Abelson family kinases regulate Frizzled planar cell polarity signaling via Dsh phosphorylation

Abelson (Abl) family tyrosine kinases have been implicated in cell morphogenesis, adhesion, motility, and oncogenesis. Using a candidate approach for genes involved in planar cell polarity (PCP) signaling, we identified Drosophila Abl (dAbl) as a modulator of Frizzled(Fz)/PCP signaling. We demonstrate that dAbl positively regulates the Fz/Dishevelled (Dsh) PCP pathway without affecting canonical Wnt/Wg–Fz signaling. Genetic dissection suggests that Abl functions via Fz/Dsh signaling in photoreceptor R3 specification, a well-established Fz–PCP signaling readout. Molecular analysis shows that dAbl binds and phosphorylates Dsh on Tyr473 within the DEP domain. This phosphorylation event on Dsh is functionally critical, as the equivalent DshY473F mutant is nonfunctional in PCP signaling and stable membrane association, although it rescues canonical Wnt signaling. Strikingly, mouse embryonic fibroblasts (MEFs) deficient for Abl1 and Abl2/Arg genes also show reduced Dvl2 phosphorylation as compared with control MEFs, and this correlates with a change in subcellular localization of endogenous Dvl2. As in Drosophila, such Abl-deficient MEFs show no change in canonical Wnt signaling. Taken together, our results argue for a conserved role of Abl family members in the positive regulation of Dsh activity toward Fz–Dsh/PCP signaling by Dsh phosphorylation.     

Development of a pentylenetetrazole-induced seizure model to evaluate kinase inhibitor efficacy in the central nervous system

c-Jun N-terminal kinases (JNKs) are implicated in cell death in neurodegenerative disorders. Therefore, JNK inhibitors could act as neuroprotective agents. To evaluate potential candidates, reproducible and quantitative CNS in vivo models are required. To that end, a pentylenetetrazole-induced seizure model was explored. c-Jun phosphorylation was detected in hippocampal extracts by blotting c-Jun immunoprecipitates with phosphorylation-specific antibodies. Pentylenetetrazole administration induced rapid and reproducible increases in c-Jun phosphorylation. However, special attention had to be paid to the composition of the extraction buffer to ensure stabilization of protein phosphorylation, as demonstrated using internal standards of phosphorylated recombinant c-Jun. As JNK and its upstream activator MKK4 are activated by phosphorylation, these events were also evaluated. In principle, kinase inhibitors could act at the level of JNK or upstream kinases to inhibit c-Jun phosphorylation. MKK4 phosphorylation was dramatically increased in response to pentylenetetrazole but, again, only when appropriate phosphatase inhibitors were in the extraction buffer. In contrast, JNK was found to be constitutively phosphorylated and unaltered upon pentylenetetrazole treatment. The JNK inhibitor SP600125 was shown to inhibit c-Jun phosphorylation without affecting MKK4 phosphorylation. Our procedures enable analysis of JNK pathway signalling in a CNS model and, also, should be applicable to that of other protein phosphorylation events in vivo.     

Up-regulation of MKK4, MKK6 and MKK7 during prostate cancer progression: an important role for SAPK signalling in prostatic neoplasia

Identification of the signalling cascades that are differentially activated during prostatic tumourigenesis is a crucial step in the search for future molecular targets in this disease. The stress-activated protein kinase (SAPK) signalling cascade culminates in the phosphorylation of the JNK and p38 mitogen-activated protein kinases (MAPKs). Recently, the upstream activators of these proteins, the MAPK kinases (MKKs), have been implicated as inhibitors of tumour progression in a variety of clinical and experimental tumour models. This study evaluates MKK4, MKK6 and MKK7 expression during prostate cancer progression in humans and in the transgenic adenocarcinoma of a mouse prostate (TRAMP) model of prostate tumourigenesis. Benign prostate, prostatic intraepithelial neoplasia (PIN) lesions and tumour tissues were collected from 37 TRAMP mice. Additionally, six tissue microarrays were constructed with tumours from a matched group of 102 men who underwent radical prostatectomy. Tissues from 20 patients with extensive high-grade prostatic intraepithelial neoplasia (HGPIN) were also analysed. For all samples, immunohistochemical staining for MKK4, MKK6 and MKK7 was scored in normal and neoplastic glands. Staining intensities of MKK4, MKK6 and MKK7 were significantly increased in HGPIN and prostate cancer compared to surrounding normal glands in both the TRAMP and human samples (p < 0.0001 for all markers). Increased levels of MKK4 or MKK7 correlated with higher pathological stage at prostatectomy (p = 0.01 and p = 0.04). Using multivariate analysis, there was no association between protein levels and time to biochemical recurrence in the human samples. The up-regulation of MKK4, MKK6 and MKK7 during prostate cancer progression in both TRAMP and human tissues highlights an important role for the SAPK signalling cascade in prostatic neoplasia. The finding that higher MKK4 and MKK7 expression is associated with higher-stage prostatic tumours underscores the dynamic regulation of these proteins during prostatic tumourigenesis.     

Effects of metabotropic glutamate mGlu5 receptor antagonist on tyrosine phosphorylation of NMDA receptor subunits and cell death in the hippocampus after brain ischemia in rats

Tyrosine phosphorylation of the N-methyl-D-aspartate (NMDA) receptor appears to be associated with the regulation of the receptor's ion channel. This study focused on the effect of a metabotropic glutamate mGlu5 receptor antagonist on tyrosine phosphorylation of NMDA receptor subunits and cell death in the hippocampal CA1 region after transient global ischemia and sought to explore their mechanisms. Pretreatment with the mGlu5 receptor antagonist reduced cell death in the hippocampal CA1 region on day 3 after the transient ischemia. Transient ischemia increased the tyrosine phosphorylation of NMDA receptor subunits, which are a major target of Src family tyrosine kinases. Therefore, we investigated the effect of the antagonist on tyrosine phosphorylation of the NMDA receptor subunits after transient ischemia. Tyrosine phosphorylation of the NR2A subunit, but not that of the NR2B one, was inhibited by the mGlu5 receptor antagonist. The administration of the antagonist also attenuated the increase in the amount of active form of Src after the reperfusion. We further demonstrated that the administration of a Src-family kinase inhibitor prevented cell death in the hippocampal CA1 region and attenuated the increase in the tyrosine phosphorylation of the NMDA receptor subunits after the reperfusion. These findings suggest that mGlu5 receptor in the hippocampal CA1 region after transient ischemia is involved in the activation of Src and subsequent tyrosine phosphorylation of NMDA receptor subunits, which actions may contribute to alterations of properties of the NMDA receptor and may be related to pathogenic events leading to neuronal cell death.     

Physiological roles of ASK1-mediated signal transduction in oxidative stress- and endoplasmic reticulum stress-induced apoptosis: advanced findings from ASK1 knockout mice

Apoptosis, a molecularly regulated form of cell death, is essential for the normal functioning and homeostasis of most multicellular organisms, and can be induced by a range of environmental, physical, and chemical stresses. As the cellular decision to live or to die is made by the coordinated action and balancing of many different pro- and antiapoptotic factors, defects in control of this coordination and balance may contribute to a variety of human diseases, including cancer and autoimmune and neurodegenerative conditions. In recent years, multiple factors associated with the execution of apoptosis, such as caspases and Bcl-2 family members, have been discovered and their complicated signaling and molecular interactions have been demonstrated; however, the precise mechanistic basis for intracellular and/or extracellular stress-induced apoptosis remains to be fully characterized. Protein kinases contribute to regulation of life and death decisions made in response to various stress signals, and the actions of pro- and antiapoptotic factors are often affected by modulation of the phosphorylation status of key elements in the execution of apoptosis. Apoptosis signal-regulating kinase 1 (ASK1) is a member of the mitogen-activated protein (MAP) kinase kinase kinase family, which activates both the MKK4/MKK7-JNK and MKK3/MKK6-p38 MAP kinase pathways and constitutes a pivotal signaling pathway in various types of stress-induced apoptosis. We have recently shown through ASK1 gene ablation in mice that ASK1 plays essential roles in oxidative stress- and endoplasmic reticulum (ER) stress-induced apoptosis. These stresses are closely linked to physiological phenomena in the control of cell fate, and the resultant apoptosis is implicated in the pathophysiology of a broad range of human diseases. This article reviews our new findings on the physiological roles of ASK1-mediated signal transduction in stress responses and the molecular mechanisms by which ASK1 determines cell fate such as survival, differentiation, or apoptosis, with special focus on the regulatory mechanisms of ASK1-mediated apoptosis induced by oxidative stress and ER stress.     

Dimerization-driven degradation of C. elegans and human E proteins

E proteins are conserved regulators of growth and development. We show that the Caenorhabditis elegans E-protein helix–loop–helix-2 (HLH-2) functions as a homodimer in directing development and function of the anchor cell (AC) of the gonad, the critical organizer of uterine and vulval development. Our structure–function analysis of HLH-2 indicates that dimerization drives its degradation in other uterine cells (ventral uterine precursor cells [VUs]) that initially have potential to be the AC. We also provide evidence that this mode of dimerization-driven down-regulation can target other basic HLH (bHLH) dimers as well. Remarkably, human E proteins can functionally substitute for C. elegans HLH-2 in regulating AC development and also display dimerization-dependent degradation in VUs. Our results suggest that dimerization-driven regulation of bHLH protein stability may be a conserved mechanism for differential regulation in specific cell contexts.     

PI3K-mediated PDGFRα signaling regulates survival and proliferation in skeletal development through p53-dependent intracellular pathways

Previous studies have identified phosphatidylinositol 3-kinase (PI3K) as the main downstream effector of PDGFRα signaling during murine skeletal development. Autophosphorylation mutant knock-in embryos in which PDGFRα is unable to bind PI3K (Pdgfra^PI3K/PI3K) exhibit skeletal defects affecting the palatal shelves, shoulder girdle, vertebrae, and sternum. To identify proteins phosphorylated by Akt downstream from PI3K-mediated PDGFRα signaling, we immunoprecipitated Akt phosphorylation substrates from PDGF-AA-treated primary mouse embryonic palatal mesenchyme (MEPM) lysates and analyzed the peptides by nanoliquid chromatography coupled to tandem mass spectrometry (nano-LC-MS/MS). Our analysis generated a list of 56 proteins, including 10 that regulate cell survival and proliferation. We demonstrate that MEPM cell survival is impaired in the presence of a PI3K inhibitor and that Pdgfra^PI3K/PI3K-derived MEPMs do not proliferate in response to PDGF-AA treatment. Several of the identified Akt phosphorylation targets, including Ybox1, mediate cell survival through regulation of p53. We show that Ybox1 binds both the Trp53 promoter and the p53 protein and that expression of Trp53 is significantly decreased upon PDGF-AA treatment in MEPMs. Finally, we demonstrate that introduction of a Trp53-null allele attenuates the vertebral defects found in Pdgfra^PI3K/PI3K neonates. Our findings identify p53 as a novel effector downstream from PI3K-engaged PDGFRα signaling that regulates survival and proliferation during skeletal development in vivo.     

Dematin,a human erythrocyte cytoskeletal protein,is a substrate for a recombinant FIKK kinase from Plasmodium falciparum

P. falciparum causes the most deadly form of malaria, resulting from the adherence of infected red blood cells to blood vessels. During the blood stage of infection, the parasite secretes a large number of proteins into the host erythrocyte. The secretion of a 20-member family of protein kinases known as FIKK kinases, after a conserved Phe-Ile-Lys-Lys sequence motif, is unique to P. falciparum. Identification of physiological substrates of these kinases may provide perspective on the importance of FIKK kinase activity to P. falciparum virulence. We demonstrate, for the first time, the heterologous expression and purification of a FIKK kinase (PfFk4.1, PFD1165w). The recombinant kinase is active against general substrates and phosphorylates itself. Having demonstrated kinase activity, we incubated recombinant Fk4.1 with parasite and human erythrocyte lysates. No parasite-derived substrates were identified. However, treatment of erythrocyte ghosts shows that the FIKK kinase Fk4.1 phosphorylates dematin, a cytoskeletal protein found at the red blood cell spectrin–actin junction.     

SEL-10/Fbw7-dependent negative feedback regulation of LIN-45/Braf signaling in C. elegans via a conserved phosphodegron

The conserved E3 ubiquitin ligase component named SEL-10 in Caenorhabditis elegans and Fbw7 in mammals targets substrates for ubiquitin-mediated degradation through a high-affinity binding site called a Cdc4 phosphodegron (CPD). As many known substrates of Fbw7 are oncoproteins, the identification of new substrates may offer insight into cancer biology as well as aspects of proteome regulation. Here, we evaluated whether the presence of an evolutionarily conserved CPD would be a feasible complement to proteomics-based approaches for identifying new potential substrates. For functional assessments, we focused on LIN-45, a component of the signal transduction pathway underlying vulval induction and the ortholog of human Braf, an effector of Ras in numerous cancers. Our analysis demonstrates that LIN-45 behaves as a bona fide substrate of SEL-10, with mutation of the CPD or loss of sel-10 resulting in increased activity and protein stability in vivo. Furthermore, during vulval induction, the downstream kinase MPK-1/ERK is also required for LIN-45 protein degradation in a negative feedback loop, resulting in degradation of LIN-45 where ERK is highly active. As the CPD consensus sequence is conserved in human Braf, we propose that Fbw7 may also regulate Braf stability in some cell contexts. We discuss the implications of our findings for vulval development in C. elegans, the potential applicability to human Braf, and the value of a CPD-based predictive approach for human Fbw7 substrates.     

A novel homozygous Fas ligand mutation leads to early protein truncation,abrogation of death receptor and reverse signaling and a severe form of the autoimmune lymphoproliferative syndrome

We report a novel type of mutation in the death ligand FasL that was associated with a severe phenotype of the autoimmune lymphoproliferative syndrome in two patients. A frameshift mutation in the intracellular domain led to complete loss of FasL expression. Cell death signaling via its receptor and reverse signaling via its intracellular domain were completely abrogated. In vitro lymphocyte proliferation induced by weak T cell receptor stimulation could be blocked and cell death was induced by engagement of FasL in T cells derived from healthy individuals and a heterozygous carrier, but not in FasL-deficient patient derived cells. Expression of genes implicated in lymphocyte proliferation and activation (CCND1, NFATc1, NF-κB1) was increased in FasL-deficient T cells and could not be downregulated by FasL engagement as in healthy cells. Our data thus suggest, that deficiency in FasL reverse signaling may contribute to the clinical lymphoproliferative phenotype of ALPS.     

Antioxidant N-acetylcysteine inhibits the activation of JNK3 mediated by the GluR6–PSD95–MLK3 signaling module during cerebral ischemia in rat hippocampus

Cerebral ischemia induces kainate receptor glutamate receptor 6 (GluR6) binding to the postsynaptic density protein 95 (PSD95), which in turn anchors mixed lineage kinase 3 (MLK3) via SH3 domain in rat brain. MLK3 subsequently activates c-Jun NH₂-terminal kinase (JNK) via MAP kinase kinases (MKKs). In this study, we investigated the association of PSD95 with GluR6 and MLK3, the autophosphorylation of MLK3, the combination of MLK3 with JNK3, and the phosphorylation of JNK3 during cerebral ischemia in rat hippocampus CA1. Our results indicate that the GluR6–PSD95–MLK3 complex quickly enhanced at 5 min of ischemia and peaked at 10 min of ischemia, and then gradually reduced with the prolonged time of ischemia. Interestingly, the combination of MLK3 and JNK3 gradually increased from 5 min to 30 min of ischemia. JNK3 phosphorylation first increased and then attenuated in cytosol, suggesting the translocation of activated JNK3 to nucleus during ischemia. To further investigate the possible mechanism of JNK3 activation, antioxidant N-acetylcysteine (NAC) was given to the rats 20 min prior to ischemia. Results indicate that NAC distinctly inhibited the association of PSD95 with GluR6 and MLK3, the autophosphorylation of MLK3, the combination of MLK3 with JNK3 and JNK3 activation. Taken together, these finding indicate that ischemic stimulation results in JNK3 activation through the GluR6–PSD95–MLK3 signaling module, and that the activation of JNK3 is closely related to oxidative stress.     

CDKL5 deficiency predisposes neurons to cell death through the deregulation of SMAD3 signaling

CDKL5 deficiency disorder (CDD) is a rare encephalopathy characterized by early onset epilepsy and severe intellectual disability. CDD is caused by mutations in the X‐linked cyclin‐dependent kinase‐like 5 (CDKL5) gene, a member of a highly conserved family of serine‐threonine kinases. Only a few physiological substrates of CDKL5 are currently known, which hampers the discovery of therapeutic strategies for CDD. Here, we show that SMAD3, a primary mediator of TGF‐β action, is a direct phosphorylation target of CDKL5 and that CDKL5‐dependent phosphorylation promotes SMAD3 protein stability. Importantly, we found that restoration of the SMAD3 signaling through TGF‐β1 treatment normalized defective neuronal survival and maturation in Cdkl5 knockout (KO) neurons. Moreover, we demonstrate that Cdkl5 KO neurons are more vulnerable to neurotoxic/excitotoxic stimuli. In vivo treatment with TGF‐β1 prevents increased NMDA‐induced cell death in hippocampal neurons from Cdkl5 KO mice, suggesting an involvement of the SMAD3 signaling deregulation in the neuronal susceptibility to excitotoxic injury of Cdkl5 KO mice. Our finding reveals a new function for CDKL5 in maintaining neuronal survival that could have important implications for susceptibility to neurodegeneration in patients with CDD.     

Drosophila Minus is required for cell proliferation and influences Cyclin E turnover

Turnover of cyclins plays a major role in oscillatory cyclin-dependent kinase (Cdk) activity and control of cell cycle progression. Here we present a novel cell cycle regulator, called minus, which influences Cyclin E turnover in Drosophila. minus mutants produce defects in cell proliferation, some of which are attributable to persistence of Cyclin E. Minus protein can interact physically with Cyclin E and the SCF Archipelago/Fbw7/Cdc4 ubiquitin–ligase complex. Minus does not affect dMyc, another known SCF^Ago substrate in Drosophila. We propose that Minus contributes to cell cycle regulation in part by selectively controlling turnover of Cyclin E.     

Notch signaling regulates the phosphorylation of Akt and survival of lipopolysaccharide-activated macrophages via regulator of G protein signaling 19 (RGS19)

Macrophages play critical roles in innate immune defense by sensing microbes using pattern-recognition receptors. Lipopolysaccharide (LPS) stimulates macrophages via TLR, which leads to activation of downstream signaling cascades. In this study, we investigated the roles of a conserved signaling pathway, Notch signaling, in regulating the downstream signaling cascades of the LPS/TLR4 pathways in macrophages. Using a phospho-proteomic approach and a gamma-secretase inhibitor (GSI) to suppress the processing and activation of Notch signaling, we identified regulator of G protein signaling 19 (RGS19) as a target protein whose phosphorylation was affected by GSI treatment. RGS19 is a guanosine triphosphatase (GTPase)-activating protein that functions to negatively regulate G protein-coupled receptors via G_αi/G_αq-linked signaling. Stimulation of RAW264.7 cells with LPS increased the level of the phosphorylated form of RGS19, while LPS stimulation in the presence of GSI decreased its level. GSI treatment did not alter the mRNA level of rgs19. Treatment with GSI or silencing of rgs19 in macrophages impaired the phosphorylation of Akt Thr³⁰⁸ upon LPS stimulation. Furthermore, targeted deletion of a DNA-binding protein and binding partner of the Notch receptor, RBP-Jκ/CSL, in macrophages resulted in delayed and decreased Akt phosphorylation. Because the PI3K/Akt pathway regulates cell survival in various cell types, the cell cycle and cell death were assayed upon GSI treatment, phosphatidylinositol 3 kinase (PI3K) inhibitor treatment or silencing of rgs19. GSI treatment resulted in decreased cell populations in the G1 and S phases, while it increased the cell population of cell death. Similarly, silencing of rgs19 resulted in a decreased cell population in the G1 phase and an increased cell population in the subG1 phase. Inhibition of Akt phosphorylation by PI3K inhibitor in LPS-stimulated macrophages increased cell population in G1 phase, suggesting a possible cell cycle arrest. Taken together, these results indicate that Notch signaling positively regulates phosphorylation of Akt, possibly via phosphorylation of RGS19, and inhibition of both molecules affects the cell survival and cell cycle of macrophages upon LPS stimulation.     

Signaling through ephrin-A ligand leads to activation of Src-family kinases, Akt phosphorylation, and inhibition of antigen receptor-induced apoptosis

Eph receptor tyrosine kinases and ephrins play important roles in diverse biological processes such as migration, adhesion, and angiogenesis. Forward and reverse signaling has been reported in receptor- and ligand-bearing cells. The ligands can be divided into the transmembrane ephrin-B family and the GPI-anchored ephrin-A family. Here, we show expression of ephrin-A ligands on CD4+ T cells cultured in medium with human serum and the T cell line Jurkat TAg and on cells isolated from patients with T cell lymphomas and T cell leukemias. Functional role and identification of proteins involved in ephrin-A signaling were investigated here in the T cell line Jurkat TAg. Signaling through ephrin-A induces phosphorylation of several proteins, including the Src kinases Lck and Fyn. In addition, PI-3K is activated, shown by induced phosphorylation of the Akt kinase. An ephrin-A signaling complex could be isolated, containing several phosphorylated proteins including Lck and Fyn. Interestingly, we show that signaling through ephrin-A in Jurkat TAg cells, initiated by interaction with the EphA2 receptor, leads to inhibition of activation-induced cell death. To conclude, ephrin-A signaling in Jurkat TAg cells leads to induced phosphorylation of several proteins including Lck, Fyn, and Akt. A consequence of ephrin-A signaling is inhibition of antigen receptor-induced apoptosis.     

MKK3, an upstream activator of p38, contributes to formalin phase 2 and late allodynia in mice

Spinal p38 mitogen activated (MAP) kinase plays a key role in chronic pain behavior. However, clinical development of p38 inhibitors has been hindered by significant toxicity. To evaluate alternative strategies of p38 regulation, we determined if known upstream activators of p38 (mitogen activated kinase kinase [MKK] 3 and MKK6), are involved in development and maintenance of pain and spinal p38 phosphorylation. Acute pain behaviors were not altered in MKK3 or MKK6 deficient mice. The phase 2 formalin response was delayed in MKK3-/- mice, but unchanged in magnitude, while the response remained normal in MKK6-/- mice. More striking, late formalin allodynia (3–18 days post-injection) was prominent in wild type and MKK6-/- mice, but was delayed for several days in MKK3-/- mice. In wild type, but not MKK3-/- mice, intraplantar formalin elicited increases in ipsilateral spinal MKK3/6 phosphorylation acutely and again at 9 days postinjection. Phosphorylation of MKK3/6 correlated with phase 2 formalin behavior. Wild type (WT) and MKK3-/- mice both expressed increases in spinal phosphorylated p38, however in WT mice this response began several days earlier, and was of higher magnitude and duration than in MKK3-/- mice. This phosphorylation correlated with the late allodynia. Phosphorylated MKK3/6 was detected only in astrocytes, given that phosphorylated p38 (P-p38) is usually not seen in astrocytes this argues for astrocytic release of soluble mediators that affect p38 phosphorylation in microglia. Taking these data together, MKK3, but not MKK6, is necessary for normal development of chronic pain behavior and phosphorylation of spinal p38.