Mice lacking phosphatase PP2A subunit PR61/B'delta (Ppp2r5d) develop spatially restricted tauopathy by deregulation of CDK5 and GSK3beta.

Functional diversity of protein phosphatase 2A (PP2A) enzymes mainly results from their association with distinct regulatory subunits. To analyze the functions of one such holoenzyme in vivo, we generated mice lacking PR61/B'δ (B56δ), a subunit highly expressed in neural tissues. In PR61/B'δ-null mice the microtubule-associated protein tau becomes progressively phosphorylated at pathological epitopes in restricted brain areas, with marked immunoreactivity for the misfolded MC1-conformation but without neurofibrillary tangle formation. Behavioral tests indicated impaired sensorimotor but normal cognitive functions. These phenotypical characteristics were further underscored in PR61/B'δ-null mice mildly overexpressing human tau. PR61/B'δ-containing PP2A (PP2A(T61δ)) poorly dephosphorylates tau in vitro, arguing against a direct dephosphorylation defect. Rather, the activity of glycogen synthase kinase-3ß, a major tau kinase, was found increased, with decreased phosphorylation of Ser-9, a putative cyclin-dependent kinase 5 (CDK5) target. Accordingly, CDK5 activity is decreased, and its cellular activator p35, strikingly absent in the affected brain areas. As opposed to tau, p35 is an excellent PP2A(T61δ) substrate. Our data imply a nonredundant function for PR61/B'δ in phospho-tau homeostasis via an unexpected spatially restricted mechanism preventing p35 hyperphosphorylation and its subsequent degradation.

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.

The Menkes and Wilson disease genes counteract in copper toxicosis in Labrador retrievers: a new canine model for copper-metabolism disorders.

The deleterious effects of a disrupted copper metabolism are illustrated by hereditary diseases caused by mutations in the genes coding for the copper transporters ATP7A and ATP7B. Menkes disease, involving ATP7A, is a fatal neurodegenerative disorder of copper deficiency. Mutations in ATP7B lead to Wilson disease, which is characterized by a predominantly hepatic copper accumulation. The low incidence and the phenotypic variability of human copper toxicosis hamper identification of causal genes or modifier genes involved in the disease pathogenesis. The Labrador retriever was recently characterized as a new canine model for copper toxicosis. Purebred dogs have reduced genetic variability, which facilitates identification of genes involved in complex heritable traits that might influence phenotype in both humans and dogs. We performed a genome-wide association study in 235 Labrador retrievers and identified two chromosome regions containing ATP7A and ATP7B that were associated with variation in hepatic copper levels. DNA sequence analysis identified missense mutations in each gene. The amino acid substitution ATP7B:p.Arg1453Gln was associated with copper accumulation, whereas the amino acid substitution ATP7A:p.Thr327Ile partly protected against copper accumulation. Confocal microscopy indicated that aberrant copper metabolism upon expression of the ATP7B variant occurred because of mis-localization of the protein in the endoplasmic reticulum. Dermal fibroblasts derived from ATP7A:p.Thr327Ile dogs showed copper accumulation and delayed excretion. We identified the Labrador retriever as the first natural, non-rodent model for ATP7B-associated copper toxicosis. Attenuation of copper accumulation by the ATP7A mutation sheds an interesting light on the interplay of copper transporters in body copper homeostasis and warrants a thorough investigation of ATP7A as a modifier gene in copper-metabolism disorders. The identification of two new functional variants in ATP7A and ATP7B contributes to the biological understanding of protein function, with relevance for future development of therapy.

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.

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.

A contracted DNA repeat in LHX3 intron 5 is associated with aberrant splicing and pituitary dwarfism in German shepherd dogs.

Dwarfism in German shepherd dogs is due to combined pituitary hormone deficiency of unknown genetic cause. We localized the recessively inherited defect by a genome wide approach to a region on chromosome 9 with a lod score of 9.8. The region contains LHX3, which codes for a transcription factor essential for pituitary development. Dwarfs have a deletion of one of six 7 bp repeats in intron 5 of LHX3, reducing the intron size to 68 bp. One dwarf was compound heterozygous for the deletion and an insertion of an asparagine residue in the DNA-binding homeodomain of LHX3, suggesting involvement of the gene in the disorder. An exon trapping assay indicated that the shortened intron is not spliced efficiently, probably because it is too small. We applied bisulfite conversion of cytosine to uracil in RNA followed by RT-PCR to analyze the splicing products. The aberrantly spliced RNA molecules resulted from either skipping of exon 5 or retention of intron 5. The same splicing defects were observed in cDNA derived from the pituitary of dwarfs. A survey of similarly mutated introns suggests that there is a minimal distance requirement between the splice donor and branch site of 50 nucleotides. In conclusion, a contraction of a DNA repeat in intron 5 of canine LHX3 leads to deficient splicing and is associated with pituitary dwarfism.

Spatial control of protein phosphatase 2A (de)methylation.

Reversible methylation of the protein phosphatase 2A catalytic subunit (PP2A(C)(1)) is an important regulatory mechanism playing a crucial role in the selective recruitment of regulatory B subunits. Here, we investigated the subcellular localization of leucine carboxyl methyltransferase (LCMT1) and protein phosphatase methylesterase (PME-1), the two enzymes catalyzing this process. The results show that PME-1 is predominantly localized in the nucleus and harbors a functional nuclear localization signal, whereas LCMT1 is underrepresented in the nucleus and mainly localizes to the cytoplasm, Golgi region and late endosomes. Indirect immunofluorescence with methylation-sensitive anti-PP2A(C) antibodies revealed a good correlation with the methylation status of PP2A(C), demethylated PP2A(C) being substantially nuclear. Throughout mitosis, demethylated PP2A(C) is associated with the mitotic spindle and during cytokinesis with the cleavage furrow. Overexpression of PME-1, but not of an inactive mutant, results in increased demethylation of PP2A(C) in the nucleus, whereas overexpression of a cytoplasmic PME-1 mutant lacking the NLS results in increased demethylation in the cytoplasm-in all cases, however, without any obvious functional consequences. PME-1 associates with an inactive PP2A population, regardless of its esterase activity or localization. We propose that stabilization of this inactive, nuclear PP2A pool is a major in vivo function of PME-1.

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.

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.

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.