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)

G protein-coupled receptor kinases are well characterized for their ability to phosphorylate and desensitize G protein-coupled receptors (GPCRs). In addition to phosphorylating the beta2-adrenergic receptor (beta2AR) and other receptors, G protein-coupled receptor kinase 2 (GRK2) can also phosphorylate tubulin, a nonreceptor substrate. To identify novel nonreceptor substrates of GRK2, we used two-dimensional gel electrophoresis to find cellular proteins that were phosphorylated upon agonist-stimulation of the beta2AR in a GRK2-dependent manner. The ribosomal protein P2 was identified as an endogenous HEK-293 cell protein whose phosphorylation was increased following agonist stimulation of the beta2AR under conditions where tyrosine kinases, PKC and PKA, were inhibited. P2 along with its other family members, P0 and P1, constitutes a part of the elongation factor-binding site connected to the GTPase center in the 60S ribosomal subunit. Phosphorylation of P2 is known to regulate protein synthesis in vitro. Further, P2 and P1 are shown to be good in vitro substrates for GRK2 with K(M) values approximating 1 microM. The phosphorylation sites in GRK2-phosphorylated P2 are identified (S102 and S105) and are identical to the sites known to regulate P2 activity. When the 60S subunit deprived of endogenous P1 and P2 is reconstituted with GRK2-phosphorylated P2 and unphosphorylated P1, translational activity is greatly enhanced. These findings suggest a previously unrecognized relationship between GPCR activation and the translational control of gene expression mediated by GRK2 activation and P2 phosphorylation and represent a potential novel signaling pathway responsible for P2 phosphorylation in mammals.
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PMID:Beta 2-adrenergic receptor stimulated, G protein-coupled receptor kinase 2 mediated, phosphorylation of ribosomal protein P2. 1237 28

Properties and regulation of the human organic cation (OC) transporter type 2 (hOCT2) expressed in HEK-293 cells were extensively characterized using the fluorescent OC 4-[4-(dimethylamino)styryl]-N-methylpyridinium (ASP(+)). ASP(+) uptake was electrogenic and inhibited by TPA(+) (EC(50) = 2.7 microM), tetraethylammonium (EC(50) = 35 microM), cimetidine (EC(50) = 36 microM), or quinine (EC(50) = 6.7 microM). Stimulation with carbachol or ATP decreased initial uptake by 44 +/- 3 (n = 14) and 34 +/- 4% (n = 21), respectively, independently of PKC but dependent on phosphatidylinositol 3-kinase (PI3K). PKA stimulation decreased uptake by 18 +/- 4% (n = 40). Inhibition of calmodulin (CaM), Ca(2+)/CaM-dependent kinase II, or myosin light chain kinase decreased uptake by 63 +/- 2 (n = 15), 40 +/- 4 (n = 30), and 31 +/- 4% (n = 16), respectively. Inhibition of CaM resulted in a significant change in the EC(50) value for the inhibition of ASP(+) uptake by tetraethylammonium. In conclusion, we demonstrate that the hOCT2 is inhibited by PI3K and PKA and activated by a CaM-dependent signaling pathway, probably via a change in substrate affinity.
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PMID:Regulation of human organic cation transporter hOCT2 by PKA, PI3K, and calmodulin-dependent kinases. 1238 97

Cystic fibrosis transmembrane conductance regulator (CFTR) regulates both HCO(3)(-) secretion and HCO(3)(-) salvage in secretory epithelia. At least two luminal transporters mediate HCO(3)(-) salvage, the Na(+)/H(+) exchanger (NHE3) and the Na(+)-HCO(3)(-) cotransport (NBC3). In a previous work, we show that CFTR interacts with NHE3 to regulate its activity (Ahn, W., Kim, K. W., Lee, J. A., Kim, J. Y., Choi, J. Y., Moe, O. M., Milgram, S. L., Muallem, S., and Lee, M. G. (2001) J. Biol. Chem. 276, 17236-17243). In this work, we report that transient or stable expression of human NBC3 (hNBC3) in HEK cells resulted in a Na(+)-dependent, DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonic acid)- and 5-ethylisopropylamiloride-insensitive HCO(3)(-) transport. Stimulation of CFTR with forskolin markedly inhibited NBC3 activity. This inhibition was prevented by the inhibition of protein kinase A. NBC3 and CFTR could be reciprocally coimmunoprecipitated from transfected HEK cells and from the native pancreas and submandibular and parotid glands. Precipitation of NBC3 or CFTR from transfected HEK293 cells and from the pancreas and submandibular gland also coimmunoprecipitated EBP50. Glutathione S-transferase-EBP50 pulled down CFTR and hNBC3 from cell lysates when expressed individually and as a complex when expressed together. Notably, the deletion of the C-terminal PDZ binding motifs of CFTR or hNBC3 prevented coimmunoprecipitation of the proteins and inhibition of hNBC3 activity by CFTR. We conclude that CFTR and NBC3 reside in the same HCO(3)(-)-transporting complex with the aid of PDZ domain-containing scaffolds, and this interaction is essential for regulation of NBC3 activity by CFTR. Furthermore, these findings add additional evidence for the suggestion that CFTR regulates the overall trans-cellular HCO(3)(-) transport by regulating the activity of all luminal HCO(3)(-) secretion and salvage mechanisms of secretory epithelial cells.
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PMID:The cystic fibrosis transmembrane conductance regulator interacts with and regulates the activity of the HCO3- salvage transporter human Na+-HCO3- cotransport isoform 3. 1240 79

Phosducin-like protein (PhLP) is a member of the phosducin family of G-protein betagamma-regulators and exists in two splice variants. The long isoform PhLP(L) and the short isoform PhLP(S) differ by the presence or absence of an 83-amino acid N terminus. In isolated biochemical assay systems, PhLP(L) is the more potent Gbetagamma-inhibitor, whereas the functional role of PhLP(S) is still unclear. We now report that in intact HEK 293 cells, PhLP(S) inhibited Gbetagamma-induced inositol phosphate generation with approximately 20-fold greater potency than PhLP(L). Radiolabeling of transfected HEK 293 cells with [(32)P] revealed that PhLP(L) is constitutively phosphorylated, whereas PhLP(S) is not. Because PhLP(L) has several consensus sites for the constitutively active kinase casein kinase 2 (CK2) in its N terminus, we tested the phosphorylation of the recombinant proteins by either HEK cell cytosol in the presence or absence of kinase inhibitors or by purified CK2. PhLP(L) was a good CK2 substrate, whereas PhLP(S) and phosducin were not. Progressive truncation and serine/threonine to alanine mutations of the PhLP(L) N terminus identified a serine/threonine cluster (Ser-18/Thr-19/Ser-20) within a small N-terminal region of PhLP(L) (amino acids 5-28) as the site in which PhLP(L) function was modified in HEK 293 cells. In native tissue, PhLP(L) also seems to be regulated by phosphorylation because phosphorylated and non-phosphorylated forms of PhLP(L) were detected in mouse brain and adrenal gland. Moreover, the alternatively spliced isoform PhLP(S) was also found in adrenal tissue. Therefore, the physiological control of G-protein regulation by PhLP seems to involve phosphorylation by CK2 and alternative splicing of the regulator.
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PMID:Regulation of phosducin-like protein by casein kinase 2 and N-terminal splicing. 1246 82

G(s)alpha is the G protein subunit that stimulates adenylyl cyclase activity in the myometrium during pregnancy, raising intracellular levels of the smooth muscle relaxant cAMP. The promoter region of the gene encoding G(s)alpha is GC rich and contains multiple putative binding sites for the specificity protein (Sp) transcription factor family. In electrophoretic mobility shift assays, four of these Sp sites were bound by recombinant Sp1 protein. Binding was dependent on phosphorylation of Sp1 by protein kinase A. Phosphorylated Sp1-4 proteins were observed in extracts of cultured human myometrial cells, but in electrophoretic mobility shift assays G(s)alpha promoter sequence binding by Sp1 was not apparent. Instead, these assays showed phosphorylation-dependent G(s)alpha promoter binding by lower molecular weight myometrial proteins that could not be supershifted by antibodies specific to Sp1-4 proteins. To investigate the regulation of G(s)alpha expression, the GC-rich promoter region was used to direct transcription of a firefly luciferase reporter gene in transient transfection assays of primary human myometrial cell cultures, COS-7 and HEK 293 cells. Reporter gene expression was found to follow a biphasic response to forskolin and 8-bromo-cAMP, with an initial, concentration-dependent increase in luciferase activity, followed by a prolonged decrease. In myometrial cells, this pattern was also seen in response to treatment with human chorionic gonadotropin.
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PMID:Differential expression of the adenylyl cyclase-stimulatory guanosine triphosphate-binding protein G(s)alpha in the human myometrium during pregnancy and labor involves transcriptional regulation by cyclic adenosine 3',5'-monophosphate and binding of phosphorylated nuclear proteins to multiple GC boxes within the promoter. 1246 71

In the present study we investigated long-term interactions between opioid and cannabinoid drugs at several steps along their cellular signal transduction pathways. For this purpose we co-transfected HEK-293 and COS-7 cells with delta-opioid (DOR) and CB1-cannabinoid receptors, and examined the effect of prolonged exposure to either opioid (etorphine) or cannabinoid (DALN) agonists on DOR and CB-1 receptor density and on the ability of subsequent application of the agonists to activate G-proteins (as measured by [35S]GTPgammaS binding) and to inhibit cAMP production. In HEK-293 cells, etorphine induced both homologous and heterologous desensitization, while DALN induced only homologous desensitization. This asymmetric cross-desensitization coincided with asymmetric cross downregulation: etorphine downregulated the binding of the cannabinoid ligand [3H]CP55,940, while DALN failed to reduce the binding of the opioid ligand [3H]diprenorphine. In contrast to the asymmetric desensitization in HEK-293 cells, COS-7 cells presented a two-way cross-desensitization between opioid and cannabinoid agonists, and DALN downregulated the binding of [3H]diprenorphine in these cells. Thus, a complete correlation was found between downregulation and reduction in cell responsiveness ('desensitization'). Moreover, when opioid downregulation in HEK-293 cells was inhibited by either hypertonic sucrose solution or protein kinase inhibitors, desensitization was suppressed to the same extent. These results suggest that, under the present experimental conditions, the reduction in cell responsiveness resulted primarily from downregulation of the receptors.
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PMID:Long-term interactions between opioid and cannabinoid agonists at the cellular level: cross-desensitization and downregulation. 1250 72

1 In fura 2-loaded HEK-293 cells stably expressing human type 1 parathyroid hormone (PTH) receptors, PTH potentiated the Ca(2+) mobilization evoked by carbachol by >4 fold without itself increasing the intracellular [Ca(2+)]. 2 PTH potentiated the Ca(2+) release evoked by a cell-permeant analogue of inositol 1,4,5-trisphosphate (InsP(3)BM). 3 Prolonged incubation with InsP(3)BM emptied the Ca(2+) stores as effectively as PTH in combination with a maximal concentration of carbachol, indicating that PTH did not increase the size of the InsP(3)-sensitive Ca(2+) pool. 4 Responses to PTH were unaffected by disruption of the cytoskeleton. 5 The EC(50) for carbachol-evoked Ca(2+) release and InsP(3) formation were indistinguishable (approximately 40 microM), consistent with even the highest concentrations of carbachol generating insufficient InsP(3) to release the entire InsP(3)-sensitive Ca(2+) pool. 6 Inhibition of cyclic AMP-dependent protein kinase A (PKA), using H89 or CMIQ, did not affect potentiation of carbachol-evoked Ca(2+) signals by PTH. 7 SQ22536 or DDA, inhibitors of adenylyl cyclase, inhibited PTH-evoked cyclic AMP formation and IBMX, an inhibitor of cyclic nucleotide phosphodiesterase, increased the amount of cyclic AMP detected after stimulation by PTH. None of these drugs affected the potentiation of Ca(2+) signals by maximal or submaximal concentrations of PTH. 8 We conclude that PTH potentiates the Ca(2+) release evoked by receptors that stimulate InsP(3) formation by sensitizing InsP(3) receptors through a cyclic AMP-independent mechanism.
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PMID:Parathyroid hormone increases the sensitivity of inositol trisphosphate receptors by a mechanism that is independent of cyclic AMP. 1252 76

The voltage-gated potassium channel Kv1.1 contains phosphorylation sites for protein kinase A (PKA) and protein kinase C (PKC). To study Kv1.1 protein expression and cellular distribution in regard to its level of phosphorylation, the effects of PKA and PKC activation on Kv1.1 were investigated in HEK 293 cells stably transfected with Kv1.1 (HEK 293/1). Without kinase activation, HEK 293/1 cells carry unphosphorylated Kv1.1 protein in the plasma membranes, whereas large amounts of phosphorylated and unphosphorylated Kv1.1 protein were located intracellularly. Activation of PKA resulted in phosphorylation of intracellular Kv1.1 protein, followed by a rapid translocation of Kv1.1 into the plasma membrane. Patch-clamp analysis revealed an increase in current amplitude upon PKA activation and demonstrated differences in the voltage dependence of current activation between unphosphorylated and phosphorylated Kv1.1 channels. In contrast to PKA, even prolonged activation of PKC did not lead to direct phosphorylation of Kv1.1, but induced Kv1.1 protein synthesis. Thus, protein kinases have direct and indirect effects on the functional expression of voltage-gated potassium channels. Our data suggest that the synergistic action of protein kinases may play an important role in the fine-tuning of Kv channel function.
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PMID:Analysis of phosphorylation-dependent modulation of Kv1.1 potassium channels. 1268 81

Outer nuclear membrane is endowed with a SERCA type Ca(2+)-ATPase which pumps calcium into the nuclear envelope lumen and creates calcium stores. Variation in this calcium pool, among other things, regulates nuclear transport. The transport of Nuclear Localization Signal (NLS)-containing molecules into the nucleus is well established. Intermediate size molecules lacking an NLS translocate to the nucleus and its mechanism remains obscure. It is observed here that the treatment of HEK 293 cells in culture with dibutyryl cyclic AMP (db-cAMP) or forskolin (FK) triggered transport of Calcium Green 10 kDa dextran into the nucleus. Under similar conditions Fluo-3-AM accumulated around the nuclei. cAMP-dependent protein kinase phosphorylated 105 kDa nuclear Ca(2+)-ATPase (NCA) which served as a trigger for NLS-independent transport into the nucleus.
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PMID:In vivo nuclear Ca2+-ATPase phosphorylation triggers intermediate size molecular transport to the nucleus. 1268 66

In HEK-293 cells, serotonin (5-hydroxytryptamine, 5-HT) was found to induce cAMP production showing pharmacological characteristics consistent with the 5-HT(7) receptor. The presence of 5-HT(7) (and 5-HT(6)) receptor mRNA was confirmed by RT-PCR. Stable HEK-293 cell lines expressing either wild-type or haemagglutinin (HA)-tagged human 5-HT transporter (SERT) were selected and SERT function was confirmed using [3H]5-HT transport. The presence of SERT caused a 10-fold reduction in the potency of 5-HT-induced cAMP production compared to control cells. Downstream signalling by 5-HT(6/7) receptors could be detected as 5-HT-induced protein kinase A activation and phosphorylation of MAP kinase and CREB using phospho-specific antibodies. SERT inhibitors reversed the reduction in potency of 5-HT-induced cAMP production caused by the presence of SERT, resulting in a concentration-dependent left shift in EC(50) values but also a progressive decrease in the maximal response. Thus, when antidepressants were used to block SERT activity, 5-HT receptor signalling was effectively clamped within a mid-range.
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PMID:Functional interactions between native Gs-coupled 5-HT receptors in HEK-293 cells and the heterologously expressed serotonin transporter. 1278 73


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