Ki-67 and RepoMan have key roles during mitotic exit. Previously, we showed that Ki-67 organizes the mitotic chromosome periphery and recruits protein phosphatase 1 (PP1) to chromatin at anaphase onset, in a similar manner as RepoMan (Booth et al., 2014). Here we show how Ki-67 and RepoMan form mitotic exit phosphatases by recruiting PP1, how they distinguish between distinct PP1 isoforms and how the assembly of these two holoenzymes are dynamically regulated by Aurora B kinase during mitosis. Unexpectedly, our data also reveal that Ki-67 and RepoMan bind PP1 using an identical, yet novel mechanism, interacting with a PP1 pocket that is engaged only by these two PP1 regulators. These findings not only show how two distinct mitotic exit phosphatases are recruited to their substrates, but also provide immediate opportunities for the design of novel cancer therapeutics that selectively target the Ki-67:PP1 and RepoMan:PP1 holoenzymes.
Calcineurin is a ubiquitously expressed calcium-dependent phosphatase that is inhibited by the immunosuppressant drugs cyclosporine and tacrolimus. Measuring calcineurin activity in transplant patients has been complicated by a lack of consistent correlation between drug level and enzyme activity, particularly with chronic use. Data from mice lacking the CnAα or CnAβ isoform of the catalytic subunit of calcineurin demonstrate that loss of CnAβ results in immunosuppression, whereas loss of CnAα does not. As such, methods to examine activity of the CnAβ isoform may be more clinically relevant than nonspecific assays.
Okadaic acid (OKA) is one of the main polyether toxins produced by marine microalgae which causes diarrhetic shellfish poisoning. It is a selective and potent inhibitor of serine/threonine phosphatases 1 and 2A induces hyperphosphorylation of tau in vitro and in vivo. The reduced activity of phosphatases like, protein phosphatase 2A (PP2A) has been implicated in the brain of Alzheimer’s disease (AD) patients. It is reported that AD is a complex multifactorial neurodegenerative disorder and hyperphosphorylated tau proteins is a major pathological hallmark of AD. The molecular pathogenesis of AD includes an extracellular deposition of beta amyloid (Aβ), accumulation of intracellular neurofibrillary tangles (NFT), GSK3β activation, oxidative stress, altered neurotransmitter and inflammatory cascades. Several lines of evidence suggested that the microinfusion of OKA into the rat brain causes cognitive deficiency, NFTs-like pathological changes and oxidative stress as seen in AD pathology via tau hyperphosphorylation caused by inhibition of protein phosphatases. So, communal data and information inferred that OKA induces neurodegeneration along with tau hyperphosphorylation; GSK3β activation, oxidative stress, neuroinflammation and neurotoxicity which is a characteristic feature of AD pathology. Through this collected evidence, it is suggested that OKA induced neurotoxicity may be a novel tool to study Alzheimer’s disease pathology and helpful in development of new therapeutic approach.
We provide evidence that AtDBP1 promotes flowering by regulating the transcript levels of several important integrators and floral meristem identity genes, including FLC, CO, SOC1, LFY, FT and FD. DNA-binding protein phosphatases (DBP) which exhibit both sequence specific DNA-binding and protein phosphatase 2C activities are important regulators that are involved in both the transcriptional and post-translational regulations. DBP factors are known to mediate susceptibility to potyviruses; however, whether they are involved in other processes is still unclear. In this study, under both long day (LD) and short day conditions, AtDBP1 overexpressing plants displayed early flowering, while the knock out mutants, atdbp1, exhibited a delay in flowering relative to the wild-type plants; both the overexpressing lines and atdbp1 mutants remained photoperiodic sensitive, indicating that AtDBP1 was involved in the autonomous pathway. AtDBP1 does not respond to vernalization at transcript level, and both AtDBP1 overexpressing plants and atdbp1 mutants remain responsive to vernalization, indicating that AtDBP1 may not be directly involved in vernalization. Real-time PCR analysis showed that AtDBP1 can suppress FLOWERING LOCUC C (FLC) expression, a key integrator of the autonomous and vernalization pathways, and enhance the expression levels of CONSTANS and FLOWERING LOCUC T, key regulators of the LD pathway. Furthermore, expression of floral meristem identity genes including SUPPRESSOR OF OVEREXPRESSION OF CO 1, LEAFY and FD was also promoted in AtDBP1overexpressing plants. AtDBP1 transcription can be detected in root, leaf, stem, flower and silique. AtDBP1-GFP and YFP-AtDBP1 fusion protein were localized in the cytosol and nucleus. Our results provide the evidence demonstrating the effective role of AtDBP1 for flowering time regulation and report a novel function of DBP factors in planta besides in plant defense.
Protein phosphatases are the essential opposite to protein kinases; together, these enzymes regulate all protein phosphorylation and most cellular processes. To better understand the global roles of protein phosphorylation, we cataloged the human protein phosphatome, composed of 189 known and predicted human protein phosphatase genes. We also identified 79 protein phosphatase pseudogenes or retrogenes, some of which may have residual function. We traced the origin and diversity of phosphatases by building protein phosphatomes for eight other eukaryotes, from the protist Dictyostelium to the sea urchin. We classified protein phosphatases from all nine species into a hierarchy of 10 protein folds, 21 families, and 178 subfamilies. We found that >80% of the 101 human subfamilies were conserved across the animal kingdom, but show substantial differences in evolution, including losses and expansions of individual subfamilies and changes in accessory domains. Protein phosphatases show similar evolutionary dynamics to those of kinases, with substantial losses in major model organisms. Sequence analysis predicts that 26 human protein phosphatase domains are catalytically disabled and that this disability is mostly conserved across orthologs. This genomic and evolutionary perspective on protein phosphatases provides a framework for global analysis of protein phosphorylation throughout the animal kingdom.
Previous evidence from post-mortem Alzheimer disease (AD) brains and drug (especially rapamycin)-oriented in vitro and in vivo models implicated an aberrant accumulation of the mammalian target of rapamycin (mTor) signaling in tangle bearing neurons in AD brains and its role in the formation of abnormally hyperphosphorylated tau. Compelling evidence indicated that the sequential molecular events such as the synthesis and phosphorylation of tau can be regulated through p70S6 kinase, the well-characterized immediate down-stream target of mTor. In the present study, we further identified that active form of mTor per se accumulates in tangle-bearing neu-rons, in particular those at early stages in AD brains. By using mass spectrometry and Western blotting we identified 3 phospho-epitopes of tau directly phos-phorylated by mTor. We have developed a variety of stable cell lines with genetic modification of mTor activity using SH-SY5Y neuroblastoma cells as background. In these cellular systems, we not only confirmed the tau phosphorylation sites found in vitro, but also found that mTor mediates the synthesis and aggregation of tau, resulting in compromised microtubule stability. Changes of mTor activity cause fluctuation of the level of a battery of tau kinases such as protein kinase A (PKA), v-Akt Murine Thymoma Viral Oncogene Homolog-1 (Akt), glycogen synthase kinase 3β (GSK-3β), cyclin-dependent kinase 5 (Cdk5) and tau protein phosphatase 2A (PP2A). These results im-plicate mTor in promoting an imbalance of tau homeostasis-a condition required for neurons to maintain physiological function.
Signaling pathways controlled by reversible protein phosphorylation (catalyzed by kinases and phosphatases) in the malaria parasite Plasmodium are of great interest, for both increased understanding of parasite biology and identification of novel drug targets. Here, we report a functional analysis in Plasmodium of an ancient bacterial Shewanella-like protein phosphatase (SHLP1) found only in bacteria, fungi, protists, and plants. SHLP1 is abundant in asexual blood stages and expressed at all stages of the parasite life cycle. shlp1 deletion results in a reduction in ookinete (zygote) development, microneme formation, and complete ablation of oocyst formation, thereby blocking parasite transmission. This defect is carried by the female gamete and can be rescued by direct injection of mutant ookinetes into the mosquito hemocoel, where oocysts develop. This study emphasizes the varied functions of SHLP1 in Plasmodium ookinete biology and suggests that it could be a novel drug target for blocking parasite transmission.
Concurrent activation of RAS/ERK and PI3K/AKT pathways is implicated in prostate cancer progression. The negative regulators of these pathways, including sprouty2 (SPRY2), protein phosphatase 2A (PP2A), and phosphatase and tensin homolog (PTEN), are commonly inactivated in prostate cancer. The molecular basis of cooperation between these genetic alterations is unknown. Here, we show that SPRY2 deficiency alone triggers activation of AKT and ERK, but this is insufficient to drive tumorigenesis. In addition to AKT and ERK activation, SPRY2 loss also activates a PP2A-dependent tumor suppressor checkpoint. Mechanistically, the PP2A-mediated growth arrest depends on GSK3β and is ultimately mediated by nuclear PTEN. In murine prostate cancer models, Pten haploinsufficiency synergized with Spry2 deficiency to drive tumorigenesis, including metastasis. Together, these results show that loss of Pten cooperates with Spry2 deficiency by bypassing a novel tumor suppressor checkpoint. Furthermore, loss of SPRY2 expression correlates strongly with loss of PTEN and/or PP2A subunits in human prostate cancer. This underlines the cooperation between SPRY2 deficiency and PTEN or PP2A inactivation in promoting tumorigenesis. Overall, we propose SPRY2, PTEN, and PP2A status as an important determinant of prostate cancer progression. Characterization of this trio may facilitate patient stratification for targeted therapies and chemopreventive interventions.
Protein phosphorylation and dephosphorylation (catalysed by kinases and phosphatases, respectively) are post-translational modifications that play key roles in many eukaryotic signalling pathways, and are often deregulated in a number of pathological conditions in humans. In the malaria parasite Plasmodium, functional insights into its kinome have only recently been achieved, with over half being essential for blood stage development and another 14 kinases being essential for sexual development and mosquito transmission. However, functions for any of the plasmodial protein phosphatases are unknown. Here, we use reverse genetics in the rodent malaria model, Plasmodium berghei, to examine the role of a unique protein phosphatase containing kelch-like domains (termed PPKL) from a family related to Arabidopsis BSU1. Phylogenetic analysis confirmed that the family of BSU1-like proteins including PPKL is encoded in the genomes of land plants, green algae and alveolates, but not in other eukaryotic lineages. Furthermore, PPKL was observed in a distinct family, separate to the most closely-related phosphatase family, PP1. In our genetic approach, C-terminal GFP fusion with PPKL showed an active protein phosphatase preferentially expressed in female gametocytes and ookinetes. Deletion of the endogenous ppkl gene caused abnormal ookinete development and differentiation, and dissociated apical microtubules from the inner-membrane complex, generating an immotile phenotype and failure to invade the mosquito mid-gut epithelium. These observations were substantiated by changes in localisation of cytoskeletal tubulin and actin, and the micronemal protein CTRP in the knockout mutant as assessed by indirect immunofluorescence. Finally, increased mRNA expression of dozi, a RNA helicase vital to zygote development was observed in ppkl(-) mutants, with global phosphorylation studies of ookinete differentiation from 1.5-24 h post-fertilisation indicating major changes in the first hours of zygote development. Our work demonstrates a stage-specific essentiality of the unique PPKL enzyme, which modulates parasite differentiation, motility and transmission.
Circadian clocks in eukaryotes keep time via cell-autonomous transcriptional feedback loops. A well-characterized example of such a transcriptional feedback loop is in Drosophila, where CLOCK-CYCLE (CLK-CYC) complexes activate transcription of period (per) and timeless (tim) genes, rising levels of PER-TIM complexes feedback to repress CLK-CYC activity, and degradation of PER and TIM permits the next cycle of CLK-CYC transcription. The timing of CLK-CYC activation and PER-TIM repression is regulated post-translationally, in part through rhythmic phosphorylation of CLK, PER and TIM. Previous behavioral screens identified several kinases that control CLK, PER and TIM levels, subcellular localization and/or activity, but two phosphatases that function within the clock were identified through the analysis of candidate genes from other pathways or model systems. To identify phosphatases that play a role in the clock, we screened clock cell-specific RNA interference (RNAi) knockdowns of all annotated protein phosphatases and protein phosphatase regulators in Drosophila for altered activity rhythms. This screen identified 19 protein phosphatases that lengthened or shortened circadian period by ≥ 1h (p-value: ≤ 0.05 compared to controls) or were arrhythmic. Additional RNAi lines, transposon inserts, overexpression and loss-of-function mutants were tested to independently confirm these RNAi phenotypes. Based on genetic validation and molecular analysis, 15 viable protein phosphatases remain for future studies. These candidates are expected to reveal novel features of the circadian timekeeping mechanism in Drosophila that are likely to be conserved in all animals including humans.