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.
Targeted cancer therapies, which act on specific cancer-associated molecular targets, are predominantly inhibitors of oncogenic kinases. While these drugs have achieved some clinical success, the inactivation of kinase signaling via stimulation of endogenous phosphatases has received minimal attention as an alternative targeted approach. Here, we have demonstrated that activation of the tumor suppressor protein phosphatase 2A (PP2A), a negative regulator of multiple oncogenic signaling proteins, is a promising therapeutic approach for the treatment of cancers. Our group previously developed a series of orally bioavailable small molecule activators of PP2A, termed SMAPs. We now report that SMAP treatment inhibited the growth of KRAS-mutant lung cancers in mouse xenografts and transgenic models. Mechanistically, we found that SMAPs act by binding to the PP2A Aα scaffold subunit to drive conformational changes in PP2A. These results show that PP2A can be activated in cancer cells to inhibit proliferation. Our strategy of reactivating endogenous PP2A may be applicable to the treatment of other diseases and represents an advancement toward the development of small molecule activators of tumor suppressor proteins.
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.
Identification and functional validation of oncogenic drivers are essential steps toward advancing cancer precision medicine. Here, we have presented a comprehensive analysis of the somatic genomic landscape of the widely used BRAFV600E- and NRASQ61K-driven mouse models of melanoma. By integrating the data with publically available genomic, epigenomic, and transcriptomic information from human clinical samples, we confirmed the importance of several genes and pathways previously implicated in human melanoma, including the tumor-suppressor genes phosphatase and tensin homolog (PTEN), cyclin dependent kinase inhibitor 2A (CDKN2A), LKB1, and others. Importantly, this approach also identified additional putative melanoma drivers with prognostic and therapeutic relevance. Surprisingly, one of these genes encodes the tyrosine kinase FES. Whereas FES is highly expressed in normal human melanocytes, FES expression is strongly decreased in over 30% of human melanomas. This downregulation correlates with poor overall survival. Correspondingly, engineered deletion of Fes accelerated tumor progression in a BRAFV600E-driven mouse model of melanoma. Together, these data implicate FES as a driver of melanoma progression and demonstrate the potential of cross-species oncogenomic approaches combined with mouse modeling to uncover impactful mutations and oncogenic driver alleles with clinical importance in the treatment of human cancer.
Recent evidence suggests that inhibition of protein phosphatase 2A (PP2A) tumor suppressor activity via the SET oncoprotein contributes to the pathogenesis of various cancers. Here we demonstrate that both SET and c-MYC expression are frequently elevated in T-ALL cell lines and primary samples compared to healthy T cells. Treatment of T-ALL cells with the SET antagonist OP449 restored the activity of PP2A and reduced SET interaction with the PP2A catalytic subunit, resulting in a decrease in cell viability and c-MYC expression in a dose-dependent manner. Since a tight balance between phosphatases and kinases is required for the growth of both normal and malignant cells, we sought to identify a kinase inhibitor that would synergize with SET antagonism. We tested various T-ALL cell lines against a small-molecule inhibitor screen of 66 compounds targeting two-thirds of the tyrosine kinome and found that combined treatment of T-ALL cells with dovitinib, an orally active multi-targeted small-molecule receptor tyrosine kinase inhibitor, and OP449 synergistically reduced the viability of all tested T-ALL cell lines. Mechanistically, combined treatment with OP449 and dovitinib decreased total and phospho c-MYC levels and reduced ERK1/2, AKT, and p70S6 kinase activity in both NOTCH-dependent and independent T-ALL cell lines. Overall, these results suggest that combined targeting of tyrosine kinases and activation of serine/threonine phosphatases may offer novel therapeutic strategies for the treatment of T-ALL.
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.