Regulation Of Protein Phosphatase 2A Research Articles

Protein phosphatase-2A modulates the serine and tyrosine phosphorylation of paxillin in Lewis lung carcinoma tumor variants.

Cellular adhesion and motility, processes regulated by focal adhesion assembly and disassembly, can influence a tumor cell's ability to metastasize. Focal adhesion dynamics are, in turn, influenced by the serine and tyrosine phosphorylation state of paxillin. Using Lewis lung carcinoma (LLC) tumor variants, this study shows the importance of the serine/threonine protein phosphatase-2A (PP-2A) in maintaining adherence and restricting tumor cell motility, and modulating the serine and tyrosine phosphorylation of paxillin. Treating non-metastatic LLC-C8 tumor variants with okadaic acid to inhibit PP-2A activity resulted in cell rounding and increased motility. These effects on motility and adherence were accompanied by increased serine and decreased tyrosine phosphorylation of paxillin. These results suggest PP-2A regulation of paxillin phosphorylation may have a role in controlling tumor cell adherence and motility.

An NMDA Receptor Signaling Complex with Protein Phosphatase 2A

Regulation of protein phosphatase 2A (PP2A) activity and NMDA receptor (NMDAR) phosphorylation state contribute to the modulation of synaptic plasticity, yet these two mechanisms have not been functionally linked. The NMDAR subunit NR3A is equipped with a unique carboxyl domain that is different from other NMDAR subunits. We hypothesized that the NR3A C-terminal intracellular domain might serve as synaptic anchor for the phosphatase in the developing CNS. A cDNA library was screened by the yeast two-hybrid method using the NR3A carboxyl domain as the bait. The catalytic subunit of the serine-threonine PP2A was found to be associated with the NR3A carboxyl domain. Immunoprecipitation studies indicated that the NR3A subunit formed a stable complex with PP2A in the rat brain in vivo. Association of PP2A with NMDARs led to an increase in the phosphatase activity of PP2A and the dephosphorylation of serine 897 of the NMDAR subunit NR1. Stimulation of NMDARs led to the dissociation of PP2A from the complex and the reduction of PP2A activity. A peptide corresponding to the PP2A-NR3A binding domain functioned as a negative regulator of PP2A activity. These data suggest that NMDARs are allosteric modulators of PP2A, which in turn controls their phosphorylation state. The data delineate a mechanistic model of the dynamic regulation of a PP2A-NMDAR signaling complex, mediated by the interaction of NR3A and PP2A, and suggest a novel NMDAR-mediated signaling mechanism in addition to the traditional ionotropic functions of NMDARs.

Induction of a mitosis delay and cell lysis by high-level secretion of mouse alpha-amylase from Saccharomyces cerevisiae.

Some foreign proteins are produced in yeast in a cell cycle-dependent manner, but the cause of the cell cycle dependency is unknown. In this study, we found that Saccharomyces cerevisiae cells secreting high levels of mouse alpha-amylase have elongated buds and are delayed in cell cycle completion in mitosis. The delayed cell mitosis suggests that critical events during exit from mitosis might be disturbed. We found that the activities of PP2A (protein phosphatase 2A) and MPF (maturation-promoting factor) were reduced in alpha-amylase-oversecreting cells and that these cells showed a reduced level of assembly checkpoint protein Cdc55, compared to the accumulation in wild-type cells. MPF inactivation is due to inhibitory phosphorylation on Cdc28, as a cdc28 mutant which lacks an inhibitory phosphorylation site on Cdc28 prevents MPF inactivation and prevents the defective bud morphology induced by overproduction of alpha-amylase. Our data also suggest that high levels of alpha-amylase may downregulate PPH22, leading to cell lysis. In conclusion, overproduction of heterologous alpha-amylase in S. cerevisiae results in a negative regulation of PP2A, which causes mitotic delay and leads to cell lysis.

The Saccharomyces cerevisiae Phosphotyrosyl Phosphatase Activator Proteins Are Required for a Subset of the Functions Disrupted by Protein Phosphatase 2A Mutations

In Saccharomyces cerevisiae, PTPA is encoded by two genes, YPA1 and YPA2. In order to examine the biological role of PTPA as potential regulator of protein phosphatase 2A (PP2A), we compared the phenotypes of the ypaΔ mutants with these of PP2A-deficient strains. While deletion of both YPA genes is lethal, deletion of YPA1 alone results in a phenotype resembling that of PP2A-deficient strains in specific aspects such as aberrant bud morphology, abnormal actin distribution, and similar growth defects under various growth conditions. These phenotypes were even more pronounced when YPA1 was deleted in a pph21Δ genetic background. Moreover, ypaΔ mutants are hypersensitive to nocodazole and show inappropriate mitotic spindle formation as previously described for mutants in the catalytic subunit of PP2A, suggesting that Ypa, like PP2A, has a function in mitotic spindle formation. These results are consistent with an in vivo role of Ypa as a regulator of PP2A. However, unlike a PP2A-deficient strain, ypaΔ mutants do not show a G2 arrest. Therefore, Ypa does not seem to play a role in the regulation of PP2A at this stage of the cell cycle. These results imply that Ypa regulates a specific subset of PP2A functions, possibly by controlling the subunit composition of PP2A.

The Phosphotyrosyl Phosphatase Activator Gene Is a Novel p53 Target Gene

The minimal promoter of the phosphotyrosyl phosphatase activator (PTPA) gene, encoding a regulator of protein phosphatase 2A contains two yin-yang 1 (YY1)-binding sites, positively regulating promoter activity. We now describe a role for p53 in the regulation of PTPA expression. Luciferase reporter assays in Saos-2 cells revealed that p53 could down-regulate PTPA promoter activity in a dose-dependent manner, whereas four different p53 mutants could not. The p53-responsive region mapped to the minimal promoter. Overexpression of YY1 reverses the repressive effect of p53, suggesting a functional antagonism between p53 and YY1. The latter does not involve competition for YY1 binding, but rather direct control of YY1 function. Inhibition of PTPA expression by endogenous p53 was demonstrated in UVB-irradiated HepG2 cells, both on the mRNA and protein level. Also basal PTPA levels are higher in p53-negative (Saos-2) versus p53-positive (HepG2, U2OS) cells, suggesting "latent" p53 can control PTPA expression as well. The higher PTPA levels in U2OS cells, programmed to overexpress constitutively a dominant-negative p53 mutant, corroborate this finding. Thus, PTPA expression is negatively regulated by p53 in normal conditions and in conditions where p53 is up-regulated, via an as yet unknown mechanism involving the negative control of YY1.

CORRIGENDA

CORRIGENDA

Functional analysis of the promoter region of the human phosphotyrosine phosphatase activator gene: Yin Yang 1 is essential for core promoter activity

The phosphotyrosine phosphatase activator (PTPA) has been isolated as an in vitro regulator of protein phosphatase 2A. Human PTPA is encoded by a single gene, the structure and chromosomal localization of which have been determined in our previous work. Here we describe the further isolation, sequencing and functional characterization of the PTPA promoter region. In agreement with its ubiquitous expression, the PTPA promoter displays several characteristics of housekeeping genes: it lacks both a TATA-box and a CAAT-box, it is very GC-rich and it contains an unmethylated CpG island surrounding the transcription initiation site. Transient transfection experiments in different cell types with several truncated chimaeric luciferase reporter gene plasmids revealed the importance of the region between positions -67 and -39 for basal promoter activity. This region coincides remarkably well with the determined CpG island. Further analysis of this region demonstrated the presence of a Yin Yang 1 (YY1) binding motif at positions -52 to -44. Binding of YY1 to this sequence is demonstrated in bandshift and DNase I footprinting experiments. Another YY1 binding motif is found in the 5' untranslated region, at positions +27 to +35. Mutations in either of these sites, abolishing YY1 binding in vitro, have differential effects on promoter activity. Point mutations in both sites completely abolish promoter activity. Moreover, induction of promoter activity by co-transfection with a YY1 expression plasmid is fully dependent upon the presence of both intact YY1 binding sites. Thus YY1 apparently mediates basal transcription of the human PTPA gene through two binding sites within its proximal promoter.

Regulation of Protein Phosphatase 2A Activity by Heat Shock Transcription Factor 2

Heat shock transcription factor (HSF) mediates the stress-induced expression of heat shock protein genes (hsp). However, HSF is required for normal cell function even in the absence of stress and is important for cell cycle progression, but the mechanism that mediates these effects of HSF is unknown. Here, it is shown that a member of the HSF family, HSF2, interacts with the PR65 (A) subunit of protein phosphatase 2A (PP2A). HSF2 binding to PR65 blocks its interaction with the catalytic subunit, due to competition between HSF2 and catalytic subunit for the same binding site in PR65. In addition, overexpression of HSF2 stimulates PP2A activity in cells, indicating the relevance of HSF2 as a regulator of PP2A in vivo. These results identify HSF2 as a dual function protein, capable of regulating both hsp expression and PP2A activity. This could function as a mechanism by which hsp expression is integrated with the control of cell division or other PP2A-regulated pathways.

The expression of casein kinase 2α′ and phosphatase 2A activity

Protein phosphatase 2A (PP2A) activity may be differentially regulated by the expression of proteins containing a related amino acid sequence motif such as the casein kinase 2α (CK2α) subunit or SV40 small t antigen (SVt). Expression of CK2α increases PP2A activity whereas SVt decreases its activity. In this work we have tested for the effect of the expression of a third protein containing a similar motif that could be involved in PP2A regulation, the catalytic casein kinase 2α′ subunit. Our results show that despite the structural similarity of this protein with the other CK2 catalytic (α) subunit, the function of the two subunits with respect to the modulation of PP2A activity is quite different: CK2α increases whereas CK2α′ slightly decreases PP2A activity.

Functional interaction between a novel protein phosphatase 2A regulatory subunit, PR59, and the retinoblastoma-related p107 protein.

The proteins of the retinoblastoma family are potent inhibitors of cell cycle progression. It is well documented that their growth-inhibitory activity can be abolished by phosphorylation on serine and threonine residues by cyclin dependent kinases. In contrast, very little is known about the dephosphorylation of retinoblastoma-family proteins. We report here the isolation, by virtue of its ability to associate with p107, of a novel Protein Phosphatase 2A (PP2A) regulatory subunit, named PR59. PR59 shares sequence homology with a known regulatory subunit of PP2A, PR72, but differs from PR72 in its expression pattern and its functional properties. We show that PR59 co-immunoprecipitates with the PP2A catalytic subunit, indicating that PR59 is a genuine component of PP2A holo-enzymes. In vivo, PR59 associates specifically with p107, but not with pRb. Elevated expression of PR59 results in dephosphorylation of p107, but not of pRb, and inhibits cell proliferation by causing cells to accumulate in G1. These data support a model in which the distinct PP2A regulatory subunits act to target the PP2A catalytic subunit to specific substrates and suggest a role for PP2A in regulation of p107.

Physical and functional interactions between type I transforming growth factor beta receptors and Balpha, a WD-40 repeat subunit of phosphatase 2A.

We have previously shown that a WD-40 repeat protein, TRIP-1, associates with the type II transforming growth factor beta (TGF-beta) receptor. In this report, we show that 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, 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, our observations suggest possible functional interactions between the TGF-beta receptor complex and the regulation of protein phosphatase 2A.

Molecular genetic analysis of Rts1p, a B' regulatory subunit of Saccharomyces cerevisiae protein phosphatase 2A.

The Saccharomyces cerevisiae gene RTS1 encodes a protein homologous to a variable B-type regulatory subunit of the mammalian heterotrimeric serine/threonine protein phosphatase 2A (PP2A). We present evidence showing that Rts1p assembles into similar heterotrimeric complexes in yeast. Strains in which RTS1 has been disrupted are temperature sensitive (ts) for growth, are hypersensitive to ethanol, are unable to grow with glycerol as their only carbon source, and accumulate at nonpermissive temperatures predominantly as large-budded cells with a 2N DNA content and a nondivided nucleus. This cell cycle arrest can be overcome and partial suppression of the ts phenotype of rts1-null cells occurs if the gene CLB2, encoding a Cdc28 kinase-associated B-type cyclin, is expressed on a high-copy-number plasmid. However, CLB2 overexpression has no suppressive effects on other aspects of the rts1-null phenotype. Expression of truncated forms of Rts1p can also partially suppress the ts phenotype and can fully suppress the inability of cells to grow on glycerol and the hypersensitivity of cells to ethanol. By contrast, the truncated forms do not suppress the accumulation of large-budded cells at high temperatures. Coexpression of truncated Rts1p and high levels of Clb2p fully suppresses the ts phenotype, indicating that the inhibition of growth of rts1-null cells at high temperatures is due to both stress-related and cell cycle-related defects. Genetic analyses show that the role played by Rts1p in PP2A regulation is distinctly different from that played by the other known variable B regulatory subunit, Cdc55p, a protein recently implicated in checkpoint control regulation.

Regulation of protein phosphatase 2A by direct interaction with casein kinase 2alpha.

Timely deactivation of kinase cascades is crucial to the normal control of cell signaling and is partly accomplished by protein phosphatase 2A (PP2A). The catalytic (alpha) subunit of the serine-threonine kinase casein kinase 2 (CK2) bound to PP2A in vitro and in mitogen-starved cells; binding required the integrity of a sequence motif common to CK2alpha and SV40 small t antigen. Overexpression of CK2alpha resulted in deactivation of mitogen-activated protein kinase kinase (MEK) and suppression of cell growth. Moreover, CK2alpha inhibited the transforming activity of oncogenic Ras, but not that of constitutively activated MEK. Thus, CK2alpha may regulate the deactivation of the mitogen-activated protein kinase pathway.

Developmental Expression and Localization of Protein Phosphatase 2A in the Kidney † 1649

Although a number of growth and transcription factors are known to regulate kidney growth and development, the signal transduction molecules necessary to mediate developmental signals in the kidney remain largely unknown. Therefore activity, mRNA and protein expression of the signal transduction molecule protein phosphatase 2A (PP2A) were examined in kidneys from embryonic day 17 to 90 day old adults. Northern analysis of total kidney RNA or western analysis of kidney protein homogenates demonstrated developmental regulation of both the catalytic and major 55 kDa regulatory subunits of PP2A with the highest levels in the embryonic and newborn kidneys. Similarly PP2A enzyme activity was highest in the embryonic and newborn kidney. In situ hybridization with a digoxigenin labeled catalytic subunit cRNA probe mapped to mRNA expressing cells predominately in the nephrogenic cortex, particularly to developing glomeruli and not to developing tubular elements in the embryonic day 20 and newborn kidney. Similarly immunocytochemistry with a specific PP2A catalytic subunit polyclonal peptide antibody demonstrated localized catalytic subunit expression particularly to podocytes of developing glomeruli. Therefore the developmental regulation of PP2A during kidney development and its mapping predominately to developing glomeruli suggests a role for PP2A in the regulation of glomerular development.

The Myeloid Leukemia-associated Protein SET Is a Potent Inhibitor of Protein Phosphatase 2A

Two potent heat-stable protein phosphatase 2A (PP2A) inhibitor proteins designated I1PP2A and I2PP2A have been purified to apparent homogeneity from extracts of bovine kidney (Li, M., Guo, H., and Damuni, Z. (1995) Biochemistry 34, 1988-1996). N-terminal and internal amino acid sequencing indicated that I2PP2A was a truncated form of SET, a largely nuclear protein that is fused to nucleoporin Nup214 in acute non-lymphocytic myeloid leukemia. Experiments using purified preparations of recombinant human SET confirmed that this protein inhibited PP2A. Half-maximal inhibition of the phosphatase occurred at about 2 nM SET. By contrast, SET (up to 20 nM) did not affect the activities of purified preparations of protein phosphatases 1, 2B, and 2C. The results indicate that SET is a potent and specific inhibitor of PP2A and suggest that impaired regulation of PP2A may contribute to acute myeloid leukemogenesis.

Identification of a Novel Protein Phosphatase 2A Regulatory Subunit Highly Expressed in Muscle

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). We have isolated and characterized three novel cDNAs related to the B' subunit of bovine cardiac PP2A. 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 revealed 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 associated with the PP2A core enzyme in both in vitro and in vivo complex formation assays. Immunofluorescence microscopy revealed that epitope-tagged B'alpha was 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 was greatly enhanced by the addition of the C subunit. Expression of the B'alpha subunits in insect Sf9 cells resulted 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.

Alternative cell fate choice induced by low-level expression of a regulator of protein phosphatase 2A in the Drosophila peripheral nervous system

The Drosophila gene twins encodes the regulatory B subunit of type 2A protein phosphatase. Here we report that its partial loss-of-function mutations caused abnormal morphogenesis in the adult peripheral nervous system. In wild-type flies, the mechanoreceptor, one major class of sensory organs, is composed of four specialized cells (one neuron and three accessory cells) that are derived from a single precursor cell. The hypomorphic twins mutations did not block division of this precursor, but most likely altered cell fate in this lineage to produce only accessory cells that form sensory structures. Stepwise reductions of twins protein enhanced this transformation. In these mutants, another regulatory subunit, A, and the catalytic subunit, C, of the phosphatase were expressed at normal levels. Therefore, the modulation of the phosphatase activity by the B subunit appears to be crucial for specification of neural cell identity.

Tyrosine phosphorylation of protein phosphatase 2A in response to growth stimulation and v-src transformation of fibroblasts.

The catalytic subunit of protein phosphatase 2A (PP2A) is inactivated by in vitro phosphorylation of Tyr307 by receptor and nonreceptor protein tyrosine kinases (Chen, J., Martin, B. L., and Brautigan, D. L. (1992) Science 257, 1261-1264). Here we show the phosphorylation of PP2A in cells under different growth conditions. In lysates of nontransformed murine 10T1/2 fibroblasts, there were two forms of PP2A at 36 kDa detected after two-dimensional gel electrophoresis and immunoblotting with anti-PP2A peptide antibody. These two forms exactly comigrated with unphosphorylated purified PP2A and the PP2A 32P-labeled by in vitro phosphorylation with p60v-src kinase. The phosphorylated form of PP2A recovered from red blood cells or produced by in vitro phosphorylation was eliminated by incubation with tyrosine-specific phosphatase (PTP1B). Transformation of 10T1/2 cells by expression of p60v-src resulted in most of the PP2A in the cells being converted to a phosphorylated form that was reactive with anti-phosphotyrosine antibody. Serum starvation of cells reduced the amount of phosphorylated PP2A, whereas serum stimulation of quiescent cells caused an increase to the same relative amount of phosphorylated PP2A as in src-transformed cells. Addition of epidermal growth factor to quiescent NeoR cells (10T1/2 fibroblasts overexpressing epidermal growth factor receptors) temporarily increased the level of phosphorylation of PP2A, with a peak at 5-15 min and a return to basal level within 60 min. The results show that PP2A is phosphorylated in intact cells, and the extent of this modification is increased by growth factors or cell transformation, providing evidence for a physiological mechanism of PP2A regulation.

Regulation of Rhodopsin Dephosphorylation by Arrestin

We have characterized the opsin phosphatase activities in extracts of rod outer segments and determined their relationship to known protein phosphatases. The opsin phosphatase activity in the extracts was not due to protein phosphatases 1, 2B, or 2C because it was neither stimulated by Mg2+ or Ca2+/calmodulin nor inhibited by protein phosphatase inhibitors-1 or -2. Opsin phosphatase activity in rod outer segment extracts was potently inhibited by okadaic acid (IC50 approximately 10 nM), a preferential inhibitor of protein phosphatase 2A. Moreover, during chromatography on DEAE-Sepharose, the opsin phosphatase activity co-eluted with three peaks of protein phosphatase 2A activity, termed protein phosphatases 2A0, 2A1, and 2A2. The opsin phosphatase activity of each peak was stimulated by polylysine, a known activator of protein phosphatase 2A. Finally, treatment of rod outer segment extracts with 80% ethanol at room temperature converted the activity from a high molecular weight form characteristic of the protein phosphatase 2A0, 2A1, and 2A2 species to a low molecular weight form characteristic of the protein phosphatase 2A catalytic subunit. We conclude that protein phosphatase 2A is likely to be the physiologically relevant rhodopsin phosphatase. The 48-kDa rod outer segment protein arrestin (S-antigen) was found to inhibit the dephosphorylation of freshly photolyzed rhodopsin by protein phosphatase 2A but did not inhibit the dephosphorylation of unbleached rhodopsin. Arrestin has no effect on the dephosphorylation of phorphorylase a, indicating that the effect was substrate-directed. It appears that dephosphorylation of the photoreceptor protein phosphorhodopsin occurs only after decay of the photoactivated protein and that this may be regulated in vivo by arrestin. The binding of arrestin to photolyzed phosphorylated rhodopsin, i.e. the binding of a regulatory protein to a protein phosphatase substrate to form a complex resistant to dephosphorylation represents a novel mechanism for the regulation of protein phosphatase 2A.