Interaction between protein phosphatase 5 and the A subunit of protein phosphatase 2A: evidence for a heterotrimeric form of protein phosphatase 5

Members of the phosphoprotein phosphatase family of serine/threonine phosphatases are thought to exist in different native oligomeric complexes. Protein phosphatase 2A (PP2A) is composed of a catalytic subunit (PP2Ac) that complexes with an A subunit, which in turn also interacts with one of many B subunits that regulate substrate specificity and/or (sub)cellular localization of the enzyme. Another family member, protein phosphatase 5 (PP5), contains a tetratricopeptide repeat domain at its N terminus, which has been suggested to mediate interactions with other proteins. PP5 was not thought to interact with partners homologous to the A or B subunits that exist within PP2A. However, our results indicate that this may not be the case. A yeast two-hybrid screen revealed an interaction between PP5 and the A subunit of PP2A. This interaction was confirmed for endogenous proteins in vivo using immunoprecipitation analysis and for recombinant proteins by in vitro binding experiments. Our results also indicate that the tetratricopeptide repeat domain of PP5 is required and sufficient for this interaction. In addition, immunoprecipitated PP5 contains associated B subunits. Thus, our results suggest that PP5 can exist in a PP2A-like heterotrimeric form containing both A and B subunits.     

Genomic structure and localization of the human protein phosphatase 2A BRgamma regulatory subunit.

In the course of our analysis of genomic sequence from the human chromosome 4p16.1 region harboring both the Wolfram and Ellis van Creveld syndrome genes we have identified a sequence with high homology (98% at the amino acid level) to the rat cDNA coding for the protein phosphatase 2A BRgamma (PP2ABRgamma) regulatory subunit. Although the human cDNAs for both the BRalpha and BRbeta isoforms have been described previously, the BRgamma subunit has not yet been identified in humans. Here we describe the precise genomic organization and genetic localization of the human PP2ABRgamma gene.     

Mutation of Tyr307 and Leu309 in the protein phosphatase 2A catalytic subunit favors association with the alpha 4 subunit which promotes dephosphorylation of elongation factor-2.

The cellular location and substrate specificity of the catalytic subunit (C) of protein phosphatase 2A (PP2A) depend on its interaction with A and B subunits. The distribution of epitope-tagged wild-type or mutated C subunits was studied by transient expression in COS-7 cells. Wild-type tagged C expressed at low levels formed ABC trimer and AC dimer like the endogenous C. Single mutations of C at the site of phosphorylation (Y307F) or carboxymethylation (L309Q) resulted in recovery of only AC dimer. Double mutation of both residues resulted in association of C with alpha 4 protein (alpha 4), a novel subunit of PP2A, instead of with A and B subunits. Thus, the distribution of C between ABC trimer, AC dimer, and alpha 4C complexes can be affected by modifications of the C-terminal residues. The alpha 4 protein is a homologue of the yeast Tap42 protein that functions downstream of the TOR protein to regulate protein synthesis. Transient overexpression of FLAG-alpha 4 resulted in increased dephosphorylation of elongation factor 2, but had no effect on phosphorylation of either p70S6 kinase or PHAS-I (eIF4E-BP). Signals that affect phosphorylation or methylation of the C subunit of PP2A may promote subunit exchange and direct phosphatase activity to specific intracellular substrates.     

The RCN1-encoded A subunit of protein phosphatase 2A increases phosphatase activity in vivo

Protein phosphatase 2A (PP2A), a heterotrimeric serine/threonine-specific protein phosphatase, comprises a catalytic C subunit and two distinct regulatory subunits, A and B. The RCN1 gene encodes one of three A regulatory subunits in Arabidopsis thaliana. A T-DNA insertion mutation at this locus impairs root curling, seedling organ elongation and apical hypocotyl hook formation. We have used in vivo and in vitro assays to gauge the impact of the rcn1 mutation on PP2A activity in seedlings. PP2A activity is decreased in extracts from rcn1 mutant seedlings, and this decrease is not due to a reduction in catalytic subunit expression. Roots of mutant seedlings exhibit increased sensitivity to the phosphatase inhibitors okadaic acid and cantharidin in organ elongation assays. Shoots of dark-grown, but not light-grown seedlings also show increased inhibitor sensitivity. Furthermore, cantharidin treatment of wild-type seedlings mimics the rcn1 defect in root curling, root waving and hypocotyl hook formation assays. In roots of wild-type seedlings, RCN1 mRNA is expressed at high levels in root tips, and accumulates to lower levels in the pericycle and lateral root primordia. In shoots, RCN1 is expressed in the apical hook and the basal, rapidly elongating cells in etiolated hypocotyls, and in the shoot meristem and leaf primordia of light-grown seedlings. Our results show that the wild-type RCN1-encoded A subunit functions as a positive regulator of the PP2A holoenzyme, increasing activity towards substrates involved in organ elongation and differential cell elongation responses such as root curling.     

Molecular characterization of the B' regulatory subunit gene family of Arabidopsis protein phosphatase 2A.

Type 2A serine/threonine protein phosphatases (PP2A) have been implicated as important mediators of a diverse array of reversible protein phosphorylation events in plants. We have identified a novel Arabidopsis gene (AtB' delta) which encodes a 55-kDa B' type regulatory subunit of PP2A. The protein encoded by this gene is 57-63% identical and 69-74% similar to the previously identified AtB' genes. The AtB' delta gene appears to be expressed in all Arabidopsis organs indicating its protein product has a basic housekeeping function in plant cells. Unlike certain mRNAs derived from the AtB' gamma gene, AtB' delta mRNAs do not fluctuate significantly in response to heat stress. Further analysis of cDNA sequences derived from the AtB' genes identified an alternatively spliced cDNA derived from AtB' gamma. This cDNA differs from the previously identified AtB' gamma cDNA by the absence of a 133-bp region in its 5' untranslated region. The missing 133-bp region appears to constitute an unspliced intron and its presence in the AtB' gamma gene was confirmed by PCR using Arabidopsis genomic DNA as a template. AtB' gamma mRNA containing the 133-bp intron accumulate in all Arabidopsis organs and their levels fluctuate differentially in response to heat stress. The 133-bp insert contains two short open reading frames and hence might serve as a translational control mechanism affecting AtB' gamma protein synthesis. Finally we show, using both the yeast two hybrid system and in vitro binding assays, that the B' subunit of Arabidopsis PP2A is able to associate with other PP2A subunits, supporting the notion that the B' protein serves as a regulator of PP2A activity in plants.     

Overexpression of the catalytic subunit of protein phosphatase 2A impairs cardiac function

Reversible protein phosphorylation is an essential regulatory mechanism in many cellular functions. In contrast to protein kinases, the role and regulation of protein phosphatases has remained ambiguous. To address this issue, we generated transgenic mice that overexpress the catalytic subunit alpha of protein phosphatase 2A (PP2A) (PP2Acalpha) in the heart driven by the alpha-myosin heavy chain promoter. Overexpression of the PP2Acalpha gene in the heart led to increased levels of the transgene both at RNA and protein levels. This was accompanied by a significant increase of PP2A enzyme activity in the myocardium. Morphological analysis revealed isles of necrosis and fibrosis. The phosphorylation state of phospholamban, troponin inhibitor, and eukaryotic elongation factor 2 was reduced significantly. The expression of junctional (calsequestrin) and free SR proteins (SERCA and phospholamban) was not altered. Whereas no increase in morbidity or mortality was noted, transgenic mice developed cardiac hypertrophy and reduced contractility of the heart, as well as cardiac dilatation as shown by biplane echocardiography. Taken together, these findings are indicative of the fundamental role of PP2A in cardiac function and imply that disturbances in protein phosphatases expression and activity may cause or aggravate the course of cardiac diseases.     

A mutation in protein phosphatase 2A regulatory subunit A affects auxin transport in Arabidopsis.

The phytohormone auxin controls processes such as cell elongation, root hair development and root branching. Tropisms, growth curvatures triggered by gravity, light and touch, are also auxin-mediated responses. Auxin is synthesized in the shoot apex and transported through the stem, but the molecular mechanism of auxin transport is not well understood. Naphthylphthalamic acid (NPA) and other inhibitors of auxin transport block tropic curvature responses and inhibit root and shoot elongation. We have isolated a novel Arabidopsis thaliana mutant designated roots curl in NPA (rcn1). Mutant seedlings exhibit altered responses to NPA in root curling and hypocotyl elongation. Auxin efflux in mutant seedlings displays increased sensitivity to NPA. The rcn1 mutation was transferred-DNA (T-DNA) tagged and sequences flanking the T-DNA insert were cloned. Analysis of the RCN1 cDNA reveals that the T-DNA insertion disrupts a gene for the regulatory A subunit of protein phosphatase 2A (PP2A-A). The RCN1 gene rescues the rcn1 mutant phenotype and also complements the temperature-sensitive phenotype of the Saccharomyces cerevisiae PP2A-A mutation, tpd3-1. These data implicate protein phosphatase 2A in the regulation of auxin transport in Arabidopsis.     

Disparate roles for the regulatory A subunit isoforms in Arabidopsis protein phosphatase 2A

The heterotrimeric protein phosphatase 2A (PP2A) complex comprises a catalytic subunit and regulatory A and B subunits that modulate enzyme activity and mediate interactions with other proteins. We report here the results of a systematic analysis of the Arabidopsis (Arabidopsis thaliana) regulatory A subunit gene family, which includes the ROOTS CURL IN NAPHTHYLPHTHALAMIC ACID1 (RCN1), PP2AA2, and PP2AA3 genes. All three A subunit isoforms accumulate in the organs of seedlings and adult plants, suggesting extensive overlap in expression domains. We have isolated pp2aa2 and pp2aa3 mutants and found that their phenotypes are largely normal and do not resemble that of rcn1. Whereas rcn1 pp2aa2 and rcn1 pp2aa3 double mutants exhibit striking abnormalities in all stages of development, the pp2aa2 pp2aa3 double mutant shows only modest defects. Together, these data suggest that RCN1 performs a cardinal role in regulation of phosphatase activity and that PP2AA2 and PP2AA3 functions are unmasked only when RCN1 is absent.     

Expression of the scaffolding subunit A of protein phosphatase 2A during rat testicular development

Previously, we found that the poly(A)+ RNA of the scaffolding subunit A (alpha isoform) of protein phosphatase 2A (PP2A-Aalpha) was clearly expressed by fetal gonocytes but weakly expressed by adult single (As), paired (Apr), and aligned (Aal) A spermatogonia. The scaffolding subunit A of PP2A (PP2A-A) is the major subunit in the formation of a functional PP2A holoenzyme. In this study, we investigated the expression of PP2A-A during testicular development in more detail using in situ hybridization, immunohistochemistry, and Western blot with testes of rats of various ages from 16 days postcoitum (pc) to adulthood. The expression of PP2A-A was detected in fetal proliferative gonocytes at 16 days pc, declining thereafter during the quiescent period of the gonocytes. From the day of birth to the start of spermatogenesis (Day 4 postpartum [pp]), the number of PP2A-A-immunopositive gonocytes increased. At Day 4 pp, the first A1 spermatogonia appeared along the basement membrane; all were PP2A-A positive. In the adult, PP2A-A was upregulated during the differentiation of the As, Apr, and Aal spermatogonia to the A1 spermatogonia and expressed thereafter by all other spermatogonia. Spermatocytes from the pachytene stage onward and all spermatids in the adult testis also showed clear expression of PP2A-A. In Sertoli cells, PP2A-A was detected during their proliferative period at 19 days pc to 15 days pp. The presence of a functional enzyme was confirmed by the additional detection of the catalytic subunit C of PP2A using Western blot analyses at various ages during testicular development. This apparent pattern of expression of PP2A-A during testicular development suggests that PP2A may play an important role in the proliferation of distinct populations of testicular cells and during meiosis and sperm maturation.     

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.     

Phoslactomycin targets cysteine-269 of the protein phosphatase 2A catalytic subunit in cells

According to the chemical genetic approach, small molecules that bind directly to proteins are used to analyze protein function, thereby enabling the elucidation of complex mechanisms in mammal cells. Thus, it is very important to identify the molecular targets of compounds that induce a unique phenotype in a target cell. Phoslactomycin A (PLMA) is known to be a potent inhibitor of protein Ser/Thr phosphatase 2A (PP2A); however, the inhibitory mechanism of PP2A by PLMA has not yet been elucidated. Here, we demonstrated that PLMA directly binds to the PP2A catalytic subunit (PP2Ac) in cells by using biotinylated PLMA, and the PLMA-binding site was identified as the Cys-269 residue of PP2Ac. Moreover, we revealed that the Cys-269 contributes to the potent inhibition of PP2Ac activity by PLMA. These results suggest that PLMA is a PP2A-selective inhibitor and is therefore expected to be useful for future investigation of PP2A function in cells.