Biomedical subjects

Veerle Janssens

Publications and source records attributed to Veerle Janssens.

8 recordsLinked to original sources

PubMed2005

The protein phosphatase 2A phosphatase activator is a novel peptidyl-prolyl cis/trans-isomerase.

The protein phosphatase 2A (PP2A) phosphatase activator (PTPA) is an essential protein involved in the regulation of PP2A and the PP2A-like enzymes. In this study we demonstrate that PTPA and its yeast homologues Ypa1 and Ypa2 can induce a conformational change in some model substrates. Using these model substrates in different assays with and without helper proteases, this isomerase activity is similar to the isomerase activity of FKBP12, the human cyclophilin A, and one of its yeast homologs Cpr7 but dissimilar to the isomerase activity of Pin1. However, neither FKBP12 nor Cpr7 can reactivate the inactive form of PP2A. Therefore, PTPA belongs to a novel peptidyl-prolyl cis/trans-isomerase (PPIase) family. The PPIase activity of PTPA correlates with its activating activity since both are stimulated by the presence of Mg2+ATP, and a PTPA mutant (Delta208-213) with 400-fold less activity in the activation reaction of PP2A also showed almost no PPIase activity. The point mutant Asp205 --> Gly (in Ypa1) identified this amino acid as essential for both activities. Moreover, PTPA dissociates the inactive form from the complex with the PP2A methylesterase. Finally, Pro190 in the catalytic subunit of PP2A (PP2AC) could be identified as the target Pro isomerized by PTPA/Mg2+ATP since among the 14 Pro residues present in 12 synthesized peptides representing the microenvironments of these prolines in PP2AC, only Pro190 could be isomerized by PTPA/Mg2+ATP. This Pro190 is present in a predicted loop structure near the catalytic center of PP2AC and, if mutated into a Phe, the phosphatase is inactive and can no longer be activated by PTPA/Mg2+ATP.

Adenosine Triphosphate↗

PubMed2005

Interaction of the protein phosphatase 2A with the regulatory domain of the cystic fibrosis transmembrane conductance regulator channel.

A direct interaction of the regulatory domain (R domain) of the cystic fibrosis transmembrane conductance regulator protein (CFTR) with PR65, a regulatory subunit of the protein phosphatase 2A (PP2A), was shown in yeast two hybrid, pull-down and co-immunoprecipitation experiments. The R domain could be dephosphorylated by PP2A in vitro. Overexpression of the interacting domain of PR65 in Caco-2 cells, as well as treatment with okadaic acid, showed a prolonged deactivation of the chloride channel. Taken together our results show a direct and functional interaction between CFTR and PP2A.

Amino Acid Sequence↗

PubMed2005

Specific interactions of PP2A and PP2A-like phosphatases with the yeast PTPA homologues, Ypa1 and Ypa2.

To elucidate the specific biological role of the yeast homologues of PTPA (phosphatase 2A phosphatase activator), Ypa1 and Ypa2 (where Ypa stands for yeast phosphatase activator), in the regulation of PP2A (protein phosphatase 2A), we investigated the physical interaction of both Ypa proteins with the catalytic subunit of the different yeast PP2A-like phosphatases. Ypa1 interacts specifically with Pph3, Sit4 and Ppg1, whereas Ypa2 binds to Pph21 and Pph22. The Ypa1 and Ypa2 proteins do not compete with Tap42 (PP2A associating protein) for binding to PP2A family members. The interaction of the Ypa proteins with the catalytic subunit of PP2A-like phosphatases is direct and independent of other regulatory subunits, implicating a specific function for the different PP2A-Ypa complexes. Strikingly, the interaction of Ypa2 with yeast PP2A is promoted by the presence of Ypa1, suggesting a positive role of Ypa1 in the regulation of PP2A association with other interacting proteins. As in the mammalian system, all yeast PP2A-like enzymes associate as an inactive complex with Yme (yeast methyl esterase). Ypa1 as well as Ypa2 can reactivate all these inactive complexes, except Pph22-Yme. Ypa1 is the most potent activator of PP2A activity, suggesting that there is no direct correlation between activation potential and binding capacity.

Adaptor Proteins, Signal Transducing↗

PubMed2005

PP2A: the expected tumor suppressor.

PP2A is one of the few serine/threonine-specific phosphatases in the cell, and its complex structure and regulation guarantees its many different functions. Some viruses have chosen to target this enzyme system in order to manage the host cell machinery for their own profit and to program cells into a malignant state. Suppression of PR61/B'gamma, a specific third regulatory subunit of PP2A, can substitute for the viral SV40 protein small t antigen in causing tumorigenic transformation of several human cell lines -- provided that telomerase, SV40 large T antigen and oncogenic Ras are also present. Accumulation of c-Myc seems to be the common denominator.

Animals↗

PubMed2004

An inactive protein phosphatase 2A population is associated with methylesterase and can be re-activated by the phosphotyrosyl phosphatase activator.

We have described recently the purification and cloning of PP2A (protein phosphatase 2A) leucine carboxylmethyltransferase. We studied the purification of a PP2A-specific methylesterase that co-purifies with PP2A and found that it is tightly associated with an inactive dimeric or trimeric form of PP2A. These inactive enzyme forms could be reactivated as Ser/Thr phosphatase by PTPA (phosphotyrosyl phosphatase activator of PP2A). PTPA was described previously by our group as a protein that stimulates the in vitro phosphotyrosyl phosphatase activity of PP2A; however, PP2A-specific methyltransferase could not bring about the activation. The PTPA activation could be distinguished from the Mn2+ stimulation observed with some inactive forms of PP2A, also found associated with PME-1 (phosphatase methylesterase 1). We discuss a potential new function for PME-1 as an enzyme that stabilizes an inactivated pool of PP2A.

Animals↗

PubMed2004

Genomic organisation, chromosomal localisation tissue distribution and developmental regulation of the PR61/B' regulatory subunits of protein phosphatase 2A in mice.

Protein phosphatase 2A (PP2A) is a major serine/threonine-specific phosphatase playing central roles in development, cell growth and transformation. Regulation is largely accomplished by the regulatory B subunits, which determine substrate specificity, subcellular localisation and catalytic activity. The B' family, also known as the PR61 family, is the most diverse, consisting of five genes (alpha,beta,gamma,delta and epsilon) that give rise to a number of splice variants. We deduced the sequences of the different PR61 proteins in mice and found evidence for the expression of PR61alpha, beta1, gamma1, gamma2, gamma3, delta1 and epsilon. We report the genomic organisation and localisation of the murine PR61 genes (Ppp2r5a-Ppp2r5e). This information will be useful for the future realisation of PR61 knockouts. Using Northern blotting, we examined the expression of the five PR61 isoforms in different tissues. A brain-specific function can be expected for the PR611beta protein based on the high expression levels observed in murine brain. In situ hybridisation analysis of the adult brain revealed a distinct and partially overlapping pattern of mRNA expression of the various PR61 isoforms. The PR61 mRNA expression during embryonic development was examined by Northern blotting. The PR61 transcripts were differentially expressed, suggesting a specific function for each of the PR61 proteins during embryonic development and/or adult life.

Amino Acid Sequence↗

PubMed2003

Identification and functional analysis of two Ca2+-binding EF-hand motifs in the B"/PR72 subunit of protein phosphatase 2A.

Protein phosphatase 2A (PP2A) is a multifunctional serine/threonine phosphatase that is critical to many cellular processes including cell cycle regulation and signal transduction. PP2A is a heterotrimer containing a structural (A) and catalytic (C) subunit, associated with one variable regulatory or targeting B-type subunit, of which three families have been described to date (B/PR55, B'/PR61, and B"/PR72). We identified two functional and highly conserved Ca(2+)-binding EF-hand motifs in human B"/PR72 (denoted EF1 and EF2), demonstrating for the first time the ability of Ca(2+) to interact directly with and regulate PP2A. EF1 and EF2 apparently bind Ca(2+) with different affinities. Ca(2+) induces a significant conformational change, which is dependent on the integrity of the motifs. We have further evaluated the effects of Ca(2+) on subunit composition, subcellular targeting, catalytic activity, and function during the cell cycle of a PR72-containing PP2A trimer (PP2A(T72)) by site-directed mutagenesis of either or both motifs. The results suggest that integrity of EF2 is required for A/PR65 subunit interaction and proper nuclear targeting of PR72, whereas EF1 might mediate the effects of Ca(2+) on PP2A(T72) activity in vitro and is at least partially required for the ability of PR72 to alter cell cycle progression upon forced expression.

Amino Acid Motifs↗

PubMed2003

Identification and characterization of B"-subunits of protein phosphatase 2 A in Xenopus laevis oocytes and adult tissues.

Protein phosphatase 2A is a phosphoserine/threonine phosphatase implicated in many cellular processes. The core enzyme comprises a catalytic and a PR65/A-subunit. The substrate specificity and subcellular localization are determined by a third regulatory B-subunit (PR55/B, PR61/B' and PR72/130/B"). To identify the proteins of the B" family in Xenopus laevis oocytes, a prophase Xenopus oocyte cDNA library was screened using human PR130 cDNA as a probe. Three different classes of cDNAs were isolated. One class is very similar to human PR130 and is probably the Xenopus orthologue of PR130 (XPR130). A second class of clones (XN73) is identical to the N-terminal part of XPR130 but ends a few amino acids downstream of the putative splicing site of PR130. To investigate how this occurs, the genomic structure of the human PR130 gene was determined. This novel protein does not act as a PP2A subunit but might compete with the function of PR130. The third set of clones (XPR70) is very similar to human PR48 but has an N-terminal extension. Further analysis of the human EST-database and the human PR48 gene structure, revealed that the human PR48 clone published is incomplete. The Xenopus orthologue of PR48 encodes a protein of 70 kDa which like the XPR130, interacts with the A-subunit in GST pull-down assays. XPR70 is ubiquitously expressed in adult tissues and oocytes whereas expression of XPR130 is very low in brain and oocytes. Expression of XN73 mainly parallels XPR130 with the exception of the brain.

Alternative Splicing↗