Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Protein kinase play important roles in the growth and differentiation of cells. We have isolated cDNA clones from the human megakaryocytic cell line CMK11-5 that encode a novel protein kinase, which we call SPRK (src-homology 3 (SH3) domain-containing proline-rich kinase). The gene sequence predicts an 847-amino acid protein kinase with a unique domain arrangement. An amino-terminal glycine-rich region is followed by an SH3 domain and a kinase domain that is similar to both tyrosine and serine/threonine kinases. Adjacent to the kinase domain are two closely spaced leucine/isoleucine zipper motifs and a stretch of basic amino acids that resembles karyophilic nuclear localization signals. The COOH-terminal half of SPRK is basic, and proline accounts for 24% of the COOH-terminal 216 amino acids. The sprk gene is widely expressed as a 4-kilobase transcript in adult and fetal human tissues. Transfection of 293 cells with a vector encoding an epitope-tagged SPRK results in the expression of a 95-kDa protein. The epitope-tagged SPRK becomes phosphorylated on serine and threonine residues in an in vitro kinase assay, whereas SPRK variants with point mutations in the predicted ATP-binding site fail to become phosphorylated. These data indicate that SPRK has serine/threonine kinase activity. The SH3 domain of SPRK is interrupted by a unique 5-amino acid insert whose location in the SH3 consensus sequence is the same as that of the inserts found in the SH3 domains of neuronal SRC and of the p85 subunit of phosphatidylinositol 3-kinase.
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PMID:Identification and characterization of SPRK, a novel src-homology 3 domain-containing proline-rich kinase with serine/threonine kinase activity. 819 46

To study the role of MAPK cascades in the regulation of naturally occurring human immunodeficiency virus type 1 long terminal repeats (HIV-1 LTRs), we analyzed several HIV-1 LTRs from patients at different stages of disease progression. One of these naturally occurring HIV-1 LTRs contains an insertion termed the most frequent naturally occurring length polymorphism (MFNLP) and exhibited high inducibility upon T cell activation. We found that the protein kinase mixed lineage kinase 3/src-homology 3 domain-containing proline-rich kinase, a specific activator of the stress-activated protein kinase (SAPK)/JNK signaling pathway in T lymphocytes, induces high transcriptional activation of this promoter. Promoter inducibility is inhibited by the SAPK/JNK inhibitor, the JNK binding domain of the JNK interacting protein 1, and Tam-67 (N-terminal deletion mutant of c-Jun). In electrophoretic mobility shift assay, several protein complexes were found to bind to the MFNLP sequence in T cells. We identified AP-1 factors c-Fos and JunB as MFNLP-binding proteins, whose binding is abolished by introducing point mutations in the 3'-half of the MFNLP sequence. Introduction of these point mutations into the MFNLP containing HIV-1 LTR reduced src-homology 3 domain-containing proline-rich kinase -mediated transactivation. These data indicate that the AP-1-like binding site in the MFNLP sequence gives rise to a higher inducibility of natural HIV-LTRs by the SAPK/JNK signaling pathway.
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PMID:Transactivation of naturally occurring HIV-1 long terminal repeats by the JNK signaling pathway. The most frequent naturally occurring length polymorphism sequence introduces a novel binding site for AP-1 factors. 1076 60

Mixed lineage kinase 3 (MLK3) is a serine/threonine protein kinase that functions as a mitogen-activated protein kinase kinase kinase to activate the c-Jun NH(2)-terminal kinase pathway. MLK3 has also been implicated as an I kappa B kinase kinase in the activation of NF-kappa B. Amino-terminal to its catalytic domain, MLK3 contains a Src homology 3 (SH3) domain. SH3 domains harbor three highly conserved aromatic amino acids that are important for ligand binding. In this study, we mutated one of these corresponding residues within MLK3 to deliberately disrupt the function of its SH3 domain. This SH3-defective mutant of MLK3 exhibited increased catalytic activity compared with wild type MLK3 suggesting that the SH3 domain negatively regulates MLK3 activity. We report herein that the SH3 domain of MLK3 interacts with full-length MLK3, and we have mapped the site of interaction to a region between the zipper and the Cdc42/Rac interactive binding motif. Interestingly, the SH3-binding region contains not a proline-rich sequence but, rather, a single proline residue. Mutation of this sole proline abrogates SH3 binding and increases MLK3 catalytic activity. Taken together, these data demonstrate that MLK3 is autoinhibited through its SH3 domain. The critical proline residue in the SH3-binding site of MLK3 is conserved in the closely related family members, MLK1 and MLK2, suggesting a common autoinhibitory mechanism among these kinases. Our study has revealed the first example of SH3 domain-mediated autoinhibition of a serine/threonine kinase and provides insight into the regulation of the mixed lineage family of protein kinases.
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PMID:Autoinhibition of mixed lineage kinase 3 through its Src homology 3 domain. 1159 Jan 55

Nuclear factor-kappaB (NF-kappaB) is the main target of anti-inflammatory therapies in human chronic inflammatory bowel diseases (IBD), Crohn disease, and ulcerative colitis. This study investigates the molecular anti-inflammatory mechanisms of SB203580, an inhibitor of the mitogen-activated protein kinase p38. The murine trinitrobenzene sulfonic acid (TNBS)-induced colitis was used as an established model of human Crohn disease. Here we show that SB203580 improved the clinical condition, reduced intestinal inflammation, and suppressed mRNA levels of pro-inflammatory cytokines elevated upon induction of colitis. Besides p38 kinase activity, the "classical" IkappaB-dependent NF-kappaB pathway was strongly up-regulated during colitis induction, whereas the "alternative" was not. SB203580 treatment resulted in a drastic down-regulation of p38 and NF-kappaB activity. The molecular analysis of NF-kappaB activation revealed that Rip-like interacting caspase-like apoptosis-regulatory protein kinase (RICK), a key component of a pathway leading to NF-kappaB induction, is also strongly inhibited by SB203580. In contrast, SB203580 had no effect on the colitis-induced activation of other potential NF-kappaB-activating kinases such as protein kinase C (PKC), mixed lineage kinase 3, and the oncogene product Cot/TPL2. Thus, the inhibitory effect of SB203580 on NF-kappaB activation is to a large extent mediated by RICK inhibition. RICK is the effector kinase of the intracellular receptor of bacterial peptidoglycan NOD. Because bacterial products are suggested to be the key pathogenic agents triggering IBD, inhibition of the NOD/RICK pathway may serve as a novel target of future therapies in human IBD.
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PMID:Inhibition of RICK/nuclear factor-kappaB and p38 signaling attenuates the inflammatory response in a murine model of Crohn disease. 1569 43

Our previous studies have demonstrated that the JNK signaling pathway plays an important role in ischemic brain injury and is mediated via glutamate receptor 6. Others studies have shown that N-methyl-d-aspartate (NMDA) receptor is involved in the neuroprotection of ischemic preconditioning. Here we examined whether ischemic preconditioning down-regulates activation of the mixed lineage kinase-JNK signaling pathway via NMDA receptor-mediated Akt1 activation. In our present results, ischemic preconditioning could not only inhibit activations of mixed lineage kinase 3, JNK1/2, and c-Jun but also enhanced activation of Akt1. In addition, both NMDA (an agonist of NMDA receptor) and preconditioning showed neuroprotective effects. In contrast, ketamine, an antagonist of NMDA receptor, prevented the above effects of preconditioning. Further studies indicated that LY294002, an inhibitor of phosphoinositide 3-kinase that is an upstream signaling protein of Akt1, could block neuroprotection of preconditioning, and KN62, an inhibitor of calmodulin-dependent protein kinase, also achieved the same effects as LY294002. Therefore, both phosphoinositide 3-kinase and calmodulin-dependent protein kinase are involved in the activation of Akt1 in ischemic tolerance. Taken together, our results indicate that preconditioning can inhibit activation of JNK signaling pathway via NMDA receptor-mediated Akt1 activation and induce neuroprotection in hippocampal CA1 region.
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PMID:Neuroprotective effects of preconditioning ischemia on ischemic brain injury through down-regulating activation of JNK1/2 via N-methyl-D-aspartate receptor-mediated Akt1 activation. 1579 68

MLK3 (mixed lineage kinase 3) is a widely expressed, mammalian serine/threonine protein kinase that activates multiple MAPK pathways. Previously our laboratory used in vivo labeling/mass spectrometry to identify phosphorylation sites of activated MLK3. Seven of 11 identified sites correspond to the consensus motif for phosphorylation by proline-directed kinases. Based on these results, we hypothesized that JNK, or another proline-directed kinase, phosphorylates MLK3 as part of a feedback loop. Herein we provide evidence that MLK3 can be phosphorylated by JNK in vitro and in vivo. Blockade of JNK results in dephosphorylation of MLK3. The hypophosphorylated form of MLK3 is inactive and redistributes to a Triton-insoluble fraction. Recovery from JNK inhibition restores MLK3 solubility and activity, indicating that the redistribution process is reversible. This work describes a novel mode of regulation of MLK3, by which JNK-mediated feedback phosphorylation of MLK3 regulates its activation and deactivation states by cycling between Triton-soluble and Triton-insoluble forms.
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PMID:Dynamic positive feedback phosphorylation of mixed lineage kinase 3 by JNK reversibly regulates its distribution to Triton-soluble domains. 1668 4

Mixed lineage kinase 3 (MLK3) is a mitogen-activated protein kinase kinase kinase member that activates the c-Jun N-terminal kinase (JNK) pathway. Aberrant activation of MLK3 has been implicated in neurodegenerative diseases. Similarly, glycogen synthase kinase (GSK)-3beta has also been shown to activate JNK and contribute to neuronal apoptosis. Here, we show a functional interaction between MLK3 and GSK-3beta during nerve growth factor (NGF) withdrawal-induced cell death in PC-12 cells. The protein kinase activities of GSK-3beta, MLK3, and JNK were increased upon NGF withdrawal, which paralleled increased cell death in NGF-deprived PC-12 cells. NGF withdrawal-induced cell death and MLK3 activation were blocked by a GSK-3beta-selective inhibitor, kenpaullone. However, the MLK family inhibitor, CEP-11004, although preventing PC-12 cell death, failed to inhibit GSK-3beta activation, indicating that induction of GSK-3beta lies upstream of MLK3. In GSK-3beta-deficient murine embryonic fibroblasts, ultraviolet light was unable to activate MLK3 kinase activity, a defect that was restored upon ectopic expression of GSK-3beta. The activation of MLK3 by GSK-3beta occurred via phosphorylation of MLK3 on two amino acid residues, Ser(789) and Ser(793), that are located within the C-terminal regulatory domain of MLK3. Furthermore, the cell death induced by GSK-3beta was mediated by MLK3 in a manner dependent on its phosphorylation of the specific residues within the C-terminal domain by GSK-3beta. Taken together, our data provide a direct link between GSK-3beta and MLK3 activation in a neuronal cell death pathway and identify MLK3 as a direct downstream target of GSK-3beta. Inhibition of GSK-3 is thus a potential therapeutic strategy for neurodegenerative diseases caused by trophic factor deprivation.
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PMID:Glycogen synthase kinase-3beta induces neuronal cell death via direct phosphorylation of mixed lineage kinase 3. 1771 61

S-nitrosylation, as a post-translational protein modification, recently has been paid more and more attention in stroke research. S-nitrosylation regulates protein function by the mechanisms of covalent attachment that control the addition or the removal of nitric oxide (NO) from a cysteine thiol. The derivation of NO is established by the demonstration that, in cerebral neurons, NO mainly generates from neuronal nitric oxide synthase (nNOS) during the early stages of reperfusion. In the past researches, we demonstrate that global ischemia-reperfusion facilitates the activation of glutamate receptor 6 (GluR6) -mediated c-Jun N-terminal kinase (JNK) signaling pathway. The objective of this study is primarily to determine, during the early stages of reperfusion in rat four-vessel occlusion (4-VO) ischemic model, whether nNOS-derived NO affects the GluR6-mediated JNK signaling route via S-nitrosylation which is performed mainly by the biotin switch assay. Here, we show that administration of 7-nitroindazole, an inhibitor of nNOS, or ketamine, an antagonist of N-methyl-d-aspartate receptor (NMDAR), diminishes the increased S-nitrosylation of GluR6 induced by cerebral ischemia-reperfusion. In contrast, 2-amion-5,6-dihydro-6-methyl-4H-1,3-thiazine, an inhibitor of inducible NO synthase does not affect S-nitrosylation of GluR6. Moreover, treatment with sodium nitroprusside (SNP), an exogenous NO donor, increases the S-nitrosylation and phosphorylation of nNOS, leading to the attenuation of the increased S-nitrosylation of GluR6 and the assembling of GluR6* postsynaptic density protein 95 (PSD95)* mixed lineage kinase 3 (MLK3) signaling module induced by cerebral ischemia-reperfusion. The results also show that GluR6 downstream MLK3* mitogen activated protein kinase kinase 4/7* JNK signaling module and nuclear or non-nuclear apoptosis pathways are involved in the above signaling route. However, dithiothreitol (DTT) antagonizes the neuroprotection of SNP. Treatment with DTT alone, as a negative control, prevents S-nitrosylation of proteins, which indicates the existence of endogenously produced S-nitrosylation. These data suggest that GluR6 is S-nitrosylated by endogenous NO in cerebral ischemia-reperfusion, which is possibly correlated with NMDAR* PSD95* nNOS signaling module, and further activates GluR6* PSD95* MLK3 signaling module and JNK signaling pathway. In contrast, exogenous NO donor antagonizes the above action of endogenous NO generated from nNOS. Thus, our results provide the coupling of nNOS with GluR6 by S-nitrosylation during the early stages of ischemia-reperfusion, which can be a new approach for stroke therapy.
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PMID:Coupling between neuronal nitric oxide synthase and glutamate receptor 6-mediated c-Jun N-terminal kinase signaling pathway via S-nitrosylation contributes to ischemia neuronal death. 1867 85

Mixed lineage kinase 3 (MLK3) is a mitogen-activated protein kinase kinase kinase (MAP3K) that activates multiple mitogen-activated protein kinase (MAPK) pathways in response to growth factors, stresses and the pro-inflammatory cytokine, tumor necrosis factor (TNF). MLK3 is required for optimal activation of stress activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) signaling by TNF, however, the mechanism by which MLK3 is recruited and activated by the TNF receptor remains poorly understood. Here we report that both TNF and interleukin-1 beta (IL-1 beta) stimulation rapidly activate MLK3 kinase activity. We observed that TNF stimulates an interaction between MLK3 and TNF receptor associated factor (TRAF) 2 and IL-1 beta stimulates an interaction between MLK3 and TRAF6. RNA interference (RNAi) of traf2 or traf6 dramatically impairs MLK3 activation by TNF indicating that TRAF2 and TRAF6 are critically required for MLK3 activation. We show that TNF also stimulates ubiquitination of MLK3 and MLK3 can be conjugated with lysine 48 (K48)- and lysine 63 (K63)-linked polyubiquitin chains. Our results suggest that K48-linked ubiquitination directs MLK3 for proteosomal degradation while K63-linked ubiquitination is important for MLK3 kinase activity. These results reveal a novel mechanism for MLK3 activation by the pro-inflammatory cytokines TNF and IL-1 beta.
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PMID:Cytokine-induced activation of mixed lineage kinase 3 requires TRAF2 and TRAF6. 1958 14

Mixed lineage kinase 3 (MLK3) is a mitogen activated protein kinase kinase kinase (MAP3K) that activates multiple MAPK signaling pathways. Nuclear factor kappa B (NF-kappaB) is a transcription factor that has important functions in inflammation, immunity and cell survival. We found that silencing mlk3 expression with RNA interference (RNAi) in SKOV3 human ovarian cancer epithelial cells and NIH-3T3 murine fibroblasts led to a reduction in the level of the inhibitor of kappa B alpha (IkappaBalpha) protein. In addition, we observed enhanced basal IkappaB kinase (IKK) activity in HEK293 cells transiently transfected with MLK3 siRNA and in NIH3T3 cells stably expressing MLK3 shRNA (shMLK3). Furthermore, the basal level of NF-kappaB-dependent gene transcription was elevated in shMLK3 cells. Silencing mlk3 expression conferred resistance of cells to etoposide-induced apoptotic cell death and overexpression of wild type MLK3 (MLK3-WT) or kinase-dead MLK3 (MLK3-KD) promoted apoptotic cell death and cleavage of poly (ADP-ribose) polymerase (PARP). Overexpression of MLK3-WT or MLK3-KD enhanced etoposide-induced apoptotic cell death and cleavage of PARP. These data suggest that MLK3 functions to limit IKK activity, and depleting MLK3 helps protect cells from etoposide-induced cell death through activation of IKK-dependent signaling.
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PMID:Mixed lineage kinase 3 negatively regulates IKK activity and enhances etoposide-induced cell death. 1978 5


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