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)

Monocytes-macrophages which serve as host immune cells to kill pathogens can often be "activated" after exposing to viruses, bacteria, cytokines as well as chemical substances, However, it is paradoxical that highly activated macrophages can be induced to become the suppressor ones by live microbes, microbial products, tumor, and autoimmune disease, although the mechanism remains unknown. Our previous experimental studies have shown that immuno-suppressor activities of suppressor macrophages on T, B and NK cells can be prevented by the treatment with LPS or supernatant in vitro from mitogen-stimulated lymphocytes, while, at the same time, the tumoricidal activities of those macrophages can be kept or even enhanced following the same treatment. This phenomenon was then termed as "immune modulation" For the understanding of its mechanism, we are now undertaking signal transduction in modulated macrophages. Since mitogen-activated protein kinase (MAPK) is an integration point of different signal transduction pathways, its cascade and regulation of activation are being investigated extensively by the assay of electrophoresis mobility shift. Recent results suggested that interaction of ligand-receptor triggers protein tyrosine kinase(PTK) activation leading to Ras-GTP binding with Raf-1 to phosphorylate MAPK kinase (MAPKK), the specific activator of MAPK. It is reported that PKC-alpha can directly phosphorylate or activate Raf-1 in NIH3 T3 cells. Raf-1 (74 KDa), with an intrinsic serine (Ser)-threonine (The) kinase activity, becomes hyperphosphorylated after activation which can be followed by gel mobility shift test. It has also been shown that a variety of extracellular factors stimulate a pair of MAPK p44 and MAPK p42 of MAPK family members. A significant property of activation of ERK 1 and ERK 2 is the requirement for the phosphorylation of both Thr-183 and Tyr-185 (at TEY motif) within in its protein kinase subdomain VIII. More recently, two other MAPK subtypes, p38 MAPK (mammalian equivalents of HOG1 in yeast) and JNK MAPK have been discovered. The requirement for activation of p38 MAPK for both Thr-180 and Tyr-182 (at TGY motif) has been shown. p38 MAPK is important in certain transcriptional regulatory pathways, since it can phosphorylate the following transcriptional factors: 1) Elk at Ser 383/389 for binding with SRE motif; 2). ATF 2 at Ser 69/71, forming a complex with Myc for DNA binding at CRE motif; 3) Max at Ser-62 to combine DNA of E-Box motif. p38 MAPK can be activated by LPS, inflammatory cytokines, such as TNF and IL-1, osmolarity. To examine the possibility that whether activation of Raf-1 and ERK 1, ERK2 and p38 MAPK can be regulated directly or/and differently by PKC and PKA pathways, herbimycin A (Ki = 0.9 mumol/L), a potent PTK inhibitor (J. Immunol. 155:3944-4003, 1995) at 2 mumol/L concentration was utilized to block Ras/Raf-1/MAPK cascade. After pre-incubation of macrophages with herbimycin A for 30 min or 90 min, cells were treated with LPS (10 micrograms/ml) and PMA (100 nmol/L) for 15 min. No inhibition of phosphorylation of Raf-1, MAPK p44 and MAPK p42 in response to LPS and PMA was observed (Fig. 1 and 3). However, forskolin, a cAMP inducer for protein kinase A (PKA) activation, inhibited the phosphorylation of LPS- and PMA-stimulated Raf-1, MAPK p44 and MAPK p42 (Fig. 2 and 4). Similarly, in agreement with a very recent report from David, M et al in NIH, in which they indicated that forskolin (30 mumol/L) inhibited IFN-beta-stimulated ERK activity by U 266 cells (J. Biol. Chem. 271: 4585-4588 1996), we found that the levels of phosphorylations of Raf-1 and ERK1 and ERK2 were declined when forskolin (30 mumol/L) was added to macrophages for 20 min at 37 degrees C prior to the stimulation by LPS and PMA. Interestingly, under the same condition, forskolin (30 mumol/L) stimulated the phosphorylation of LPS- and PMA-triggered p38 MAPK of murine peritoneal suppressor macrophages, suggesting that activatio
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PMID:[Studies on cell signaling immunomodulated murine peritoneal suppressor macrophages: LPS and PMA mediate the activation of RAF-1, MAPK p44 and MAPK p42 and p38 MAPK]. 1068 11

A third calcium-dependent protein kinase (CDPK) gene has been isolated from the human malaria parasite Plasmodium falciparum by vectorette technology. The gene consists of five exons and four introns. The open reading frame resulting from removal of the four introns encodes a protein of 562 amino acid residues with a predicted molecular mass of 65.3 kDa. The encoded protein, termed PfCDPK3, consists of four distinct domains characteristic of a member of the CDPK family and displays the highest homology (46% identity and 69% similarity) to PfCDPK2, the second CDPK of P. falciparum. The N-terminal variable domain is rich in serine/threonine and lysine and contains multiple consensus phosphorylation sites for a range of protein kinases. The catalytic domain possesses all conserved motifs of the protein kinase family except for the highly conserved glutamic acid residue in subdomain VIII, which is replaced by a glutamine residue. The sequence of the junction domain comprising 31 amino acid residues is less conserved. The calmodulin-like regulatory domain contains four EF-hand calcium-binding motifs, each consisting of a loop of 12 amino acid residues which is flanked by two alpha-helices. Southern blotting of genomic DNA digests showed that the Pfcdpk3 gene is present as a single copy per haploid genome. A 2900 nucleotide transcript of this gene is expressed specifically in the sexual erythrocytic stage, indicating that PfCDPK3 is involved in sexual stage-specific events. It is proposed that PfCDPK3 may serve as a link between calcium and gametogenesis of P. falciparum.
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PMID:Sexual stage-specific expression of a third calcium-dependent protein kinase from Plasmodium falciparum. 1076 Jun 1

Kinesin-like calmodulin-binding protein (KCBP) is a novel member of the kinesin superfamily that is involved in cell division and trichome morphogenesis. KCBP is unique among all known kinesins in having a myosin tail homology-4 region in the N-terminal tail and a calmodulin-binding region following the motor domain. Calcium, through calmodulin, has been shown to negatively regulate the interaction of KCBP with microtubules. Here we have used the yeast two-hybrid system to identify the proteins that interact with the tail region of KCBP. A protein kinase (KCBP-interacting protein kinase (KIPK)) was found to interact specifically with the tail region of KCBP. KIPK is related to a group of protein kinases specific to plants that has an additional sequence between subdomains VII and VIII of the conserved C-terminal catalytic domain and an extensive N-terminal region. The catalytic domain alone of KIPK interacted weakly with the N-terminal KCBP protein but strongly with full-length KCBP, whereas the noncatalytic region did not interact with either protein. The interaction of KCBP with KIPK was confirmed using coprecipitation assays. Using bacterially expressed full-length and truncated proteins, we have shown that the catalytic domain is capable of phosphorylating itself. The association of KIPK with KCBP suggests regulation of KCBP or KCBP-associated proteins by phosphorylation and/or that KCBP is involved in targeting KIPK to its proper cellular location.
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PMID:Interaction of a kinesin-like calmodulin-binding protein with a protein kinase. 1078 94

Our recent data suggest that during auto- and allograft recognition in sponges (Porifera), cytokines are differentially expressed. Since the mitogen-activated protein kinase (MAPK) signal transduction modulates the synthesis and release of cytokines, we intended to identify one key molecule of this pathway. Therefore, a cDNA from the marine sponge Suberites domuncula encoding the MAPK was isolated and analyzed. Its encoded protein is 366 amino acids long (calculated Mr 42 209), has a TGY dual phosphorylation motif in protein kinase subdomain VIII and displays highest overall similarity to the mammalian p38 stress activated protein kinase (SAPK2), one subfamily of MAPKs. The sponge protein was therefore termed p38_SD. The overall homology (identity and similarity) between p38_SD and human p38alpha (CSBP2) kinase is 82%. One feature of the sponge kinase is the absence of threonine at position 106. In human p38alpha MAPK this residue is involved in the interaction with the specific pyridinyl-imidazole inhibitor; T106 is replaced in p38_SD by methionine. Inhibition studies with the respective inhibitor SB 203580 showed that it had no effect on the phosphorylation of the p38 substrate myelin basic protein. A stress responsive kinase Krs_SD similar to mammalian Ste20 kinases, upstream regulators of p38, had already previously been found in S. domuncula. The S. domuncula p38 MAPK is phosphorylated after treatment of the animal in hypertonic medium. In contrast, exposure of cells to hydrogen peroxide, heat shock and ultraviolet light does not cause any phosphorylation of p38. It is concluded that sponges, the oldest and most simple multicellular animals, utilize the conserved p38 MAPK signaling pathway, known to be involved in stress and immune (inflammatory) responses in higher animals.
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PMID:The mitogen-activated protein kinase p38 pathway is conserved in metazoans: cloning and activation of p38 of the SAPK2 subfamily from the sponge Suberites domuncula. 1087 30

The Raf kinase family serves as a central intermediate to relay signals from Ras to ERK. The precise molecular mechanism for Raf activation is still not fully understood. Here we report that phosphorylation of Thr598 and Ser601, which lie between kinase subdomains VII and VIII, is essential for B-Raf activation by Ras. Substitution of these residues by alanine (B-RafAA) abolished Ras-induced B-Raf activation without altering the association of B-Raf with other signaling proteins. Phosphopeptide mapping and immunoblotting with phospho-specific antibodies confirmed that Thr598 and Ser601 are in vivo phosphorylation sites induced by Ras. Furthermore, replacement of these two sites by acidic residues (B-RafED) renders B-Raf constitutively active. Con sistent with these data, B-RafAA and B-RafED exhibited diminished and enhanced ability, respectively, to stimulate ERK activation and Elk-dependent transcription. Moreover, functional studies revealed that B-RafED was able to promote NIH 3T3 cell transformation and PC12 cell differentiation. Since Thr598 and Ser601 are conserved in all Raf family members from Caenorhabditis elegans to mammals, we propose that phosphorylation of these two residues may be a general mechanism for Raf activation.
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PMID:Activation of B-Raf kinase requires phosphorylation of the conserved residues Thr598 and Ser601. 1103 10

The mitogen-activated protein kinases (MAPKs) and the cyclin-dependent kinases (CDKs) are key mediators of cell proliferation in response to extracellular signals. Recent additions to each of these families and the identification of kinases with structural features of both have provided insights into fundamental processes, such as cell division and differentiation. To identify novel serine kinases with features of MAPKs or CDKs, a degenerate PCR-based amplification approach was undertaken. The 57- and 52-kDa isoforms of a novel protein kinase, termed NKIATRE, were molecularly cloned from rat brain and jejunum cDNA libraries. Like the MAPKs, NKIATRE has a Thr-Xaa-Tyr motif in kinase subdomain VIII. NKIATRE also shows close homology to the cyclin-dependent kinase class of protein kinases and the cdc2-related kinases NKIAMRE, KKIALRE, and KKIAMRE, containing both conserved inhibitory phosphorylation sites and a putative cyclin-binding domain. Two isoforms of NKIATRE that differ in their carboxy-terminal ends have been identified. A functional nuclear localization signal is specific to the longer 57-kDa alpha isoform. Sequence similarity to the putative human tumor suppressor gene NKIAMRE, which is lost in leukemic patients with chromosome 5q deletions, suggests that NKIATRE may have a role in restricting cell growth or maintaining differentiation.
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PMID:NKIATRE is a novel conserved cdc2-related kinase. 1116 6

Various proteins/enzymes obtained commercially were tested for the presence of endogenously nitrated tyrosine by Western blot analysis omitting reducing agent in the step of SDS-PAGE. Histones II-S and VIII-S, IgG, cAMP-dependent protein kinase (PKA), phosphorylase b, and phosphorylase kinase exhibited strong immunoreactive bands. Histone VI-S, glycogen synthase, lactate dehydrogenase, actin, thyroglobulin, and macroglobulin exhibited moderate immunoreactivity. Histone III-S, casein, acetyl cholinesterase, DNase I, and lipase had only traceable immunoreactivity. Whereas histone VII-S, pyruvate kinase, trypsin, pepsin, chymotrypsin, protease IV, and protease XIII, and glutathione S-transferase lacked immunoreactivity. A variation of immunoreactivity between hypertensive and normaltensive rat hearts was found in the histone-agarose fractions of crude extracts. Additionally, nitrotyrosine immunoreactivity was observed in non-mammalian organisms including Eschericia coli, Saccharomyces cerevisiae and Triticum vulgaris. Upon the treatment of 15 microM peroxynitrite (PN), strong oxidant derived from nitric oxide (NO), the apparent Km of PKA for cAMP increased from approximately 10(-8) to 10(-6) M. The results imply that the varied nitration of tyrosine residues in proteins/enzymes may occur as a post-translational modification in vivo, and such discriminative nitration may be vital in PN/NO-regulated signal transduction cascade.
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PMID:Protein nitration. 1119 83

Granulosa cells play an essential role in follicular development and formation of corpora lutea. Many functions of granulosa-lutein cells are controlled by activation of G protein-coupled receptors and the formation of cyclic AMP (cAMP) by adenylyl cyclase. There are at least nine mammalian adenylyl cyclase isoenzymes, which show different sensitivities towards other signalling systems. The aim of this study was to identify the types of adenylyl cyclase present in human granulosa cells and to investigate its functional regulation by G proteins, calcium and the protein kinase C and A pathways. Granulosa cells were obtained from women undergoing IVF. The cells were maintained in primary culture and they consistently expressed mRNA coding for adenylyl cyclase I, III, VI, VII and IX. The signals for adenylyl cyclase V and VIII were more variable among patients and there was no signal for adenylyl cyclase II. The expression of multiple adenylyl cyclase proteins was confirmed by immunochemistry with subtype-specific antibodies. The formation of cAMP in cultured cells was stimulated many times by hCG (EC(50) value 4.2 iu ml(-1)) and by prostaglandin E(2) (PGE(2); EC(50) = 0.75 micromol l(-1)) in a concentration-dependent manner, thus confirming the presence of receptors coupled positively to G(s). The diterpene forskolin, which stimulates all isoforms of adenylyl cyclase except for adenylyl cyclase IX, increased cAMP formation to higher levels than hCG or PGE(2). The strong stimulation by forskolin indicates that adenylyl cyclase IX is unlikely to be the major source of cyclase activity in these cells. Basal and forskolin- or PGE(2)-stimulated adenylyl cyclase activity was amplified 1.5-2.0 times by phorbol-12,13-dibutyrate, indicating that protein kinase C-sensitive enzymes (for example, adenylyl cyclase types IV, V, VI or VII) may be active in the cells. In contrast, hCG-stimulated activity was inhibited (76 +/- 6%) by phorbol ester. Stimulation of G(i) with the alpha-adrenoceptor agonist clonidine inhibited hCG-induced cyclase activity. This finding indicates that adenylyl cyclase II and IV subtypes, which are stimulated by betagamma subunits released from G(i), are not predominant. Increases in intracellular free calcium concentrations by the ionophore A23187, the calcium-ATPase inhibitor thapsigargin or by fluprostenol, a selective prostanoid FP receptor agonist, which is known to open calcium channels in granulosa cells, or removal of calcium by EGTA, had no significant effects on basal or forskolin-stimulated formation of cAMP. These results indicate that subtypes adenylyl cyclase I, III and VIII, which are activated by calcium, and adenylyl cyclase V and VI, which are inhibited by calcium, are not dominant isoforms in granulosa-lutein cells. The protein kinase A inhibitor H89 had no effects on formation of cAMP; this finding rules out the involvement of adenylyl cyclase V and VI subtypes, which are subjected to negative feedback by protein kinase A. These results indicate that adenylyl cyclase VII is the dominant functional isoenzyme in human granulosa-lutein cells.
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PMID:Characterization of adenylyl cyclases in cultured human granulosa cells. 1122 46

The Raf serine/threonine kinase plays an essential role to relay intracellular signals from the protooncogene Ras to activation of the mitogen-activated protein kinase (MAPK) cascade. The Raf kinase family consists of C-Raf (Raf-1), B-Raf, and A-Raf. Extensive efforts have been made in the last decade to study Raf regulation; however, precise molecular mechanism for Raf activation is still not fully understood. In this report, we discuss the current model of Raf regulation. Here we also report our recent findings that phosphorylation of Thr598 and Ser601, which lie between kinase subdomains VII and VIII, is essential for B-Raf activation by Ras. Substitution of these residues to alanine (B-RafAA) abolished Ras-induced B-Raf activation, without altering the association of B-Raf with other signaling proteins. Phosphopeptide mapping and immunoblotting with phosphospecific antibodies, which selectively recognize Thr598 and Ser601, phosphorylated B-Raf, confirmed that Thr598 and Ser601 are in vivo phosphorylation sites induced by Ras. Further, replacement of these two sites with acidic residues (B-RafED) renders B-Raf constitutively active. Consistent with these data, B-RafAA and B-RafED exhibited diminished and enhanced ability, respectively, to stimulate extracellular signal-regulated kinase (ERK) and Elk-dependent transcription. Moreover, functional studies revealed that B-RafED was able to promote NIH3T3 cell transformation and PC12 cell differentiation. Because Thr598 and Ser601 are conserved in all Raf family members, from Caenorhabditis elegans to mammals, we propose that phosphorylation of these two residues may be a general mechanism for Raf activation.
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PMID:Regulation of the Raf kinase by phosphorylation. 1129 29

The Arabidopsis thaliana somatic embryogenesis receptor kinase 1 (AtSERK1) gene encodes a receptor-like protein kinase that is transiently expressed during embryogenesis. To determine the intrinsic biochemical properties of the AtSERK1 protein, we have expressed the intracellular catalytic domain as a glutathione S-transferase fusion protein in Escherichia coli. The AtSERK1-glutathione S-transferase fusion protein mainly autophosphorylates on threonine residues (K(m) for ATP, 4 x 10(-6) m), and the reaction is Mg(2+) dependent and inhibited by Mn(2+). A K330E substitution in the kinase domain of AtSERK1 abolishes all kinase activity. The active AtSERK1(kin) can phosphorylate inactive AtSERK1(K330E) protein, suggesting an intermolecular mechanism of autophosphorylation. The AtSERK1 kinase protein was modeled using the insulin receptor kinase as a template. On the basis of this model, threonine residues in the AtSERK1 activation loop of catalytic subdomain VIII are potential targets for phosphorylation. AtSERK1 phosphorylation on myelin basic protein and casein showed tyrosine, serine, and threonine as targets, demonstrating that AtSERK1 is a dual specificity kinase. Replacing Thr-468 with either alanine or glutamic acid not only obliterated the ability of the AtSERK1 protein to be phosphorylated but also inhibited phosphorylation on myelin basic protein and casein, suggesting that Thr-468 is essential for AtSERK-mediated signaling.
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PMID:Role of threonines in the Arabidopsis thaliana somatic embryogenesis receptor kinase 1 activation loop in phosphorylation. 1150 54


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