Rare variant analysis in eczema identifies exonic variants in DUSP1, NOTCH4 and SLC9A4.

Previous genome-wide association studies revealed multiple common variants involved in eczema but the role of rare variants remains to be elucidated. Here, we investigate the role of rare variants in eczema susceptibility. We meta-analyze 21 study populations including 20,016 eczema cases and 380,433 controls. Rare variants are imputed with high accuracy using large population-based reference panels. We identify rare exonic variants in DUSP1, NOTCH4, and SLC9A4 to be associated with eczema. In DUSP1 and NOTCH4 missense variants are predicted to impact conserved functional domains. In addition, five novel common variants at SATB1-AS1/KCNH8, TRIB1/LINC00861, ZBTB1, TBX21/OSBPL7, and CSF2RB are discovered. While genes prioritized based on rare variants are significantly up-regulated in the skin, common variants point to immune cell function. Over 20% of the single nucleotide variant-based heritability is attributable to rare and low-frequency variants. The identified rare/low-frequency variants located in functional protein domains point to promising targets for novel therapeutic approaches to eczema.

GP78 Cooperates with Dual-Specificity Phosphatase 1 To Stimulate Epidermal Growth Factor Receptor-Mediated Extracellular Signal-Regulated Kinase Signaling.

GP78 is an autocrine motility factor (AMF) receptor (AMFR) with E3 ubiquitin ligase activity that plays a significant role in tumor cell proliferation, motility, and metastasis. Aberrant extracellular signal-regulated kinase (ERK) activation via receptor tyrosine kinases promotes tumor proliferation and invasion. The activation of GP78 leads to ERK activation, but its underlying mechanism is not fully understood. Here, we show that GP78 is required for epidermal growth factor receptor (EGFR)-mediated ERK activation. On one hand, GP78 interacts with and promotes the ubiquitination and subsequent degradation of dual-specificity phosphatase 1 (DUSP1), an endogenous negative regulator of mitogen-activated protein kinases (MAPKs), resulting in ERK activation. On the other hand, GP78 maintains the activation status of EGFR, as evidenced by the fact that EGF fails to induce EGFR phosphorylation in GP78-deficient cells. By the regulation of both EGFR and ERK activation, GP78 promotes cell proliferation, motility, and invasion. Therefore, this study identifies a previously unknown signaling pathway by which GP78 stimulates ERK activation via DUSP1 degradation to mediate EGFR-dependent cancer cell proliferation and invasion.

MBP-binding DARPins facilitate the crystallization of an MBP fusion protein.

The production of high-quality crystals is the main bottleneck in determining the structures of proteins using X-ray crystallography. In addition to being recognized as a very effective solubility-enhancing fusion partner, Escherichia coli maltose-binding protein (MBP) has also been successfully employed as a `fixed-arm' crystallization chaperone in more than 100 cases. Here, it is reported that designed ankyrin-repeat proteins (DARPins) that bind with high affinity to MBP can promote the crystallization of an MBP fusion protein when the fusion protein alone fails to produce diffraction-quality crystals. As a proof of principle, three different co-crystal structures of MBP fused to the catalytic domain of human dual-specificity phosphatase 1 in complex with DARPins are reported.

Crystal structure of the human dual specificity phosphatase 1 catalytic domain.

The dual specificity phosphatase DUSP1 was the first mitogen activated protein kinase phosphatase (MKP) to be identified. It dephosphorylates conserved tyrosine and threonine residues in the activation loops of mitogen activated protein kinases ERK2, JNK1 and p38-alpha. Here, we report the crystal structure of the human DUSP1 catalytic domain at 2.49 Å resolution. Uniquely, the protein was crystallized as an MBP fusion protein in complex with a monobody that binds to MBP. Sulfate ions occupy the phosphotyrosine and putative phosphothreonine binding sites in the DUSP1 catalytic domain.

Comparative analysis of dual specificity protein phosphatase genes 1, 2 and 5 in response to immune challenges in Japanese flounder Paralichthys olivaceus.

Dual-specificity MAP kinase (MAPK) phosphatases (DUSPs) are well-established negative modulators in regulating MAPK signaling in mammalian cells and tissues. Our previous studies have shown the involvement of DUSP6 in regulating innate immunity in Japanese flounder Paralichthys olivaceus. In order to gain a better understanding of the role of DUSPs in fish innate immunity, in the present study we identified and characterized three additional DUSP genes including DUSP1, 2 and 5 in P. olivaceus. The three Japanese flounder DUSP proteins share common domain structures composed of a conserved N-terminal Rhodanase/CDC25 domain and a C-terminal catalytic phosphatase domain, while they show only less than 26% sequence identities, indicating that they may have different substrate selectivity. In addition, mRNA transcripts of all the three DUSP genes are detected in all examined Japanese flounder tissues; however, DUSP1 is dominantly expressed in spleen while DUSP2 and 5 are primarily expressed in skin. Furthermore, all the three DUSP genes are constitutively expressed in the Japanese flounder head kidney macrophages (HKMs) and peripheral blood leucocytes (PBLs) with unequal distribution patterns. Moreover, all the three DUSPs gene expression was induced differently in response to the LPS and double-stranded RNA mimic poly(I:C) stimulations both in the Japanese flounder HKMs and PBLs, suggesting an association of DUSPs with TLR signaling in fish. Taken together, the co-expression of various DUSPs members together with their different responses to the immune challenges indicate that the DUSP members may operate coordinately in regulating the MAPK-dependent immune responses in the Japanese flounder.

Regulation of the wheat MAP kinase phosphatase 1 by 14-3-3 proteins.

Plant MAP kinase phosphatases (MKPs) are major regulators of MAPK signaling pathways and play crucial roles in controlling growth, development and stress responses. The presence of several functional domains in plant MKPs such as a dual specificity phosphatase catalytic domain, gelsolin, calmodulin-binding and serine-rich domains, suggests that MKPs can interact with distinct cellular partners, others than MAPKs. In this report, we identified a canonical mode I 14-3-3-binding motif (574KLPSLP579) located at the carboxy-terminal region of the wheat MKP, TMKP1. We found that this motif is well-conserved among other MKPs from monocots including Hordeum vulgare, Brachypodium distachyon and Aegilops taushii. Using co-immunoprecipitation assays, we provide evidence for interaction between TMKP1 and 14-3-3 proteins in wheat. Moreover, the phosphatase activity of TMKP1 is increased in a phospho-dependent manner by either Arabidopsis or yeast 14-3-3 isoforms. TMKP1 activation by 14-3-3 proteins is enhanced by Mn2+, whereas in the presence of Ca2+ ions, TMKP1 activation was limited to Arabidopsis 14-3-3φ (phi), an isoform harboring an EF-hand motif. Such findings strongly suggest that 14-3-3 proteins, in conjunction with specific divalent cations, may stimulate TMKP1 activity and point-out that 14-3-3 proteins bind and regulate the activity of a MKP in eukaryotes.

The Atypical Dual Specificity Phosphatase hYVH1 Associates with Multiple Ribonucleoprotein Particles.

Human YVH1 (hYVH1), also known as dual specificity phosphatase 12 (DUSP12), is a poorly characterized atypical dual specificity phosphatase widely conserved throughout evolution. Recent findings have demonstrated that hYVH1 expression affects cellular DNA content and is a novel cell survival phosphatase preventing both thermal and oxidative stress-induced cell death, whereas studies in yeast have established YVH1 as a novel 60S ribosome biogenesis factor. In this study, we have isolated novel hYVH1-associating proteins from human U2OS osteosarcoma cells using affinity chromatography coupled to mass spectrometry employing ion mobility separation. Numerous ribosomal proteins were identified, confirming the work done in yeast. Furthermore, proteins known to be present on additional RNP particles were identified, including Y box-binding protein 1 (YB-1) and fragile X mental retardation protein, proteins that function in translational repression and stress granule regulation. Follow-up studies demonstrated that hYVH1 co-localizes with YB-1 and fragile X mental retardation protein on stress granules in response to arsenic treatment. Interestingly, hYVH1-positive stress granules were significantly smaller, whereas knocking down hYVH1 expression attenuated stress granule breakdown during recovery from arsenite stress, indicating a possible role for hYVH1 in stress granule disassembly. These results propagate a role for dual specificity phosphatases at RNP particles and suggest that hYVH1 may affect a variety of fundamental cellular processes by regulating messenger ribonucleoprotein (mRNP) dynamics.

AMPK Suppresses Vascular Inflammation In Vivo by Inhibiting Signal Transducer and Activator of Transcription-1.

Activation of AMPK suppresses inflammation, but the underlying mechanisms remain poorly understood. This study was designed to characterize the molecular mechanisms by which AMPK suppresses vascular inflammation. In cultured human aortic smooth muscle cells, pharmacologic or genetic activation of AMPK inhibited the signal transducer and activator of transcription-1 (STAT1), while inhibition of AMPK had opposite effects. Deletion of AMPKα1 or AMPKα2 resulted in activation of STAT1 and in increases in proinflammatory mediators, both of which were attenuated by administration of STAT1 small interfering RNA or fludarabine, a selective STAT1 inhibitor. Moreover, AMPK activation attenuated the proinflammatory actions induced by STAT1 activators such as interferon-γ and angiotensin II (AngII). Mechanistically, we found that AMPK activation increased, whereas AMPK inhibition decreased, the levels of mitogen-activated protein kinase phosphatase-1 (MKP-1), an inducible nuclear phosphatase, by regulating proteasome-dependent degradation of MKP-1. Gene silencing of MKP-1 increased STAT1 phosphorylation and prevented 5-aminoimidazole-4-carboxyamide ribonucleoside-reduced STAT1 phosphorylation. Finally, we found that infusion of AngII caused a more severe inflammatory response in AMPKα2 knockout mouse aortas, all of which were suppressed by chronic administration of fludarabine. We conclude that AMPK activation suppresses STAT1 signaling and inhibits vascular inflammation through the upregulation of MKP-1.

Mitogen-activated protein kinase phosphatase-1: a critical phosphatase manipulating mitogen-activated protein kinase signaling in cardiovascular disease (review).

Mitogen-activated protein kinase (MAPK) cascades are important players in the overall representation of cellular signal transduction pathways, and the deregulation of MAPKs is involved in a variety of diseases. The activation of MAPK signals occurs through phosphorylation by MAPK kinases at conserved threonine and tyrosine (Thr-Xaa-Tyr) residues. The mitogen-activated protein kinase phosphatases (MKPs) are a major part of the dual-specificity family of phosphatases and specifically inactivate MAPKs by dephosphorylating both phosphotyrosine and phosphoserine/phosphothreonine residues within the one substrate. MAPKs binding to MKPs can enhance MKP stability and activity, providing an important negative-feedback control mechanism that limits the MAPK cascades. In recent years, accumulating and compelling evidence from studies mainly employing cultured cells and mouse models has suggested that the archetypal MKP family member, MKP-1, plays a pivotal role in cardiovascular disease as a major negative modulator of MAPK signaling pathways. In the present review, we summarize the current knowledge on the pathological properties and the regulation of MKP-1 in cardiovascular disease, which may provide valuable therapeutic options.

The activity of the wheat MAP kinase phosphatase 1 is regulated by manganese and by calmodulin.

MAPK phosphatases (MKPs) are negative regulators of MAPKs in eukaryotes and play key roles in the regulation of different cellular processes. However in plants, little is known about the regulation of these Dual Specific Phosphatases (DSPs) by Ca(2+) and calmodulin (CaM). Here, we showed that the wheat MKP (TMKP1) harboring a calmodulin (CaM) binding domain, binds to CaM in a Ca(2+)-dependent manner. In addition, TMKP1 exhibited a phosphatase activity in vitro that is specifically enhanced by Mn(2+) and to a lesser extent by Mg(2+), but without any synergistic effect between the two bivalent cations. Most interestingly, CaM/Ca(2+) complex inhibits the catalytic activity of TMKP1 in a CaM-dose dependent manner. However, in the presence of Mn(2+) this activity is enhanced by CaM/Ca(2+) complex. These dual regulatory effects seem to be mediated via interaction of CaM/Ca(2+) to the CaM binding domain in the C-terminal part of TMKP1. Such effects were not reported so far, and raise a possible role for CaM and Mn(2+) in the regulation of plant MKPs during cellular response to external signals.

In silico characterization of an atypical MAPK phosphatase of Plasmodium falciparum as a suitable target for drug discovery.

Plasmodium falciparum, the causative agent of malaria, contributes to significant morbidity and mortality worldwide. Forward genetic analysis of the blood-stage asexual cycle identified the putative phosphatase from PF3D7_1305500 as an important element of intraerythrocytic development expressed throughout the life cycle. Our preliminary evaluation identified it as an atypical mitogen-activated protein kinase phosphatase. Additional bioinformatic analysis delineated a conserved signature motif and three residues with potential importance to functional activity of the atypical dual-specificity phosphatase domain. A homology model of the dual-specificity phosphatase domain was developed for use in high-throughput in silico screening of the available library of antimalarial compounds from ChEMBL-NTD. Seven compounds from this set with predicted affinity to the active site were tested against in vitro cultures, and three had reduced activity against a ∆PF3D7_1305500 parasite, suggesting PF3D7_1305500 is a potential target of the selected compounds. Identification of these compounds provides a novel starting point for a structure-based drug discovery strategy that moves us closer toward the discovery of new classes of clinical antimalarial drugs. These data suggest that mitogen-activated protein kinase phosphatases represent a potentially new class of P. falciparum drug target.

Phosphorylation and stabilization of Arabidopsis MAP kinase phosphatase 1 in response to UV-B stress.

MAP kinase phosphatases (MKPs) are important regulators of the activation levels and kinetics of MAP kinases. This is crucial for a large number of physiological processes during development and growth, as well as interactions with the environment, including the response to ultraviolet-B (UV-B) stress. Arabidopsis MKP1 is a key regulator of MAP kinases MPK3 and MPK6 in response to UV-B stress. However, virtually nothing is presently known about the post-translational regulation of plant MKPs in vivo. Here, we provide evidence that MKP1 is a phosphoprotein in vivo and that MKP1 accumulates in response to UV-B stress. Moreover, proteasome inhibitor experiments suggest that MKP1 is constantly turned-over under non-stress conditions and that MKP1 is stabilized upon stress treatment. Stress-responsive phosphorylation and stabilization of MKP1 demonstrate the post-translational regulation of a plant MKP in vivo, adding an additional regulatory layer to MAP kinase signaling in plants.

Protein S-nitrosylation and cancer.

Protein S-nitrosylation is a covalent post-translational modification through coupling of a nitric oxide (NO) moiety with the reactive thiol group of a protein cysteine residue to form an S-nitrosothiol (SNO). S-nitrosylation is a key mechanism in the transmission of NO-based cellular signals in the vital cellular processes, including transcription regulation, DNA repair, and apoptosis. Contemporary research has implicated dysregulation of S-nitrosylation in severe pathological events, including cancer onset, progression, and treatment resistance. The S-nitrosylation status may be directly linked to many cancer therapy outcomes as well as therapeutic-resistance, emphasizing the need to develop S-nitrosylation-related anti-cancer therapeutics. The role of S-nitrosylated proteins in the development and progression of cancer are varied, generating a critical need for a thorough review of the current dynamic research in this area.

Identification of novel inhibitors of mitogen-activated protein kinase phosphatase-1 with structure-based virtual screening.

Mitogen-activated protein kinase phosphatase-1 (MKP-1) has proved to be an attractive target for the development of therapeutics for the treatment of cancer. We report the first example for a successful application of the structure-based virtual screening to identify the novel inhibitors of MKP-1. It is shown that the efficiency of virtual screening can be enhanced significantly by the incorporation of a new solvation energy term in the scoring function. The newly found inhibitors have desirable physicochemical properties as a drug candidate and reveal a moderate potency with IC(50) values ranging from 20 to 50 µM. Therefore, they deserve a consideration for further development by structure-activity relationship studies to optimize the inhibitory activities. Structural features relevant to the stabilization of the inhibitors in the active site of MKP-1 are discussed in detail.

Gold nanoparticle enrichment method for identifying S-nitrosylation and S-glutathionylation sites in proteins.

We present a simple method by which gold nanoparticles (AuNPs) are used to simultaneously isolate and enrich for free or modified thiol-containing peptides, thus facilitating the identification of protein S-modification sites. Here, protein disulfide isomerase (PDI) and dual specificity phosphatase 12 (DUSP12 or hYVH1) were S-nitrosylated or S-glutathionylated, their free thiols differentially alkylated, and subjected to proteolysis. AuNPs were added to the digests, and the AuNP-bound peptides were isolated by centrifugation and released by thiol exchange. These AuNP-bound peptides were analyzed by MALDI-TOF mass spectrometry revealing that AuNPs result in a significant enrichment of free thiol-containing as well as S-nitrosylated, S-glutathionylated, and S-alkylated peptides, leading to the unequivocal assignment of thiols susceptible to modification.

Structural studies of soybean calmodulin isoform 4 bound to the calmodulin-binding domain of tobacco mitogen-activated protein kinase phosphatase-1 provide insights into a sequential target binding mode.

The calcium regulatory protein calmodulin (CaM) binds in a calcium-dependent manner to numerous target proteins. The calmodulin-binding domain (CaMBD) region of Nicotiana tabacum MAPK phosphatase has an amino acid sequence that does not resemble the CaMBD of any other known Ca(2+)-CaM-binding proteins. Using a unique fusion protein strategy, we have been able to obtain a high resolution solution structure of the complex of soybean Ca(2+)-CaM4 (SCaM4) and this CaMBD. Complete isotope labeling of both parts of the complex in the fusion protein greatly facilitated the structure determination by NMR. The 12-residue CaMBD region was found to bind exclusively to the C-lobe of SCaM4. A specific Trp and Leu side chain are utilized to facilitate strong binding through a novel "double anchor" motif. Moreover, the orientation of the helical peptide on the surface of Ca(2+)-SCaM4 is distinct from other known complexes. The N-lobe of Ca(2+)-SCaM4 in the complex remains free for additional interactions and could possibly act as a calcium-dependent adapter protein. Signaling through the MAPK pathway and increases in intracellular Ca(2+) are both hallmarks of the plant stress response, and our data support the notion that coordination of these responses may occur through the formation of a unique CaM-MAPK phosphatase multiprotein complex.

Redox regulation of the human dual specificity phosphatase YVH1 through disulfide bond formation.

YVH1 was one of the first eukaryotic dual specificity phosphatases cloned, and orthologues poses a unique C-terminal zinc-coordinating domain in addition to a cysteine-based phosphatase domain. Our recent results revealed that human YVH1 (hYVH1) protects cells from oxidative stress. This function requires phosphatase activity and the zinc binding domain. This current study provides evidence that the thiol-rich zinc-coordinating domain may act as a redox sensor to impede the active site cysteine from inactivating oxidation. Furthermore, using differential thiol labeling and mass spectrometry, it was determined that hYVH1 forms intramolecular disulfide bonds at the catalytic cleft as well as within the zinc binding domain to avoid irreversible inactivation during severe oxidative stress. Importantly, zinc ejection is readily reversible and required for hYVH1 activity upon returning to favorable conditions. This inimitable mechanism provides a means for hYVH1 to remain functionally responsive for protecting cells during oxidative stimuli.

The dual-specificity phosphatase hYVH1 interacts with Hsp70 and prevents heat-shock-induced cell death.

hYVH1 [human orthologue of YVH1 (yeast VH1-related phosphatase)] is an atypical dual-specificity phosphatase that is widely conserved throughout evolution. Deletion studies in yeast have suggested a role for this phosphatase in regulating cell growth. However, the role of the human orthologue is unknown. The present study used MS to identify Hsp70 (heat-shock protein 70) as a novel hYVH1-binding partner. The interaction was confirmed using endogenous co-immunoprecipitation experiments and direct binding of purified proteins. Endogenous Hsp70 and hYVH1 proteins were also found to co-localize specifically to the perinuclear region in response to heat stress. Domain deletion studies revealed that the ATPase effector domain of Hsp70 and the zinc-binding domain of hYVH1 are required for the interaction, indicating that this association is not simply a chaperone-substrate complex. Thermal phosphatase assays revealed hYVH1 activity to be unaffected by heat and only marginally affected by non-reducing conditions, in contrast with the archetypical dual-specificity phosphatase VHR (VH1-related protein). In addition, Hsp70 is capable of increasing the phosphatase activity of hYVH1 towards an exogenous substrate under non-reducing conditions. Furthermore, the expression of hYVH1 repressed cell death induced by heat shock, H2O2 and Fas receptor activation but not cisplatin. Co-expression of hYVH1 with Hsp70 further enhanced cell survival. Meanwhile, expression of a catalytically inactive hYVH1 or a hYVH1 variant that is unable to interact with Hsp70 failed to protect cells from the various stress conditions. The results suggest that hYVH1 is a novel cell survival phosphatase that co-operates with Hsp70 to positively affect cell viability in response to cellular insults.

Histone H3 as a novel substrate for MAP kinase phosphatase-1.

Mitogen-activated protein (MAP) kinase phosphatase-1 (MKP-1) is a nuclear, dual-specificity phosphatase that has been shown to dephosphorylate MAP kinases. We used a "substrate-trap" technique involving a mutation in MKP-1 of the catalytically critical cysteine to a serine residue ("CS" mutant) to capture novel MKP-1 substrates. We transfected the MKP-1 (CS) mutant and control (wild-type, WT) constructs into phorbol 12-myristate 13-acetate (PMA)-activated COS-1 cells. MKP-1-substrate complexes were immunoprecipitated, which yielded four bands of 17, 15, 14, and 10 kDa with the CS MKP-1 mutant but not the WT MKP-1. The bands were identified by mass spectrometry as histones H3, H2B, H2A, and H4, respectively. Histone H3 was phosphorylated, and purified MKP-1 dephosphorylated histone H3 (phospho-Ser-10) in vitro; whereas, histone H3 (phospho-Thr-3) was unaffected. We have previously shown that thrombin and vascular endothelial growth factor (VEGF) upregulated MKP-1 in human endothelial cells (EC). We now show that both thrombin and VEGF caused dephosphorylation of histone H3 (phospho-Ser-10) and histone H3 (phospho-Thr-3) in EC with kinetics consistent with MKP-1 induction. Furthermore, MKP-1-specific small interfering RNA (siRNA) prevented VEGF- and thrombin-induced H3 (phospho-Ser-10) dephosphorylation but had no effect on H3 (phospho-Thr-3 or Thr-11) dephosphorylation. In summary, histone H3 is a novel substrate of MKP-1, and VEGF- and thrombin-induced H3 (phospho-Ser-10) dephosphorylation requires MKP-1. We propose that MKP-1-mediated H3 (phospho-Ser-10) dephosphorylation is a key regulatory step in EC activation by VEGF and thrombin.