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)

Two-dimensional (2-D) gel electrophoresis has been used to map proteins from various cell types in an effort to eventually link such maps to the sequencing of the entire human genome. While this analysis indicates the cellular disposition and expression of proteins, another application of 2-D gels, the analysis of phosphoproteins, can provide much information as to the assembly and "wiring" of the signal transduction circuits within cells which appear to be enervated by phosphate exchange. The preparation and separation of 32P-labeled proteins is described, as well as various analytical methods, including: the variety of gel systems available for specialist types of analyses, comparing 33P- and 32P-labeling of proteins, imaging techniques, phosphoamino analysis, phosphopeptide separation, identifying the amino acid groups that are phosphorylated, and the identification of phosphoproteins on 2-D gels by immunoprecipitation, corunning of purified proteins, comparative mapping and microsequencing, and by Western blotting. Examples (in brackets) are given of applications in which 2-D phosphogels can be applied, which offer advantages over other techniques. These include: (i) identifying in vivo substrates for kinases (protein kinase C activated by phorbol myristate acetate), (ii) investigating cytokine signaling pathways (tumor necrosis factor and interleukin-1), (iii) investigating the effects of drugs on signaling pathways (okadaic acid, menadione and cyclooxygenase inhibitors), (iv) characterization of specific phosphoproteins (heat-shock protein Hsp27 and stathmin), (v) comparing normal and transformed cells (MRC-5 human lung fibroblasts and their SV-40-transformed counterparts, MRC-5 SV1 cells), (vi) purifying phosphoproteins, (vii) investigating the relationship of protein phosphorylation to stages in the cell cycle (stathmin), (viii) investigating protein/protein interactions, (ix) mapping in vitro kinase substrates (protein kinase C, protein kinase A, and mitogen activated protein kinase activated protein kinase 2), and (x) locating and identifying cellular phosphatases (Hsp27 phosphatase). It is possible that the mapping of phosphoproteins can be linked to other 2-D gel databases and that information derived from these can be used in the future to better understand the signaling mechanisms of normal and cancerous cells.
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PMID:Analysis of cellular phosphoproteins by two-dimensional gel electrophoresis: applications for cell signaling in normal and cancer cells. 805 70

It has recently been recognized that cellular stresses activate certain members of the mitogen-activated protein kinase (MAPK) superfamily. One role of these "stress-activated" MAPKs is to increase the transactivating activity of the transcription factors c-Jun, Elk1, and ATF2. These findings may be particularly relevant to hearts that have been exposed to pathological stresses. Using the isolated perfused rat heart, we show that global ischemia does not activate the 42- and 44-kD extracellular signal-regulated (protein) kinase (ERK) subfamily of MAPKs but rather stimulates a 38-kD activator of MAPK-activated protein kinase-2 (MAPKAPK2). This activation is maintained during reperfusion. The molecular characteristics of this protein kinase suggest that it is a member of the p38/reactivating kinase (RK) group of stress-activated MAPKs. In contrast, stress-activated MAPKs of the c-Jun N-terminal kinase (JNK/SAPKs) subfamily are not activated by ischemia alone but are activated by reperfusion following ischemia. Furthermore, transfection of ventricular myocytes with activated protein kinases (MEKK1 and SEK1) that may be involved in the upstream activation of JNK/ SAPKs induces increases in myocyte size and transcriptional changes typical of the hypertrophic response. We speculate that activation of multiple parallel MAPK pathways may be important in the responses of hearts to cellular stresses.
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PMID:Stimulation of the stress-activated mitogen-activated protein kinase subfamilies in perfused heart. p38/RK mitogen-activated protein kinases and c-Jun N-terminal kinases are activated by ischemia/reperfusion. 875 92

The p38 mitogen-activated protein kinases (MAPK) are activated by cellular stresses and play an important role in regulating gene expression. We have isolated a cDNA encoding a novel protein kinase that has significant homology (57% amino acid identity) to human p38alpha/CSBP. The novel kinase, p38delta, has a nucleotide sequence encoding a protein of 365 amino acids with a putative TGY dual phosphorylation motif. Dot-blot analysis of p38delta mRNA in 50 human tissues revealed a distribution profile of p38delta that differs from p38alpha. p38delta is highly expressed in salivary gland, pituitary gland, and adrenal gland, whereas p38alpha is highly expressed in placenta, cerebellum, bone marrow, thyroid gland, peripheral leukocytes, liver, and spleen. Like p38alpha, p38delta is activated by cellular stress and proinflammatory cytokines. p38delta phosphorylates ATF-2 and PHAS-I, but not MAPK-activated protein kinase-2 and -3, known in vivo and in vitro substrates of p38alpha. We also observed that p38delta was strongly activated by MKK3 and MKK6, while p38alpha was preferentially activated by MKK6. Other experiments showed that a potent p38alpha kinase inhibitor AMG 2372 minimally inhibited the kinase activity of p38delta. Taken together, these data indicate that p38delta is a new member of the p38 MAPK family and that p38delta likely has functions distinct from that of p38alpha.
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PMID:Molecular cloning and characterization of a novel p38 mitogen-activated protein kinase. 929 8

Organization of intermediate filament, a major component of cytoskeleton, is regulated by protein phosphorylation/dephosphorylation, which is a dynamic process governed by a balance between the activities of involved protein kinases and phosphatases. Blocking dephosphorylation by protein phosphatase inhibitors such as okadaic acid (OA) leads to an apparent activation of protein kinase(s) and to genuine activation of phosphatase-regulated protein kinase(s). Treatment of 9L rat brain tumor cells with OA results in a drastically increased phosphorylation of vimentin, an intermediate filament protein. In-gel renaturing assays and in vitro kinase assays using vimentin as the exogenous substrate indicate that certain protein kinase(s) is activated in OA-treated cells. With specific protein kinase inhibitors, we show the possible involvement of the cdc2 kinase- and p38 mitogen-activated protein kinase (p38MAPK)-mediated pathways in this process. Subsequent in vitro assays demonstrate that vimentin may serve as an excellent substrate for MAPK-activated protein kinase-2 (MAPKAPK-2), the downstream effector of p38MAPK, and that MAPKAPK-2 is activated with OA treatment. Comparative analysis of tryptic phosphopeptide maps also indicates that corresponding phosphopeptides emerged in vimentin from OA-treated cells and were phosphorylated by MAPKAPK-2. Taken together, the results clearly demonstrate that MAPKAPK-2 may function as a vimentin kinase in vitro and in vivo. These findings shed new light on the possible involvement of the p38MAPK signaling cascade, via MAPKAPK-2, in the maintenance of integrity and possible physiological regulation of intermediate filaments.
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PMID:Identification of mitogen-activated protein kinase-activated protein kinase-2 as a vimentin kinase activated by okadaic acid in 9L rat brain tumor cells. 977 16

Extracellular signals activate mitogen-activated protein kinase (MAPK) cascades to execute complex cellular programs, like proliferation, differentiation and apoptosis. In mammalian cells, three MAPK families have been characterized: extracellular signal-regulated kinase (ERK), which is activated by growth factors, peptide hormones and neurotransmitters, and Jun kinase (JNK) and p38 MAPK, which are activated by cellular stress stimulus as well as growth factors. This review describes the family of 90 kDa ribosomal S6 kinases (RSK; also known as p90rsk or MAPK-activated protein kinase-1, MAPKAP-K1), which were among the first substrates of ERK to be discovered and which has proven to be a ubiquitous and versatile mediator of ERK signal transduction. RSK is composed of two functional kinase domains that are activated in a sequential manner by a series of phosphorylations. Recently, a family of RSK-related kinases that are activated by ERK as well as p38 MAPK were discovered and named mitogen- and stress-activated protein kinases (MSK). A number of cellular functions of RSK have been proposed. (1) Regulation of gene expression via association and phosphorylation of transcriptional regulators including c-Fos, estrogen receptor, NFkappaB/IkappaB alpha, cAMP-response element-binding protein (CREB) and CREB-binding protein; (2) RSK is implicated in cell cycle regulation in Xenopus laevis oocytes by inactivation of the Myt1 protein kinase leading to activation of the cyclin-dependent kinase p34cdc2; (3) RSK may regulate protein synthesis by phosphorylation of polyribosomal proteins and glycogen synthase kinase-3; and (4) RSK phosphorylates the Ras GTP/GDP-exchange factor, Sos leading to feedback inhibition of the Ras-ERK pathway.
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PMID:Role and regulation of 90 kDa ribosomal S6 kinase (RSK) in signal transduction. 1041 21

p38 mitogen-activated protein kinases (p38-MAPKs) are activated by cytokines, cellular stresses, growth factors, and hormones. We show here that p38-MAPKs are activated upon stimulation by thyroid-stimulating hormone (TSH) or cAMP. TSH caused the phosphorylation of p38-MAPK in Chinese hamster ovary cells stably transfected with the human TSH receptor but not in wild-type Chinese hamster ovary cells. The effect of TSH was fully mimicked by the adenylyl cyclase activator, forskolin, and by a permeant analog of cAMP. The effect of forskolin was reproduced in FRTL5 rat thyroid cells. TSH also stimulated the phosphorylation of MAPK kinase 3 or 6, over the same time scale as that of p38-MAPKs. TSH and forskolin stimulated the activity of the alpha-isoform of p38-MAPK assayed by phosphorylation of the transcription factor ATF2. The activity of MAPK-activated protein kinase-2 was stimulated by TSH and forskolin. This stimulation was abolished by SB203580, a specific inhibitor of p38-MAPKs. The protein kinase A inhibitor H89 inhibited the stimulation of phosphorylation of p38-MAPKs by forskolin, whereas inhibitors of protein kinase C, p70(S6k), and phosphatidylinositol 3-kinase were ineffective. Expression of the dominant negative form of Rac1, but not that of Ras, blocked forskolin-induced p38-MAPK activation. Diphenylene iodonium, a potent inhibitor of NADPH oxidase(s), and ascorbic acid, an effective free radical scavenger, suppressed TSH- or forskolin-stimulated p38-MAPK phosphorylation, indicating that the generation of reactive oxygen species plays a key role in signaling from cAMP to p38-MAPKs. Inhibition of the p38-MAPK pathway with SB203580 partially but significantly, attenuates cAMP- and TSH-induced expression of the sodium iodide symporter in FRTL-5 cells. These results point to a new signaling pathway for the G(s)-coupled TSH receptor, involving cAMP, protein kinase A, Rac1, and reactive oxygen species and resulting in the activation of a signaling kinase cascade that includes MAPK kinase 3 or 6, p38-MAPK, and MAPK-activated protein kinase-2.
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PMID:Thyroid-stimulating hormone and cyclic AMP activate p38 mitogen-activated protein kinase cascade. Involvement of protein kinase A, rac1, and reactive oxygen species. 1100 68

The current study investigates the activation in vivo and regulation of the expression of components of the p38 mitogen-activated protein kinase (MAPK) pathway during gonadotropin-induced formation and development of the rat corpus luteum, employing a sequential PMSG/human CG (hCG) treatment paradigm. We postulated that the p38 MAPK pathway could serve to promote phosphorylation of key substrates during luteal maturation, since maturing luteal cells, thought to be cAMP-nonresponsive, nevertheless maintain critical phosphoproteins. Both p38 MAPK and its upstream activator MAPK kinase-6 (MKK6) were found to be chronically activated during the luteal maturation phase, with activation detected by 24 h post hCG and maintained through 4 days post hCG. The p38 MAPK downstream protein kinase target termed MAPK-activated protein kinase-3 (MAPKAPK-3) was newly induced at both mRNA and protein levels during luteal formation and maturation, while mRNA and protein expression of the closely related MAPKAPK-2 diminished. Two potential substrates for MAPKAPKs, the small heat shock protein HSP-27 and the cAMP regulatory element binding protein CREB, were monitored in vivo for phosphorylation. HSP-27 phosphorylation was not modulated during luteal maturation. In contrast, we observed sustained luteal-phase CREB phosphorylation in vivo, consistent with upstream MKK6/p38 MAPK activation and MAPKAPK-3 induction. MAPKAPK-3-specific immune complex kinase assays provided direct evidence that MAPKAPK-3 was in an activated state during luteal maturation in vivo. Cellular inhibitor studies indicated that an intact p38 MAPK path was required for CREB phosphorylation in a cellular model of luteinization, as treatment of luteinized granulosa cells with the p38 MAPK inhibitor SB 203580 strongly inhibited CREB phosphorylation. Transient transfection studies provided direct evidence that MAPKAPK-3 was capable of signaling to activate CREB transcriptional activity, as assessed by means of GAL4-CREB fusion protein construct coexpressed with GAL4-luciferase reporter construct. Introduction of wild-type, but not kinase-dead mutant, MAPKAPK-3 cDNA, into a mouse ovarian cell line stimulated GAL4-CREB- dependent transcriptional activity approximately 3-fold. Thus MAPKAPK-3 is indeed uniquely poised to support luteal maturation through the phosphorylation and activation of the nuclear transcription factor CREB.
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PMID:Developmental regulation of mitogen-activated protein kinase-activated kinases-2 and -3 (MAPKAPK-2/-3) in vivo during corpus luteum formation in the rat. 1132 54

Although mast cells accumulate within the mucosal epithelial layer of patients with allergic rhinitis and bronchial asthma, the responsible chemotactic factors are undefined. We investigated whether mast cells sensitized with Ag-specific IgE migrate toward the Ag. MC/9 mast cells sensitized with anti-DNP IgE migrated toward DNP-conjugated human serum albumin. This migration was directional, and the degree was stronger than that induced by stem cell factor. IL-3 and stem cell factor-dependent cultured mast cells derived from mouse bone marrow also migrated toward the Ag. Subsequent migration mediated by the Fc(epsilon)RI was significantly inhibited by incubating the cells with Y-27632, a Rho-associated coiled-coil-forming protein kinase inhibitor, or with SB203580, a p38 mitogen-activated protein kinase (MAPK) inhibitor. Both p38 MAPK and MAPK-activated protein kinase (MAPKAPK)2 were activated following Fc(epsilon)RI aggregation, and activation of MAPKAPK2 was almost completely inhibited by 10 microM SB203580. Wortmannin or a low concentration of SB203580 partially inhibited MAPKAPK2, but did not block mast cell migration. In contrast, Y-27632 did not affect the activation of MAPKAPK2. These results indicate that Ag works not only as a stimulant for allergic mediators from IgE-sensitized mast cells, but also as a chemotactic factor for mast cells. Both p38 MAPK activation and Rho-dependent activation of Rho-associated coiled-coil-forming protein kinase may be required for Fc(epsilon)RI-mediated cell migration.
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PMID:Sensitized mast cells migrate toward the antigen: a response regulated by p38 mitogen-activated protein kinase and Rho-associated coiled-coil-forming protein kinase. 1149 18

The p38 mitogen-activated protein kinase (MAPK) pathway plays an important role in cellular responses to inflammatory stimuli and environmental stress. p38 regulated/activated protein kinase (PRAK, also known as mitogen-activated protein kinase activated protein kinase 5 [MAPKAPK5]) functions downstream of p38alpha and p38beta in mediating the signaling of the p38 pathway. Immunostaining revealed that endogenous PRAK was predominantly localized in the cytoplasm. Interestingly, ectopically expressed PRAK was localized in the nucleus and can be redistributed by coexpression of p38alpha or p38beta to the locations of p38alpha and p38beta. Mutations in the docking groove on p38alpha/p38beta, or the p38-docking site in PRAK, disrupted the PRAK-p38 interaction and impaired the ability of p38alpha and p38beta to redistribute ectopically expressed PRAK, indicating that the location of PRAK could be controlled by its docking interaction with p38alpha and p38beta. Although the majority of PRAK molecules were detected in the cytoplasm, PRAK is consistently shuttling between the cytoplasm and the nucleus. A sequence analysis of PRAK shows that PRAK contains both a putative nuclear export sequence (NES) and a nuclear localization sequence (NLS). The shuttling of PRAK requires NES and NLS motifs in PRAK and can be regulated through cellular activation induced by stress stimuli. The nuclear content of PRAK was reduced after stimulation, which resulted from a decrease in the nuclear import of PRAK and an increase in the nuclear export of PRAK. The nuclear import of PRAK is independent from p38 activation, but the nuclear export requires p38-mediated phosphorylation of PRAK. Thus, the subcellular distribution of PRAK is determined by multiple factors including its own NES and NLS, docking interactions between PRAK and docking proteins, phosphorylation of PRAK, and cellular activation status. The p38 MAPKs not only regulate PRAK activity and PRAK activation-related translocation, but also dock PRAK to selected subcellular locations in resting cells.
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PMID:Regulation of PRAK subcellular location by p38 MAP kinases. 1280 55

Cdc14 is a member of the dual-specificity phosphatase family, which is essential for faithful cell cycle progression in eukaryotic cells of different origin. The function of human Cdc14A (hCdc14A), however, has not been fully elucidated as only few physiological substrates have been identified. To gain insight into the biological role of Cdc14A, we performed a yeast two-hybrid screen designed to isolate substrates of this human phosphatase. Using this genetic approach, we here report the identification of Erk3, an atypical mitogen-activated protein kinase (MAPK), as a specific binding partner of hCdc14A. GST pull-down assays show that Erk3 interacts directly with hCdc14A in vitro via its unique C-terminal domain. Furthermore, biochemical analysis reveals that hCdc14A can remove cyclin-dependent kinase (Cdk)-mediated phosphorylation of Erk3 in vitro raising the possibility that Erk3 may be a potential substrate for hCdc14A in vivo. Consistent with a physiologically relevant cross-talk in vivo, we find that Cdc14A forms a stable complex with Erk3 in human cells independent of its intrinsic phosphatase activity but mediated by its regulatory C-terminal domain. We show that hCdc14A impacts the emerging signaling pathway between Erk3 and MK5, a MAPK-activated protein kinase. We document that hCdc14A upregulation leads to redistribution of the Erk3 substrate MK5 from the nucleus to the cytoplasm. In addition, we find that hCdc14A stabilizes complex formation between Erk3 and its binding partner cyclin D3, a D-type cyclin implicated in both cellular proliferation and differentiation. Collectively, our findings suggest an intimate functional relationship between the Cdc14A phosphatase and the Erk3 kinase in signaling pathways that regulate key cell-fate decisions in human cells.
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PMID:A functional link between the human cell cycle-regulatory phosphatase Cdc14A and the atypical mitogen-activated kinase Erk3. 1823 25


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