twins


EVOLUTIONARY HOMOLOGS (part 1/2)

Protein phosphatase 2A (PP2A) appears to be involved in the regulation of many cellular processes. Control mechanisms that lead to the activation (and deactivation) of the various holoenzymes to initiate appropriate dephosphorylation events remain obscure. The core components of all PP2A holoenzymes are the catalytic (PP2Ac) and 63-65-kD regulatory (PR65) subunits. Monospecific and affinity-purified antibodies against both PP2Ac and PR65 show that these proteins are ubiquitously localized in the cytoplasm and the nucleus in nontransformed fibroblasts. The core subunits of PP2A are twofold more concentrated in the nucleus than in the cytoplasm. Detailed analysis of synchronized cells reveals striking changes in the nuclear to cytoplasmic ratio of PP2Ac-specific immunoreactivity, albeit the total amounts of PP2Ac and PR65 in each compartment do not alter significantly during the cell cycle. These results imply that differential methylation of PP2Ac occurs at the G0/G1 and G1/S boundaries. Specifically, a demethylated form of PP2Ac is found in the cytoplasm of G1 cells, and in the nucleus of S and G2 cells. In addition, nuclear PP2A holoenzymes appear to undergo conformational changes at the G0/G1 and G1/S boundaries. During mitosis PP2A is lost from the nuclear compartment; unlike protein phosphatase 1, PP2A shows no specific association with the condensed chromatin (Turowski, 1995).

Differential association of regulatory B subunits with a core heterodimer, composed of a catalytic (C) and a structural (A) subunit, is an important mechanism that regulates protein phosphatase 2A (PP2A). Three novel cDNAs related to the B' subunit of bovine cardiac PP2A have been isolated and characterized. Two human (B'alpha1 and B'alpha2) and a mouse (B'alpha3) cDNA encode for alternatively spliced variants of the B subunit. The deduced primary sequences of these clones contain 12 of 15 peptides derived from the purified bovine B' subunit. Differences between the deduced sequences of the B alpha splice variants and the cardiac peptide sequences suggest the existence of multiple isoforms of the B' subunit. Comparison of the protein and nucleotide sequences of the cloned cDNAs show that all three forms of B'alpha diverge at a common splice site near the 3'-end of the coding regions. Northern blot and reverse transcription-polymerase chain reaction analyses reveals that the B'alpha transcripts (4.3-4.4 kb) are widely expressed and very abundant in heart and skeletal muscle. The expressed human and mouse B'alpha proteins readily associate with the PP2A core enzyme in both in vitro and in vivo complex formation assays. Epitope-tagged B'alpha has been localized in both the cytosol and nuclei of transiently transfected cells. The efficiency of binding of all three expressed proteins to a glutathione S-transferase-A subunit fusion protein is greatly enhanced by the addition of the C subunit. Expression of the B'alpha subunits in insect Sf9 cells results in formation of AC.B'alpha heterotrimers with the endogenous insect A and C subunits. These results show that the B' subunit, which is the predominant regulatory subunit in cardiac PP2A, is a novel protein whose sequence is unrelated to other PP2A regulatory subunits. The nuclear localization of expressed B'alpha suggests that some variants of the B' subunit are involved in the nuclear functions of PP2A (Tehrani, 1998).

The phosphoprotein phosphatase 2A (PP2A) catalytic subunit contains a methyl ester on its C-terminus, which in mammalian cells is added by a specific carboxyl methyltransferase and removed by a specific carboxyl methylesterase. Genes in yeast have been identified that show significant homology to human carboxyl methyltransferase and methylesterase. The methyltransferase homolog is not present in C. elegans or D. melanogaster. Extracts of wild-type yeast cells contain carboxyl methyltransferase activity, while extracts of strains deleted for one of the methyltransferase genes, PPM1, lack all activity. Mutation of PPM1 partially disrupts the PP2A holoenzyme in vivo and ppm1 mutations exhibit synthetic lethality with mutations in genes encoding the B or B' regulatory subunit. Inactivation of PPM1 or overexpression of PPE1, the yeast gene homologous to bovine methylesterase, yields phenotypes similar to those observed after inactivation of either regulatory subunit. These phenotypes can be reversed by overexpression of the B regulatory subunit. These results demonstrate that Ppm1 is the sole PP2A methyltransferase in yeast and that its activity is required for the integrity of the PP2A holoenzyme (Wu, 2000).

In contrast to its effect on heterotrimer formation, loss of PP2A methyltransferase activity does not diminish the affinity of the C subunit for another regulatory partner in the cell, Tap42. The retention of interaction of the unmethylated C subunit with Tap42 is consistent with the rapamycin resistance of ppm1 strains, since resistance to rapamycin depends on retaining association between Tap42 and the C subunit. This difference in the effect of carboxyl methylation on affinity of the C subunit for B regulatory subunits compared with Tap42/alpha4 raises the possibility that methylation could provide a mechanism for modulating the distribution of the C subunit among these different regulatory elements. These results indicate that methylation of the PP2A C subunits is in dynamic equilibrium through the competing reactions catalyzed by Ppm1 and Ppe1. Accordingly, inhibition of PP2A methyltransferase or activation of PPME would tend to diminish PP2A activity while strengthening the Tor-mediated pathway (Tor kinase mediates phosphorylation of Tap42). Thus, PPMT or PPME could serve as a locus through which internal or external signals impinge on cellular proliferation (Wu, 2000).

Phosphoprotein phosphatase 2A (PP2A) is a major phosphoserine/threonine protein phosphatase in all eukaryotes. It has been isolated as a heterotrimeric holoenzyme composed of a 65 kDa A subunit, which serves as a scaffold for the association of the 36 kDa catalytic C subunit, and a variety of B subunits that control phosphatase specificity. The C subunit is reversibly methyl esterified by specific methyltransferase and methylesterase enzymes at a completely conserved C-terminal leucine residue. Methylation plays an essential role in promoting PP2A holoenzyme assembly and demethylation has an opposing effect. Changes in methylation indirectly regulate PP2A phosphatase activity by controlling the binding of regulatory B subunits to AC dimers. Since PP2A has been implicated in the regulation of cell proliferation, it seems likely that changes in methylation function in cell cycle regulation. Results with mammalian fibroblasts in tissue culture indicate that methylation levels may depend on the cell cycle, with different patterns of regulation in the nucleus and cytoplasm. Cytoplasmic PP2A appears to become demethylated at the G0/G1 boundary and remethylated as cells entered S phase, at which point nuclear PP2A is demethylated (Tolstykh, 2000).

Okadaic acid (OA) enhances the resumption of meiosis in mouse oocytes, indicating that serine/threonine protein phosphatase-1 (PP1) and/or PP2A is involved. However, specific identification of PP1 and/or PP2A in mouse oocytes has not been reported. Fully grown germinal vesicle-intact (GVI) mouse oocytes contain mRNA corresponding to two isotypes of PP1: PP1alpha and PP1gamma. Also present is the transcript for PP2A. At the protein level only PP1alpha and PP2A are recognized in fully grown GVI oocytes, using Western blot analysis. Neither of the PP1gamma spliced variant proteins, PP1gamma1 and PP1gamma2, is detectable. Immunohistochemical analysis of ovarian tissue from gonadotropin-stimulated adult mice results in subcellular localization of both PP1alpha and PP2A, but not PP1gamma, in oocytes from all stages of folliculogenesis. In primordial oocytes, PP1alpha and PP2A are present in the cytoplasm. In more advanced stages of oogenesis, PP1alpha, although still present in the cytoplasm, is highly concentrated in the nucleus, whereas PP2A is predominantly cytoplasmic with a distinct reduction in the nuclear area. Both PP1alpha and PP2A are immunodetectable in oocytes during the prepubertal period. Eleven-day-old mouse oocytes, considered OA-insensitive and germinal vesicle breakdown (GVB)-incompetent, display both PP1alpha and PP2A predominantly in the cytoplasm. By 15 days of age mouse oocytes, which are beginning to acquire OA sensitivity and GVB competence, show a relocation of PP1alpha into the nucleoplasm while PP2A remains predominantly cytoplasmic. This is the first specific identification of PP1alpha and PP2A in mouse oocytes. The differential localization of PP1alpha and PP2A, in addition to the relocation of PP1alpha during the acquisition of meiotic competence, suggests that these PPs have distinct regulatory roles during the resumption of meiosis (Smith, 1998).

Structure of PP2A

The PR65/A subunit of protein phosphatase 2A serves as a scaffolding molecule to coordinate the assembly of the catalytic subunit and a variable regulatory B subunit, generating functionally diverse heterotrimers. Mutations of the beta isoform of PR65 are associated with lung and colon tumors. The crystal structure of the PR65/Aalpha subunit, at 2.3 A resolution, reveals the conformation of its 15 tandemly repeated HEAT sequences, degenerate motifs of approximately 39 amino acids present in a variety of proteins, including huntingtin and importin beta. Individual motifs are composed of a pair of antiparallel alpha helices that assemble in a mainly linear, repetitive fashion to form an elongated molecule characterized by a double layer of alpha helices. Left-handed rotations at three interrepeat interfaces generate a novel left-hand superhelical conformation. The protein interaction interface is formed from the intrarepeat turns that are aligned to form a continuous ridge (Groves, 1999).

Alpha4 is a ubiquitin-binding protein that regulates protein serine/threonine phosphatase 2A ubiquitination

Multiple regulatory mechanisms control the activity of the protein serine/threonine phosphatase 2A catalytic subunit (PP2Ac), including post-translational modifications and its association with regulatory subunits and interacting proteins. Alpha4 is a PP2Ac-interacting protein that is hypothesized to play a role in PP2Ac ubiquitination via its interaction with the E3 ubiquitin ligase Mid1, which targets phosphatase 2A for degradation. This report shows that murine alpha4 serves as a necessary adaptor protein that provides a binding platform for both PP2Ac and Mid1. Aa novel ubiquitin-interacting motif (UIM) was identified within alpha4 (amino acid residues 46-60), and the interaction between alpha4 and ubiquitin was analyze using NMR. Consistent with other UIM-containing proteins, alpha4 is monoubiquitinated. Interestingly, deletion of the UIM within alpha4 enhances its association with polyubiquitinated proteins. Lastly, it was demonstrated that addition of wild-type alpha4 but not an alpha4 UIM deletion mutant suppresses PP2Ac polyubiquitination. Thus, the polyubiquitination of PP2Ac is inhibited by the UIM within alpha4. These findings reveal direct regulation of PP2Ac polyubiquitination by a novel UIM within the adaptor protein alpha4 (McConnell, 2010).

PP2A mutation

Protein phosphatase 2A (PP2A) is a multimeric enzyme, containing a catalytic subunit complexed with two regulatory subunits. The catalytic subunit PP2A C is encoded by two distinct and unlinked genes, termed Calpha and Cbeta. The specific function of these two catalytic subunits is unknown. To address the possible redundancy between PP2A and related phosphatases as well as between Calpha and Cbeta, the Calpha subunit gene was deleted by homologous recombination. Homozygous null mutant mice are embryonically lethal, demonstrating that the Calpha subunit gene is an essential gene. Because PP2A exerts a range of cellular functions, including cell cycle regulation and cell fate determination, it was surprising to find that these embryos develop normally until postimplantation, around embryonic day 5.5/6.0. While no Calpha protein is expressed, comparable expression levels of PP2A C are found at a time when the embryo is degenerating. Despite a 97% amino acid identity, Cbeta cannot completely compensate for the absence of Calpha. Degenerated embryos can be recovered even at embryonic day 13.5, indicating that although embryonic tissue is still capable of proliferating, normal differentiation is significantly impaired. While the primary germ layers (ectoderm and endoderm) are formed, mesoderm is not formed in degenerating embryos (Gotz, 1998).

The PPP2R1B gene, which encodes the beta isoform of the A subunit of the serine/threonine protein phosphatase 2A (PP2A), has been identified as a putative human tumor suppressor gene. Sequencing of the PPP2R1B gene, located on human chromosome 11q22-24, reveals somatic alterations in 15% (5 out of 33) of primary lung tumors, 6% (4 out of 70) of lung tumor-derived cell lines, and 15% (2 out of 13) of primary colon tumors. One deletion mutation generated a truncated PP2A-Abeta protein that is unable to bind to the catalytic subunit of the PP2A holoenzyme. The PP2R1B gene product may suppress tumor development through its role in cell cycle regulation and cellular growth control (Wang, 1998).

Lipids have been implicated in signal transduction and in several stages of membrane trafficking, but these two functions have not been functionally linked. In yeast, sphingoid base synthesis is required for the internalization step of endocytosis and organization of the actin cytoskeleton. Inactivation of a protein phosphatase 2A (PP2A) or overexpression of one of two kinases, Yck2p or Pkc1p, can specifically suppress the sphingoid base synthesis requirement for endocytosis. The two kinases have an overlapping function because only a mutant with impaired function of both kinases is defective in endocytosis. An ultimate target of sphingoid base synthesis may be the actin cytoskeleton, because overexpression of the kinases and inactivation of PP2A substantially corrected the actin defect due to the absence of sphingoid base. These results suggest that sphingoid base controls protein phosphorylation, perhaps by activating a signal transduction pathway that is required for endocytosis and proper actin cytoskeleton organization in yeast (Friant, 2000).

PP2A in yeast

Tor proteins, homologous to DNA-dependent protein kinases, participate in a signal transduction pathway in yeast that regulate protein synthesis and cell wall expansion in response to nutrient availability. The anti-inflammatory drug rapamycin inhibits yeast cell growth by inhibiting Tor protein signaling. This leads to diminished association of a protein, Tap42, with two different protein phosphatase catalytic subunits; one encoded redundantly by PPH21 and PPH22, and one encoded by SIT4. Inactivation of either Cdc55 or Tpd3, which regulate Pph21/22 activity, results in rapamycin resistance and this resistance correlates with an increased association of Tap42 with Pph21/22. Tor-dependent phosphorylation of Tap42 is shown both in vivo and in vitro and this phosphorylation is rapamycin sensitive. Inactivation of Cdc55 or Tpd3 enhances in vivo phosphorylation of Tap42. It is concluded that Tor phosphorylates Tap42 and that phosphorylated Tap42 effectively competes with Cdc55/Tpd3 for binding to the phosphatase 2A catalytic subunit. Furthermore, Cdc55 and Tpd3 promote dephosphorylation of Tap42. Thus, Tor stimulates growth-promoting association of Tap42 with Pph21/22 and Sit4, while Cdc55 and Tpd3 inhibit this association both by direct competition and by dephosphorylation of Tap42. These results establish Tap42 as a target of Tor and add further refinement to the Tor signaling pathway (Jiang, 1999).

Protein phosphatase 2A (PP2A) is an essential intracellular serine/threonine phosphatase containing a catalytic subunit that possesses the potential to dephosphorylate promiscuously tyrosine-phosphorylated substrates in vitro. How PP2A acquires its intracellular specificity and activity for serine/threonine-phosphorylated substrates is unknown. A novel and phylogenetically conserved mechanism is reported to generate active phospho-serine/threonine-specific PP2A in vivo. Phosphotyrosyl phosphatase activator (PTPA), a protein of so far unknown intracellular function, is required for the biogenesis of active and specific PP2A. Deletion of the yeast PTPA homologs generates a PP2A catalytic subunit with a conformation different from the wild-type enzyme, as indicated by its altered substrate specificity, reduced protein stability, and metal dependence. Complementation and RNA-interference experiments show that PTPA fulfills an essential function conserved from yeast to man (Fellner, 2003).

Homologue segregation during the first meiotic division requires the proper spatial regulation of sister chromatid cohesion and its dissolution along chromosome arms, but its protection at centromeric regions. This protection requires the conserved MEI-S332/Sgo1 proteins that localize to centromeric regions and also recruit the PP2A phosphatase by binding its regulatory subunit, Rts1. Centromeric Rts1/PP2A then locally prevents cohesion dissolution possibly by dephosphorylating the protein complex cohesin. This study shows that Aurora B kinase in Saccharomyces cerevisiae (Ipl1) is also essential for the protection of meiotic centromeric cohesion. Coupled with a previous study in Drosophila, this meiotic function of Aurora B kinase appears to be conserved among eukaryotes. Furthermore, Sgo1 recruits Ipl1 to centromeric regions. In the absence of Ipl1, Rts1 can initially bind to centromeric regions but disappears from these regions after anaphase I onset. It is suggested that centromeric Ipl1 ensures the continued centromeric presence of active Rts1/PP2A, which in turn locally protects cohesin and cohesion (Yu, 2007).

In budding yeast, a surveillance mechanism known as the spindle position checkpoint (SPOC) ensures accurate genome partitioning. In the event of spindle misposition, the checkpoint delays exit from mitosis by restraining the activity of the mitotic exit network (MEN). To date, the only component of the checkpoint to be identified is the protein kinase Kin4. Furthermore, how the kinase is regulated by spindle position is not known. This study identified the protein phosphatase 2A (PP2A) in complex with the regulatory subunit Rts1 as a component of the SPOC. Loss of PP2A-Rts1 function abrogates the SPOC but not other mitotic checkpoints. The protein phosphatase functions upstream of Kin4, regulating the kinase's phosphorylation and localization during an unperturbed cell cycle and during SPOC activation, thus defining the phosphatase as a key regulator of SPOC function (Chan, 2009).

Tension-dependent removal of pericentromeric shugoshin is an indicator of sister chromosome biorientation

During mitosis and meiosis, sister chromatid cohesion resists the pulling forces of microtubules, enabling the generation of tension at kinetochores upon chromosome biorientation. How tension is read to signal the bioriented state remains unclear. Shugoshins form a pericentromeric platform that integrates multiple functions to ensure proper chromosome biorientation. This study shows that budding yeast shugoshin Sgo1 (see Drosophila Mei-S332) dissociates from the pericentromere reversibly in response to tension. The antagonistic activities of the kinetochore-associated Bub1 kinase and the Sgo1-bound phosphatase protein phosphatase 2A (PP2A)-Rts1 underlie a tension-dependent circuitry that enables Sgo1 removal upon sister kinetochore biorientation. Sgo1 dissociation from the pericentromere triggers dissociation of condensin and Aurora B (see Drosophila Aurora B) from the centromere, thereby stabilizing the bioriented state. Conversely, forcing sister kinetochores to be under tension during meiosis I leads to premature Sgo1 removal and precocious loss of pericentromeric cohesion. Overall, this study shows that the pivotal role of shugoshin is to build a platform at the pericentromere that attracts activities that respond to the absence of tension between sister kinetochores. Disassembly of this platform in response to intersister kinetochore tension signals the bioriented state. Therefore, tension sensing by shugoshin is a central mechanism by which the bioriented state is read (Nerusheva, 2014).

PP2A regulates vulval induction in C. elegans

Protein phosphatase 2A (PP2A) can both positively and negatively influence the Ras/Raf/MEK/ERK signaling pathway, but its relevant substrates are largely unknown. In C. elegans, the PR55/B regulatory subunit of PP2A, encoded by sur-6, positively regulates Ras-mediated vulval induction and acts at a step between Ras and Raf. The catalytic subunit (C) of PP2A, encoded by let-92, also positively regulates vulval induction. Therefore SUR-6/PR55 and LET-92/PP2A-C probably act together to dephosphorylate a Ras pathway substrate. PP2A has been proposed to activate the Raf kinase by removing inhibitory Ser259 phosphates from Raf-1 or from equivalent Akt phosphorylation sites in other Raf family members. However, mutant forms of C. elegans LIN-45 RAF that lack these sites still require sur-6. Therefore, SUR-6 must influence Raf activity via a different mechanism. SUR-6 and KSR (kinase suppressor of Ras) function at a similar step in Raf activation but genetic analysis suggests that KSR activity is intact in sur-6 mutants. The kinase PAR-1 has been identified as a negative regulator of vulval induction; it is shown to act in opposition to SUR-6 and KSR-1. In addition to their roles in Ras signaling, SUR-6/PR55 and LET-92/PP2A-C cooperate to control mitotic progression during early embryogenesis (Kao, 2004).

In other systems, PR55/B and PP2A have been found to have both positive and negative effects on Ras signaling. For example, in Drosophila a positive role for PR55/PP2A is supported by findings that mutations in tws/PR55 suppress the lethality caused by activated Sevenless receptor and activated Ras, and mutations in the PP2A catalytic subunit enhance photoreceptor defects caused by a hypomorphic Draf allele. However, a negative role for PR55/PP2A is supported by findings that RNAi against tws/PR55 elevates the level of phospho-ERK in cultured S2 cells, and mutations in the PP2A catalytic subunit enhance photoreceptor defects caused by activated Ras. Thus, in Drosophila the role of PR55/PP2A appears complex, and PP2A may act on multiple substrates within the Ras pathway. Similarly, in mammalian cells PP2A has been suggested to positively regulate Ras signaling by removing inhibitory phosphates from Raf and to negatively regulate Ras signaling by removing activating phosphates from MEK or ERK. By contrast, no evidence was found for a negative role of SUR-6/PR55 or LET-92/PP2A in C. elegans, despite having tested sur-6 and let-92 mutations in numerous genetic backgrounds. Therefore either PP2A lacks a negative role in C. elegans, or its negative role is masked by its stronger positive role (Kao, 2004 and references therein).

The sur-6 maternal effect lethal phenotype reveals that in addition to Ras signaling, sur-6 is required for mitotic progression. sur-6(sv30) and sur-6(RNAi) embryos display a variety of mitotic defects such as ectopic and aberrant cytokinesis, the collapse and re-elaboration of well-extended anaphase spindles, abnormally shaped spindles and chromatin bridges during anaphase. Similar mitotic defects have been observed in Drosophila tws/PR55 mutants. Premature sister chromatid separation and cytokinesis defects have also been observed in S. cerevisiae cdc55/PR55 mutants. Thus, the mitotic role of PR55 appears to be evolutionarily conserved. The early C. elegans embryo is a particularly tractable system for further study of this poorly understood mitotic role of PR55 (Kao, 2004 and references therein).

A PP2A regulatory subunit regulates C. elegans insulin/IGF-1 signaling by modulating AKT-1 phosphorylation

The C. elegans insulin/IGF-1 signaling (IIS) cascade plays a central role in regulating life span, dauer, metabolism, and stress. The major regulatory control of IIS is through phosphorylation of its components by serine/threonine-specific protein kinases. An RNAi screen for serine/threonine protein phosphatases that counterbalance the effect of the kinases in the IIS pathway identified pptr-1, a B56 regulatory subunit of the PP2A holoenzyme. Modulation of pptr-1 affects IIS pathway-associated phenotypes including life span, dauer, stress resistance, and fat storage. PPTR-1 functions by regulating worm AKT-1 phosphorylation at Thr 350. With striking conservation, mammalian B56beta regulates Akt phosphorylation at Thr 308 in 3T3-L1 adipocytes. In C. elegans, this ultimately leads to changes in subcellular localization and transcriptional activity of the forkhead transcription factor DAF-16. This study reveals a conserved role for the B56 regulatory subunit in regulating insulin signaling through AKT dephosphorylation, thereby having widespread implications in cancer and diabetes research (Padmanabhan, 2009).

Protein phosphatase 2A cooperates with the autophagy-related kinase UNC-51 to regulate axon guidance in Caenorhabditis elegans

UNC-51 is a serine/threonine protein kinase conserved from yeast to humans. The yeast homolog Atg1 regulates autophagy (catabolic membrane trafficking) required for surviving starvation. In C. elegans, UNC-51 regulates the axon guidance of many neurons by a different mechanism than it and its homologs use for autophagy. UNC-51 regulates the subcellular localization (trafficking) of UNC-5, a receptor for the axon guidance molecule UNC-6/Netrin; however, the molecular details of the role for UNC-51 are largely unknown. This study reports that UNC-51 physically interacts with LET-92, the catalytic subunit of serine/threonine protein phosphatase 2A (PP2A-C), which plays important roles in many cellular functions. A low allelic dose of LET-92 partially suppresses axon guidance defects of weak, but not severe, unc-51 mutants, and a low allelic dose of PP2A regulatory subunits A (PAA-1/PP2A-A) and B (SUR-6/PP2A-B) partially enhance the weak unc-51 mutants. It was also found that LET-92 can work cell-non-autonomously on axon guidance in neurons, and that LET-92 colocalizes with UNC-51 in neurons. In addition, PP2A dephosphorylates phosphoproteins that have been phosphorylated by UNC-51. These results suggest that, by forming a complex, PP2A cooperates with UNC-51 to regulate axon guidance by regulating phosphorylation. This is the first report of a serine/threonine protein phosphatase functioning in axon guidance in vivo (Ogura, 2010).

par-1, atypical pkc and PP2A/B55 sur-6 are implicated in the regulation of exocyst-mediated membrane trafficking in Caenorhabditis elegans

The exocyst is a conserved protein complex that is involved in tethering secretory vesicles to the plasma membrane and regulating cell polarity. Despite a large body of work little is known how exocyst function is controlled. To identify regulators for exocyst function, a targeted RNAi screen was performed in Caenorhabditis elegans to uncover kinases and phosphatases that genetically interact with the exocyst. Six kinase and seven phosphatase genes were found that display enhanced phenotypes when combined with hypomorphic alleles of exoc-7 (exo70), exoc-8 (exo84), or an exoc-7;exoc-8 double mutant. In line with its reported role in exocytotic membrane trafficking, a defective exoc-8 caused accumulation of exocytotic SNARE proteins in both intestinal and neuronal cells in C. elegans. Down-regulation of the PP2A phosphatase regulatory subunit sur-6/B55 gene resulted in accumulation of exocytic SNARE proteins SNB-1 and SNAP-29 in wild-type and in exoc-8 mutant animals. In contrast, RNAi of the kinase par-1 caused reduced intracellular GFP signal for the same proteins. Double RNAi experiments for par-1, pkc-3 and sur-6/B55 in C. elegans suggest a possible cooperation and involvement in post-embryo lethality, developmental timing, as well as SNARE protein trafficking. Functional analysis of the homologous kinases and phosphatases in Drosophila median neurosecretory cells showed that aPKC kinase and phosphatase PP2A regulate exocyst-dependent insulin-like peptide secretion. Collectively, these results characterize kinases and phosphatases implicated in the regulation of exocyst function, and suggest the possibility for interplay between the par-1 and pkc-3 kinases and the PP2A phosphatase regulatory subunit sur-6 in this process (Jiu, 2013).

PP2A and receptor function

Leptin exerts its weight-reducing effects by binding to its receptor and activating signal transduction in hypothalamic neurons and other cell types. To identify the components of the leptin signal transduction pathway, an approach was developed in which bacterially expressed phosphorylated fragments of Ob receptor b (Ob-Rb) were used as affinity agents. Leptin binding to the Ob-Rb form of the leptin receptor leads to tyrosyl phosphorylation of the cytoplasmic domain of its receptor. Two of the three cytoplasmic tyrosines of Ob-Rb, at positions 985 and 1138, are phosphorylated after leptin treatment. Affinity chromatography using a tyrosine-phosphorylated fragment spanning Tyr 985 of Ob-Rb was used to identify proteins that bind to this site. The SH2 domain containing protein tyrosine phosphatase 2 (SHP-2) was isolated from bovine and mouse hypothalamus by using this method. After cotransfection into 293T cells of Ob-Rb, Janus kinase 2 (JAK2), and SHP-2, leptin treatment results in direct binding of SHP-2 to the phosphorylated Tyr 985. The bound SHP-2 is itself tyrosine phosphorylated after leptin treatment. SHP-2 is not phosphorylated after leptin treatment when a Y to F 985 receptor mutant is cotransfected. In the absence of SHP-2 phosphorylation, the level of JAK2 phosphorylation is increased. Tyrosyl phosphorylation of the leptin receptor and signal transducer and activator of transcription 3 (STAT3) are not affected by phosphorylation of SHP-2. These data suggest that activation of SHP-2 by the leptin receptor results in a decreased phosphorylation of JAK2 and may act to attenuate leptin signal transduction. The data also suggest that the dephosphorylation of JAK2 is a direct action of SHP-2. Thus, a point mutation that ablates SHP-2 phosphatase activity also ablates its effects on the state of JAK2 phosphorylation. Although SHP-2 does have intrinsic phosphatase activity, it also could lead to dephosphorylation of JAK2 indirectly by functioning as an adapter protein. For example, binding of SHP-2 to the activated platelet-derived growth factor receptor leads to its own phosphorylation at position Tyr 584, which in turn leads to binding of Grb2. Grb2 then activates ras and the mitogen-activated protein kinase signaling pathway. Previous studies have shown that leptin can activate mitogen-activated protein kinase. Indeed the available data are consistent with the possibility that SHP-2 could both decrease JAK2 phosphorylation and stimulate signaling via the mitogen-activated protein kinase or other pathways (Li, 1999).

PP2A and DNA replication

Protein phosphatase 2A (PP2A) is an abundant, multifunctional serine/threonine-specific phosphatase that stimulates simian virus 40 DNA replication. The question as to whether chromosomal DNA replication also depends on PP2A was addressed by using a cell-free replication system derived from Xenopus laevis eggs. Immunodepletion of PP2A from Xenopus egg extract results in strong inhibition of DNA replication. PP2A is required for the initiation of replication but not for the elongation of previously engaged replication forks. Therefore, the initiation of chromosomal DNA replication depends not only on phosphorylation by protein kinases but also on dephosphorylation by PP2A (Lin, 1998).

PP2A, Pin1 and proline isomerization

The reversible protein phosphorylation on serine or threonine residues that precede proline (pSer/Thr-Pro) is a key signaling mechanism for the control of various cellular processes, including cell division. The pSer/Thr-Pro moiety in peptides exists in the two completely distinct cis and trans conformations whose conversion is catalyzed specifically by the essential prolyl isomerase Pin1 (Drosophila homolog dodo). Previous results suggest that Pin1 might regulate the conformation and dephosphorylation of its substrates. However, it is not known whether phosphorylation-dependent prolyl isomerization occurs in a native protein and/or affects dephosphorylation of pSer/Thr-Pro motifs. The major Pro-directed phosphatase PP2A is conformation-specific and effectively dephosphorylates only the trans pSer/Thr-Pro isomer. Furthermore, Pin1 catalyzes prolyl isomerization of specific pSer/Thr-Pro motifs both in Cdc25C and tau to facilitate their dephosphorylation by PP2A. Moreover, Pin1 and PP2A show reciprocal genetic interactions, and prolyl isomerase activity of Pin1 is essential for cell division in vivo. Thus, phosphorylation-specific prolyl isomerization catalyzed by Pin1 is a novel mechanism essential for regulating dephosphorylation of certain pSer/Thr-Pro motifs (Zhou, 2000).

PP2A, mitosis and cell cycle arrest

A temperature-sensitive S. cerevisiae type 2A phosphatase (PP2A) mutant, pph21-102 arrests predominantly with small or aberrant buds, with abnormal actin cytoskeleton and chitin deposition. The involvement of PP2A in bud growth may be due to the role of PP2A in actin distribution during the cell cycle. Moreover, after a shift to the non-permissive temperature, the pph21-102 cells are blocked in G2 and have low activity of Clb2-Cdc28 kinase. Expression of Clb2 from the S.cerevisiae ADH promoter in pph21-102 cells is able to partially bypass the G2 arrest in the first cell cycle, but is not able to stimulate passage through a second mitosis. These cells have higher total amounts of Clb2-Cdc28 kinase activity, but the Clb2-normalized specific activity is lower in the pph21-102 cells compared with wild-type cells. Unlike wild-type strains, a PP2A-deficient strain is sensitive to the loss of MIH1, which is a homolog of the S. pombe mitotic inducer cdc25+. Furthermore, the cdc28F19 mutation cures the synthetic defects of a PP2A-deficient strain containing a deletion of MIH1. These results suggest that PP2A is required during G2 for the activation of Clb-Cdc28 kinase complexes for progression into mitosis (Lin, 1995).

The giant, unicellular alga Acetabularia is a well known experimental model for the study of actin-dependent intracellular organelle motility. In the cyst stage, however, which is equivalent to the gametophytic stage, organelles are immobile, even though an actin cytoskeleton is present. To test the hypothesis that organelle motility could be under the control of posttranslational modification by protein phosphorylation, cysts were treated with submicromolar concentrations of okadaic acid or calyculin A, both potent inhibitors of serine/threonine protein phosphatases (ser/thr-PPases). The effects were dramatic: Instead of linear actin bundles typical for control cysts, circular arrays of actin bundles form in the cortical cyst cytoplasm. Concomitant with the formation of these actin rings, the cytoplasmic layers beneath the rings begin to slowly rotate in a continuous and uniform counter-clockwise fashion. This effect suggests that protein phosphorylation acts on the actin cytoskeleton at two levels: (1) it changes the assembly properties of the actin filament system to the extent that novel cytoskeletal configurations are formed and (2) it raises the activity of putative motor proteins involved in the rotational movements to levels sufficiently high to support motility at a stage when organelle motility does not normally occur. PP2A is strongly expressed at this developmental stage whereas PP1 is not detectable, suggesting that PP2A is the likely target of the protein phosphatase inhibitors (Menzel, 1995).

Mitosis is regulated by MPF (maturation promoting factor), the active form of Cdc2/28-cyclin B complexes. Increasing levels of cyclin B abundance and the loss of inhibitory phosphates from Cdc2/28 drives cells into mitosis, whereas cyclin B destruction inactivates MPF and drives cells out of mitosis. Cells with defective spindles are arrested in mitosis by the spindle-assembly checkpoint, which prevents the destruction of mitotic cyclins and the inactivation of MPF. The relationship between the spindle-assembly checkpoint, cyclin destruction, inhibitory phosphorylation of Cdc2/28, and exit from mitosis has been investigated. Budding yeast mad mutants lack the spindle-assembly checkpoint. Spindle depolymerization does not arrest them in mitosis because they cannot stabilize cyclin B. In contrast, a newly isolated mutant in the budding yeast CDC55 gene, which encodes a protein phosphatase 2A (PP2A) regulatory subunit, shows a different checkpoint defect. In the presence of a defective spindle, these cells separate their sister chromatids and leave mitosis without inducing cyclin B destruction. Despite the persistence of B-type cyclins, cdc55 mutant cells inactivate MPF. Two experiments show that this inactivation is due to inhibitory phosphorylation on Cdc28: phosphotyrosine accumulates on Cdc28 in cdc55 delta cells whose spindles have been depolymerized, and a cdc28 mutant that lacks inhibitory phosphorylation sites on Cdc28 allows spindle defects to arrest cdc55 mutants in mitosis with active MPF and unseparated sister chromatids. It is concluded that perturbations of protein phosphatase activity allow MPF to be inactivated by inhibitory phosphorylation instead of by cyclin destruction. Under these conditions, sister chromatid separation appears to be regulated by MPF activity rather than by protein degradation. The role of PP2A and Cdc28 phosphorylation in cell-cycle control is discussed; it is possibile that the novel mitotic exit pathway plays a role in adaptation to prolonged activation of the spindle-assembly checkpoint (Minshull, 1996).

Saccharomyces cerevisiae, like most eukaryotic cells, can prevent the onset of anaphase until chromosomes are properly aligned on the mitotic spindle. Cdc55p (regulatory B subunit of protein phosphatase 2A [PP2A]) is required for the kinetochore/spindle checkpoint regulatory pathway in yeast. ctf13 cdc55 double mutants could not maintain a ctf13-induced mitotic delay, as determined by antitubulin staining and levels of histone H1 kinase activity. In addition, cdc55::LEU2 mutants and tpd3::LEU2 mutants (regulatory A subunit of PP2A) are nocodazole sensitive and exhibit the phenotypes of previously identified kinetochore/spindle checkpoint mutants. Inactivating CDC55 does not simply bypass the arrest that results from inhibiting ubiquitin-dependent proteolysis because cdc16-1 cdc55::LEU2 and cdc23-1 cdc55::LEU2 double mutants arrest normally at elevated temperatures. CDC55 is specific for the kinetochore/spindle checkpoint because cdc55 mutants show normal sensitivity to gamma radiation and hydroxyurea. The conditional lethality and the abnormal cellular morphogenesis of cdc55::LEU2 are suppressed by cdc28F19, suggesting that the cdc55 phenotypes are dependent on the phosphorylation state of Cdc28p. In contrast, the nocodazole sensitivity of cdc55::LEU2 is not suppressed by cdc28F19. Therefore, the mitotic checkpoint activity of CDC55 (and TPD3) is independent of regulated phosphorylation of Cdc28p. Finally, cdc55::LEU2 suppresses the temperature sensitivity of cdc20-1, suggesting additional roles for CDC55 in mitosis (Wang, 1997).

PPAR gamma is an adipose-selective nuclear hormone receptor that plays a key role in the control of adipocyte differentiation. Previous studies have indicated that activation of ectopically expressed PPAR gamma induces differentiation when cells have ceased growth because of confluence. Ligand activation of PPAR gamma is sufficient to induce growth arrest in fibroblasts and SV40 large T-antigen transformed, adipogenic HIB1B cells. Cell cycle withdrawal is accompanied by a decrease in the DNA-binding and transcriptional activity of the E2F/DP complex (See Drosophila E2F), which is attributable to an increase in the phosphorylation of these proteins, especially DP-1. This effect is a consequence of decreased expression of the catalytic subunit of the serine-threonine phosphatase PP2A. These data suggest an important role for PP2A in the control of E2F/DP activity and a new mode of cell cycle control in differentiation (Altiok, 1997).

MPF, a protein kinase complex consisting of cyclin and p34cdc2 subunits, promotes the G2 to M phase transition in eukaryotic cells. The pathway of activation and inactivation of MPF is not well understood, although there is strong evidence that removal of phosphate from a tyrosine residue on p34cdc2 is part of the activation process. INH was originally identified as an activity that could inhibit the posttranslational activation of a latent form of MPF, called pre-MPF, in immature (G2 phase-arrested) Xenopus oocytes. INH is a form of protein phosphatase 2A. Both INH and the catalytic subunit of protein phosphatase 2A can directly inactivate an isolated p34cdc2-cyclin complex. Both cyclin and p34cdc2 become dephosphorylated; the rate of inactivation closely parallels the removal of phosphate from a specific site on p34cdc2. It is proposed that INH opposes MPF activation by reversing this critical phosphorylation (Lee, 1991).

INH, a type 2A protein phosphatase (PP2A), negatively regulates entry into M phase and the cyclin B-dependent activation of cdc2 in Xenopus extracts. INH appears to be central to the mechanism of the trigger for mitotic initiation, as it prevents the premature activation of cdc2. INH is a conventional form of PP2A with a B alpha regulatory subunit. Although PP2A inhibits the switch in tyrosine kinase and tyrosine phosphatase activities accompanying mitosis, this switch is a consequence of the inhibition of some other rate-limiting event. In the preactivation phase, PP2A inhibits the pathway leading to T161 phosphorylation, suggesting that this activity may be one of the rate-limiting events for transition. However, these results also suggest that the accumulation of active cdc2/cyclin complexes during the lag is only one of the events required for triggering entry into mitosis (Lee, 1994).

A WD-40 repeat protein, TRIP-1, associates with the type II transforming growth factor beta (TGF-beta) receptor. Another WD-40 repeat protein, the Balpha subunit of protein phosphatase 2A, associates with the cytoplasmic domain of type I TGF-beta receptors. This association depends on the kinase activity of the type I receptor; it is increased by coexpression of the type II receptor (which is known to phosphorylate and activate the type I receptor) and allows the type I receptor to phosphorylate Balpha. Furthermore, Balpha enhances the growth inhibition activity of TGF-beta in a receptor-dependent manner. Because Balpha has been characterized as a regulator of phosphatase 2A activity, these observations suggest possible functional interactions between the TGF-beta receptor complex and the regulation of protein phosphatase 2A (Griswold-Prenner, 1998).

The effect of Balpha on the growth inhibition response of TGF-beta complements the role of Smads as effectors of TGF-beta receptor signaling. Smads function as transcriptional activators that induce the expression of various genes. Since the transcription of several genes is induced by Smads, Smads may induce growth inhibition by inducing transcription of the cdk inhibitors p15 and p21 in response to TGF-beta. Overexpression of Balpha induces growth inhibition to a level comparable to that of overexpression of Smads and, like the Smads, the effect of Balpha on growth inhibition depends on receptor activity. Furthermore, the antiproliferative effect of Balpha does not depend on Smad4, suggesting that TGF-beta receptor activation may induce two parallel pathways that lead to the antiproliferative response, one propagated by Smad proteins and the other one propagated through Balpha. Although the mechanism of the receptor-dependent growth inhibition by Balpha is not known, one possibility is that it acts through the ability of PP2A to regulate MAP kinase activity, especially since PP2A is a major enzyme involved in dephosphorylating MAP kinase. Therefore, altered PP2A activity following TGF-beta receptor activation might contribute to growth inhibition by deactivating this growth stimulatory pathway, theregy complementing the direct induction of growth inhibition by Smads. Moreover, a possible regulation of PP2A activity by TGF-beta may also directly affect the cell cycle, which would be consistent with the observed role of PP2A in cell cycle control (Griswold-Prenner, 1998 and references).

The activation of Cdc2 kinase induces the entry into M-phase of all eukaryotic cells. A cell-free system prepared from prophase-arrested Xenopus oocytes has been developed to analyze the mechanism initiating the all-or-none activation of Cdc2 kinase. Inhibition of phosphatase 2A, the major okadaic acid-sensitive Ser/Thr phosphatase in these extracts, provokes Cdc2 kinase amplification and concomitant hyperphosphorylation of Cdc25 phosphatase, with a lag of about 1 h. Polo-like kinase (Plx1 kinase) is activated slightly after Cdc2. All these events are totally inhibited by the cdk inhibitor p21(Cip1), demonstrating that Plx1 kinase activation depends on Cdc2 kinase activity. Addition of a threshold level of recombinant Cdc25 induces a linear activation of Cdc2 and Plx1 kinases and a partial phosphorylation of Cdc25. It is proposed that the Cdc2 positive feedback loop involves two successive phosphorylation steps of Cdc25 phosphatase: the first one is catalyzed by Cdc2 kinase and/or Plx1 kinase but it does not modify Cdc25 enzymatic activity; the second one requires a new kinase counteracted by phosphatase 2A. Under the conditions of this assay, Cdc2 amplification and MAP kinase activation are two independent events (Kara, 1998).

The initiation of anaphase and exit from mitosis depend on the anaphase-promoting complex (APC), which mediates the ubiquitin-dependent proteolysis of anaphase-inhibiting proteins and mitotic cyclins. An investigation was carried out using Xenopus egg extracts to see if protein phosphatases are required for mitotic APC activation. In Xenopus egg extracts, APC activation occurs normally in the presence of protein phosphatase 1 inhibitors, suggesting that the anaphase defects caused by protein phosphatase 1 mutation in several organisms are not due to a failure to activate the APC. Contrary to this, the initiation of mitotic cyclin B proteolysis is prevented by inhibitors of protein phosphatase 2A, such as okadaic acid. Okadaic acid induces an activity that inhibits cyclin B ubiquitination. This activity is referred to as inhibitor of mitotic proteolysis because it also prevents the degradation of other APC substrates. A similar activity exists in extracts of Xenopus eggs that are arrested at the second meiotic metaphase by the cytostatic factor activity of the protein kinase mos. In Xenopus eggs, the initiation of anaphase II may therefore be prevented by an inhibitor of APC-dependent ubiquitination (Vorlaufer, 1998).

Efficient translation of the mRNA encoding the 65-kDa regulatory subunit (PR65 alpha) of protein phosphatase 2A (PP2A) is prevented by an out of frame upstream AUG and a stable stem-loop structure (delta G = -55.9 kcal/mol) in the 5'-untranslated region (5'-UTR). Deletion of the 5'-UTR allows efficient translation of the PR65 alpha message in vitro and overexpression in COS-1 cells. Insertion of the 5'-UTR into the beta-galactosidase leader sequence dramatically inhibits translation of the beta-galactosidase message in vitro and in vivo, confirming that this sequence functions as a potent translation regulatory sequence. Cells transfected or microinjected with a PR65 alpha expression vector lacking the 5'-UTR, express high levels of PR65 alpha, accumulating in both nucleus and cytoplasm. PR65 alpha overexpressing rat embryo fibroblasts (REF-52 cells) become multinucleated. These data suggest that PP2A participates in the regulation of both mitosis and cytokinesis (Wera, 1995).

Protein phosphatase 2A (PP2A) is present on microtubules in neuronal and nonneuronal cells. Interphase and mitotic spindle microtubules, as well as centrosomes, were all labeled with antibodies against individual PP2A subunits, showing that the AB alpha C holoenzyme is associated with microtubules. PP2A can be reversibly bound to microtubules in vitro; approximately 75% of the PP2A in cytosolic extracts can interact with microtubules. The activity of microtubule-associated PP2A is differentially regulated during the cell cycle. Enzymatic activity is high during S phase and intermediate during G1, while the activity in G2 and M is 20-fold lower than during S phase. The amount of microtubule-bound PP2A remains constant throughout the cell cycle, implying that cell cycle regulation of its enzymatic activity involves factors other than microtubules. These results raise the possibility that PP2A regulates cell cycle-dependent microtubule functions, such as karyokinesis and membrane transport (Sontag, 1995).

Assembly of a mitotic spindle requires the accurate regulation of microtubule dynamics; this is accomplished, at least in part, by phosphorylation-dephosphorylation reactions. The role of serine-threonine phosphatases in the control of microtubule dynamics has been investigated using specific inhibitors in Xenopus egg extracts. Type 2A phosphatases are required to maintain the short steady-state length of microtubules in mitosis by regulating the level of microtubule catastrophes, in part by controlling the the microtubule-destabilizing activity and phosphorylation of Op18/stathmin. Type 1 phosphatases are only required for control of microtubule dynamics during the transitions into and out of mitosis. Thus, although both type 2A and type 1 phosphatases are involved in the regulation of microtubule dynamics, they have distinct, non-overlapping roles (Tournebize, 1997).

Most cancer cells have increased levels of telomerase activity implicated in cell immortalization. Activation of telomerase, a ribonucleoprotein complex, catalyzes the elongation of the ends of mammalian chromosomal DNA (telomeres), the length of which regulates cell proliferation. Currently, how telomerase is regulated in cancer is not yet established. The present study shows that telomerase activity is regulated by protein phosphorylation in human breast cancer cells. Incubation of cell nuclear telomerase extracts with protein phosphatase 2A (PP2A) abolishes the telomerase activity; in contrast, cytoplasmic telomerase activity is unaffected, and protein phosphatases 1 and 2B are ineffective. Inhibition of telomerase activity by PP2A is both concentration- and time-dependent and is prevented by the protein phosphatase inhibitor okadaic acid. In addition, nuclear telomerase inhibited by PP2A is reactivated by endogenous protein kinase(s) in the presence of ATP, but not in the presence of ATPgammaS. Telomerase activity in cultured human breast cancer PMC42 cells is stimulated by okadaic acid, consistent with a role for PP2A in the regulation of telomerase activity in intact cells. These findings suggest that protein phosphorylation reversibly regulates the function of telomerase and that PP2A is a telomerase inhibitory factor in the nucleus of human breast cancer cells (Li, 1997).

The auto-catalytic activation of the cyclin-dependent kinase Cdc2 or MPF (M-phase promoting factor) is an irreversible process responsible for the entry into M phase. In Xenopus oocyte, a positive feed-back loop between Cdc2 kinase and its activating phosphatase Cdc25 allows the abrupt activation of MPF and the entry into the first meiotic division. The Cdc2/Cdc25 feed-back loop was studied using cell-free systems derived from Xenopus prophase-arrested oocyte. The findings support the following two-step model for MPF amplification: during the first step, Cdc25 acquires a basal catalytic activity resulting in a linear activation of Cdc2 kinase. In turn, Cdc2 partially phosphorylates Cdc25 but no amplification takes place; under this condition Plx1 kinase and its activating kinase Plkk1 are activated. However, their activity is not required for the partial phosphorylation of Cdc25. This first step occurs independent of PP2A or Suc1/Cks-dependent Cdc25/Cdc2 association. On the contrary, the second step involves the full phosphorylation and activation of Cdc25 and the initiation of the amplification loop. It depends both on PP2A inhibition and Plx1 kinase activity. Suc1-dependent Cdc25/Cdc2 interaction is required for this process (Karaiskou, 1999).

On TGF-beta binding, the TGF-beta receptor directly phosphorylates and activates the transcription factors Smad2/3, leading to G1 arrest. Evidence is presented for a second, parallel, TGF-beta-dependent pathway for cell cycle arrest, achieved via inhibition of p70s6k. TGF-beta induces association of its receptor with protein phosphatase-2A (PP2A)-Balpha. Concomitantly, three PP2A-subunits, Balpha, Abeta, and Calpha, associate with p70s6k, leading to its dephosphorylation and inactivation. Although either pathway is sufficient to induce G1 arrest, abrogation of both, the inhibition of p70s6k, and transcription through Smad proteins is required for release of epithelial cells from TGF-beta-induced G1 arrest. TGF-beta thereby modulates the translational and posttranscriptional control of cell cycle progression (Petritsch, 2000).

On receptor activation, PP2A-Balpha specifically binds the activated TbetaRI and is catalytically activated by TGF-beta. PP2A-Balpha then recruits PP2A-Abeta and PP2A-C to bind and dephosphorylate p70s6k. Complexes containing at the same time PP2A, TbetaRI and p70s6k, could not be detected, indicating that on activation the phosphatase is released from the receptor to bind to the target molecule. Immunolocalization of the endogenous proteins supports this model. p70s6k activity controls the translational upregulation of proteins important for G1/S progression and is itself essential for cell cycle progression. Most of the transcripts isolated to date represent ribosomal proteins and elongation factors of protein synthesis. TGF-beta-induced inactivation of p70s6k leads to the translational regulation of a group of cell cycle regulators for G1 progression. It remains unclear, however, if the repression of those cell cycle regulators result from global repression of protein translation or represent a class of specifically translationally repressed mRNAs. It is conceivable that the regulation of crucial components of the cell cycle machinery is mediated at the transcriptional, translational, and posttranslational levels (Petritsch, 2000).

Expression of the regulatory subunit PP2A-Balpha itself appears to be a prerequisite for the PP2A-mediated inhibition of p70s6k by TGF-beta. Cells with nondetectable PP2A-Balpha expression remain solely responsive to TGF-beta-mediated transcriptional responses. p70s6k is not inhibited by TGF-beta in these cells; this reflects the differential sensitivity of epithelial cells and mesenchymal cells to growth inhibitory effects of TGF-beta. The chromosomal localization of PP2A-Balpha has not been investigated; PP2A-Abeta, however, has been mapped to a human tumor suppressor locus on 11q22-24 and appears to be mutated in a subset of human lung tumors. It is tempting to speculate that mutations of the regulatory subunits of PP2A in human tumors abolish the regulation of p70s6k to TGF-beta and confer a selective advantage to growing tumors (Petritsch, 2000).

Protein phosphatase 2A (PP2A) holoenzymes consist of a catalytic C subunit, a scaffolding A subunit, and one of several regulatory B subunits that recruit the AC dimer to substrates. PP2A is required for chromosome segregation, but PP2A's substrates in this process remain unknown. To identify PP2A substrates, a two-hybrid screen was carried out with the regulatory B/PR55 subunit. A human homolog of C. elegans HCP6, a protein distantly related to the condensin subunit hCAP-D2 was isolated, and this homolog was named hHCP-6. Both C. elegans HCP-6 and condensin are required for chromosome organization and segregation. HCP-6 binding partners are unknown, whereas condensin is composed of the structural maintenance of chromosomes proteins SMC2 and SMC4 and of three non-SMC subunits. hHCP-6 becomes phosphorylated during mitosis and its dephosphorylation by PP2A in vitro depends on B/PR55, suggesting that hHCP-6 is a B/PR55-specific substrate of PP2A. Unlike condensin, hHCP-6 is localized in the nucleus in interphase, but similar to condensin, hHCP-6 associates with chromosomes during mitosis. hHCP-6 is part of a complex that contains SMC2, SMC4, kleisin-ß, and the previously uncharacterized HEAT repeat protein FLJ20311. hHCP-6 is therefore part of a condensin-related complex that associates with chromosomes in mitosis and may be regulated by PP2A (Yeong, 2003).

The success of cell division relies on the activation of its master regulator Cdc2-cyclin B, and many other kinases controlling cellular organization, such as Aurora-A. Most of these kinase activities are regulated by phosphorylation. Despite numerous studies showing that okadaic acid-sensitive phosphatases regulate both Cdc2 and Aurora-A activation, their identity has not yet been established in Xenopus oocytes and the importance of their regulation has not been evaluated. Using an oocyte cell-free system, it has been demonstrated that PP2A depletion is sufficient to lead to Cdc2 activation, whereas Aurora-A activation depends on Cdc2 activity. The activity level of PP1 does not affect Cdc2 kinase activation promoted by PP2A removal. PP1 inhibition is also not sufficient to lead to Aurora-A activation in the absence of active Cdc2. It is therefore conclude that in Xenopus oocytes, PP2A is the key phosphatase that negatively regulates Cdc2 activation. Once this negative regulator is removed, endogenous kinases are able to turn on the activator Cdc2 system without any additional stimulation. In contrast, Aurora-A activation is indirectly controlled by Cdc2 activity independently of either PP2A or PP1. This strongly suggests that in Xenopus oocytes, Aurora-A activation is mainly controlled by the specific stimulation of kinases under the control of Cdc2 and not by downregulation of phosphatase (Maton, 2005).

DNA-responsive checkpoints prevent cell-cycle progression following DNA damage or replication inhibition. The mitotic activator Cdc25 is suppressed by checkpoints through inhibitory phosphorylation at Ser287 (Xenopus numbering) and docking of 14-3-3. Ser287 phosphorylation is a major locus of G2/M checkpoint control, although several checkpoint-independent kinases can phosphorylate this site. Mitotic entry requires 14-3-3 removal and Ser287 dephosphorylation. DNA-responsive checkpoints also activate PP2A/B56Δ phosphatase complexes to dephosphorylate Cdc25 at a site distinct from Ser287 (T138), the phosphorylation of which is required for 14-3-3 release. However, phosphorylation of T138 is not sufficient for 14-3-3 release from Cdc25. These data suggest that creation of a 14-3-3 'sink,' consisting of phosphorylated 14-3-3 binding intermediate filament proteins, including keratins, coupled with reduced Cdc25-14-3-3 affinity, contribute to Cdc25 activation. These observations identify PP2A/B56Δ as a central checkpoint effector and suggest a mechanism for controlling 14-3-3 interactions to promote mitosis (Margolis, 2006).

A role for Cdc2- and PP2A-mediated regulation of Emi2 in the maintenance of CSF arrest

Vertebrate oocytes are arrested in metaphase II of meiosis prior to fertilization by cytostatic factor (CSF). CSF enforces a cell-cycle arrest by inhibiting the anaphase-promoting complex (APC), an E3 ubiquitin ligase that targets Cyclin B for degradation. Although Cyclin B synthesis is ongoing during CSF arrest, constant Cyclin B levels are maintained. To achieve this, oocytes allow continuous slow Cyclin B degradation, without eliminating the bulk of Cyclin B, which would induce release from CSF arrest. However, the mechanism that controls this continuous degradation is not understood. This study reports the molecular details of a negative feedback loop wherein Cyclin B promotes its own destruction through Cdc2/Cyclin B-mediated phosphorylation and inhibition of the APC inhibitor Emi2. Emi2 binds to the core APC, and this binding is disrupted by Cdc2/Cyclin B, without affecting Emi2 protein stability. Cdc2-mediated phosphorylation of Emi2 is antagonized by PP2A, which can bind to Emi2 and promote Emi2-APC interactions. It is concluded that constant Cyclin B levels are maintained during a CSF arrest through the regulation of Emi2 activity. A balance between Cdc2 and PP2A controls Emi2 phosphorylation, which in turn controls the ability of Emi2 to bind to and inhibit the APC. This balance allows proper maintenance of Cyclin B levels and Cdc2 kinase activity during CSF arrest (Wu, 2007)

Phosphorylation of mammalian Sgo2 by Aurora B recruits PP2A and MCAK to centromeres

Shugoshin (Sgo) is a conserved centromeric protein. Mammalian Sgo1 collaborates with protein phosphatase 2A (PP2A) to protect mitotic cohesin from the prophase dissociation pathway. Although another shugoshin-like protein, Sgo2, is required for the centromeric protection of cohesion in germ cells, its precise molecular function remains largely elusive. This study demonstrates that hSgo2 plays a dual role in chromosome congression and centromeric protection of cohesion in HeLa cells, while the latter function is exposed only in perturbed mitosis. These functions partly overlap with those of Aurora B, a kinase setting faithful chromosome segregation. Accordingly, phosphorylation of hSgo2 by Aurora B was identified at the N-terminal coiled-coil region and the middle region, and these phosphorylations were shown to separately promote binding of hSgo2 to PP2A and MCAK, factors required for centromeric protection and chromosome congression, respectively. Furthermore, these phosphorylations are essential for localizing PP2A and MCAK to centromeres. This mechanism seems applicable to germ cells as well. Thus, this study identifies Sgo2 as a hitherto unknown crucial cellular substrate of Aurora B in mammalian cells (Tanno, 2010).

Formation of stable attachments between kinetochores and microtubules depends on the B56-PP2A phosphatase

Error-free chromosome segregation depends on the precise regulation of phosphorylation to stabilize kinetochore-microtubule attachments (K-fibres) on sister chromatids that have attached to opposite spindle poles (bi-oriented). In many instances, phosphorylation correlates with K-fibre destabilization. Consistent with this, multiple kinases, including Aurora B and Plk1, are enriched at kinetochores of mal-oriented chromosomes when compared with bi-oriented chromosomes, which have stable attachments. Paradoxically, however, these kinases also target to prometaphase chromosomes that have not yet established spindle attachments and it is therefore unclear how kinetochore-microtubule interactions can be stabilized when kinase levels are high. This study shows, using human retinal pigment epithelial cells and HeLa cells, that the generation of stable K-fibres depends on the B56-PP2A phosphatase, which is enriched at centromeres/kinetochores of unattached chromosomes. When B56-PP2A is depleted, K-fibres are destabilized and chromosomes fail to align at the spindle equator. Strikingly, B56-PP2A depletion increases the level of phosphorylation of Aurora B and Plk1 kinetochore substrates as well as Plk1 recruitment to kinetochores. Consistent with increased substrate phosphorylation, it was found that chemical inhibition of Aurora or Plk1 restores K-fibres in B56-PP2A-depleted cells. These findings reveal that PP2A, an essential tumour suppressor, tunes the balance of phosphorylation to promote chromosome-spindle interactions during cell division (Foley, 2011).

PP2A-B56gamma is required for an efficient spindle assembly checkpoint

The Spindle Assembly Checkpoint (SAC) is part of a complex feedback system designed to ensure that cells do not proceed through mitosis unless all chromosomal kinetochores have attached to spindle microtubules. The formation of the kinetochore complex and the implementation of the SAC are regulated by multiple kinases and phosphatases. BubR1 is a phosphoprotein that is part of the Cdc20 containing mitotic checkpoint complex that inhibits the APC/C so that Cyclin B1 and Securin are not degraded, thus preventing cells going into anaphase. This study found that PP2A in association with its B56gamma regulatory subunit, are needed for the stability of BubR1 during nocodazole induced cell cycle arrest. In primary cells that lack B56gamma, BubR1 is prematurely degraded and the cells proceed through mitosis. The reduced SAC efficiency results in cells with abnormal chromosomal segregation, a hallmark of transformed cells. Previous studies on PP2A's role in the SAC and kinetochore formation were done using siRNAs to all 5 of the B56 family members. This study shows that inactivation of only the PP2A-B56gamma subunit can affect the efficiency of the SAC. Data is provided that show the intracellular locations of the B56 subunits varies between family members, which is consistent with the hypothesis that they are not completely functionally redundant (Varadkar, 2017).

PP2A, Mdm2, Cyclin G and p53

A fuller understanding of the function of cyclin G, a commonly induced p53 target, has remained elusive. Cyclin G forms a quaternary complex in vivo and in vitro with enzymatically active phosphatase 2A (PP2A) holoenzymes containing B' subunits. Interestingly, cyclin G also binds in vivo and in vitro to Mdm2 and markedly stimulates the ability of PP2A to dephosphorylate Mdm2 at T216. Consistent with these data, cyclin G null cells have both Mdm2 that is hyperphosphorylated at T216 and markedly higher levels of p53 protein when compared to wild-type cells. Cyclin G expression also results in reduced phosphorylation of human Hdm2 at S166. Thus, these data suggest that cyclin G recruits PP2A in order to modulate the phosphorylation of Mdm2 and thereby to regulate both Mdm2 and p53 (Okamoto, 2002).

Eukaryotic proteins are frequently regulated through their state of phosphorylation. Although protein kinases frequently recognize sequence motifs to target them to their sites in substrates, there is often less specificity in the sequence requirements of the major cellular phosphatases. Therefore, other mechanisms are needed for direction of phosphatases to their substrates, and these results suggest that cylin G serves such a role. In fact, PP2A is likely to be extensively regulated. Individual PP2A complexes have been shown to differ in some cases in their roles, localization, and substrate specificity. Thus, the apparently exclusive association of cyclin G with the B' subfamily is tantalizing. Cyclin G is of course not the only protein that has been shown to be able to interact with PP2A. Among the proteins shown to associate with PP2A and regulate its activity are the small t antigens encoded by SV40 and polyomavirus, the adenovirus E4orf4 protein, casein kinase II, Hox II, PKR, and several others. In some cases, the interaction results in negative regulation of PP2A activity. Although the effect of recruitment of cyclin G on the specific activity of PP2A is not known, cyclin G clearly does not block PP2A enzymatic activity, supporting the possibility that cyclin G serves to recruit PP2A to specific substrates (Okamoto, 2002).

The discovery that cyclin G binds to Mdm2 provided the impetus for testing whether Mdm2 might serve as such a substrate. The data strongly support the conclusion that at least two phosphorylation sites (Mdm2 T216 and Hdm2 S166) are substrates of cyclin G-directed PP2A. Since Mdm2 can associate with a host of cellular proteins, a future challenge will be to determine whether such interactions are regulated by phosphorylation, and if so, which of these are regulated by the cyclin G-PP2A complex. It is, of course, also possible that the cyclin G-PP2A interaction is relevant to other potential substrates, and therefore, the identification of cellular proteins that can interact with cyclin G may prove to be very interesting (Okamoto, 2002).

Mouse cells lacking cyclin G contain both Mdm2 that is hyperphosphorylated at T216 and higher p53 levels when compared to wild-type cells. These two observations are very likely interrelated. Although it is still not fully understood how modification of p53 affects its functions in vivo, phosphorylation of p53 at some N-terminal residues (that are modified in cells in response to DNA damage) decreases the ability of p53 to bind to Mdm2 in vitro. Modification of Mdm2 also impacts on its interactions with p53; phosphorylation of human Hdm2 by DNA PK (at S17 within the N terminus) and phosphorylation of murine Mdm2 by cyclin A/CDK2 (at T216 within the acidic domain) block and attenuate, respectively, the ability of either protein to bind to p53. Moreover, phosphorylation of human Mdm2 (Hdm2) at S395, a process that can be accomplished by ATM kinase in vitro, counteracts Mdm2's ability to target p53 for degradation in vivo. It can thus be speculated that in general, activated p53 and deactivated Mdm2 are the more phosphorylated forms of each protein. The data imply that the function of cyclin G is to serve as a negative regulator of p53 by activating Mdm2 through dephosphorylation. When seen in this context, it becomes less surprising that many previous studies have indicated that cyclin G expression is associated with growth promotion rather than arrest. Most exciting is the evidence that cyclin G null mice have fewer and smaller carcinogen-induced liver tumors, consistent with the hypothesis that cyclin G serves to negatively regulate the tumor suppressor function of p53 (Okamoto, 2002).

Structure and function of the PP2A-shugoshin interaction

Accurate chromosome segregation during mitosis and meiosis depends on shugoshin proteins that prevent precocious dissociation of cohesin from centromeres. Shugoshins associate with PP2A, which is thought to dephosphorylate cohesin and thereby prevent cleavage by separase during meiosis I. A crystal structure of a complex between a fragment of human Sgo1 and an AB'C PP2A holoenzyme reveals that Sgo1 forms a homodimeric parallel coiled coil that docks simultaneously onto PP2A's C and B' subunits. Sgo1 homodimerization is a prerequisite for PP2A binding. While hSgo1 interacts only with the AB'C holoenzymes, its relative, Sgo2, interacts with all PP2A forms and may thus lead to dephosphorylation of distinct substrates. Mutant shugoshin proteins defective in the binding of PP2A cannot protect centromeric cohesin from separase during meiosis I or support the spindle assembly checkpoint in yeast. Finally, evidence is provided that PP2A's recruitment to chromosomes may be sufficient to protect cohesin from separase in mammalian oocytes (Xu, 2009).

Sgol2 provides a regulatory platform that coordinates essential cell cycle processes during meiosis I in oocytes: Sgol2's ability to protect cohesin depends on its interaction with PP2A

Accurate chromosome segregation depends on coordination between cohesion resolution and kinetochore-microtubule interactions (K-fibers), a process regulated by the spindle assembly checkpoint (SAC). How these diverse processes are coordinated remains unclear. This study shows that in mammalian oocytes Shugoshin-like protein 2 (Sgol2; Drosophila homolog Mei-S332) in addition to protecting cohesin, plays an important role in turning off the SAC, in promoting the congression and bi-orientation of bivalents on meiosis I spindles, in facilitating formation of K-fibers and in limiting bivalent stretching. Sgol2's ability to protect cohesin depends on its interaction with PP2A, as is its ability to silence the SAC, with the latter being mediated by direct binding to Mad2. In contrast, its effect on bivalent stretching and K-fiber formation is independent of PP2A and mediated by recruitment of MCAK and inhibition of Aurora C kinase activity respectively. By virtue of its multiple interactions, Sgol2 links many of the processes essential for faithful chromosome segregation (Rattani, 2013).

The production of haploid gametes from diploid germ cells depends on two rounds of chromosome segregation (meiosis I and II) without an intervening round of DNA replication. Defects during the first or second meiotic division in oocytes lead to formation of aneuploid eggs, which in humans occurs with a frequency between 10 and 30% and is a major cause of fetal miscarriage. Understanding the causes of meiotic chromosome missegregation will require clarifying not only the forces and regulatory mechanisms governing meiotic chromosome segregation but also how these are coordinated (Rattani, 2013).

At the heart of this process are two opposing forces. The pulling forces produced by kinetochore-microtubules attachments (K-fibers) and resisting forces generated by sister chromatid cohesion, which counteracts K-fiber forces if and when kinetochores attach to microtubules with different polarities. During meiosis I, cohesion along chromosome arms holds bivalent chromosomes together following the creation of chiasmata produced by reciprocal recombination between homologous non-sister chromatids. This cohesion must persist during the attachment of maternal and paternal kinetochores to microtubules from opposite poles (bi-orientation) and resist the resulting spindle forces. The degree of traction exerted by meiotic spindles must create sufficient tension to facilitate bi-orientation. During the bi-orientation process, the initial attachment of kinetochores to the surface of the microtubule lattice (lateral attachment) is converted to attachments to their plus ends (end on attachment), creating K-fibers. Because this process is intrinsically error prone, inappropriate attachments, for example those that connect maternal and paternal kinetochores to the same pole, must be disrupted through phosphorylation of kinetochore proteins by Aurora B/C kinases. However, because these kinases disrupt K-fibers, they must subsequently be down-regulated once bivalents bi-orient correctly (Rattani, 2013).

The first meiotic division is eventually triggered by activation of a gigantic ubiquitin protein ligase called the anaphase-promoting complex or cyclosome (APC/C) whose destruction of securin and cyclin B activates a thiol protease called separase that cleaves the kleisin subunit of the cohesin complex holding sister chromatids together. This process converts chromosomes from bivalents to dyads. It is delayed until all bivalents have bi-oriented by the production, at kinetochores that have not yet come under tension, of a potent inhibitor of the APC/C called the mitotic checkpoint complex (MCC) whose Mad2 subunit binds tightly to the APC/C's Cdc20 co-activator protein. This regulatory mechanism, called the spindle assembly checkpoint (SAC), must be turned off before APC/CCdc20 can direct destruction of securin and cyclin B and thereby activate separase. Another pre-condition for cleavage, at least in yeast, is phosphorylation of cohesin's kleisin subunit by a pair of protein kinases, namely CK1δ/ε and DDK. During the first meiotic division, cohesin is phosphorylated along chromosome arms but not at centromeres, which ensures that only cohesion along arms is destroyed by separase at the onset of anaphase I. The consequent persistence of cohesion at centromeres promotes bi-orientation of dyads during meiosis II (Rattani, 2013).

Centromeric cohesin avoids phosphorylation and therefore cleavage during the first meiotic division because separase activation is preceded by the recruitment to centromeres of orthologues of the Drosophila MEI-S332 protein, called shugoshins. Members of this family contain a conserved C-terminal basic region and an N-terminal homodimeric parallel coiled coil, which provides a docking site for protein phosphatase 2A's (PP2A) C and B' subunits (Xu, 2009). In budding yeast, mutant proteins defective specifically in PP2A binding fail to confer protection of centromeric cohesion during meiosis I (Rattani, 2013).

Mammals have two members of the shugoshin family: Shugoshin-like protein 1 (Sgol1), and Shugoshin-like protein 2 (Sgol2). The former prevents centromeric cohesin from a process called the 'prophase pathway' that removes cohesin from chromosomes by a non-proteolytic mechanism soon after cells enter mitosis (McGuinness, 2005; Liu, 2013). Sgol2, on the other hand, protects centromeric cohesin from separase at the first meiotic division (Lee, 2008; Llano, 2008). Sgol2's coiled coil domain binds PP2A in vitro (Xu, 2009) but whether this is vital for protecting centromeric cohesion is not known. Sgol2 also interacts with MCAK (Huang, 2007), a microtubule depolymerizing kinesin, implicated in correcting inappropriate kinetochore-microtubule interactions (error correction), and with Mad2 an essential component of the MCC (Orth, 2011). Shugoshins, though not explicitly Sgol2, have also been implicated in recruiting to kinetochores the Aurora B kinase, necessary both for the SAC and for error correction (Tsukahara, 2010; Yamagishi, 2010). Based on its interactions, Sgol2 has been linked to cohesion protection, the spindle assembly checkpoint, and error correction pathways. However, the physiological significance of these multiple interactions remain unclear (Rattani, 2013).

This study shows that Sgol2 defective in PP2A binding fails to protect centromeric cohesin, as found for Sgo1 in yeast (Xu, 2009). However, if this were the sole function of Sgol2, then chromosome behavior during meiosis I should be unaffected. Surprisingly it was found that Sgol2 deficiency caused striking changes in chromosome and microtubule dynamics. It delayed bi-orientation of bivalents on meiosis I spindles, caused increased bivalent stretching, and greatly increased Aurora B/C kinase activity at kinetochores, which was accompanied by an increase in lateral and a decrease in end on kinetochore-microtubules attachments. Lastly, Sgol2 deficiency delayed considerably APC/CCdc20 activation, suggesting that it was required to shut off the SAC. Sgol2 helps to turn off the SAC by binding directly both PP2A and the MCC protein Mad2, it moderates chromosome stretching by recruiting to kinetochores the kinesin MCAK, and likely promotes formation of K-fibers by down-regulating the activity of Aurora B/C kinase specifically at kinetochores. Meiotic chromosome segregation is a highly complex process dependent on an array of different biochemical processes. These findings imply that through its multi-domain structure, Sgol2 has an important if not unique role in coordinating many of the key processes within this array (Rattani, 2013).

Cyclin B-Cdk1 inhibits protein phosphatase PP2A-B55 via a Greatwall kinase-independent mechanism>

Entry into M phase is governed by cyclin B-Cdk1, which undergoes both an initial activation and subsequent autoregulatory activation. A key part of the autoregulatory activation is the cyclin B-Cdk1-dependent inhibition of the protein phosphatase 2A (PP2A)-B55, which antagonizes cyclin B-Cdk1. Greatwall kinase (Gwl) is believed to be essential for the autoregulatory activation because Gwl is activated downstream of cyclin B-Cdk1 to phosphorylate and activate alpha-endosulfine (Ensa)/Arpp19, an inhibitor of PP2A-B55. However, cyclin B-Cdk1 becomes fully activated in some conditions lacking Gwl, yet how this is accomplished remains unclear. This study shows that cyclin B-Cdk1 can directly phosphorylate Arpp19 on a different conserved site, resulting in inhibition of PP2A-B55. Importantly, this novel bypass is sufficient for cyclin B-Cdk1 autoregulatory activation. Gwl-dependent phosphorylation of Arpp19 is nonetheless necessary for downstream mitotic progression because chromosomes fail to segregate properly in the absence of Gwl. Such a biphasic regulation of Arpp19 results in different levels of PP2A-B55 inhibition and hence might govern its different cellular roles (Okumura, 2014).

PP2A and the cytoskeleton

Both F10 and BL6 sublines of B16 mouse melanoma cells are metastatic after intravenous injection, but only BL6 cells are metastatic after subcutaneous injection. Retrotransposon insertion produces an N-terminally truncated form (Deltagamma1) of the B56gamma1 regulatory subunit isoform of protein phosphatase (PP) 2A in BL6 cells, but not in F10 cells. An interaction of paxillin is found with PP2A C and B56gamma subunits by co-immunoprecipitation. B56gamma1 co-localizes with paxillin at focal adhesions, suggesting a role for this isoform in targeting PP2A to paxillin. In this regard, Deltagamma1 behaves similarly to B56gamma1. However, the Deltagamma1-containing PP2A heterotrimer is insufficient for the dephosphorylation of paxillin. Transfection with Deltagamma1 enhances paxillin phosphorylation on serine residues and recruitment into focal adhesions, and cell spreading with an actin network. In addition, Deltagamma1 renders F10 cells as highly metastatic as BL6 cells. These results suggest that mutations in PP2A regulatory subunits may cause malignant progression (Ito, 2000).

PP2A and organelle movement

Melanophores, cells specialized for regulated organelle transport, were used to study signaling pathways involved in the regulation of transport. Immortalized Xenopus melanophores were transfected with plasmids encoding epitope-tagged inhibitors of protein phosphatases and protein kinases or control plasmids encoding inactive analogs of these inhibitors. Expression of a recombinant inhibitor of protein kinase A (PKA) results in spontaneous pigment aggregation. alpha-Melanocyte-stimulating hormone (MSH), a stimulus that increases intracellular cAMP, cannot disperse pigment in these cells. However, melanosomes in these cells can be partially dispersed by PMA, an activator of protein kinase C (PKC). When a recombinant inhibitor of PKC is expressed in melanophores, PMA-induced pigment dispersion is inhibited, but not dispersion induced by MSH. It is concluded that PKA and PKC activate two different pathways for melanosome dispersion. When melanophores express the small t antigen of SV-40 virus, a specific inhibitor of protein phosphatase 2A (PP2A), aggregation is completely prevented. Conversely, overexpression of PP2A inhibits pigment dispersion by MSH. Inhibitors of protein phosphatase 1 and protein phosphatase 2B (PP2B) do not affect pigment movement. Therefore, melanosome aggregation is mediated by PP2A (Reilein, 1998).

PP2A regulates transcription factor phosphorylation

The bHLH factors HAND1 and HAND2 are required for heart, vascular, neuronal, limb, and extraembryonic development. Unlike most bHLH proteins, HAND factors exhibit promiscuous dimerization properties. Phosphorylation/dephosphorylation via PKA, PKC, and a specific heterotrimeric protein phosphatase 2A (PP2A) modulates HAND function. The PP2A targeting-subunit B56delta specifically interacts with HAND1 and -2, but not other bHLH proteins. PKA and PKC phosphorylate HAND proteins in vivo, and only B56delta-containing PP2A complexes reduce levels of HAND1 phosphorylation. During RCHOI trophoblast stem cell differentiation, B56delta expression is downregulated and HAND1 phosphorylation increases. Mutations in phosphorylated residues result in altered HAND1 dimerization and biological function. Taken together, these results suggest that site-specific phosphorylation regulates HAND factor functional specificity (Firulli, 2003).

Acquisition of epidermal barrier function, that serves to prevent water loss, occurs late in mouse gestation. Several days before birth a wave of barrier acquisition sweeps across murine fetal skin, converging on dorsal and ventral midlines. The molecular pathways active during epidermal barrier formation were investigated. Akt signaling increased as the barrier wave crossed epidermis and Jun was transiently dephosphorylated. Inhibitor experiments on embryonic explants showed that the dephosphorylation of Jun was dependent on both Akt and protein phosphatase 2A (Pp2a). Inhibition of Pp2a and Akt signaling also caused defects in epidermal barrier formation. These data are compatible with a model for developmental barrier acquisition mediated by Pp2a regulation of Jun dephosphorylation, downstream of Akt signaling. Support for this model was provided by siRNA-mediated knockdown of Ppp2r2a (Pr55alpha or B55alpha), a regulatory subunit of Pp2a expressed in an Akt-dependent manner in epidermis during barrier formation. Ppp2r2a reduction caused significant increase in Jun phosphorylation and interfered with the acquisition of barrier function, with barrier acquisition being restored by inhibition of Jun phosphorylation. These data provide strong evidence that Ppp2r2a is a regulatory subunit of Pp2a that targets this phosphatase to Jun, and that Pp2a action is necessary for barrier formation. This study therefore describes a novel Akt-dependent Pp2a activity that acts at least partly through Jun to affect initial barrier formation during late embryonic epidermal development (O'Shaughnessy, 2009).

PP2A and differentiation

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