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)

We report the first RNAi-induced phenotypes in mammalian cultured cells using RNA interference mediated by duplexes of 21-nt RNAs. The 21 gene products studied have different functions and subcellular localizations. Knockdown experiments monitored by immunofluorescence and immunoblotting show that even major cellular proteins such as actin and vimentin can be silenced efficiently. Genes were classified as essential or nonessential depending on impaired cell growth after RNA silencing. Phenotypes also involved altered cell morphology and aberrant mitotic arrest. Among the essential genes identified by RNAi for which such information was previously not available are lamin B1, lamin B2, NUP153, GAS41, ARC21, cytoplasmic dynein, the protein kinase cdk1 and both beta- and gamma-actin. Newly defined nonessential genes are emerin and zyxin. Several genes previously characterized by other methods such as knockout of murine genes are included as internal controls and gave identical results when RNAi was used. In the case of two nonessential genes (lamin A/C and zyxin) RNAi provides a recognizable phenotype. Our results complete the characterization of the mammalian nuclear lamins. While lamins A/C appear as nonessential proteins in the mouse embryo and in RNAi treated cultured cells, the two other lamins, B1 and B2, are now identified as essential proteins. Interestingly the inner nuclear membrane protein emerin, thought to be a ligand of lamin A/C, is also a nonessential protein in tissue culture cells.
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PMID:Identification of essential genes in cultured mammalian cells using small interfering RNAs. 1179 20

Aurora-B is an evolutionally conserved protein kinase that regulates several mitotic events including cytokinesis. We previously demonstrated the possible existence of a protein kinase that phosphorylates at least Ser-72 on vimentin, the most widely expressed intermediate filament protein, in the cleavage furrow-specific manner. Here we showed that vimentin-Ser-72 phosphorylation occurred specifically at the border of the Aurora-B-localized area from anaphase to telophase. Expression of a dominant-negative mutant of Aurora-B led to a reduction of this vimentin-Ser-72 phosphorylation. In vitro analyses revealed that Aurora-B phosphorylates vimentin at approximately 2 mol phosphate/mol of substrate for 30 min and that this phosphorylation dramatically inhibits vimentin filament formation. We further identified eight Aurora-B phosphorylation sites, including Ser-72 on vimentin, and then constructed the mutant vimentin in which these identified sites are changed into Ala. Cells expressing this mutant formed an unusually long bridge-like intermediate filament structure between unseparated daughter cells. We then identified important phosphorylation sites for the bridge phenotype. Our findings indicate that Aurora-B regulates the cleavage furrow-specific vimentin phosphorylation and controls vimentin filament segregation in cytokinetic process.
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PMID:Aurora-B regulates the cleavage furrow-specific vimentin phosphorylation in the cytokinetic process. 1245

Previous studies have demonstrated that functional interaction between endothelin (ET)-1 and nitric oxide (NO) involves changes in Ca(2+) mobilization and cytoskeleton in human brain microvascular endothelial cells. The focus of this investigation was to examine the possible existence of analogous interplay between these vasoactive substances and elucidate their signal transduction pathways in human brain capillary endothelial cells. The results indicate that ET-1-stimulated Ca(2+) mobilization in these cells is dose-dependently inhibited by NOR-1 (an NO donor). This inhibition was prevented by ODQ (an inhibitor of guanylyl cyclase) or Rp-8-CPT-cGMPS (an inhibitor of protein kinase G). Treatment of endothelial cells with 8-bromo-cGMP reduced ET-1-induced Ca(2+) mobilization in a manner similar to that observed with NOR-1 treatment. In addition, NOR-1 or cGMP reduced Ca(2+) mobilization induced by mastoparan (an activator of G protein), inositol 1,4,5-trisphosphate, or thapsigargin (an inhibitor of Ca(2+)-ATPase). Interestingly, alterations in endothelial cytoskeleton (actin and vimentin) were associated with these effects. The data indicate for the first time that the cGMP-dependent protein kinase colocalizes with actin. These changes were accompanied by altered levels of phosphorylated vasodilator-stimulated phosphoprotein, which were elevated in endothelial cells incubated with NOR-1 and significantly reduced by ODQ or Rp-8-CPT-cGMPS. The findings indicate a potential mechanism by which the functional interrelationship between ET-1 and NO plays a role in regulating capillary tone, microcirculation, and blood-brain barrier function.
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PMID:ET-1- and NO-mediated signal transduction pathway in human brain capillary endothelial cells. 1252 47

The following study was undertaken to elucidate the cytoskeletal phenotype of neonatal rat cardiac fibroblasts (NNCF) and the signaling pathways coupled to beta-adrenergic receptor stimulated DNA synthesis. The cytoskeletal proteins vimentin, and smooth muscle alpha-actin were detected in NNCF, suggestive of a myofibroblast phenotype. Isoproterenol (ISO) treatment stimulated (3)H-thymidine uptake, and concomitantly increased intracellular cyclic AMP levels. However, cyclic AMP-elevating agents markedly decreased DNA synthesis. Coincident with growth, ISO-stimulated phosphatidylinositol 3-kinase (PI3-K) activity, and the PI3-K inhibitor LY294002 abrogated enzyme activity, and DNA synthesis. Unexpectedly, the serine/threonine kinase protein kinase Balpha (PKBalpha), a putative downstream target of PI3-K, was dephosphorylated following ISO treatment. Despite PKBalpha inactivation, the phosphorylation of its putative downstream target, the pro-apoptotic enzyme glycogen synthase kinase-3alpha was significantly increased in response to ISO. These latter effects of ISO were mimicked by the cyclic AMP-elevating agent forskolin. Lastly, ISO treatment increased p70 ribosomal S6 kinase (p70S6K) phosphorylation, as reflected by an upward electrophoretic mobility shift. The pretreatment with rapamycin abrogated the ISO-mediated mobility shift of p70S6K, and DNA synthesis. Collectively, these data demonstrate that NNCF express a myofibroblast phenotype, and beta-adrenergic agonists promote DNA synthesis via a PI3-K-dependent pathway involving p70S6K. Although unable to suppress ISO-stimulated DNA synthesis, cyclic AMP can influence specific downstream targets of PI3-K highlighting a novel crosstalk between these signaling pathways.
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PMID:Beta-adrenergic receptor-mediated DNA synthesis in neonatal rat cardiac fibroblasts proceeds via a phosphatidylinositol 3-kinase dependent pathway refractory to the antiproliferative action of cyclic AMP. 1265 58

Intermediate filament (IF) networks can be regulated by phosphorylation of unit proteins, such as vimentin, by specific kinases leading to reorganization of the IF filamentous structure. Recently, we identified mitogen-activated protein kinase-activated protein kinase-2 (MAPKAP kinase-2) as a vimentin kinase (Cheng and Lai [1998] J. Cell. Biochem. 71:169-181). Herein we describe the results of further in vitro studies investigating the effects of MAPKAP kinase-2 phosphorylation on vimentin and the effects of the phosphorylation on the filamentous structure. We show that MAPKAP kinase-2 mainly phosphorylates vimentin at Ser-38, Ser-50, Ser-55, and Ser-82, residues all located in the head domain of the protein. Surprisingly, and in stark contrast to phosphorylation by most other kinases, phosphorylation of vimentin by MAPKAP kinase-2 has no discernable effect on its assembly. It suggested that structure disassembly is not the only obligated consequence of phosphorylated vimentin as regulated by other kinases. Finally, a mutational analysis of each of the phosphorylated serine residues in vimentin suggested that no single serine site was primarily responsible for structure maintenance, implying that the retention of filamentous structure may be the result of the coordinated action of several phosphorylated serine sites. This also shed new lights on the functional task(s) of vimentin that is intermediate filament proteins might provide a phosphate reservoir to accommodate the phosphate surge without any structural changes.
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PMID:Retaining of the assembly capability of vimentin phosphorylated by mitogen-activated protein kinase-activated protein kinase-2. 1276 92

Nitric oxide (NO) synthase (NOS) catalyzes the oxidation of L-arginine to NO. NO plays a crucial role in regulating various physiological functions, possibly including junction dynamics via its effects on cAMP and cGMP, which are known modulators of tight junction (TJ) dynamics. Although inducible NOS (iNOS) and endothelial NOS (eNOS) are found in the testis and have been implicated in the regulation of spermatogenesis, their role(s) in TJ dynamics, if any, is not known. When Sertoli cells were cultured at 0.5-1.2 x 10(6) cells/cm(2) on Matrigel-coated dishes or bicameral units, functional TJ barrier was formed when the barrier function was assessed by quantifying transepithelial electrical resistance across the cell epithelium. The assembly of the TJ barrier was shown to associate with a significant plummeting in the levels of iNOS and eNOS, seemingly suggesting that their presence by producing NO might perturb TJ assembly. To further confirm the role of NOS on the TJ barrier function in vitro, zinc (II) protoporphyrin-IX (ZnPP), an NOS inhibitor and a soluble guanylate cyclase inhibitor, was added to the Sertoli cell cultures during TJ assembly. Indeed, ZnPP was found to facilitate the assembly and maintenance of the Sertoli cell TJ barrier, possibly by inducing the production of TJ-associated proteins, such as occludin. Subsequent studies by immunoprecipitation and immunoblotting have shown that iNOS and eNOS are structurally linked to TJ-integral membrane proteins, such as occludin, and cytoskeletal proteins, such as actin, vimentin, and alpha-tubulin. When the cAMP and cGMP levels in these ZnPP-treated samples were quantified, a ZnPP-induced reduction of intracellular cGMP, but not cAMP, was indeed detected. Furthermore, 8-bromo-cGMP, a cell membrane-permeable analog of cGMP, could also perturb the TJ barrier dose dependently similar to the effects of 8-bromo-cAMP. KT-5823, a specific inhibitor of protein kinase G, was shown to facilitate the Sertoli cell TJ barrier assembly. Cytokines, such as TGF-beta and TNF-alpha, known to perturb the Sertoli cell TJ barrier, were also shown to stimulate Sertoli cell iNOS and eNOS expression dose dependently in vitro. Collectively, these results illustrate NOS is an important physiological regulator of TJ dynamics in the testis, exerting its effects via the NO/soluble guanylate cyclase/cGMP/protein kinase G signaling pathway.
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PMID:Regulation of Sertoli cell tight junction dynamics in the rat testis via the nitric oxide synthase/soluble guanylate cyclase/3',5'-cyclic guanosine monophosphate/protein kinase G signaling pathway: an in vitro study. 1281 May 68

Cyclic AMP-elevating agents are highly effective in preventing the loss of dopaminergic neurons that occurs spontaneously in neuronal-glial mesencephalic cultures. We demonstrate here that cAMP causes a concomitant decline in the number of dividing non-neuronal cells, suggesting that inhibition of proliferation contributes to neuroprotection. Consistent with this hypothesis, a transient treatment with the antimitotic cytosine arabinoside, at concentrations that induce long-term repression of glial cell proliferation, mimicked the neuroprotective action of cAMP and also obviated the need for the cyclic nucleotide. Treatment with cAMP-elevating agents reduced the population of OX-42-positive microglial cells and the number of immature astrocytes expressing vimentin and low levels of the astrocytic marker glial fibrillary acidic protein. The effect on the immature astrocytes, however, seemed essential for neuroprotection. Ciliary neurotrophic factor and leukemia inhibitory factor, which stimulate astrocyte differentiation without reducing cell proliferation, failed to reproduce the protective effects of the cyclic nucleotide. Cyclic AMP did not operate by counteracting the action of the astrocyte mitogen epidermal growth factor or by reducing activation of the mitogen-activated protein kinase signaling pathway. The neuroprotective and antiproliferative actions of cAMP, however, were closely mimicked by olomoucine and roscovitine, potent inhibitors of the cyclin-dependent kinase CDK1 that are structurally related to cAMP. Measurement of CDK1 activity confirmed that neuroprotection was closely correlated with inhibition of this kinase by cAMP. In summary, neuroprotection of mesencephalic dopaminergic neurons by cAMP most probably requires the repression of presumptive astrocytes through inhibition of CDK1.
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PMID:Prevention of dopaminergic neuronal death by cyclic AMP in mixed neuronal/glial mesencephalic cultures requires the repression of presumptive astrocytes. 1292 Jan 93

Human papillomavirus E6 oncoproteins induce the proteasomal degradation of several multi-PDZ (PSD95/Dlg/ZO-1) domain-containing proteins such as the human homologue of Drosophila discs large. Binding to PDZ domain-containing proteins is mediated by a PDZ-binding motif contained within the C-terminus of E6. The ability of E6 proteins to induce degradation of PDZ domain-containing proteins correlates with their oncogenic potential. Here we examined the biological effect of this region of the human papillomavirus type 18 E6 oncoprotein on keratinocyte morphology. Our results show that in simian virus 40-immortalized human keratinocytes, stable expression of E6 correlated with the induction of an exaggerated mesenchymal-like morphology and actin cytoskeleton disorganization compared with parental cells. The altered phenotype was accentuated in cells expressing an E6 protein containing a mutation (Arg153Leu) within a protein kinase A recognition motif that abrogates protein kinase A's negative regulation of the activity of the PDZ-binding domain. The E6-induced changes indicated an epithelial-mesenchymal transition and were supported by the finding that E6-expressing cells contained vimentin. Changes to the epithelial phenotype of cells expressing a mutant E6 protein (Thr156Glu) that is unable to degrade discs large was significantly less marked, although they did show evidence of epithelial-mesenchymal transition. These observations imply that the activity of the E6 PDZ-binding motif contributes only to a part of the transition. Further analysis of the E6 cell lines showed a decrease in adherens junction and desmosome formation. Cells expressing a functional PDZ-binding motif showed the greatest disruption of intercellular junction formation, but this did not correlate with a decrease in total cellular levels of the individual components of adhesion junctions. This suggests that the activity of the PDZ-binding motif may have influenced either the assembly or integrity of functional adhesion complexes. An E6-mediated decrease in peripheral membrane levels of PDZ proteins like discs large could be the basis for the enhanced morphological transformation of immortalized keratinocytes.
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PMID:Activity of the human papillomavirus E6 PDZ-binding motif correlates with an enhanced morphological transformation of immortalized human keratinocytes. 1462 86

Intermediate filaments (IFs) continuously exchange between a small, depolymerized fraction of IF protein and fully polymerized IFs. To elucidate the possible role of phosphorylation in regulating this equilibrium, we disrupted the exchange of phosphate groups by specific inhibition of dephosphorylation and by specific phosphorylation and site-directed mutagenesis of two of the major in vivo phosphorylation sites determined in this study. Inhibition of type-1 (PP1) and type-2A (PP2A) protein phosphatases in BHK-21 fibroblasts with calyculin-A, induced rapid vimentin phosphorylation in concert with disassembly of the IF polymers into soluble tetrameric vimentin oligomers. This oligomeric composition corresponded to the oligopeptides released by cAMP-dependent kinase (PKA) following in vitro phosphorylation. Characterization of the (32)P-labeled vimentin phosphopeptides, demonstrated Ser-4, Ser-6, Ser-7, Ser-8, Ser-9, Ser-38, Ser-41, Ser-71, Ser-72, Ser-418, Ser-429, Thr-456, and Ser-457 as significant in vivo phosphorylation sites. A number of the interphase-specific high turnover sites were shown to be in vitro phosphorylation sites for PKA and protein kinase C (PKC). The effect of presence or absence of phosphate groups on individual subunits was followed in vivo by microinjecting PKA-phosphorylated (primarily S38 and S72) and mutant vimentin (S38:A, S72:A), respectively. The PKA-phosphorylated vimentin showed a clearly decelerated filament formation in vivo, whereas obstruction of phosphorylation at these sites by site-directed mutagenesis had no significant effect on the incorporation rates of subunits into assembled polymers. Taken together, our results suggest that elevated phosphorylation regulates IF assembly in vivo by changing the equilibrium constant of subunit exchange towards a higher off-rate.
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PMID:Specific in vivo phosphorylation sites determine the assembly dynamics of vimentin intermediate filaments. 1476 6

Shiga toxin is a bacterial toxin consisting of A and B subunits. Generally, the essential cytotoxicity of the toxin is thought to be mediated by the A subunit, which possesses RNA cleavage activity and thus induces protein synthesis inhibition. We previously reported, however, that the binding of the Shiga toxin 1-B subunit to globotriaosyl ceramide, a functional receptor for Shiga toxin, induces intracellular signals in a manner that is dependent on glycolipid-enriched membrane domains, or lipid rafts. Although the precise role of this signaling mechanism is not known, here we report that Shiga-toxin-mediated intracellular signals induce cytoskeleton remodeling in ACHN cells derived from renal tubular epithelial carcinoma. Using confocal laser scanning microscopy, we observed that Shiga toxin 1-B treatment induces morphological changes in ACHN cells in a time-dependent manner. In addition, the morphological changes were accompanied by the redistribution of a number of proteins, including actin, ezrin, CD44, vimentin, cytokeratin, paxillin, FAK, and alpha- and gamma-tubulins, all of which are involved in cytoskeletal organization. The transient phosphorylation of ezrin and paxillin was also observed during the course of protein redistribution. Experiments using inhibitors for a variety of kinases suggested the involvement of lipid rafts, Src family protein kinase, PI 3-kinase, and RHO-associated kinase in Shiga toxin 1-B-induced ezrin phosphorylation. Shiga toxin 1-B-induced cytoskeletal remodeling should provide an in vitro model that can be used to increase our understanding of the pathogenesis of Shiga-toxin-mediated cell injury and the role of lipid-raft-mediated cell signaling in cytoskeletal remodeling.
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PMID:Shiga toxin binding to globotriaosyl ceramide induces intracellular signals that mediate cytoskeleton remodeling in human renal carcinoma-derived cells. 1526 87


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