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C Van Hoof

Publications and source records attributed to C Van Hoof.

16 recordsLinked to original sources
PubMed2001

Inhibition of the Wnt signaling pathway by the PR61 subunit of protein phosphatase 2A.

Axin, a negative regulator of the Wnt signaling pathway, forms a complex with glycogen synthase kinase-3beta (GSK-3beta), beta-catenin, adenomatous polyposis coli (APC) gene product, and Dvl, and it regulates GSK-3beta-dependent phosphorylation in the complex and the stability of beta-catenin. Using yeast two-hybrid screening, we found that regulatory subunits of protein phosphatase 2A, PR61beta and -gamma, interact with Axin. PR61beta or -gamma formed a complex with Axin in intact cells, and their interaction was direct. The binding site of PR61beta on Axin was different from those of GSK-3beta, beta-catenin, APC, and Dvl. Although PR61beta did not affect the stability of beta-catenin, it inhibited Dvl- and beta-catenin-dependent T cell factor activation in mammalian cells. Moreover, it suppressed beta-catenin-induced axis formation and expression of siamois, a Wnt target gene, in Xenopus embryos, suggesting that PR61beta acts either at the level of beta-catenin or downstream of it. Taken together with the previous observations that PR61 interacts with APC and functions upstream of beta-catenin, these results demonstrate that PR61 regulates the Wnt signaling pathway at various steps.

Animals↗
PubMed2001

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 ypaDelta 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 pph21Delta genetic background. Moreover, ypaDelta 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, ypaDelta 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.

Actins↗
PubMed2000

The Saccharomyces cerevisiae homologue YPA1 of the mammalian phosphotyrosyl phosphatase activator of protein phosphatase 2A controls progression through the G1 phase of the yeast cell cycle.

The Saccharomyces cerevisiae gene YPA1 encodes a protein homologous to the phosphotyrosyl phosphatase activator, PTPA, of the mammalian protein phosphatase type 2A (PP2A). In order to examine the biological role of PTPA, we disrupted YPA1 and characterised the phenotype of the ypa1Delta mutant. Comparison of the growth rate of the wild-type strain and the ypa1Delta mutant on glucose-rich medium after nutrient depletion showed that the ypa1Delta mutant traversed the lag period more rapidly. This accelerated progression through "Start" was also observed after release from alpha-factor-induced G1 arrest as evidenced by a higher number of budding cells, a faster increase in CLN2 mRNA expression and a more rapid reactivation of Cdc28 kinase activity. This phenotype was specific for deletion of YPA1 since it was not observed when YPA2, the second PTPA gene in budding yeast was deleted. Reintroduction of YPA1 or the human PTPA cDNA in the ypa1Delta mutant suppressed this phenotype as opposed to overexpression of YPA2. Disruption of both YPA genes is lethal, since sporulation of heterozygous diploids resulted in at most three viable spores, none of them with a ypa1Delta ypa2Delta genotype. This observation indicates that YPA1 and YPA2 share some essential functions. We compared the ypa1Delta mutant phenotype with a PP2A double deletion mutant and a PP2A temperature-sensitive mutant. The PP2A-deficient yeast strain also showed accelerated progression through the G1 phase. In addition, both PP2A and ypa1Delta mutants show similar aberrant bud morphology. This would support the notion that YPA1 may act as a positive regulator of PP2A in vivo.

CDC28 Protein Kinase, S cerevisiae↗
PubMed2000

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.

Binding Sites↗
PubMed1999

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.

Animals↗
PubMed1999

Purification of porcine brain protein phosphatase 2A leucine carboxyl methyltransferase and cloning of the human homologue.

The carboxyl methyltransferase, which is claimed to exclusively methylate the carboxyl group of the C-terminal leucine residue of the catalytic subunit of protein phosphatase 2A (Leu(309)), was purified from porcine brain. On the basis of tryptic peptides, the cDNA encoding the human homologue was cloned. The cDNA of this gene encodes for a protein of 334 amino acids with a calculated M(r) of 38 305 and a predicted pI of 5.72. Database screening reveals the presence of this protein in diverse phyla. Sequence analysis shows that the novel methyltransferase is distinct from other known protein methyltransferases, sharing only sequence motifs supposedly involved in the binding of adenosylmethionine. The recombinant protein expressed in bacteria is soluble and the biophysical, catalytic, and immunological properties are indistinguishable from the native enzyme. The methylation of PP2A by the recombinant protein is restricted to Leu(309) of PP2A(C). No direct effects on phosphatase activity changes were observed upon methylation of the dimeric or trimeric forms of PP2A.

Amino Acid Sequence↗
PubMed1998

Functional analysis of conserved domains in the phosphotyrosyl phosphatase activator. Molecular cloning of the homologues from Drosophila melanogaster and Saccharomyces cerevisiae.

Phosphotyrosyl phosphatase activator (PTPA), a 37 kDa cytosolic protein that specifically activates the phosphotyrosyl phosphatase activity of the dimeric form of PP2A, was cloned from Drosophila melanogaster and Saccharomyces cerevisiae. Sequence alignment of PTPA from yeast to human revealed highly conserved regions including the type B fragment of the putative PTPA ATP binding site. We generated PTPA deletion mutants of these conserved regions as well as point mutations within regions that were suggested to be functionally important. The recombinant proteins were expressed in E. coli and subsequently purified. Activity measurements, linked with immunological detection, revealed that most of the well-conserved regions are essential for PTPA activity. However, neither the type A fragment of the putative ATP binding site nor the cysteine-rich region, present in all but the Drosophila and yeast homologues, appeared to be essential for PTPA activity. Moreover, we observed that PTPA truncated at glycine266 behaves as a dominant negative mutant since it is inhibitory to the wild-type PTPA.

Adenosine Triphosphate↗
PubMed1996

The catalytic subunit of protein phosphatase 2A associates with the translation termination factor eRF1.

By a number of criteria, we have demonstrated that the translation termination factor eRF1 (eukaryotic release factor 1) associates with protein phosphatase 2A (PP2A). Trimeric PP2A1 was purified from rabbit skeletal muscle using an affinity purification step. In addition to the 36 kDa catalytic subunit (PP2Ac) and established regulatory subunits of 65 kDa (PR65) and 55 kDa (PR55), purified preparations contained two proteins with apparent Mrs of 54 and 55 kDa. Protein microsequencing revealed that the 55 kDa component is a novel protein, whereas the 54 kDa protein was identified as eRF1, a protein that functions in translational termination as a polypeptide chain release factor. Using the yeast two-hybrid system, human eRF1 was shown to interact specifically with PP2Ac, but not with the PR65 or PR55 subunits. By deletion analysis, the binding domains were found to be located within the 50 N-terminal amino acids of PP2Ac, and between amino acid residues 338 and 381 in the C-terminal part of human eRF1. This association also occurs in vivo, since PP2A can be co-immunoprecipitated with eRF1 from mammalian cells. We observed a significant increase in the amount of PP2A associated with the polysomes when eRF1 was transiently expressed in COS1 cells, and eRF1 immunoprecipitated from those fractions contained associated PP2A. Since we did not observe any dramatic effects of PP2A on the polypeptide chain release activity of eRF1 (or vice versa), we postulate that eRF1 also functions to recruit PP2A into polysomes, thus bringing the phosphatase into contact with putative targets among the components of the translational apparatus.

Amino Acid Sequence↗
PubMed1996

The variable subunit associated with protein phosphatase 2A0 defines a novel multimember family of regulatory subunits.

Two protein phosphatase 2A (PP2A) holoenzymes were isolated from rabbit skeletal muscle containing, in addition to the catalytic and PR65 regulatory subunits, proteins of apparent molecular masses of 61 and 56 kDa respectively. Both holoenzymes displayed low basal phosphorylase phosphatase activity, which could be stimulated by protamine to an extent similar to that of previously characterized PP2A holoenzymes. Protein micro-sequencing of tryptic peptides derived from the 61 kDa protein, termed PR61, yielded 117 residues of amino acid sequence. Molecular cloning by enrichment of specific mRNAs, followed by reverse transcription-PCR and cDNA library screening, revealed that this protein exists in multiple isoforms encoded by at least three genes, one of which gives rise to several splicing variants. Comparisons of these sequences with the available databases identified one more human gene and predicted another based on a rabbit cDNA-derived sequence, thus bringing the number of genes encoding PR61 family members to five. Peptide sequences derived from PR61 corresponded to the deduced amino acid sequences of either alpha or beta isoforms, indicating that the purified PP2A preparation was a mixture of at least two trimers. In contrast, the 56 kDa subunit (termed PR56) seems to correspond to the epsilon isoform of PR61. Several regulatory subunits of PP2A belonging to the PR61 family contain consensus sequences for nuclear localization and might therefore target PP2A to nuclear substrates.

Alternative Splicing↗
PubMed1996

A comparative study of the phosphotyrosyl phosphatase specificity of protein phosphatase type 2A and phosphotyrosyl phosphatase type 1B using phosphopeptides and the phosphoproteins p50/HS1, c-Fgr and Lyn.

The phosphotyrosyl phosphatase (PTPase) specificity of phosphotyrosyl-phosphatase-activator-(PTPA)-stimulated protein phosphatase (PP)2A(D) (rabbit muscle) and a bona fide PTP-1B (Xenopus laevis oocytes) were examined in vitro using phosphotyrosine-containing peptides, derived from the phosphorylation sites of p34cdc2, p50/HS1 protein, Abl, c-Src and c-Fgr, as well as the intact phosphoprotein p50/HS1 and the Src-related tyrosine kinases, Lyn and c-Fgr. The local specificity determinants were found to be different for both PTPases. The length of the phosphopeptides is more important for PP2A(D) than for PTP-1B, C-terminal acidic residues adjacent to the phosphotyrosine are detrimental for the PTPase activity of PP2A(D), but they do not affect the PTP-1B activity. Acidic residues at the --2 and --3 position relative to Tyr(P) primarily dictate dephosphorylation by PTP-1B. The higher-order structure of the protein substrates also differentially influences both enzymes: the phospho-octapeptide KDDEYpNPA, which reproduces the autophosphorylation site in c-Fgr (Tyr400), is only dephosphorylated by PP2A(D) if embedded in the intact protein, whereas the opposite is true for PTP-1B. Both the intact p50/HS1 phosphoprotein and the derived phosphopeptide are substrates only for PTP-1B and not for PP2A(D). Lyn and c-Fgr phosphorylated by C-terminal Src kinase (CSK) at their down-regulatory site are resistant to the action of both PTPases while the [Phe6]Src-(514-533) phosphopeptide, representing the highly similar site affected by CSK in c-Src, is readily dephosphorylated by both PTPases, although to a different extent. In vitro dephosphorylation of the c-Fgr Tyr400 site by PP2A(D) is correlated with a decreased tyrosine kinase activity towards exogenous substrates. Under experimental conditions in which both Tyr400 (autophosphorylation site) and Tyr511 (down-regulatory site) of c-Fgr are phosphorylated, PP2A(D) can reverse both phosphorylations.

Amino Acid Sequence↗
PubMed1995

Molecular cloning and developmental regulation of expression of two isoforms of the catalytic subunit of protein phosphatase 2A from Xenopus laevis.

Two types of PP2A catalytic subunit cDNA clones were isolated from a Xenopus oocyte library. One type corresponds to the Xenopus C36 alpha (XC36 alpha) isoform as published by Cormier et al. (1991) and another type encodes for a novel isoform of PP2AC36 (XC36 beta), more homologous to the C36 beta isoform cloned from Mammalia. Northern blot analysis with isoform specific probes showed that the XC36 alpha mRNA transcript of 2 kb is more abundant than the XC36 beta mRNA of 1.9 kb during embryogenesis and in adult tissues. Both transcripts are highly expressed in ovary and heart relative to the other adult tissues examined. The high expression levels of both XC36 alpha and XC36 beta transcripts in the ovary decrease during the first mitotic embryonic divisions. Resumption of zygotic expression starts at about the same time for both mRNAs, during the tailbud stage, but the XC36 beta transcript shows only a moderate and transient increase and declines again during the tadpole stage whereas expression of XC36 alpha increases further during the same period until the onset of metamorphosis.

Aging↗
PubMed1995

Structure and chromosomal localization of the human gene of the phosphotyrosyl phosphatase activator (PTPA) of protein phosphatase 2A.

The PTPA gene encodes a specific phosphotyrosyl phosphatase activator of the dimeric form of protein phosphatase 2A. PTPA, cloned from human genomic libraries, is encoded by one single-copy gene, composed of 10 exons and 9 introns with a total length of about 60 kb. The transcription start site was determined, and the 5' flanking sequence was analyzed for its potential as a promotor. This region lacks a TATA sequence in the appropriate position relative to the transcription start, is very GC-rich, and contains upstream of the transcription start four Sp1 sites, a feature common to many TATA-less promotors. Based on the homology with DNA binding consensus sequences of transcription factors, we identified in this promotor region several putative DNA binding sites for transcription factors, such as NF-kappa B, Myb, Ets-1, Myc, and ATF. Transfection experiments with a construct containing the PTPA promotor region inserted 5' of a luciferase reporter gene revealed that the 5' flanking sequence of the PTPA gene indeed displayed promotor activity that seems to be cell-line dependent. By fluorescence in situ hybridization and G-banding, the PTPA gene was localized to the 9q34 region. The PTPA gene is positioned centromeric of c-abl in a region embracing several genes implicated in oncogenesis.

Amino Acid Sequence↗
PubMed1995

The PR55 and PR65 subunits of protein phosphatase 2A from Xenopus laevis. molecular cloning and developmental regulation of expression.

cDNA clones encoding the 65-kDa (PR65) and 55-kDa (PR55) regulatory subunits of protein phosphatase 2A from Xenopus laevis were isolated by homology screening with the corresponding human cDNAs, and used to analyze the developmental expression patterns of these genes. The PR65 subunit was found to be encoded by two genes, termed XPR65 alpha and XPR65 beta. The open reading frames of the alpha and beta cDNAs both span 1767 bp, and predict proteins of 64.4 kDa and 65.3 kDa, respectively, that are 87% identical. The predicted amino acid sequence of XPR65 alpha showed 95% and 84% identity with human PR65 alpha and PR65 beta proteins, respectively, whereas the identity of XPR65 beta with the same proteins was 87% and 86.5%, respectively. Only one type of Xenopus PR55 (XPR55) was isolated that showed 93% and 84% similarity to human PR55 alpha and PR55 beta, respectively. Analysis of the N-terminal region of XPR55 with the same regions of human PR55 alpha and PR55 beta, indicates that the XPR55 is the Xenopus homolog of the human PR55 alpha isoform. Despite the overall similarity with PR55 from other species, XPR55 has an N-terminal extention of at least 24 amino acids. In the ovary, a transcript of 2.8 kb, encoding the XPR65 beta, was predominantly expressed and these XPR65 beta mRNAs are present at a constant level during oogenesis until late embryogenesis. Expression of the 2.4-kb XPR65 alpha was low until the larval stage, then dramatically increased. In all adult tissues except ovary, the 2.4-kb alpha-specific mRNA was more abundant than the 2.8-kb beta transcript. Two transcripts of 2.4 kb and 2.5 kb, encoding the XPR55 subunit, were detected at a constant level throughout Xenopus oogenesis and during embryogenesis. Both transcripts were also expressed at similar levels in all adult tissues, but in a tissue-specific manner. Analysis of the XPR55 and XPR65 proteins using antibodies to recombinant proteins revealed that the overall levels of the two proteins were constant, in good agreement with mRNA data.

Amino Acid Sequence↗
PubMed1994

The phosphotyrosyl phosphatase activator of protein phosphatase 2A. A novel purification method, immunological and enzymic characterization.

A simple, improved procedure for the isolation of the phosphotyrosyl phosphatase activator (PTPA) from rabbit skeletal muscle has been developed. The majority of the protein phosphatase 2A (PP2A) was separated from PTPA at an early stage in the procedure. The procedure yields approximately 1 mg essentially pure PTPA/kg rabbit skeletal muscle; it was also applied to porcine brain and the yeast Saccharomyces cerevisiae. The physico-chemical properties of PTPA obtained from all sources are very similar. The pure rabbit skeletal muscle protein was used to raise polyclonal goat antibodies and to affinity purify these antibodies. Immunological studies revealed the presence of PTPA in all mammalian tissues and cell lines examined with differences in tissue distribution, brain showing the highest concentration. PTPA could only be detected in cytosolic fractions. Using a semi-quantitative immunological assay (Western blot), the in vivo concentration could be estimated to be micromolar, which is in the same range as the PP2A target. The purified Xenopus oocyte PTPA showed only a weak cross reactivity, whereas yeast PTPA was not recognised by the antibody indicating some evolutionary diversity of the protein. In a PTPA-affinity column chromatography, the weak interaction with PP2A was independent of the presence of ATP.Mg, a necessary cofactor in the activation process. Interaction of PTPA with PP2A in a 1:1 ratio induces a low (kcat = 3 min-1) ATPase activity that is inhibited by okadaic acid, ADP and non-hydrolysable ATP analogues.

Adenosine Triphosphatases↗
PubMed1994

Molecular cloning, expression, and characterization of PTPA, a protein that activates the tyrosyl phosphatase activity of protein phosphatase 2A.

PTPA, or phosphotyrosyl phosphatase activator, is a protein that stimulates the tyrosyl phosphatase activity of protein phosphatase 2A in an ATP, Mg(2+)-requiring reaction (Cayla, X., Goris, J., Hermann, J., Hendrix, P., Ozon, R., and Merevede, W. (1990) Biochemistry 29, 658-667). We constructed oligonucleotide probes based on the amino acid sequences of peptides isolated from purified PTPA and used them to probe rabbit muscle and human heart cDNA libraries. A putative full-length clone was isolated from the rabbit skeletal muscle as well as from the human heart library. The nucleotide sequence of both clones contains an open reading frame of 969 nucleotides starting from an assigned initial ATG codon and encodes for a protein of 323 amino acids. The predicted rabbit and human PTPA protein sequences show an identity of 96.6%. The predicted protein matched all the peptide sequences obtained from the rabbit skeletal muscle protein. Bacterially expressed protein, as well as the in vitro reticulocyte lysate translation product, comigrated with the purified 37-kDa protein on sodium dodecyl sulfate-polyacrylamide gels. Both proteins reacted with immunopurified, anti-PTPA polyclonal antiserum. The recombinant protein was a soluble and active protein. Northern blot analysis revealed two transcripts of 2.8 and 4 kilobases, respectively, in human placenta but only one 2.8-kilobase transcript in rabbit and rat tissues. High levels of PTPA mRNA were detected in testis, which contrasted with the low levels present in skeletal muscle.

Amino Acid Sequence↗