RNA-binding proteins (RBPs) play a major role during control of mRNA localization, stability, and translation and are central to most cellular processes. In the fission yeast Schizosaccharomyces pombe, the multiple K homology (KH) domain RBP Rnc1 downregulates the activity of the cell integrity pathway (CIP) via stabilization of pmp1+ mRNA, which encodes the Pmp1 phosphatase that inactivates Pmk1, the mitogen-activated protein kinase (MAPK) component of this signaling cascade. However, Rnc1 likely regulates the half-life/stability of additional mRNAs. We show that Rnc1 downregulates the activity of Sty1, the MAPK of the stress-activated MAPK pathway (SAPK), during control of cell length at division and recovery in response to acute stress. Importantly, this control strictly depends on Rnc1's ability to bind mRNAs encoding activators (Wak1 MAPKKK, Wis1 MAPKK) and downregulators (Atf1 transcription factor, Pyp1 and Pyp2 phosphatases) of Sty1 phosphorylation through its KH domains. Moreover, Sty1 is responsible for Rnc1 phosphorylation in vivo at multiple phosphosites during growth and stress, and these modifications trigger Rnc1 for proper binding and destabilization of the above mRNA targets. Phosphorylation by Sty1 prompts Rnc1-dependent mRNA destabilization to negatively control SAPK signaling, thus revealing an additional feedback mechanism that allows precise tuning of MAPK activity during unperturbed cell growth and stress.IMPORTANCE Control of mRNA localization, stability, turnover, and translation by RNA-binding proteins (RBPs) influences essential processes in all eukaryotes, including signaling by mitogen-activated protein kinase (MAPK) pathways. We describe that in the fission yeast Schizosaccharomyces pombe the RBP Rnc1 negatively regulates cell length at division during unperturbed growth and recovery after acute stress by reducing the activity of the MAPK Sty1, which regulates cell growth and differentiation during environmental cues. This mechanism relies on Rnc1 binding to specific mRNAs encoding both enhancers and negative regulators of Sty1 activity. Remarkably, multiple phosphorylation of Rnc1 by Sty1 favors RBP binding and destabilization of the above mRNAs. Thus, posttranscriptional modulation of MAP kinase signaling by RNA-binding proteins emerges as a major regulatory mechanism that dictates the growth cycle and cellular adaptation in response to the changing environment in eukaryotic organisms.

Neprilysin is one of the major amyloid-β peptide (Aβ)-degrading enzymes, the expression of which declines in the brain during aging. The decrease in neprilysin leads to a metabolic Aβ imbalance, which can induce the amyloidosis underlying Alzheimer disease. Pharmacological activation of neprilysin during aging therefore represents a potential strategy to prevent the development of Alzheimer disease. However, the regulatory mechanisms mediating neprilysin activity in the brain remain unclear. To address this issue, we screened for pharmacological regulators of neprilysin activity and found that the neurotrophic factors brain-derived neurotrophic factor, nerve growth factor, and neurotrophins 3 and 4 reduce cell surface neprilysin activity. This decrease was mediated by MEK/ERK signaling, which enhanced phosphorylation at serine 6 in the neprilysin intracellular domain (S6-NEP-ICD). Increased phosphorylation of S6-NEP-ICD in primary neurons reduced the levels of cell surface neprilysin and led to a subsequent increase in extracellular Aβ levels. Furthermore, a specific inhibitor of protein phosphatase-1a, tautomycetin, induced extensive phosphorylation of the S6-NEP-ICD, resulting in reduced cell surface neprilysin activity. In contrast, activation of protein phosphatase-1a increased cell surface neprilysin activity and lowered Aβ levels. Taken together, these results indicate that the phosphorylation status of S6-NEP-ICD influences the localization of neprilysin and affects extracellular Aβ levels. Therefore, maintaining S6-NEP-ICD in a dephosphorylated state, either by inhibition of protein kinases involved in its phosphorylation or by activation of phosphatases catalyzing its dephosphorylation, may represent a new approach to prevent reduction of cell surface neprilysin activity during aging and to maintain physiological levels of Aβ in the brain.

The activation of extracellular receptor kinase (ERK) is one of the checkpoints to assess the activation of the classical Ras/mitogen-activated protein kinase (MAPK) cascade. Therefore, we tested more than 100 selenium-containing compounds for their ability to activate the MAPK signal pathway. Among them, we found that three selenazoles, 5-chloroacetyl-2-piperidino-1,3-selenazole (CS1), 5-chloroacetyl-2-morpholino-1,3-selenazole (CS2), and 5-chloroacetyl-2-dimethylamino-1,3-selenazole (CS3), induced the phosphorylation of ERK. These compounds also enhanced the phosphorylation of Akt, a signal transducing protein kinase for cell survival; and this phosphorylation was followed by suppression of cell death, thus suggesting that they had anti-apoptotic effects. Moreover, CSs 1-3 induced neurite outgrowth and facilitated the expression of neurofilament-M of PC12 cells, demonstrating that they induced neuronal differentiation of these cells. On the other hand, the CS-induced phosphorylation of MAPK was enhanced by buthionine sulfoximine (BSO), an activator of protein tyrosine phosphatases (PTPs), but inhibited by N-acetyl-l-cysteine (NAC), an inhibitor of receptor tyrosine kinase. These results imply that activation of some receptor tyrosine kinase(s) is involved in the mechanism of action of CSs 1-3. The activation of MAPK by CSs 1-3 was suppressed by U0126, a MEK inhibitor, but not by K252a, an inhibitor of TrkA; AG1478, an antagonist of epidermal growth factor receptor (EGFR); or by pertussis toxin. These results demonstrate that the CS-induced phosphorylation of Akt and MAP kinase (receptor tyrosine kinase(s)-MEK1/2-ERK1/2) cascades was responsible for suppression of apoptosis and facilitation of neuronal differentiation of PC12 cells, respectively. Our results suggest that CSs 1-3 are promising candidates as neuroprotective and/or neurotrophic agents for the treatment of various neurodegenerative neurological disorders.

Src homology 2 (SH2) domain-containing phosphotyrosine phosphatases (SHPs) are increasingly being shown to play critical roles in protein tyrosine kinase-mediated signaling pathways. The role of SHP-1 as a negative regulator of T cell receptor (TCR) signaling has been established. To further explore the function of the other member of this family, SHP-2, in TCR-mediated events, a catalytically inactive mutant SHP-2 was expressed under an inducible promoter in Jurkat T cells. Expression of the mutant phosphatase significantly inhibited TCR-induced activation of the extracellular-regulated kinase (ERK)-2 member of the mitogen-activated protein kinase (MAPK) family, but had no effect on TCR-zeta chain tyrosine phosphorylation or TCR-elicited Ca2+ transients. Inactive SHP-2 was targeted to membranes resulting in the selective increase in tyrosine phosphorylation of three membrane-associated candidate SHP-2 substrates of 110 kD, 55-60 kD, and 36 kD, respectively. Analysis of immunoprecipitates containing inactive SHP-2 also indicated that the 110-kD and 36-kD Grb-2-associated proteins were putative substrates for SHP-2. TCR-stimulation of Jurkat T cells expressing wild-type SHP-2 resulted in the formation of a multimeric cytosolic complex composed of SHP-2, Grb-2, phosphatidylinositol (PI) 3'-kinase, and p110. A significant proportion of this complex was shown to be membrane associated, presumably as a result of translocation from the cytosol. Catalytically inactive SHP-2, rather than the wild-type PTPase, was preferentially localized in complex with Grb-2 and the p85 subunit of PI 3'-kinase, suggesting that the dephosphorylating actions of SHP-2 may regulate the association of these signaling molecules to the p110 complex. Our results show that SHP-2 plays a critical role in linking the TCR to the Ras/MAPK pathway in Jurkat T cells, and also provide some insight into the molecular interactions of SHP-2 that form the basis of this signal transduction process.

The filamentous fungus Aspergillus fumigatus can cause a distinct set of clinical disorders in humans. Invasive aspergillosis (IA) is the most common life-threatening fungal disease of immunocompromised humans. The mitogen-activated protein kinase (MAPK) signaling pathways are essential to the adaptation to the human host. Fungal cell survival is highly dependent on the organization, composition, and function of the cell wall. Here, an evaluation of the global A. fumigatus phosphoproteome under cell wall stress caused by the cell wall-damaging agent Congo red (CR) revealed 485 proteins potentially involved in the cell wall damage response. Comparative phosphoproteome analyses with the ΔsakA, ΔmpkC, and ΔsakA ΔmpkC mutant strains from the osmotic stress MAPK cascades identify their additional roles during the cell wall stress response. Our phosphoproteomics allowed the identification of novel kinases and transcription factors (TFs) involved in osmotic stress and in the cell wall integrity (CWI) pathway. Our global phosphoproteome network analysis showed an enrichment for protein kinases, RNA recognition motif domains, and the MAPK signaling pathway. In contrast to the wild-type strain, there is an overall decrease of differentially phosphorylated kinases and phosphatases in ΔsakA, ΔmpkC, and ΔsakA ΔmpkC mutants. We constructed phosphomutants for the phosphorylation sites of several proteins differentially phosphorylated in the wild-type and mutant strains. For all the phosphomutants, there is an increase in the sensitivity to cell wall-damaging agents and a reduction in the MpkA phosphorylation upon CR stress, suggesting these phosphosites could be important for the MpkA modulation and CWI pathway regulation.IMPORTANCEAspergillus fumigatus is an opportunistic human pathogen causing allergic reactions or systemic infections, such as invasive pulmonary aspergillosis in immunocompromised patients. The mitogen-activated protein kinase (MAPK) signaling pathways are essential for fungal adaptation to the human host. Fungal cell survival, fungicide tolerance, and virulence are highly dependent on the organization, composition, and function of the cell wall. Upon cell wall stress, MAPKs phosphorylate multiple target proteins involved in the remodeling of the cell wall. Here, we investigate the global phosphoproteome of the ΔsakA and ΔmpkCA. fumigatus and high-osmolarity glycerol (HOG) pathway MAPK mutants upon cell wall damage. This showed the involvement of the HOG pathway and identified novel protein kinases and transcription factors, which were confirmed by fungal genetics to be involved in promoting tolerance of cell wall damage. Our results provide understanding of how fungal signal transduction networks modulate the cell wall. This may also lead to the discovery of new fungicide drug targets to impact fungal cell wall function, fungicide tolerance, and virulence.

Mitogen-activated protein kinases (MAPKs) play critical roles in the induction of numerous cytokines, chemokines, and inflammatory mediators that mobilize the immune system to counter pathogenic infections. Dual-specificity phosphatase 1 (DUSP1) is a member of the dual-specificity phosphatases that inactivates MAPKs through a negative-feedback mechanism. Here, we report that in response to viral and bacterial infections, not only the DUSP1 transcript but also its N6-methyladenosine (m6A) levels rapidly increase together with that of the m6A reader protein YTHDF2, resulting in enhanced YTHDF2-mediated DUSP1 transcript degradation. The knockdown of DUSP1 promotes p38 and Jun N-terminal kinase (JNK) phosphorylation and activation, thus increasing the expression of innate immune response genes, including the interleukin-1β (IL-1β), colony-stimulating factor 3 (CSF3), transglutaminase 2 (TGM2), and proto-oncogene tyrosine-protein kinase Src (SRC) genes. Similarly, the knockdown of the m6A eraser ALKBH5 increases the DUSP1 transcript m6A level, resulting in accelerated transcript degradation, the activation of p38 and JNK, and the enhanced expression of IL-1β, CSF3, TGM2, and SRC. These results demonstrate that m6A and the reader protein YTHDF2 orchestrate optimal innate immune responses during viral and bacterial infections by downregulating the expression of a negative regulator, DUSP1, of the p38 and JNK pathways that are central to innate immune responses against pathogenic infections. IMPORTANCE Innate immunity is central to controlling pathogenic infections and maintaining the homeostasis of the host. In this study, we have revealed a novel mechanism regulating innate immune responses during viral and bacterial infections. We have found that N6-methyladenosine (m6A) and the reader protein YTHDF2 regulate dual-specificity phosphatase 1, a negative regulator of the mitogen-activated protein kinases p38 and JNK, to maximize innate immune responses during viral and bacterial infections. These results provide novel insights into the mechanism regulating innate immunity, which could help in the development of novel approaches for controlling pathogenic infections.

Protein phosphorylation cascades are universal in cell signaling. While kinome diversity allows specific phosphorylation events, relatively few phosphatases dephosphorylate key signaling proteins. Fungal mitogen activated protein kinases (MAPK), in contrast to their mammalian counterparts, often show detectable basal phosphorylation levels. Dephosphorylation, therefore, could act as a signal. In Cochliobolus heterostrophus, the Dothideomycete causing Southern corn leaf blight, ferulic acid (FA)-an abundant phenolic found in plant host cell walls-acts as a signal to rapidly dephosphorylate the stress-activated MAP kinase Hog1 (High Osmolarity Glycerol 1). In order to identify the protein phosphatases responsible, we constructed mutants in Hog1 phosphatases predicted from the genome by homology to yeast and other species. We found that Cochliobolus heterostrophus mutants lacking PtcB, a member of the PP2C family, exhibited altered growth, sporulation, and attenuated dephosphorylation in response to FA. The loss of the dual-specificity phosphatase CDC14 led to slow growth, decreased virulence, and attenuated dephosphorylation. Mutants in two predicted tyrosine phosphatase genes PTP1 and PTP2 showed normal development and virulence. Our results suggest that a network of phosphatases modulate Hog1's dual phosphorylation levels. The mutants we constructed in this work provide a starting point to further unravel the signaling hierarchy by which exposure to FA leads to stress responses in the pathogen.

Phosphorylation and dephosphorylation events play an important role in the transmission of the ABA signal. Although SnRK2 [sucrose non-fermenting1-related kinase2] protein kinases and group A protein phosphatase type 2C (PP2C)-type phosphatases constitute the core ABA pathway, mitogen-activated protein kinase (MAPK) pathways are also involved in plant response to ABA. However, little is known about the interplay between MAPKs and PP2Cs or SnRK2 in the regulation of ABA pathways. In this study, an effort was made to elucidate the role of MAP kinase kinase kinase18 (MKKK18) in relation to ABA signaling and response. The MKKK18 knockout lines showed more vigorous root growth, decreased abaxial stomatal index and increased stomatal aperture under normal growth conditions, compared with the control wild-type Columbia line. In addition to transcriptional regulation of the MKKK18 promoter by ABA, we demonstrated using in vitro and in vivo kinase assays that the kinase activity of MKKK18 was regulated by ABA. Analysis of the cellular localization of MKKK18 showed that the active kinase was targeted specifically to the nucleus. Notably, we identified abscisic acid insensitive 1 (ABI1) PP2C as a MKKK18-interacting protein, and demonstrated that ABI1 inhibited its activity. Using a cell-free degradation assay, we also established that MKKK18 was unstable and was degraded by the proteasome pathway. The rate of MKKK18 degradation was delayed in the ABI1 knockout line. Overall, we provide evidence that ABI1 regulates the activity and promotes proteasomal degradation of MKKK18.

The mitogen activated protein kinase (MAPK) pathway has been reported to be involved in many cancer developments. Normally, MAPK activity is self-limited between rapid phosphorylation and dephosphorylation. In abnormal conditions, however, this dynamic equilibrium is broken, trigging tumor-suppressing or -promoting roles. While dual-specificity MAPK phosphatases (MKP/DUSPs) are important for cascade control in MAPK pathway, their role in colorectal cancer (CRC) remains largely unknown. Here, we investigated lnc-FAM84B-4 and DUSP1 to systematically elucidate their underlying roles in MAPK singling pathway and functions in CRC. Upregulated lnc-FAM84B-4 was identified by re-mining CRC microarray. Functional assays were performed in vitro and in vivo. RNA-Seq, RNA pull-down, and RIP assays were used to investigate the mechanisms of Lnc-FAM84B-4 in regulating expression of DUSP1. The results indicated that Lnc-FAM84B-4 regulates MAPK pathway by restraining DUSP1 expression. Mechanistically, RNA pull-down followed by mass spectrum determined hnRNPK functions as a binding partner of lnc-FAM84B-4 in mediating DUSP1 expression. Our findings demonstrate the important role of lnc-FAM84B-4-hnRNPK-DUSP1 axis in CRC development, and suggest a therapeutic target for CRC treatment.

A series of protein kinases and phosphatases (PKPs) have been linked to contextual fear conditioning (cFC) but information is mainly derived from immunochemical studies. It was therefore decided to use an explorative label-free quantitative proteomics approach to concomitantly determine PKPs in hippocampi of mice in the individual phases of cFC. C57BL/6J mice were divided into four groups: three training groups representing the acquisition, consolidation and retrieval phases of cFC and a foot shock control group. Using this approach we identified 32 protein kinases or phosphatases/phosphatase subunits with significantly changed protein levels in one or more training groups as compared to foot shock control. These include members of PKP signalling modules of mitogen-activated protein kinase (MAP3K10, RAF1, KSR2), Ca2+/calmodulin-dependent protein kinase (CaMKIIα, DAPK1), protein kinase C (PRKCD) and protein phosphatases 1, 2A, 2B(3) previously implicated in various learning paradigms. In addition, our analysis showed protein kinases WNK1, LYN, VRK1, ABL1, CDK4, CDKL3, SgK223 and ADCK1, and protein phosphatases PTPRF, ACP1, DNAJC6, SSH2 and UBASH3B that have not been directly linked to fear memory processes so far. Determination of PKPs in the individual cFC phases represents a valuable resource for interpretation of previous and design of future studies on PKPs in memory mechanisms.

Mitogen-activated protein kinase (MAPK) cascades transmit environmental signals and induce stress and defence responses in plants. These signalling cascades are negatively controlled by specific Ser/Thr protein phosphatases of the type 2C (PP2C) and dual-specificity phosphatase (DSP) families that inactivate stress-induced MAPKs; however, the interplay between phosphatases of these different types has remained unknown. This work reveals that different Arabidopsis MAPK phosphatases, the PP2C-type AP2C1 and the DSP-type MKP1, exhibit both specific and overlapping functions in plant stress responses. Each single mutant, ap2c1 and mkp1, and the ap2c1 mkp1 double mutant displayed enhanced stress-induced activation of the MAPKs MPK3, MPK4, and MPK6, as well as induction of a set of transcription factors. Moreover, ap2c1 mkp1 double mutants showed an autoimmune-like response, associated with increased levels of the stress hormones salicylic acid and ethylene, and of the phytoalexin camalexin. This phenotype was reduced in the ap2c1 mkp1 mpk3 and ap2c1 mkp1 mpk6 triple mutants, suggesting that the autoimmune-like response is due to MAPK misregulation. We conclude that the evolutionarily distant MAPK phosphatases AP2C1 and MKP1 contribute crucially to the tight control of MAPK activities, ensuring appropriately balanced stress signalling and suppression of autoimmune-like responses during plant growth and development.

Angiopoietin-1 (Ang-1) promotes survival and migration of endothelial cells, in part through the activation of mitogen-activated protein kinase (MAPK) pathways downstream of Tie-2 receptors. Dual-specificity phosphatases (DUSPs) dephosphorylate phosphotyrosine and phosphoserine/phosphothreonine residues on target MAPKs. The mechanisms by which DUSPs modulate MAPK activation in Ang-1/Tie-2 receptor signaling are unknown in endothelial cells.

Protein phosphatases (PPs) and protein kinases (PKs) regulate numerous developmental, defense, and phytohormone signaling processes in plants. However, the underlying regulatory mechanism governing biosynthesis of specialized metabolites, such as alkaloids, by the combined effects of PPs and PKs, is insufficiently understood. Here, we report the characterization of a group B protein phosphatase type 2C, NtPP2C2b, that likely acts upstream of the NICOTINE2 locus APETALA 2/Ethylene Response Factors (AP2/ERFs), to regulate nicotine biosynthesis in tobacco. Similar to the nicotine pathway genes, NtPP2C2b is highly expressed in roots and induced by jasmonic acid (JA). Overexpression of NtPP2C2b in transgenic hairy roots or stable transgenic tobacco plants repressed nicotine pathway gene expression and reduced nicotine accumulation. Additionally, transient overexpression of NtPP2C2b, together with the NtERF221, repressed transactivation of the quinolinate phosphoribosyltransferase promoter in tobacco cells. We further demonstrate that the JA-responsive tobacco mitogen-activated protein kinase (MAPK) 4 interacts with NtPP2C2b in yeast and plant cells. Conditional overexpression of NtMPK4 in tobacco hairy roots up-regulated nicotine pathway gene expression and increased nicotine accumulation. Our findings suggest that a previously uncharacterized PP-PK module acts to modulate alkaloid biosynthesis, highlighting the importance of post-translational control in the biosynthesis of specialized plant metabolites.

LIN28A, an RNA-binding protein, plays an important role in porcine induced pluripotent stem cells (piPSCs). However, the molecular mechanism underlying the function of LIN28A in the maintenance of pluripotency in piPSCs remains unclear. Here, we explored the function of LIN28A in piPSCs based on its overexpression and knockdown. We performed total RNA sequencing (RNA-seq) of piPSCs and detected the expression levels of relevant genes by quantitative real-time polymerase chain reaction (qRT-PCR), western blot analysis, and immunofluorescence staining. Results indicated that piPSC proliferation ability decreased following LIN28A knockdown. Furthermore, when LIN28A expression in the shLIN28A2 group was lower (by 20%) than that in the negative control knockdown group ( shNC), the pluripotency of piPSCs disappeared and they differentiated into neuroectoderm cells. Results also showed that LIN28A overexpression inhibited the expression of DUSP (dual-specificity phosphatases) family phosphatases and activated the mitogen-activated protein kinase (MAPK) signaling pathway. Thus, LIN28A appears to activate the MAPK signaling pathway to maintain the pluripotency and proliferation ability of piPSCs. Our study provides a new resource for exploring the functions of LIN28A in piPSCs.

The formation of erythroid progenitor cells depends sharply upon erythropoietin (EPO), its cell surface receptor (erythropoietin receptor, EPOR), and Janus kinase 2 (JAK2). Clinically, recombinant human EPO (rhEPO) additionally is an important anti-anemia agent for chronic kidney disease (CKD), myelodysplastic syndrome (MDS) and chemotherapy, but induces hypertension, and can exert certain pro-tumorigenic effects. Cellular signals transduced by EPOR/JAK2 complexes, and the nature of EPO-modulated signal transduction factors, therefore are of significant interest. By employing phospho-tyrosine post-translational modification (p-Y PTM) proteomics and human EPO- dependent UT7epo cells, we have identified 22 novel kinases and phosphatases as novel EPO targets, together with their specific sites of p-Y modification. New kinases modified due to EPO include membrane palmitoylated protein 1 (MPP1) and guanylate kinase 1 (GUK1) guanylate kinases, together with the cytoskeleton remodeling kinases, pseudopodium enriched atypical kinase 1 (PEAK1) and AP2 associated kinase 1 (AAK1). Novel EPO- modified phosphatases include protein tyrosine phosphatase receptor type A (PTPRA), phosphohistidine phosphatase 1 (PHPT1), tensin 2 (TENC1), ubiquitin associated and SH3 domain containing B (UBASH3B) and protein tyrosine phosphatase non-receptor type 18 (PTPN18). Based on PTPN18's high expression in hematopoietic progenitors, its novel connection to JAK kinase signaling, and a unique EPO- regulated PTPN18-pY389 motif which is modulated by JAK2 inhibitors, PTPN18's actions in UT7epo cells were investigated. Upon ectopic expression, wt-PTPN18 promoted EPO dose-dependent cell proliferation, and survival. Mechanistically, PTPN18 sustained the EPO- induced activation of not only mitogen-activated protein kinases 1 and 3 (ERK1/2), AKT serine/threonine kinase 1-3 (AKT), and signal transducer and activator of transcription 5A and 5B (STAT5), but also JAK2. Each effect further proved to depend upon PTPN18's EPO- modulated (p)Y389 site. In analyses of the EPOR and the associated adaptor protein RHEX (regulator of hemoglobinization and erythroid cell expansion), wt-PTPN18 increased high molecular weight EPOR forms, while sharply inhibiting the EPO-induced phosphorylation of RHEX-pY141. Each effect likewise depended upon PTPN18-Y389. PTPN18 thus promotes signals for EPO-dependent hematopoietic cell growth, and may represent a new druggable target for myeloproliferative neoplasms.

Transforming growth factor-beta (TGFβ) proteins induce an epithelial-mesenchymal transition (EMT) programme that is associated with increased invasive and drug-resistant phenotype of carcinoma cells. In addition to the canonical pathway involving SMAD proteins, the mitogen-activated kinase (MAPK) pathway via extracellular signal-regulated kinases ½ (ERK1/2) is also involved in promoting and maintaining a mesenchymal phenotype by tumor cells following TGFβ signal activation. As dual-specificity phosphatases (DUSPs) regulate ERK1/2 activity by dephosphorylation, we aimed to examine DUSPs' expression upon TGFβ stimulation and whether DUSPs play a role in the EMT and related phenotypes promoted by TGFβ1 in A549 cells. We found that TGFβ1 stimulation led to marked changes in several DUSP proteins, including significant decreases in DUSP4 and DUSP13 expressions. We then showed that the ectopic co-expression of DUSP4/13 suppresses TGFβ1-induced ERK1/2 phosphorylation and protein levels of the EMT transcription factors Snail and Slug proteins. We then demonstrated that DUSP4/13 co-expression partially inhibited TGFβ1-promoted migration, invasion, and chemoresistance in A549 cells. Collectively, this report provides data for the involvement of DUSP4/13 in malignant phenotypes regulated by TGFβ1 in A549 cells.

Growth factors regulate ovarian follicle development and they signal through intracellular pathways including mitogen-activated protein kinase (MAPK) phosphorylation, which is negatively regulated by a subfamily of 23 dual-specificity phosphatases (DUSP). Using sheep granulosa cells as a model, we detected mRNA encoding 16 DUSPs in vivo and in vitro. Stimulation of cells in vitro with FGF2 increased (p < 0.05) abundance of DUSP1, DUSP2, DUSP5 and DUSP6 mRNA, and abundance of DUSP1 and DUSP6 proteins (p < 0.05). In contrast, neither FGF8b nor FGF18 had any major effect on DUSP mRNA abundance. Inhibition of DUSP6 action with the inhibitor BCI significantly increased (p < 0.05) MAPK8 (JNK) phosphorylation but not phosphoMAPK14 (p38) or MAPK3/1 (ERK1/2) abundance. This study suggests that FGFs stimulate DUSP protein abundance, that DUSP6 regulates MAPK8 phosphorylation in granulosa cells, and DUSPs are involved in the differential MAPK signaling of individual FGF ligands.

Protein phosphorylation plays critical roles in the regulation of protein activity and cell signaling. The level of protein phosphorylation is controlled by protein kinases and protein tyrosine phosphatases (PTPs). Disturbance of the equilibrium between protein kinase and PTP activities results in abnormal protein phosphorylation, which has been linked to the etiology of several diseases, including cancer. In this study, we screened protein tyrosine phosphatases (PTPs) by in vitro phosphatase assays to identify PTPs that are inhibited by bis (4-trifluoromethyl-sulfonamidophenyl, TFMS)-1,4-diisopropylbenzene (PTP inhibitor IV). PTP inhibitor IV inhibited DUSP14 phosphatase activity. Kinetic studies with PTP inhibitor IV and DUSP14 revealed a competitive inhibition, suggesting that PTP inhibitor IV binds to the catalytic site of DUSP14. PTP inhibitor IV effectively and specifically inhibited DUSP14-mediated dephosphorylation of JNK, a member of the mitogen-activated protein kinase (MAPK) family.

The three layer mitogen activated protein kinase (MAPK) signaling cascade exhibits different designs of interactions between its kinases and phosphatases. While the sequential interactions between the three kinases of the cascade are tightly preserved, the phosphatases of the cascade, such as MKP3 and PP2A, exhibit relatively diverse interactions with their substrate kinases. Additionally, the kinases of the MAPK cascade can also sequester their phosphatases. Thus, each topologically distinct interaction design of kinases and phosphatases could exhibit unique signal processing characteristics, and the presence of phosphatase sequestration may lead to further fine tuning of the propagated signal.

MAPK phosphatases (MKP) downregulate the activity of mitogen-activated protein kinases (MAPK), such as ERK1/2, and modulate the processes regulated by these kinases. ERK1/2 participate in a wide range of processes including tissue-specific hormone-stimulated steroidogenesis. H295R cells are a suitable model for the study of human adrenal cortex functions, particularly steroid synthesis, and respond to angiotensin II (Ang II) triggering ERK1/2 phosphorylation in a transient fashion. MKP-3 dephosphorylates ERK1/2 and, as recently reported, forkhead box protein 1 (FOXO1). Here, we analyzed MKP-3 expression in H295R cells and its putative regulation by Ang II. Results showed the expression of MKP-3 full length (L) and a short splice variant (S), and the upregulation of both isoforms by Ang II. L and S messenger and protein levels increased 30 min after Ang II stimulation and declined over the next 3 h, a temporal frame compatible with ERK1/2 dephosphorylation. In addition, FOXO1 activation is known to include its dephosphorylation and nuclear translocation. Therefore, we analyzed the effect of Ang II on FOXO1 modulation. Ang II induced FOXO1 transient phosphorylation and translocation and also the induction of p21, a FOXO1-dependent gene, whereas MKP-3 knock-down reduced both FOXO1 translocation and p21 induction. These data suggest that, through MKP-3, Ang II counteracts its own effects on ERK1/2 activity and also triggers the activation of FOXO-1 and the induction of cell cycle inhibitor p21. Taken together, the current findings reveal the participation of MKP-3 not only in turn-off but also in turn-on signals which control important cellular processes.