahn and Weinberg 2002). Particularly, PP2A-B56 trimers function as major tumor suppressive PP2A complexes. PP2A is genetically altered or functionally inactivated (e.g., by overexpression of cellular PP2A inhibitors) in a large variety of human cancers (Sangodkar et al. 2016; Ruvolo 2016), resulting in uncontrolled activation of specific oncogenes (e.g., MYC, β-catenin) or signaling pathways downstream of activated oncogenes (e.g., of RAS or tyrosine kinase receptors). Pharmacologic PP2A reactivation is intensively being explored as a promising anticancer strategy, either through de novo discovery of small, PP2A-modulating molecules or through drug repurposing (Sangodkar et al. 2016).
PP2A Inactivation by Pathogenic Viruses
PP2A is a privileged cellular target for proteins encoded by DNA (tumor) viruses (e.g., adenovirus, SV40, polyomavirus), RNA viruses (e.g., West Nile virus, hepatitis C virus), and retroviruses (e.g., HIV, HTLV-1) (Guergnon et al. 2011). These viruses inhibit or subvert PP2A function to accomplish their life cycle, further underscoring the role of PP2A as a master controller of cell proliferation, division, and survival.
In Alzheimer’s disease (AD), PP2A dysfunction has been linked to tau hyperphosphorylation and neurofibrillary tangle formation, amyloidogenesis, and synaptic deficits (Sontag and Sontag 2014). The deregulation of PP2A methylation in AD is especially interesting because it leads to loss of PP2A-B55, the primary tau phosphatase, and can be induced by dietary folate and vitamin B deficiency. Although there is little evidence for PP2A dysfunction in Parkinson’s disease (PD), PP2A-B55 dephosphorylates α-synuclein at a site that is hyperphosphorylated in the aggregates constituting the Lewy bodies. Additionally, PP2A is involved in leucine-rich repeat kinase-2 (LRRK2) dephosphorylation (Taymans and Baekelandt 2014). PP2A reactivation is considered a promising strategy for AD and, potentially, PD treatment (Voronkov et al. 2011).
De Novo PP2A Mutations in Intellectual Disability and Overgrowth
Single de novo point mutations in PP2A genes PPP2R1A, PPP2R5D, PPP2R5C, and PPP2R5B have been causally linked to intellectual disability and overgrowth (Houge et al. 2015; Loveday et al. 2015). The resulting amino acid substitutions all disrupt PP2A holoenzyme formation or result in formation of catalytically incompetent complexes that act as dominant negatives (Houge et al. 2015). The precise neurological consequences of these “PP2A” dysfunctions remain yet to be determined.
Diabetes and Other Metabolic Diseases
Within pancreatic β-cells, glucose stimulates PP2A methylation and activity, thereby promoting insulin secretion. Chronic exposure to saturated fatty acids (lipotoxicity) or high glucose (glucotoxicity) results however in PP2A hyperactivation, which decreases β-cell survival through dephosphorylation of pro-survival proteins Akt and Bcl-2 (Kowluru and Matti 2012). PP2A hyperactivation is also seen in insulin target tissues (liver, muscle) in several in vitro and in vivo models of insulin resistance and diabetes. Liver-specific knockout of PP2A-B56γ leads to increased glucose uptake and de novo lipogenesis, resulting in improved systemic glucose tolerance and insulin sensitivity, but elevated circulating triglycerides. In diabetics, hepatic levels of B56γ are elevated and correlate with obesity and insulin resistance (Cheng et al. 2015).
PP2A is a major phosphatase in the heart, modulating excitation-contraction coupling through regulation of intracellular Ca2+ transient. PP2A thereby largely antagonizes the effects of β-adrenergic stimulation through dephosphorylation of L-type Ca2+ channels, ryanodine type 2 receptors (RyR2), SERCA pumps, Na+/Ca2+ exchangers (NCX), and their respective regulators (e.g., phospholamban). Connexin-43, cardiac myosin binding protein-C (cMyBP-C) and the inhibitory subunit of troponin (TnI), all mediating (synchronous) cardiomyocyte contractility, are targets of PP2A-B56α, a major cardiac PP2A complex (Kirchhefer et al. 2014). Intriguingly, models of Cα or B56α overexpression as well as inhibition display cardiac abnormalities, suggesting a balance in PP2A activity and targeting is vital to avoid heart failure (Lubbers and Mohler 2016).
The cellular and physiologic importance of PP2A phosphatases can hardly be underestimated, given they act as Ser/Thr kinase counterparts in a large variety of signaling cascades. Despite increasing evidence that pharmacologic PP2A modulation in human disease may be feasible, PP2A remains a challenging therapeutic target, not the least because of its complex structure and regulation, and our incomplete understanding of PP2A holoenzyme functions in different cells and tissues in vivo. This knowledge should enable us in the near future to modulate activity of just a selective group of PP2A holoenzymes, a specific PP2A-regulator interaction or a specific PP2A-substrate interaction.
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