Dietary supplementation of cystinotic mice by lysine inhibits the megalin pathway and decreases kidney cystine content.

Megalin/LRP2 is a major receptor supporting apical endocytosis in kidney proximal tubular cells. We have previously reported that kidney-specific perinatal ablation of the megalin gene in cystinotic mice, a model of nephropathic cystinosis, essentially blocks renal cystine accumulation and partially preserves kidney tissue integrity. Here, we examined whether inhibition of the megalin pathway in adult cystinotic mice by dietary supplementation (5x-fold vs control regular diet) with the dibasic amino-acids (dAAs), lysine or arginine, both of which are used to treat patients with other rare metabolic disorders, could also decrease renal cystine accumulation and protect cystinotic kidneys. Using surface plasmon resonance, we first showed that both dAAs compete for protein ligand binding to immobilized megalin in a concentration-dependent manner, with identical inhibition curves by L- and D-stereoisomers. In cystinotic mice, 2-month diets with 5x-L-lysine and 5x-L-arginine were overall well tolerated, while 5x-D-lysine induced strong polyuria but no weight loss. All diets induced a marked increase of dAA urinary excretion, most prominent under 5x-D-lysine, without sign of kidney insufficiency. Renal cystine accumulation was slowed down approx. twofold by L-dAAs, and totally suppressed by D-lysine. We conclude that prolonged dietary manipulation of the megalin pathway in kidneys is feasible, tolerable and can be effective in vivo.

Loss of protein phosphatase 2A regulatory subunit B56δ promotes spontaneous tumorigenesis in vivo.

Protein Phosphatase 2A (PP2A) enzymes counteract diverse kinase-driven oncogenic pathways and their function is frequently impaired in cancer. PP2A inhibition is indispensable for full transformation of human cells, but whether loss of PP2A is sufficient for tumorigenesis in vivo has remained elusive. Here, we describe spontaneous tumor development in knockout mice for Ppp2r5d, encoding the PP2A regulatory B56δ subunit. Several primary tumors were observed, most commonly, hematologic malignancies and hepatocellular carcinomas (HCCs). Targeted immunoblot and immunohistochemistry analysis of the HCCs revealed heterogeneous activation of diverse oncogenic pathways known to be suppressed by PP2A-B56. RNA sequencing analysis unveiled, however, a common role for oncogenic c-Myc activation in the HCCs, independently underscored by c-Myc Ser62 hyperphosphorylation. Upstream of c-Myc, GSK-3ß Ser9 hyperphosphorylation occurred both in the HCCs and non-cancerous B56δ-null livers. Thus, uncontrolled c-Myc activity due to B56δ-driven GSK-3ß inactivation is the likely tumor predisposing factor. Our data provide the first compelling mouse genetics evidence sustaining the tumor suppressive activity of a single PP2A holoenzyme, constituting the final missing incentive for full clinical development of PP2A as cancer biomarker and therapy target.

Hematopoietic Stem Cells Transplantation Can Normalize Thyroid Function in a Cystinosis Mouse Model.

Hypothyroidism is the most frequent and earliest endocrine complication in cystinosis, a multisystemic lysosomal storage disease caused by defective transmembrane cystine transporter, cystinosin (CTNS gene). We recently demonstrated in Ctns(-/-) mice that altered thyroglobulin biosynthesis associated with endoplasmic reticulum stress, combined with defective lysosomal processing, caused hypothyroidism. In Ctns(-/-) kidney, hematopoietic stem cell (HSC) transplantation provides long-term functional and structural protection. Tissue repair involves transfer of cystinosin-bearing lysosomes from HSCs differentiated as F4/80 macrophages into deficient kidney tubular cells, via tunneling nanotubes that cross basement laminae. Here we evaluated the benefit of HSC transplantation for cystinotic thyroid and investigated the underlying mechanisms. HSC engraftment in Ctns(-/-) thyroid drastically decreased cystine accumulation, normalized the TSH level, and corrected the structure of a large fraction of thyrocytes. In the thyroid microenvironment, HSCs differentiated into a distinct, mixed macrophage/dendritic cell lineage expressing CD45 and major histocompatibility complex II but low CD11b and F4/80. Grafted HSCs closely apposed to follicles and produced tunneling nanotube-like extensions that crossed follicular basement laminae. HSCs themselves further squeezed into follicles, allowing extensive contact with thyrocytes, but did not transdifferentiate into Nkx2.1-expressing cells. Our observations revealed significant differences of basement lamina porosity between the thyroid and kidney and/or intrinsic macrophage invasive properties once in the thyroid microenvironment. The contrast between extensive thyrocyte protection and low HSC abundance at steady state suggests multiple sequential encounters and/or remanent impact. This is the first report demonstrating the potential of HSC transplantation to correct thyroid disease and supports a major multisystemic benefit of stem cell therapy for cystinosis.

A mouse model suggests two mechanisms for thyroid alterations in infantile cystinosis: decreased thyroglobulin synthesis due to endoplasmic reticulum stress/unfolded protein response and impaired lysosomal processing.

Thyroid hormones are released from thyroglobulin (Tg) in lysosomes, which are impaired in infantile/nephropathic cystinosis. Cystinosis is a lysosomal cystine storage disease due to defective cystine exporter, cystinosin. Cystinotic children develop subclinical and then overt hypothyroidism. Why hypothyroidism is the most frequent and earliest endocrine complication of cystinosis is unknown. We here defined early alterations in Ctns(-/-) mice thyroid and identified subcellular and molecular mechanisms. At 9 months, T4 and T3 plasma levels were normal and TSH was moderately increased (∼4-fold). By histology, hyperplasia and hypertrophy of most follicles preceded colloid exhaustion. Increased immunolabeling for thyrocyte proliferation and apoptotic shedding indicated accelerated cell turnover. Electron microscopy revealed endoplasmic reticulum (ER) dilation, apical lamellipodia indicating macropinocytic colloid uptake, and lysosomal cystine crystals. Tg accumulation in dilated ER contrasted with mRNA down-regulation. Increased expression of ER chaperones, glucose-regulated protein of 78 kDa and protein disulfide isomerase, associated with alternative X-box binding protein-1 splicing, revealed unfolded protein response (UPR) activation by ER stress. Decreased Tg mRNA and ER stress suggested reduced Tg synthesis. Coordinated increase of UPR markers, activating transcription factor-4 and C/EBP homologous protein, linked ER stress to apoptosis. Hormonogenic cathepsins were not altered, but lysosome-associated membrane protein-1 immunolabeling disclosed enlarged vesicles containing iodo-Tg and impaired lysosomal fusion. Isopycnic fractionation showed iodo-Tg accumulation in denser lysosomes, suggesting defective lysosomal processing and hormone release. In conclusion, Ctns(-/-) mice showed the following alterations: 1) compensated primary hypothyroidism and accelerated thyrocyte turnover; 2) impaired Tg production linked to ER stress/UPR response; and 3) altered endolysosomal trafficking and iodo-Tg processing. The Ctns(-/-) thyroid is useful to study disease progression and evaluate novel therapies.

The role and therapeutic potential of Ser/Thr phosphatase PP2A in apoptotic signalling networks in human cancer cells.

A block in apoptotic cell death is a likely requirement for cancer maintenance. Likewise, drug resistance, one of the key clinical problems in oncology, can often be explained by apoptotic resistance following drug administration. Several signalling pathways can commit cells to death, including intrinsic mitochondrial pathways controlled by the Bcl-2-like proteins, extrinsic Death Receptor-triggered pathways, and Dependence Receptor-initiated pathways. In addition, depending on the cell type, external stimulus and context, various other pro- or anti-survival signalling pathways may become repressed or activated. Proper coordination and conversion into a common cellular response is ensured by various ways of inter-pathway crosstalk. As for most signalling cascades, post-translational control of the signalling proteins involved is mainly achieved by reversible phosphorylation and thus by the coordinated actions of protein kinases and phosphatases. Despite increasing interest in phosphatases as potential tumour suppressors, their role in controlling apoptotic signalling remains poorly understood. Here we review current knowledge about the regulatory functions of Protein Phosphatase type 2A (PP2A) phosphatases in these apoptotic signalling networks. PP2A represents an abundant class of structurally complex Ser/Thr phosphatases which are of particular interest in this context because of their recently established role as genuine tumour suppressors. In line with these tumour suppressive characteristics, PP2A predominantly displays pro-apoptotic functions, although some PP2A complexes also clearly counteract apoptotic cell death. Finally, we speculate how this knowledge might be exploited for therapeutic purposes, in light of pre-clinical pharmacological approaches, currently demonstrated to target PP2A in cancer cells.

The Accelerating Access Initiative: experience with a multinational workplace programme in Africa.

PROBLEM: A multinational company with operations in several African countries was committed to offer antiretroviral treatment to its employees and their dependants. APPROACH: The Accelerating Access Initiative (AAI), an initiative of six pharmaceutical companies and five United Nations' agencies, offered the possibility of obtaining brand antiretroviral drugs (ARVs) at 10% of the commercial price. PharmAccess, a foundation aimed at removing barriers to AIDS treatment in Africa, helped to establish an HIV policy and treatment guidelines, and a workplace programme was rolled out from September 2001. LOCAL SETTING: Private sector employers in Africa are keen to take more responsibility in HIV prevention and AIDS care. An important hurdle for African employers remains the price and availability of ARVs. RELEVANT CHANGES: The programme encountered various hurdles, among them the need for multiple contracts with multiple companies, complex importation procedures, taxes levied on ARVs, lack of support from pharmaceutical companies in importation and transportation, slow delivery of the drugs, lack of institutional memory in pharmaceutical companies and government policies excluding the company from access to ARVs under the AAI. LESSONS LEARNED: The launch of the AAI enabled this multinational company to offer access to ARVs to its employees and dependants. The private sector should have access to these discounted drugs under the AAI. A network of local AAI offices should be created to assist in logistics of drugs ordering, purchase and clearance. No taxes should be levied on ARVs.

Task shifting in HIV/AIDS: opportunities, challenges and proposed actions for sub-Saharan Africa.

Sub-Saharan Africa is facing a crisis in human health resources due to a critical shortage of health workers. The shortage is compounded by a high burden of infectious diseases; emigration of trained professionals; difficult working conditions and low motivation. In particular, the burden of HIV/AIDS has led to the concept of task shifting being increasingly promoted as a way of rapidly expanding human resource capacity. This refers to the delegation of medical and health service responsibilities from higher to lower cadres of health staff, in some cases non-professionals. This paper, drawing on Médecins Sans Frontières' experience of scaling-up antiretroviral treatment in three sub-Saharan African countries (Malawi, South Africa and Lesotho) and supplemented by a review of the literature, highlights the main opportunities and challenges posed by task shifting and proposes specific actions to tackle the challenges. The opportunities include: increasing access to life-saving treatment; improving the workforce skills mix and health-system efficiency; enhancing the role of the community; cost advantages and reducing attrition and international 'brain drain'. The challenges include: maintaining quality and safety; addressing professional and institutional resistance; sustaining motivation and performance and preventing deaths of health workers from HIV/AIDS. Task shifting should not undermine the primary objective of improving patient benefits and public health outcomes.

RheoDSC: a hyphenated technique for the simultaneous measurement of calorimetric and rheological evolutions.

A newly developed hyphenated technique is presented combining an existing rheometer and differential scanning calorimeter into a single experimental setup. Through the development of a fixation accessory for differential scanning calorimeter (DSC) crucibles and a novel rotor, the simultaneous measurement is performed inside the well-controlled thermal environment of a Tzero DSC cell. Hence, the evolution of thermal and flow properties of a material can be simultaneously measured using steady or oscillatory shear measurements and regular or modulated temperature DSC measurements. Along with the construction of a prototype, a validation of the design was performed. The technique offers interesting opportunities for the investigation of flow-induced transitions, for instance, crystallization or phase separation, and provides an asset for high-throughput screening of materials. The potential of the novel technique is demonstrated by two case studies: the chemorheology during the cure of a thermosetting epoxy-amine system and the flow-induced crystallization of syndiotactic polypropylene.

Database-supported teleconferencing: an additional clinical mentoring tool to assist a multinational company HIV/AIDS treatment program in Africa.

BACKGROUND: The lack of human resources for health is presently recognized as a major factor limiting scale-up of antiretroviral treatment (ART) programs in resourcelimited settings. The mobilization of public and private partners, the decentralization of care, and the training of non-HIV specialist nurses and general practitioners could help increase the number of HIV-infected patients receiving ART. In addition to other forms of training, scheduled teleconferences (TCs) have been organized to support a comprehensive HIV treatment program delivered by a private company's health team. OBJECTIVE: To describe the role of the TC as an additional tool in mentoring a company's health care workers (HCWs). METHOD: For this study, all TC reports were retrospectively reviewed and the questions classified by topic. Participating Heineken physicians evaluated the technical quality and scientific relevance of the TCs through an anonymous survey. RESULTS: From October 2001 to December 2003, 10 HCWs working in 14 operating companies in 5 African countries raised 268 problems during 45 TCs. A total of 79 questions (29%) were asked about antiretroviral (ARV) therapy, 53 (20%) about the diagnosis and treatment of opportunistic infection, 43 (16%) about ARV toxicity, 40 (15%) about care organization and policy, 32 (12%) about laboratory or drug supply, and 21 (8%) about biological parameters. The mean TC attendance rate was 70%. The level of satisfaction among local company physicians was 65% for logistics, 89% for scientific relevance, 84% for applicability of advice, and 85% overall. The most common complaints concerned the poor quality of the telephone connection and language problems for francophone participants. CONCLUSION: Database-supported teleconferencing could be an additional tool to mentor company HCWs in their routine care of HIV-infected workers and family members. The role and costeffectiveness of telemedicine in improving health outcomes should be further studied.

Relationships between visceral, trunk and whole-body adipose tissue weights by cadaver dissection.

BACKGROUND: Despite the important association of central adiposity and cardiovascular and other risk factors, there are only three reported values for directly weighed visceral adipose tissue (AT). All other reported values are based on medical imaging techniques. OBJECTIVE: The study aimed to investigate the relationships between visceral, trunk and total AT weights in older men and women. METHODS: Data was obtained from the combination of two studies involving the complete dissection of 15 male and 16 female cadavers (age range 55-94 years) and allowed for compartmentation into skin, AT, muscle, bone and a residual component, divided over six body segments: head, trunk, legs and arms. Visceral AT was separated from trunk subcutaneous AT. All tissues were weighed. RESULTS: Visceral AT weights ranged from 0.3 to 5.8 kg. Mean values were 3.00 +/- 1.52 kg (mean +/- SE) for the men and 3.24 +/- 1.67 kg for the women. These were not significantly different (p = 0.68), but visceral AT weight, expressed as a percentage of total body AT weight was significantly greater (p = 0.02) in the men (16.8 +/- 5.4%) than in the women (12.9 +/- 3.5%). Correlations between visceral AT weight and the weight of subcutaneous AT of the trunk were highly significant (men, r = 0.70, women, r = 0.81, p < 0.005), with similar slopes for the two sexes. The correlation coefficients of visceral with total body AT weights were even greater (men, r = 0.83 and women, r = 0.96, p < 0.0001). CONCLUSIONS: In this sample of older Belgians, visceral AT is strongly related to total body adiposity, corresponding to an increment of about 200 g of visceral AT for every kilogram of total AT in men and about 180 g in women. Because of this relationship, techniques such as skinfold calipers and ultrasound for assessing whole body fatness from measurement of only the subcutaneous layer are thus able to account for visceral adiposity.

Observation of a supernumerary hamstring muscle: a state of the art on its incidence and clinical relevance.

During routine anatomic dissection of the lower extremities of a 67-year-old male body, a supernumerary ishiocrural muscle was observed. This supernumerary muscle had similarities to a rare variant of the semimembranous muscle. On the left side it arose from the lateral dorsal side of the femur between the short head of the biceps femoris muscle and the origin of the adductor magnus muscle. It inserted on the medial condyle deep to the normal insertion of the semimembranous at the posterior aspect of the articular capsule. This muscle can be regarded either as a short deep semimembranous muscle (M. semimembranosus profondus) or as a short belly of a semitendinous biceps as known in birds. The muscle was situated closely to the vessels and nerves of the popliteal region. On the right side a similar but somewhat fainter muscle was observed whose origin emanated from the fascia of the adductor magnus muscle. The muscle probably has no major clinical importance but might be important to the surgeon who has to intervene in the popliteal region.

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.

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.

Protein phosphatase 2A: a highly regulated family of serine/threonine phosphatases implicated in cell growth and signalling.

Protein phosphatase 2A (PP2A) comprises a family of serine/threonine phosphatases, minimally containing a well conserved catalytic subunit, the activity of which is highly regulated. Regulation is accomplished mainly by members of a family of regulatory subunits, which determine the substrate specificity, (sub)cellular localization and catalytic activity of the PP2A holoenzymes. Moreover, the catalytic subunit is subject to two types of post-translational modification, phosphorylation and methylation, which are also thought to be important regulatory devices. The regulatory ability of PTPA (PTPase activator), originally identified as a protein stimulating the phosphotyrosine phosphatase activity of PP2A, will also be discussed, alongside the other regulatory inputs. The use of specific PP2A inhibitors and molecular genetics in yeast, Drosophila and mice has revealed roles for PP2A in cell cycle regulation, cell morphology and development. PP2A also plays a prominent role in the regulation of specific signal transduction cascades, as witnessed by its presence in a number of macromolecular signalling modules, where it is often found in association with other phosphatases and kinases. Additionally, PP2A interacts with a substantial number of other cellular and viral proteins, which are PP2A substrates, target PP2A to different subcellular compartments or affect enzyme activity. Finally, the de-regulation of PP2A in some specific pathologies will be touched upon.

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.

Identification and characterization of alternative splice products encoded by the human phosphotyrosyl phosphatase activator gene.

The phosphotyrosyl phosphatase activator (PTPA), a protein phosphatase 2A (PP2A) regulatory protein, specifically stimulates the phosphotyrosyl phosphatase activity of PP2A in vitro. Human PTPA is encoded by a single gene, the structure and chromosomal localization of which have been determined in our previous work. In this paper, we report the identification and characterization of six additional splice variants, termed PTPAbeta to PTPAeta, in addition to the originally identified PTPAalpha form. Interestingly, PTPAbeta and PTPAgamma contain a novel exon that had been overlooked in the formerly identified gene structure. As revealed by nested PCR, all these PTPA transcripts are expressed in various human cDNA libraries and cell lines. However, a quantitative approach, using a single PCR reaction followed by detection of the reaction products with a radioactively labeled probe, revealed only PTPAalpha, beta and delta, suggesting that the other transcripts are expressed very poorly. In vitro transcription-translation revealed that only PTPAalpha, beta, delta and epsilon are translated into functional proteins, whereas translation of PTPAgamma, zeta and eta is stopped prematurely due to a frameshift resulting from skipping exon 2, suggesting that the latter isoforms may result from splicing errors. By western analysis of HepG2 and Saos-2 cell extracts, only PTPAalpha and beta were detected. PTPAalpha and beta were expressed as GST fusion proteins in bacteria, and were found to contain the same specific phosphotyrosyl phosphatase stimulatory activity towards PP2A. The identification of this family of PTPA variants adds another level of complexity to the in vivo function(s) of PTPA, opening up the possibility that different isoforms may perform different functions.

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.

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.

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.