twins
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).
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).
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).
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).
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).
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).
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).
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).
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).
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)
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).
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).
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).
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).
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