Loss of LCMT1 and biased protein phosphatase 2A heterotrimerization drive prostate cancer progression and therapy resistance.

Loss of the tumor suppressive activity of the protein phosphatase 2A (PP2A) is associated with cancer, but the underlying molecular mechanisms are unclear. PP2A holoenzyme comprises a heterodimeric core, a scaffolding A subunit and a catalytic C subunit, and one of over 20 distinct substrate-directing regulatory B subunits. Methylation of the C subunit regulates PP2A heterotrimerization, affecting B subunit binding and substrate specificity.

PP2A Phospho-Tyr Antibodies Are Not Specific for this Modification but Are Sensitive to Other PP2A Modifications Including Leu Methylation.

Protein phosphatase 2A (PP2A) is an important regulator of signal transduction pathways and a tumor suppressor. Phosphorylation of the PP2A catalytic subunit (PP2A) at tyrosine 307 has been claimed to inactivate PP2A and was examined in more than 180 studies using commercial antibodies, but this modification was never identified using mass spectrometry. Here we show that the most cited pTyr monoclonal antibodies, E155 and F-8, are not specific for phosphorylated Tyr but instead are hampered by PP2A methylation at leucine 309 or phosphorylation at threonine 304.

The Myc tag monoclonal antibody 9E10 displays highly variable epitope recognition dependent on neighboring sequence context.

Epitope tags are short, linear antibody recognition sequences that enable detection of tagged fusion proteins by antibodies. Epitope tag position and neighboring sequences potentially affect its recognition by antibodies, and such context-dependent differences in tag binding may have a wide-ranging effect on data interpretation. We tested by Western blotting six antibodies that recognize the c-Myc epitope tag, including monoclonal antibodies 9E10, 4A6, 9B11, and 71D10 and polyclonal antibodies 9106 and A-14.

Antibodies recognizing the C terminus of PP2A catalytic subunit are unsuitable for evaluating PP2A activity and holoenzyme composition.

The methyl-esterification of the C-terminal leucine of the protein phosphatase 2A (PP2A) catalytic (C) subunit is essential for the assembly of specific trimeric PP2A holoenzymes, and this region of the C subunit also contains two threonine and tyrosine phosphorylation sites. Most commercial antibodies-including the monoclonal antibody 1D6 that is part of a frequently used, commercial phosphatase assay kit-are directed toward the C terminus of the C subunit, raising questions as to their ability to recognize methylated and phosphorylated forms of the enzyme. Here, we tested several PP2A C antibodies, including monoclonal antibodies 1D6, 7A6, G-4, and 52F8 and the polyclonal antibody 2038 for their ability to specifically detect PP2A in its various modified forms, as well as to coprecipitate regulatory subunits.

[Osteotomy after Distal Radius Fractures - Five-Year Clinical and Radiological Outcomes].

PURPOSE OF THE STUDY The purpose of our retrospective study is to evaluate 5-year functional and radiological outcomes in patients following corrective osteotomy of the distal radius and ulnar osteotomy for malposition after a distal radius fracture, to identify differences in the outcomes of corrective osteotomies depending on the type of the original fracture according to the AO classification, the grade of arthritis of radiocarpal (RC) joint, surgical approach and the way of stabilisation of the osteotomy. MATERIAL AND METHODS The followed-up group of 22 patients (8 men and 14 women) underwent osteotomy for malposition of distal radius in the period 2007-2011. The age of patients in the followed-up group ranged from 21 to 72 years, with the mean age of 51 years at the time of surgery.

Anti-RAINBOW dye-specific antibodies as universal tools for the visualization of prestained protein molecular weight markers in Western blot analysis.

Western blotting is one of the most widely used techniques in molecular biology and biochemistry. Prestained proteins are used as molecular weight standards in protein electrophoresis. In the chemiluminescent Western blot analysis, however, these colored protein markers are invisible leaving researchers with the unsatisfying situation that the signal for the protein of interest and the signal for the markers are not captured simultaneously and have to be merged in an error-prone step.

Protein Phosphatase Methyl-Esterase PME-1 Protects Protein Phosphatase 2A from Ubiquitin/Proteasome Degradation.

Protein phosphatase 2A (PP2A) is a conserved essential enzyme that is implicated as a tumor suppressor based on its central role in phosphorylation-dependent signaling pathways. Protein phosphatase methyl esterase (PME-1) catalyzes specifically the demethylation of the C-terminal Leu309 residue of PP2A catalytic subunit (PP2Ac). It has been shown that PME-1 affects the activity of PP2A by demethylating PP2Ac, but also by directly binding to the phosphatase active site, suggesting loss of PME-1 in cells would enhance PP2A activity.

Budding yeast greatwall and endosulfines control activity and spatial regulation of PP2A(Cdc55) for timely mitotic progression.

Entry into mitosis is triggered by cyclinB/Cdk1, whose activity is abruptly raised by a positive feedback loop. The Greatwall kinase phosphorylates proteins of the endosulfine family and allows them to bind and inhibit the main Cdk1-counteracting PP2A-B55 phosphatase, thereby promoting mitotic entry. In contrast to most eukaryotic systems, Cdc14 is the main Cdk1-antagonizing phosphatase in budding yeast, while the PP2A(Cdc55) phosphatase promotes, instead of preventing, mitotic entry by participating to the positive feedback loop of Cdk1 activation.

M-Track: detecting short-lived protein-protein interactions in vivo.

We developed a protein-proximity assay in yeast based on fusing a histone lysine methyltransferase onto a bait and its substrate onto a prey. Upon binding, the prey is stably methylated and detected by methylation-specific antibodies. We applied this approach to detect varying interaction affinities among proteins in a mitogen-activated protein kinase pathway and to detect short-lived interactions between protein phosphatase 2A and its substrates that have so far escaped direct detection.

Generation of active protein phosphatase 2A is coupled to holoenzyme assembly.

Protein phosphatase 2A (PP2A) is a prime example of the multisubunit architecture of protein serine/threonine phosphatases. Until substrate-specific PP2A holoenzymes assemble, a constitutively active, but nonspecific, catalytic C subunit would constitute a risk to the cell. While it has been assumed that the severe proliferation impairment of yeast lacking the structural PP2A subunit, TPD3, is due to the unrestricted activity of the C subunit, we recently obtained evidence for the existence of the C subunit in a low-activity conformation that requires the RRD/PTPA proteins for the switch into the active conformation.

Expression of protein phosphatase 2A mutants and silencing of the regulatory B alpha subunit induce a selective loss of acetylated and detyrosinated microtubules.

Carboxymethylation and phosphorylation of protein phosphatase 2A (PP2A) catalytic C subunit are evolutionary conserved mechanisms that critically control PP2A holoenzyme assembly and substrate specificity. Down-regulation of PP2A methylation and PP2A enzymes containing the B alpha regulatory subunit occur in Alzheimer's disease. In this study, we show that expressed wild-type and methylation- (L309 Delta) and phosphorylation- (T304D, T304A, Y307F, and Y307E) site mutants of PP2A C subunit differentially bind to B, B', and B''-type regulatory subunits in NIH 3T3 fibroblasts and neuro-2a (N2a) neuroblastoma cells.

Protein phosphatase 2A protects centromeric sister chromatid cohesion during meiosis I.

Segregation of homologous maternal and paternal centromeres to opposite poles during meiosis I depends on post-replicative crossing over between homologous non-sister chromatids, which creates chiasmata and therefore bivalent chromosomes. Destruction of sister chromatid cohesion along chromosome arms due to proteolytic cleavage of cohesin's Rec8 subunit by separase resolves chiasmata and thereby triggers the first meiotic division. This produces univalent chromosomes, the chromatids of which are held together by centromeric cohesin that has been protected from separase by shugoshin (Sgo1/MEI-S332) proteins.

Downregulation of protein phosphatase 2A carboxyl methylation and methyltransferase may contribute to Alzheimer disease pathogenesis.

ABalphaC, a major protein phosphatase 2A (PP2A) heterotrimeric enzyme, binds to and regulates the microtubule cytoskeleton and tau. We have shown that ABalphaC protein expression levels are selectively reduced in Alzheimer disease (AD). Notably, the carboxyl methylation of PP2A catalytic subunit (PP2A(C)) is critically required for ABalphaC holoenzyme assembly, and catalyzed by a specific methyltransferase (PPMT).

Altered expression levels of the protein phosphatase 2A ABalphaC enzyme are associated with Alzheimer disease pathology.

The formation of amyloid-containing senile plaques and tau-rich neurofibrillary tangles are central events in Alzheimer disease (AD) pathogenesis. Significantly, ABalphaC, a major protein phosphatase 2A (PP2A) holoenzyme, specifically binds to and dephosphorylates tau. Deregulation of PP2A results in tau hyperphosphorylation in vivo.

Identification of a subunit of a novel Kleisin-beta/SMC complex as a potential substrate of protein phosphatase 2A.

Protein phosphatase 2A (PP2A) holoenzymes consist of a catalytic C subunit, a scaffolding A subunit, and one of several regulatory B subunits that recruit the AC dimer to substrates. PP2A is required for chromosome segregation, but PP2A's substrates in this process remain unknown. To identify PP2A substrates, we carried out a two-hybrid screen with the regulatory B/PR55 subunit.

[A Rare Fracture-dislocation of Talus.].

In the introduction the authors analyze the issues of the fractures of talus which is a key connection between the foot and the lower limb. For the prognosis of the healing of fractures of the talus they consider the most important its retrograde vascularization. Further they decribe types of fractures of the talus according to Hawkins.

Catalytically inactive protein phosphatase 2A can bind to polyomavirus middle tumor antigen and support complex formation with pp60(c-src).

Interaction between the heterodimeric form of protein phosphatase 2A (PP2A) and polyomavirus middle T antigen (MT) is required for the subsequent assembly of a transformation-competent MT complex. To investigate the role of PP2A catalytic activity in MT complex formation, we undertook a mutational analysis of the PP2A 36-kDa catalytic C subunit. Several residues likely to be involved in the dephosphorylation mechanism were identified and mutated.

A common regulation of genes encoding enzymes of the deoxynucleotide metabolism is lost after neoplastic transformation.

We determined the cell cycle-dependent fluctuation of mRNAs that encode different enzymes of the deoxynucleotide metabolism in permanent cell lines of human and murine origin. In normal growing cells, dihydrofolate reductase, thymidine kinase, and both subunits of ribonucleotide reductase all show exactly the same variation. The mRNAs rise near the G1-S boundary, peak in early S phase, and return in G2 to approximately the level of early G1.

Coordinated trans activation of DNA synthesis- and precursor-producing enzymes by polyomavirus large T antigen through interaction with the retinoblastoma protein.

Previously constructed Swiss mouse 3T3 fibroblasts producing polyomavirus large T antigen after addition of dexamethasone were used to study the transcriptional activation by the viral protein of five genes coding for enzymes involved in DNA synthesis and precursor production, namely, dihydrofolate reductase, thymidine kinase, thymidylate synthase, DNA polymerase alpha, and proliferating-cell nuclear antigen. It was found that all these genes, whose expression is stimulated at the G1/S boundary of the cell cycle after growth stimulation by serum addition, are coordinately trans activated when T antigen is induced in cells previously growth arrested by serum withdrawal. Cell lines carrying the information for a mutant form of large T antigen, in which a glutamic acid residue in the binding site for the retinoblastoma protein was changed into aspartic acid, were constructed to test the involvement of an interaction of T antigen with the retinoblastoma protein in this reaction.

Distinct amounts of polyomavirus large T antigen are required for different functions of the protein.

Many cellular and some virally coded proteins regulating key events in higher cells have been found to be multifunctional. One example of such a protein is the polyomavirus large T antigen, which is involved not only in viral DNA replication and gene expression but also in the induction of the S phase in host cells, in the immortalization of various cell types and in the transactivation of some cellular genes. We recently constructed cell lines in which T antigen was synthesized under the control of a hormone-inducible promoter.

A binding site for transcription factor E2F is a target for trans activation of murine thymidine kinase by polyomavirus large T antigen and plays an important role in growth regulation of the gene.

The promoter of the murine thymidine kinase gene contains a binding site for transcription factor E2F. Using cell lines (3T3-LT) conditionally expressing polyomavirus large T antigen from a hormone-responsive promoter and reporter gene constructs carrying the thymidine kinase promoter with intact or mutated E2F sites, we show that this E2F site is the target for trans activation by the viral protein. Transcription of the growth-regulated endogenous thymidine kinase gene can be activated in serum-starved, quiescent 3T3-LT cells by induction of T antigen.

Polyomavirus large and small T antigens cooperate in induction of the S phase in serum-starved 3T3 mouse fibroblasts.

The induction of an S phase in the host cell is a prerequisite for the lytic replication cycle of polyomavirus. This function was attributed to proteins coded for by the early region of the viral DNA, the T antigens. A consideration of the role of the T antigens in the initiation of a mitogenic response of the host cell has to take into account the recent discovery that virus adsorption is sufficient to induce the synthesis of proteins which are known to appear early after quiescent cells are stimulated by the addition of serum, namely fos, jun, and myc (J.

Sodium butyrate inhibits the synthesis of the transformation related protein p 53 in 3T6 mouse fibroblasts.

Sodium butyrate, which blocks the cell cycle of many cell types in the G1 phase, strongly inhibits the synthesis of the transformation related, 53 kDa protein in 3T6 fibroblasts but much less so in SV 40 transformed mouse cells. By several criteria, this effect of the fatty acid appears to be indirect; p 53 synthesis takes place several hours after the butyrate-sensitive step in G1. The results are discussed in the light of a putative role of p 53 in growth control.