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)

The HIV-1 Vpu protein induces the proteolysis of CD4 in the endoplasmic reticulum (ER) and enhances the release of virus particles from the plasma membrane. The two biological activities of HIV-1 Vpu appear to be reconstituted in distinct membrane compartments of the mammalian cell. We carried out experiments to understand the role of Vpu sequences in membrane trafficking of the Vpu protein and to gain insights into Vpu-mediated proteolytic reactions. To this end, we generated CD4/Vpu hybrid proteins and analyzed their biochemical and biological properties in HeLa cells. We show here that all hybrid proteins are delivered to the plasma membrane undergoing endo-H-resistant modifications in the Golgi complex. Importantly, a hybrid protein bearing the CD4 extracellular domain and full-length Vpu induced the degradation of HIV envelope glycoproteins bearing the transmembrane and cytoplasmic domains of CD4 (Vpu-responsive elements, VRE). Glycoproteins lacking the VRE are stable under these conditions. In addition, a hybrid protein having the extracellular-transmembrane domains of CD4 and the Vpu cytoplasmic domain was only partially active in inducing the degradation of Vpu-sensitive proteins. These results suggest that the Vpu transmembrane domain is capable of regulating Vpu activity in the cell. Mutational studies have further demonstrated that casein kinase-2 phosphorylation is critically important in the degradation reaction, but does not regulate membrane trafficking of the CD4/Vpu hybrid proteins. We also show that the CD4 extracellular domain appended to the Vpu protein is protected from degradation while existing in a complex with Vpu-sensitive ectodomains. Taken together, these studies have revealed that the Vpu protein does not possess sequences that have the ability to sequester CD4 in the intracellular compartments of mammalian cells and that the Vpu protein tethered to the CD4 extracellular domain was biologically active in inducing the degradation of VRE-bearing glycoproteins in the ER.
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PMID:The human immunodeficiency virus type 1 Vpu protein tethered to the CD4 extracellular domain is localized to the plasma membrane and is biologically active in the secretory pathway of mammalian cells: implications for the mechanisms of Vpu function. 865 6

Calcium is a second messenger that controls a wide variety of cellular functions. Because of its multiple actions, there is a stringent requirement for calcium homeostasis, and this is achieved in part by a system of transport and storage proteins such as calreticulin located in the endoplasmic reticulum. Calreticulin is also found in the nucleus, suggesting that it may have a role in transcriptional regulation. It has been reported that calreticulin can inhibit steroid-regulated gene transcription by preventing receptor binding to DNA. Here we report that overexpression of the calreticulin gene in B16 mouse melanoma cells resulted in a decrease in retinoic acid (RA)-stimulated reporter gene expression. Gel shift analysis showed that purified calreticulin inhibited the binding of endogenous RAR to a beta-RA response element oligonucleotide, only if added prior to the addition of the oligonucleotide. Co-immunoprecipitation studies suggest a physical interaction between RAR and calreticulin. Transfection of the calreticulin gene into B16 cells inhibited the RA induction of protein kinase Calpha, a marker of RA-induced differentiation. We also found that cyclic AMP increased the expression of calreticulin. Cyclic AMP may act to antagonize RA action by both decreasing RAR expression (Y. Xiao, D. Desai, T. Quick, and R. M. Niles, J. Cell Physiol., in press) and stimulating calreticulin levels.
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PMID:Inhibition of retinoic acid receptor function and retinoic acid-regulated gene expression in mouse melanoma cells by calreticulin. A potential pathway for cyclic AMP regulation of retinoid action. 866 62

The effects of expression of the Drosophila melanogaster Trpl protein, which is thought to encode a putative Ca2+ channel [Phillips, Bull and Kelly (1992) Neuron 8, 631-642], on divalent cation inflow in Xenopus laevis oocytes were investigated. The addition of extracellular Ca2+ ([Ca2+]0) to oocytes injected with trpl cRNA and to mock-injected controls, both loaded with the fluorescent Ca2+ indicator fluo-3, induced a rapid initial and a slower sustained rate of increase in fluorescence, which were designated the initial and sustained rates of Ca2+ inflow respectively. Compared with mock-injected oocytes, trpl-cRNA-injected oocytes exhibited a higher resting cytoplasmic free Ca2+ concentration ([Ca2+]i), and higher initial and sustained rates of Ca2+ inflow in the basal (no agonist) states. The basal rate of Ca2+ inflow in trpl-cRNA-injected oocytes increased with (1) an increase in the time elapsed between injection of trpl cRNA and the measurement of Ca2+ inflow, (2) an increase in the amount of trpl cRNA injected and (3) an increase in [Ca2+]0. Gd3+ inhibited the trpl cRNA-induced basal rate of Ca2+ inflow, with a concentration of approx. 5 microM Gd3+ giving half-maximal inhibition. Expression of trpl cRNA also caused an increase in the basal rate of Mn2+ inflow. The increases in resting [Ca2+]1 and in the basal rate of Ca2+ inflow induced by expression of trpl cRNA were inhibited by the calmodulin inhibitors W13, calmodazolium and peptide (281-309) of (Ca2+ and calmodulin)-dependent protein kinase II. A low concentration of exogenous calmodulin (introduced by microinjection) activated, and a higher concentration inhibited, the trpl cRNA-induced increase in basal rate of Ca2+ inflow. The action of the high concentration of exogenous calmodulin was reversed by W13 and calmodazolium. When rates of Ca2+ inflow in trpl-cRNA-injected oocytes were compared with those in mock-injected oocytes, the guanosine 5'-[beta-thio]diphosphate-stimulated rate was greater, the onset of thapsigargin-stimulated initial rate somewhat delayed and the inositol 1,4,5-trisphosphate-stimulated initial rate markedly inhibited. It is concluded that (1) the divalent cation channel activity of the Drosophila Trpl protein can be detected in Xenopus oocytes: (2) in the environment of the Xenopus oocyte the Trpl channel admits some Mn2+ as well as Ca2+, is activated by cytoplasmic free Ca2+ (through endogenous calmodulin) and by a trimeric GTP-binding regulatory protein, but does not appear to be activated by depletion of Ca2+ in the endoplasmic reticulum; and (3) expression of the Trpl protein inhibits the process by which the release of Ca2+ from intracellular stores activates endogenous store-activated Ca2+ channels.
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PMID:Expression of Drosophila trpl cRNA in Xenopus laevis oocytes leads to the appearance of a Ca2+ channel activated by Ca2+ and calmodulin, and by guanosine 5'[gamma-thio]triphosphate. 867 Jan 54

Ca2+/calmodulin- and cAMP-dependent protein kinase activities were characterized in two subcellular membrane samples. Membranes from rat lacrimal gland were isolated by differential and density gradient centrifugation into six density windows. The present study focused on membranes from density windows III and V which contain mixtures of apical, Golgi, endosomal, and endoplasmic reticulum membranes in different proportions. Phosphorylation of membrane proteins was measured by incubating the samples in [g-32P]ATP and separating the proteins by discontinuous SDS-PAGE followed by autoradiography. The amount of phosphate incorporated into specific peptide bands was quantified by densitometry. Ca2+/calmodulin-dependent protein kinase phosphorylated a 52,000 MW peptide in membranes from both density windows with a maximal increase from 0.3 to 66 microM free Ca2+. Trifluoperazine and promethazine, two inhibitors of Ca2+/calmodulin-dependent protein kinases, inhibited this phosphorylation. cAMP-dependent protein kinase phosphorylated a 22,000 MW peptide and a 91,000 MW peptide which were present in membranes from density window III only. We conclude that a Ca2+/calmodulin-dependent protein kinase activity is present in membranes from both density window III and V whereas a cAMP-dependent protein kinase activity is present only in membranes from density window III.
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PMID:Protein phosphorylation in Golgi, endosomal, and endoplasmic reticulum membrane fractions of lacrimal gland. 867 Jul 24

Osteopontin is an acidic phosphoprotein containing casein kinase II (CKII) phosphorylatable sites and an acidic amino acid cluster. The metabolically 32P-labelings of both serines and threonines in vitro in osteopontin immunoprecipitated from rat osteoblast-like ROS 17/2.8 cells may suggest that casein kinase II catalyzes this modification. The enzyme occurs in microsomal fractions of rat osteoblast-like ROS 17/2.8 cells. Subcellular fractions containing endoplasmic reticulum and Golgi apparatus were isolated by differential centrifugation and were identified according to their ultrastructures and the presence of marker enzymes such as glucose-6-phosphatase and thiamine pyrophosphatase, respectively. both fractions phosphorylated the partially dephosphorylated osteopontin and the specific substrate peptide RRREEETEEE. Endoplasmic reticulum-catalyzed peptide phosphorylation was 2.7 times lower than that of Golgi although both endoplasmic reticulum- and Golgi-catalyzed peptide reactions were 50% inhibited by 20 and 100 ng/ml heparin, respectively. Western blot analysis revealed that both fractions contained osteopontin and microsomal CKII. Furthermore, microsomal CKII was immunogold-labeled in endoplasmic reticulum and Golgi apparatus. Heparin inhibition and utilization of [gamma-32P]GTP as a phosphate donor by both fractions confirmed their capacity to phosphorylate osteopontin. The results suggest that microsomal CKII modifies the acidic matrix proteins during transportation. These matrix phosphoproteins may participate in the mineralization process of hard tissues.
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PMID:Microsomal casein kinase II in endoplasmic reticulum- and Golgi apparatus-rich fractions of ROS 17/2.8 osteoblast-like cells: an enzyme that modifies osteopontin. 867 66

We have extensively purified three of the hepatic microsomal intralumenal Ca2+-binding proteins, CBP1, CBP2, and CBP3, which were originally described by Van et al. [(1989) J. Biol. Chem. 264, 17494-17501]. These apparently homogeneous preparations showed only single 45Ca2+ binding bands. On the basis of the peptide sequence, CBP2 was found to be highly homologous with the previously described protein ERp72. Similarly, CBP3 was identical to calreticulin and CBP1 had some homology to calmodulin. Contrary to the report of Van et al. (1989), we found that CBP2 had little thiol:protein disulfide oxidoreductase activity. Of the three purified preparations, only CBP2 exhibited apparent intrinsic protein kinase activity. This activity was found to be due to contamination of the CBP2 preparation by an extremely low concentration of tightly bound casein kinase 2 (CK2). In line with this observation, the phosphorylation was inhibited by heparin, removed by antibody to CK2, and stimulated by spermine. Furthermore, CBP2 was readily phosphorylated in vitro by added CK2 but only slowly phosphorylated by several other protein kinases. Thus, the persistence of CK2 in a highly purified preparation of CBP2 along with several other lines of evidence presented in this study might suggest that the protein CBP2 is a physiologically relevant substrate for CK2. Furthermore, these data suggest that CK2 might be localized in the lumen of the endoplasmic reticulum and that the phosphorylation of CBP2 in the lumen may play a role in the chaperone activity attributed to this protein.
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PMID:Evidence that casein kinase 2 phosphorylates hepatic microsomal calcium-binding proteins 1 and 2 but not 3. 867 86

Recent studies have demonstrated that opioid agonists affect the cytosolic Ca2+ concentration ([Ca2+]i) either by regulating plasma membrane Ca(2+)-channel activity or by mobilizing intracellular Ca2+ stores. The present report documents the [Ca2+]i increase induced by opioid agonists in a human neuroblastoma cell line, SK-N-BE, expressing delta-opioid receptors. In the presence, as well as in the absence, of extracellular Ca2+, opioid agonists enhanced significantly [Ca2+]i, whereas carbachol, known to mobilize specifically inositol 1,4,5-trisphosphate-sensitive intracellular Ca2+ stores, acted only in the presence of extracellular Ca2+. The opioid-induced increase in [Ca2+]i was not affected by treatments modifying the trimeric Gl, Go, and Gs protein transduction mechanisms or the activity of adenylyl cyclase. The Ca(2+)-ATPase pump-inhibiting sesquiterpene lactone, thapsigargin, did not modify the opioid-induced [Ca2+]i response, whereas it abolished the effects of carbachol. The Ryana speciosa alkaloid, ryanodine, at concentrations known to block endoplasmic reticulum ryanodine receptors, decreased significantly the response to opioids without affecting the effects of carbachol. Thus, our results suggest that, in SK-N-BE cells, delta-opioid receptors mobilize Ca2+ from intracellular ryanodine-sensitive stores and the mechanism involved is independent of Gl/Go Gs proteins and protein kinase A activation.
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PMID:The delta-opioid receptor regulates activity of ryanodine receptors in the human neuroblastoma cell line SK-N-BE. 893 79

Calcium ions (Ca2+) are involved in the regulation of many cellular activities. The Ca-ATPase(s) of the plasma membrane and of the endoplasmic reticulum play an important role in controlling the intracellular Ca2+ concentration. Therefore, it is not unexpected that these enzymes are modulated by different factors. The activity of the plasma membrane Ca-ATPase is modified by the amount of negatively charged phospholipids surrounding the enzyme. The regulation of the endoplasmic reticulum Ca-ATPase depends on the phosphorylation of phospholamban by cAMP- and cGMP-dependent protein kinase. These two different Ca2+ transport ATPases are present in both visceral and vascular smooth muscle, but tissue- and species-dependent differences in their relative amount have been observed. In this article we will review the characteristics of Ca-ATPases of the smooth muscle.
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PMID:Plasma membrane and sarcoplasmic reticulum Ca-ATPase and smooth muscle. 893 5

The influence of protein kinase A activity on transport of newly synthesized vesicular stomatitis virus G glycoprotein along the exocytic pathway was examined. Transport of vesicular stomatitis virus G glycoprotein to the cell surface was inhibited by N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89), a selective inhibitor of protein kinase A. This block occurred at the exit of the Golgi complex, whereas transport through the Golgi compartments or from the endoplasmic reticulum to the Golgi was decreased in the presence of H-89. As judged by immunofluorescence endoplasmic reticulum to Golgi transport was accelerated in cells incubated with activators of protein kinase A such as isobutylmethylxanthine (IBMX) or forskolin (FK). Treatment with IBMX and FK also increased transport from the trans-Golgi network to the cell surface. During incubation with IBMX and FK, the organization of the Golgi complex was altered showing intercisternae fusion and miscompartmentalization of resident proteins. These structural changes affected both the kinetics of acquisition of endoglycosidase H resistance and transport activities. These data support a differential regulatory role for protein kinase A in different transport steps along the exocytic pathway. In particular, transport from the trans-Golgi network to the cell surface was dependent on protein kinase A activity. In addition, the results suggest the involvement of this enzyme on the maintenance of the Golgi complex organization.
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PMID:A regulatory role for cAMP-dependent protein kinase in protein traffic along the exocytic route. 894 80

Rat hepatic Golgi apparatus-rich fractions were utilized in an in vitro phosphorylation system containing [gamma-32P]ATP to investigate the phosphorylation of apolipoproteins (apo) B48 and B100. Our results demonstrate that the Golgi apparatus contains a kinase(s) that phosphorylates both apoB48 and apoB100 as well as 290- and 460-kDa proteins recognized by antibody to apoB. We refer to the latter proteins as apoB57 and apoB90, respectively. Phosphorylations in the presence of Triton X-100, which increases the permeability of the membranes, or alamethicin, an ionophore that facilitates transmembrane diffusion of ATP, indicate that the active site of the kinase is on the luminal side of the membranes. However, studies with EDTA and EGTA, which are inhibitory to the kinase, suggest binding sites for Mg2+ and perhaps Ca2+ on the cytosolic membrane face. Phosphorylation of apoB was not stimulated by cAMP nor inhibited by protein kinase inhibitor peptide (5-24), indicating that cAMP dependent protein kinase was not involved in the phosphorylation process. Sodium carbonate treatment of the phosphorylated fraction, which permits separation of membrane and luminal contents, revealed that phosphorylated apoB90 and apoB57 are associated primarily with the membrane, whereas phosphorylated apoB48 is found in luminal contents as well as with the membranes. Phosphorylated apoB100 was found primarily with the membrane fraction. No evidence was found for phosphorylation of apoB in rough endoplasmic reticulum fractions. These studies demonstrate the importance of the Golgi apparatus as a subcellular site for the phosphorylation of apoB and suggest that apoB phosphorylation may be important in the assembly and secretion of apoB-containing lipoproteins.
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PMID:Role of the Golgi apparatus in the phosphorylation of apolipoprotein B. 894 Jan 63


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