JKAP serves as a potential biomarker for the evaluation of inflammatory condition, disease activity, and treatment response to TNF inhibitor in ankylosing spondylitis patients.

OBJECTIVE: This study aimed to evaluate the value of JKAP as a biomarker in estimating treatment response to TNF inhibitor in AS patients. METHODS: Totally, 63 AS patents who planned to receive adalimumab (TNF inhibitor) treatment were enrolled. Baseline JKAP level was determined in serum samples. All patients received 40 mg adalimumab every two weeks for 12 weeks. At W2, W4, W8, and W12, ASAS40 response rates were evaluated. RESULTS: JKAP was negatively correlated with CRP (P = 0.032), BASDAI score (P = 0.021), BASFI score (P = 0.045), ASDASCRP score (P = 0.038), TNF-α (P = 0.031), IL-6 (P = 0.025) and IL-17A (P = 0.022). The ASAS40 response rates were 17.5%, 31.7%, 44.4% and 55.5% at W2, W4, W8 and W12, respectively. Baseline JKAP level was lower in patients with ASAS40 response than those without ASAS40 response (25.8 (13.2-42.7) pg/mL vs. 47.3 (26.7-71.2) pg/mL, P = 0.003). Multivariate logistic regression disclosed that JKAP level (P = 0.049) and CRP level (P = 0.014) independently correlated with ASAS40 response; further analyses disclosed that they exhibited acceptable to good ability in distinguishing patients with ASAS40 response from those without ASAS40 response. CONCLUSION: JKAP serves as a potential biomarker for evaluation of inflammatory condition, disease activity, especially for assessing treatment response to TNF inhibitor in AS patients.

A Single Site Phosphorylation on Hsp82 Ensures Cell Survival during Starvation in Saccharomyces cerevisiae.

Unicellular organisms live under diverse stressful conditions and must respond and adapt quickly to these stresses. When these stresses persist, cells favor a transition to quiescence. There are changes to many processes when cells begin their entry into quiescence. It has been reported that Hsp82 plays an important role in several such processes, and its distribution and activity change according to nutrient conditions. In this study, we found that the subcellular distribution of Hsp82 is regulated by its co-chaperone Ppt1. Under starvation conditions, Ppt1 expression was significantly reduced by a TOR-independent pathway. Furthermore, we found that Ppt1 regulates Hsp82 distribution in the cytoplasm and nucleus by dephosphorylating the S485 residue on Hsp82. The Hsp82S485A strain has impaired membrane-related protein transport, and its cell size did not become larger in quiescence compared to log phase, resulting in failure to survive during starvation.

Design and synthesis of fluorescent activity probes for protein phosphatases.

Protein phosphatases act in concert with protein kinases to regulate and maintain the phosphoproteome. However, the catalog of chemical tools to directly monitor the enzymatic activity of phosphatases has lagged behind their kinase counterparts. In this chapter, we provide protocols for repurposing the phosphorylation-sensitive sulfonamido-oxine fluorophore known as Sox to afford direct activity probes for phosphatases. With validated activity probes in-hand, inhibitor screens can be conducted with recombinant enzyme and the role of phosphatases in cell signaling can be investigated in unfractionated cell lysates.

In-Gel Protein Phosphatase Assay Using Fluorogenic Substrates.

Protein phosphorylation plays important roles in regulating a variety of biological processes in animals, plants, and microorganisms. Therefore, it is important to use appropriate techniques to detect and analyze protein kinases and protein phosphatases. In this chapter, we describe the method to detect protein phosphatase activities using fluorogenic substrates such as 4-methylumbelliferyl phosphate (MUP) after separating proteins by one-dimensional or two-dimensional polyacrylamide gel electrophoresis.

LIMK/cofilin pathway and Slingshot are implicated in human colorectal cancer progression and chemoresistance.

Cofilin phospho-regulation is important for actin filament turnover and is implicated in cancer. Phosphorylation of cofilin is mediated by LIM kinases (LIMKs) and dephosphorylation by Slingshot phosphatases (SSH). LIMKs and SSH promote cancer cell invasion and metastasis and represent novel anti-cancer targets. However, little is known regarding LIMK/cofilin and SSH in human colorectal cancer (CRC). In this study, we aimed to address their expression and significance in human CRC. We evaluated expression of non-phosphorylated (active) and phosphorylated cofilin, LIMK1, LIMK2, and SSH1 by immunohistochemistry in 143 human CRC samples in relation to clinicopathologic parameters, response of metastatic disease to chemotherapy, and epithelial-mesenchymal transition (EMT) markers ß-catenin, E-cadherin, and ZEB. We show that active cofilin, LIMK1, LIMK2, and SSH1 are overexpressed in human CRC and are associated with tumor progression parameters. SSH1 is an independent predictor of lymph node metastasis by multivariate analysis. LIMK1 and SSH1 expression is also higher in non-responders to chemotherapy, and SSH1 is shown by multivariate analysis to independently predict response of metastatic disease to chemotherapy. Active cofilin, LIMK1, LIMK2, and SSH1 also correlated with the EMT markers examined. In addition, immunofluorescence analysis showed increased expression of active cofilin, LIMK1, LIMK2, and SSH1 in HT29 colon cancer cells resistant to 5-fluorouracil compared to parental HT29 cells. Our results suggest that F-actin regulators LIMK/cofilin pathway and SSH1 are associated with CRC progression and chemoresistance representing promising tumor biomarkers and therapeutic targets in CRC.

The Phosphatome of Medicinal and Edible Fungus Wolfiporia cocos.

Wolfiporia cocos is an important medicinal and edible fungus that grows in association with pine trees, and its dried sclerotium has been used as a traditional medicine in China for centuries. However, the commercial production of W. cocos sclerotia is currently limited by shortages in pine wood resources. Since protein phosphatases (PPs) play significant roles in growth, signal transduction, development, metabolism, sexual reproduction, cell cycle, and environmental stress responses in fungi, the phosphatome of W. cocos was analyzed in this study by identifying PP genes, studying transcript profiles and assigning PPs to orthologous groups. Fifty-four putative PP genes were putatively identified in W. cocos genome based on homologous sequences searching using BLASTx program against the Saccharomyces cerevisiae, Fusarium graminearum, and Sclerotinia sclerotiorum databases. Based on known and presumed functions of orthologues of these PP genes found in other fungi, the putative roles of these W. cocos PPs in colonization, hyphal growth, sclerotial formation, secondary metabolism, and stress tolerance to environment were discussed in this study. And the level of transcripts from PP genes in the mycelium and sclerotium stages was also analyzed by qRT-PCR. Our study firstly identified and functional discussed the phosphatome in the medicinal and edible fungus W. cocos. The data from our study contribute to a better understanding of PPs potential roles in various cellar processes of W. cocos, and systematically provide comprehensive and novel insights into W. cocos economically important traits that could be extended to other fungi.

The three Type 2A protein phosphatases, PP2Ac, PP4c and PP6c, are differentially regulated by Alpha4.

Alpha4 is a non-canonical regulatory subunit of Type 2A protein phosphatases that interacts directly with the phosphatase catalytic subunits (PP2Ac, PP4c, and PP6c) and is upregulated in a variety of cancers. Alpha4 modulates phosphatase expression levels and activity, but the molecular mechanism of this regulation is unclear, and the extent to which the various Type 2A catalytic subunits associate with Alpha4 is also unknown. To determine the relative fractions of the Type 2A catalytic subunits associated with Alpha4, we conducted Alpha4 immunodepletion experiments in HEK293T cells and found that a significant fraction of total PP6c is associated with Alpha4, whereas a minimal fraction of total PP2Ac is associated with Alpha4. To facilitate studies of phosphatases in the presence of mutant or null Alpha4 alleles, we developed a facile and rapid method to simultaneously knockdown and rescue Alpha4 in tissue culture cells. This approach has the advantage that levels of endogenous Alpha4 are dramatically reduced by shRNA expression thereby simplifying interpretation of mutant phenotypes. We used this system to show that knockdown of Alpha4 preferentially impacts the expression of PP4c and PP6c compared to expression levels of PP2Ac.

Rhizobiales-like Phosphatase 2 from Arabidopsis thaliana Is a Novel Phospho-tyrosine-specific Phospho-protein Phosphatase (PPP) Family Protein Phosphatase.

Cellular signaling through protein tyrosine phosphorylation is well established in mammalian cells. Although lacking the classic tyrosine kinases present in humans, plants have a tyrosine phospho-proteome that rivals human cells. Here we report a novel plant tyrosine phosphatase from Arabidopsis thaliana (AtRLPH2) that, surprisingly, has the sequence hallmarks of a phospho-serine/threonine phosphatase belonging to the PPP family. Rhizobiales/Rhodobacterales/Rhodospirillaceae-like phosphatases (RLPHs) are conserved in plants and several other eukaryotes, but not in animals. We demonstrate that AtRLPH2 is localized to the plant cell cytosol, is resistant to the classic serine/threonine phosphatase inhibitors okadaic acid and microcystin, but is inhibited by the tyrosine phosphatase inhibitor orthovanadate and is particularly sensitive to inhibition by the adenylates, ATP and ADP. AtRLPH2 displays remarkable selectivity toward tyrosine-phosphorylated peptides versus serine/threonine phospho-peptides and readily dephosphorylates a classic tyrosine phosphatase protein substrate, suggesting that in vivo it is a tyrosine phosphatase. To date, only one other tyrosine phosphatase is known in plants; thus AtRLPH2 represents one of the missing pieces in the plant tyrosine phosphatase repertoire and supports the concept of protein tyrosine phosphorylation as a key regulatory event in plants.

The Groucho-associated phosphatase PPM1B displaces Pax transactivation domain interacting protein (PTIP) to switch the transcription factor Pax2 from a transcriptional activator to a repressor.

Pax genes encode developmental regulatory proteins that specify cell lineages and tissues in metazoans. Upon binding to DNA through the conserved paired domain, Pax proteins can recruit both activating and repressing complexes that imprint distinct patterns of histone methylation associated with either gene activation or silencing. How the switch from Pax-mediated activation to repression is regulated remains poorly understood. In this report, we identify the phosphatase PPM1B as an essential component of the Groucho4 repressor complex that is recruited by Pax2 to chromatin. PPM1B can dephosphorylate the Pax2 activation domain and displace the adaptor protein PTIP, thus inhibiting H3K4 methylation and gene activation. Loss of PPM1B prevents Groucho-mediated gene repression. Thus, PPM1B helps switch Pax2 from a transcriptional activator to a repressor protein. This can have profound implications for developmental regulation by Pax proteins and suggests a model for imprinting specific epigenetic marks depending on the availability of co-factors.

Quantitative phosphoproteomic analysis of IL-33-mediated signaling.

Interleukin-33 (IL-33) is a novel member of the IL-1 family of cytokines that plays diverse roles in the regulation of immune responses. IL-33 exerts its effects through a heterodimeric receptor complex resulting in the production and release of proinflammatory cytokines. A detailed understanding of the signaling pathways activated by IL-33 is still unclear. To gain insights into the IL-33-mediated signaling mechanisms, we carried out a SILAC-based global quantitative phosphoproteomic analysis that resulted in the identification of 7191 phosphorylation sites derived from 2746 proteins. We observed alterations in the level of phosphorylation in 1050 sites corresponding to 672 proteins upon IL-33 stimulation. We report, for the first time, phosphorylation of multiple protein kinases, including mitogen-activated protein kinase activated protein kinase 2 (Mapkapk2), receptor (TNFRSF) interacting serine-threonine kinase 1 (Ripk1), and NAD kinase (Nadk) that are induced by IL-33. In addition, we observed IL-33-induced phosphorylation of several protein phosphatases including protein tyrosine phosphatase, nonreceptor-type 12 (Ptpn12), and inositol polyphosphate-5-phosphatase D (Inpp5d), which have not been reported previously. Network analysis revealed an enrichment of actin binding and cytoskeleton reorganization that could be important in macrophage activation induced by IL-33. Our study is the first quantitative analysis of IL-33-regulated phosphoproteome. Our findings significantly expand the understanding of IL-33-mediated signaling events and have the potential to provide novel therapeutic targets pertaining to immune-related diseases such as asthma where dysregulation of IL-33 is observed. All MS data have been deposited in the ProteomeXchange with identifier PXD000984 (http://proteomecentral.proteomexchange.org/dataset/PXD000984).

Low expression of PHLPP1 in sacral chordoma and its association with poor prognosis.

Sacral chordoma is a rare spine tumor with a high recurrence rate even after optimal therapy. Previous studies have demonstrated that the PI3K/AKT pathway plays a pivotal role in chordoma, and high expression of pAKT is associated with poor prognosis. Recently, PHLPP was recognized to be a tumor suppressor that targets AKT. We analyzed the expression of PHLPP1 and AKT2 in 37 chordoma samples and 11 fetal nucleus pulposus samples by immunohistochemical staining. Of the chordoma cases, 40.5% (15/37) showed strong cytoplasmic staining (score ≥3) for PHLPP1, which was significantly lower than the 90.9% (10/11) of fetal nucleus pulposus samples (P = 0.004). Conversely, strong immunohistochemical staining for AKT2 was observed in 75.7% (28/37) of chordoma samples, which was significantly higher than 36.4% (4/11) of fetal nucleus pulposus (P = 0.021). Kaplan-Meier survival curves and log-rank test showed that patients with high expression of PHLPP1 experienced longer progression free survival time than those with low PHLPP1 expression (P = 0.011). Further multivariate Cox regression analysis indicated that PHLPP1 expression level and surgical approaches were independent risk factors for chordoma recurrence (P = 0.023 and P = 0.022). However, PHLPP1 expression was not statistically related to patients' total survival time. Conclusively, our results suggest that PHLPP1 plays a crucial role in sacral chordoma, and may be a promising biomarker for prognosis. Meanwhile, manipulation of PHLPP1 expression is also a potential therapeutic approach for the treatment of sacral chordoma.

Activation of cytosolic Slingshot-1 phosphatase by gelsolin-generated soluble actin filaments.

Slingshot-1 (SSH1) is a protein phosphatase that dephosphorylates and activates cofilin, an actin-severing and -disassembling protein. SSH1 is bound to and activated by F-actin, but not G-actin. SSH1 is accumulated in the F-actin-rich lamellipodium but is also diffusely distributed in the cytoplasm. It remains unknown whether SSH1 is activated by soluble (low-level polymerized) actin filaments in the cytoplasm. In this study, we show that SSH1 binds to gelsolin via actin filaments in the cytosolic fraction. Gelsolin promoted solubilization of actin filaments and SSH1 in cell-free assays and in cultured cells. SSH1 was activated by gelsolin-generated soluble actin filaments. Furthermore, gelsolin enhanced cofilin dephosphorylation in neuregulin-stimulated cells. Our results suggest that cytosolic SSH1 forms a complex with gelsolin via soluble actin filaments and is activated by gelsolin-generated soluble actin filaments and that gelsolin promotes stimulus-induced cofilin dephosphorylation through increasing soluble actin filaments, which support SSH1 activation in the cytoplasm.

IRE1 phosphatase PP2Ce regulates adaptive ER stress response in the postpartum mammary gland.

We recently reported that the PPM1l gene encodes an endoplasmic reticulum (ER) membrane targeted protein phosphatase (named PP2Ce) with highly specific activity towards Inositol-requiring protein-1 (IRE1) and regulates the functional outcome of ER stress. In the present report, we found that the PP2Ce protein is highly expressed in lactating epithelium of the mammary gland. Loss of PP2Ce in vivo impairs physiological unfolded protein response (UPR) and induces stress kinase activation, resulting in loss of milk production and induction of epithelial apoptosis in the lactating mammary gland. This study provides the first in vivo evidence that PP2Ce is an essential regulator of normal lactation, possibly involving IRE1 signaling and ER stress regulation in mammary epithelium.

Arabidopsis protein phosphatase DBP1 nucleates a protein network with a role in regulating plant defense.

Arabidopsis thaliana DBP1 belongs to the plant-specific family of DNA-binding protein phosphatases. Although recently identified as a novel host factor mediating susceptibility to potyvirus, little is known about DBP1 targets and partners and the molecular mechanisms underlying its function. Analyzing changes in the phosphoproteome of a loss-of-function dbp1 mutant enabled the identification of 14-3-3λ isoform (GRF6), a previously reported DBP1 interactor, and MAP kinase (MAPK) MPK11 as components of a small protein network nucleated by DBP1, in which GRF6 stability is modulated by MPK11 through phosphorylation, while DBP1 in turn negatively regulates MPK11 activity. Interestingly, grf6 and mpk11 loss-of-function mutants showed altered response to infection by the potyvirus Plum pox virus (PPV), and the described molecular mechanism controlling GRF6 stability was recapitulated upon PPV infection. These results not only contribute to a better knowledge of the biology of DBP factors, but also of MAPK signalling in plants, with the identification of GRF6 as a likely MPK11 substrate and of DBP1 as a protein phosphatase regulating MPK11 activity, and unveils the implication of this protein module in the response to PPV infection in Arabidopsis.

Proto-oncogene Wip1, a member of a new family of proliferative genes in NSCLC and its clinical significance.

This study aimed to analyze the expression, clinical significance of proto-oncogene in non small cell lung cancer (NSCLC), and the biological effect in its cell line by siRNA targeting wild-type p53-induced phosphatase 1 (Wip1). Immunohistochemistry and reverse transcription polymerase chain reaction (RT-PCR) were, respectively, used to analyze Wip1 protein expression in 75 cases of NSCLC and normal tissues to study the relationship between Wip1 expression and clinical parameters. Wip1 siRNA was transiently transfected into papillary NSCLC H1299 cell by liposome-mediated method and was detected by RT-PCR and Western blot. MTT assay, cell apoptosis, and cell cycle were also conducted as to the influence of the downregulated expression of Wip1 that might be found on H1299 cells biological effect. The positive rates of Wip1 protein was 69.3% in NSCLC tissues but 16.0% expressed in normal tissues (P < 0.05). The relative content of Wip1 mRNA was 0.785 ± 0.062 and 0.147 ± 0.020 in NSCLC tissues and normal tissues, respectively, with significant differences between the two types (P < 0.05). There were no significant differences between Wip1 expression and sex, age, tumor size, and pathological types (P > 0.05). However, there were significant differences between Wip1 expression and lymph node metastasis, clinical stages, and tumor differentiation (P < 0.05). Individuals with positive and negative levels of Wip1 expression showed were statistically significant differences in the 5-year overall survival rate (P < 0.05). RT-PCR and Western blot showed that H1299 cell transfected Wip1 siRNA had a lower relative expressive content than normal cell (P < 0.05). MTT assay, cell apoptosis, and cell cycles demonstrated that H1299 cell transfected Wip1 siRNA had a lower survival fraction, higher cell apoptosis, more percentage of the G0/G1 phases, and lower cells in the S phases (P < 0.05). Wip1 protein and mRNA were increased in NSCLC, specifically in lymph node metastasis, clinical stages, and tumor differentiation. Wip1 may be involved in the biological processes of NSCLC cell proliferation, cell apoptosis, and cell cycle.

Interaction of DISC1 with the PTB domain of Tensin2.

The gene for Disrupted-in-Schizophrenia 1 (DISC1) is amongst the most significant risk genes for schizophrenia. The DISC1 protein is an intracellular scaffolding molecule thought to act an important hub for protein interactions involved in signalling for neural cell differentiation and function. Tensin2 is an intracellular actin-binding protein that bridges the intracellular portion of transmembrane receptors to the cytoskeleton, thereby regulating signalling for cell shape and motility. In this study, we probed in molecular detail a novel interaction between DISC1 and Tensin2. Western blot and confocal microscopic analyses revealed widespread expression of both DISC1 and Tensin2 proteins throughout the mouse brain. Furthermore, we have developed novel anti-DISC1 antibodies that verified the predominant expression of a 105-kDa isoform of DISC1 in the rodent brain as well as in human cells. In the mouse brain, both proteins showed region-specific expression patterns, including strong expression in the pyramidal cell layer of the hippocampus and dentate gyrus. DISC1-Tensin2 colocalisation was most clearly observed in the Purkinje cells of the mouse cerebellum. Biochemical coimmunoprecipitation experiments revealed an interaction between endogenous DISC1 and Tensin2 proteins in the mouse brain. Further pulldown studies in human cells using Myc-tagged Tensin2 constructs revealed that DISC1 specifically interacts with the C-terminal PTB domain of Tensin2 in a phosphorylation-independent manner. This new knowledge on the DISC1-Tensin2 interaction, as part of the wider DISC1 interactome, should further elucidate the signalling pathways that are perturbed in schizophrenia and other mental disorders.

Human aqueous humor phosphatase activity in cataract and glaucoma.

PURPOSE: To investigate the presence and activity of protein phosphatase-2A (PPase2A), protein phosphatase-2C (PPase2C), and protein tyrosine phosphatases (PTPs) in the human aqueous humor (AH) of patients with primary open-angle glaucoma (POAG) and cataract and to study the correlation between these phosphatases and the redox state of the AH. METHODS: Eighty-six cataract patients and 29 POAG patients who were scheduled for cataract surgery with or without glaucoma surgery were enrolled in the study. PPase2A, PPase2C, and PTPs levels in AH were measured by enzyme-linked immunosorbent assays, Western blot analyses, and spectral METHODS: The redox state was measured by spectral and fluorescent methods. RESULTS: Phosphatase activity-positive results were significantly higher in AH samples from the POAG group (PP2A χ(2)(1) = 11.754, P < 0.01; PP2C χ(2)(1) = 8.754, P < 0.01; PTP χ(2)(1) = 11.073, P < 0.01). Western blot analysis revealed higher PP2C levels in the AH of glaucoma patients compared with PP2C levels in the AH of cataract patients (P = 0.012). Both oxidized/reduced glutathione ratios and superoxide dismutase levels in the AH were significantly higher in the glaucoma group than in the cataract group. Finally significant correlations were found between PP2A and PP2C, PP2A and PTP, and total antioxidant activity and PTP levels. CONCLUSIONS: There is a statistically significant difference between phosphatase levels in the AH of POAG patients and cataract patients. The phosphatase content of the AH represents tissue pathology, but their presence in the AH may be attributed to cell debris or to active signaling to other molecular events.

Functional processing of nuclear Ca2+/calmodulin-dependent protein kinase phosphatase (CaMKP-N): evidence for a critical role of proteolytic processing in the regulation of its catalytic activity, subcellular localization and substrate targeting in vivo.

Ca(2+)/calmodulin-dependent protein kinase phosphatase (CaMKP) and its nuclear homolog CaMKP-N are Ser/Thr protein phosphatases that belong to the PPM family. These phosphatases are highly specific for multifunctional CaM kinases and negatively regulate their activities. CaMKP-N is only expressed in the brain and specifically localized in the nucleus. In this study, we found that zebrafish CaMKP-N (zCaMKP-N) underwent proteolytic processing in both the zebrafish brain and Neuro2a cells. In Neuro2a cells, the proteolytic processing was effectively inhibited by the proteasome inhibitors MG-132, Epoxomicin, and Lactacystin, suggesting that the ubiquitin-proteasome pathway was involved in this processing. Using MG-132, we found that the proteolytic processing changed the subcellular localization of zCaMKP-N from the nucleus to the cytosol. Accompanying this change, the cellular targets of zCaMKP-N in Neuro2a cells were significantly altered. Furthermore, we obtained evidence that the zCaMKP-N activity was markedly activated when the C-terminal domain was removed by the processing. Thus, the proteolytic processing of zCaMKP-N at the C-terminal region regulates its catalytic activity, subcellular localization and substrate targeting in vivo.