Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.1 (protein kinase)
 81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The Na-K-Cl cotransport system of vascular endothelial cells plays a central role in maintenance and regulation of intracellular volume. Activity of the cotransporter is modulated both by hormones and by extracellular tonicity. Vasopressin and other hormones that stimulate the endothelial cotransporter act via a Ca- and calmodulin-dependent pathway. Little is known, however, about the mechanisms that mediate cell shrinkage-induced stimulation of cotransport activity. In the present study, we evaluated the Ca dependence of cell shrinkage-stimulated Na-K-Cl cotransport activity and cell volume recovery of cultured bovine aortic endothelial cells and also the effects of protein kinase and phosphatase inhibitors on these processes. In addition, to investigate the possibility that hormones and/or hypertonicity regulate endothelial Na-K-Cl cotransport via direct phosphorylation of the cotransporter protein, we employed a monoclonal antibody to the human colonic T84 epithelial cell Na-K-Cl cotransport protein (T4 antibody) for Western blot analysis and immunoprecipitation of phosphoprotein. Our studies revealed that both cell shrinkage-stimulated net K uptake and recovery of intracellular volume were Ca dependent. We also found that hypertonicity-induced stimulation of cotransport activity was blocked by several inhibitors of Ca- and calmodulin-dependent protein kinases. Furthermore, inhibitors of myosin light chain kinase blocked cell shrinkage-stimulated cotransport and recovery of intracellular volume, while having no effect on vasopressin-stimulated cotransport. Western blot analysis of bovine aortic and cerebral microvascular endothelial cell membrane preparations revealed a 170-kDa protein recognized by the T4 antibody. In addition, we found that hypertonicity induced a marked increase in phosphorylation of the endothelial cotransport protein, as did vasopressin, bradykinin, okadaic acid, and calyculin A. Our findings indicate that modulation of endothelial cell Na-K-Cl cotransport activity by hypertonicity and by stimulatory hormones occurs via pathways involving Ca- and calmodulin-dependent protein kinases and direct phosphorylation of the cotransport protein.
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PMID:Endothelial Na-K-Cl cotransport regulation by tonicity and hormones: phosphorylation of cotransport protein. 857 81

In principal cells of rat cortical collecting ducts (CCD) cellular pH (pHi) is regulated by basolateral Na+/H+ exchange. The influence of various agonists on pHi and cellular Ca2+ activity ([Ca2+]i) in freshly isolated CCD cells was examined with BCECF and fura-2 fluorescence ratios. The recovery of pHi per minute (delta pH/min) after an acid load was 0.26 +/- 0.03 (N = 53) in control conditions and was increased by the diadenosine polyphosphates Ap4A, Ap5A, Ap6A, the phorbol ester phorbol 12-myristat 13-acetate (PMA) (each 5 mumol/L) and angiotensin II (100 nmol/L) by 0.05 +/- 0.02 (N = 10), 0.11 +/- 0.05 (N = 13), 0.09 +/- 0.02 (N = 24), 0.10 +/- 0.03 (N = 7), and 0.09 +/- 0.03 (N = 8), respectively. Vasopressin (10 nmol/L) decreased delta pH/min by 0.11 +/- 0.03 (N = 9); ATP and Ap3A (each 5 mumol/L) had no significant effect. The increase in delta pH/min with Ap6A was abolished in the presence of an inhibitor of protein kinase C, calphostin C (0.1 mumol/l, N = 8). Fura-2 fluorescence ratio was not significantly changed with angiotensin II, Ap3A, or Ap4A but increased with vasopressin, ATP, Ap5A, and Ap6A by 0.08 +/- 0.02 (N = 13), 0.04 +/- 0.02 (N = 13), 0.03 +/- 0.01 (N = 14), and 0.03 +/- 0.01 (N = 10), respectively. These data indicate that Na+/H+ exchange in rat CCD is activated by the stimulation of a Ca(2+)-independent protein kinase C and inhibited by protein kinase A.
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PMID:Regulation of Na+/H+ exchange by diadenosine polyphosphates, angiotensin II, and vasopressin in rat cortical collecting duct. 858 90

Nitric oxide has a diuretic effect in vivo. We have shown that nitric oxide inhibits antidiuretic hormone-stimulated osmotic water permeability in the collecting duct; however, the mechanism by which this occurs is unknown. We hypothesized that inhibition of antidiuretic hormone-stimulated water permeability by nitric oxide in the collecting duct is the result of activation of cGMP-dependent protein kinase, which in turn decreases intracellular cAMP. To test this hypothesis, we microperfused cortical collecting ducts. Antidiuretic hormone-stimulated water permeability was 317 +/- 47 microm/s (P < .001). Addition of spermine NONOate, a nitric oxide donor, to the bath decreased water permeability to 74 +/- 38 microm/s (P < .002). In the presence of LY 83583, an inhibitor of soluble guanylate cyclase, spermine NONOate did not change water permeability. Addition of spermine NONOate increased cGMP production (P < .01). In the presence of the cGMP-dependent protein kinase inhibitor, spermine NONOate did not change water permeability. Since antidiuretic hormone increases water permeability by increasing cAMP, we hypothesized that nitric oxide inhibits water permeability by decreasing cAMP. In tubules pretreated with antidiuretic hormone, intracellular cAMP was 18.9 +/- 3.9 fmol/mm. In tubules treated with antidiuretic hormone and spermine NONOate, cAMP was 9.3 +/- 1.7 fmol/mm (P < .03). We also examined the effect of spermine NONOate on dibutyryl-cAMP-stimulated water permeability. In the presence of dibutyryl-cAMP, water permeability was 388 +/- 30 microm/s. Addition of spermine NONOate had no significant effect on water permeability. Time controls and inhibitors by themselves did not change antidiuretic hormone-stimulated water permeability. We concluded that nitric oxide decreases antidiuretic hormone-stimulated water permeability by increasing cGMP via soluble guanylate cyclase, activating cGMP-dependent protein kinase and decreasing cAMP.
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PMID:Mechanism of the nitric oxide-induced blockade of collecting duct water permeability. 861 24

Antidiuretic hormone modulates the water permeability (Pf) of epithelial cells in the rat kidney by vesicle-mediated insertion and removal of the aquaporin-2 (AQP-2) water channel. AQP-2 possesses a single consensus cAMP-dependent protein kinase A (PKA) phosphorylation site (Ser-256) hypothesized to regulate channel Pf(Kuwahara, M., Fushimi, K., Terada, Y., Bai, L., Sasaki, S., and Marumo, F. (1995) J. Biol. Chem. 270, 10384-10387). To test whether PKA phosphorylation of AQP-2 alters channel Pf, we compared the Pf values of purified AQP-2 endosomes after incubation with either PKA or alkaline phosphatase. Studies using [gamma-32P]ATP reveal that AQP-2 endosomes contain endogenous PKA and phosphatase activities that add and remove 32P label from AQP-2. However, the Pf (0.16 +/- 0.06 cm/s) of endosomes containing phosphorylated AQP-2 (0.7 +/- 0. 3 mol of PO4/mol of protein) is not significantly different from the same AQP-2 endosomes where 95 +/- 8% of the phosphate has been removed (Pf 0.14 +/- 0.06 cm/s). These data do not support a role for PKA phosphorylation in alteration of AQP-2's Pf. Instead, AQP-2 phosphorylation by PKA may modulate AQP-2's distribution between plasma membrane and intracellular vesicle compartments.
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PMID:Phosphorylation of aquaporin-2 does not alter the membrane water permeability of rat papillary water channel-containing vesicles. 862 14

Although the effects of the various secretagogues on corticotropin (ACTH) secretion have been well studied, their effects on the POMC gene expression have not been thoroughly characterized. In this study, we established a new model system using the AtT20 mouse corticotroph tumor cell line transfected stably with a plasmid containing 0.7 kb of the rat POMC 5' promoter-luciferase fusion gene. The responsiveness to exogenous CRH improved markedly when the cells were cultured with low serum medium (1% FBS) compared with serum rich medium (10%). Using this culture condition, we examined the effects of not only CRH but also other secretagogues such as catecholamines, vasopressin, and angiotensin II, upon the transcriptional activity of the POMC gene. CRH stimulated POMC promoter activity (3.5-fold increase) as well as cAMP generation and ACTH secretion in a dose- and time-dependent manner, with the maximal effect being observed 3-5 h after the start of incubation. Catecholamines, especially epinephrine (10 nM and above), also stimulated all parameters, although less potently than CRH, and the effect was mimicked by the beta-, but not alpha-adrenergic, agonist, suggesting the involvement of the beta-adrenergic receptor. The combined effects of epinephrine and CRH were greater in all parameters than those of CRH alone, and the effects of both hormones were completely blocked by H89, an inhibitor of protein kinase A. Vasopressin and angiotensin II showed minimal effects on POMC expression. Our results suggest that 1) catecholamines, as well as CRH, positively regulate the POMC gene at physiological concentrations; 2) the cAMP-PKA system is the common intracellular signaling pathway for CRH and catecholamines; and 3) vasopressin and angiotensin II also have weak but significant stimulatory effects on POMC promoter activity.
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PMID:Regulation of the rat proopiomelanocortin gene expression in AtT-20 cells. I: Effects of the common secretagogues. 911 88

Vasopressin-dependent translocation of aquaporin-2 (AQP2) between intracellular vesicles and the plasma membrane has been demonstrated in vivo and in vitro. Furthermore, the vasopressin-induced increase in apical membrane water permeability of renal principal cells is dependent on a rise in intracellular adenosine 3',5'-cyclic monophosphate and activation of protein kinase A (PKA). To determine whether trafficking of AQP2 is dependent on PKA phosphorylation, we first examined the effect of the PKA-inhibitor N-(2[[3-(4-bromophenyl)-2-propenyl]-amino]-ethyl)-5-isoquinolinesulfonam ide (H-89) on AQP2 translocation in transfected LLC-PK1 cells. Vasopressin-induced membrane insertion of AQP2 was completely inhibited by pretreatment of the cells for 60 min with H-89. This reagent also caused a dense accumulation of AQP2 in the Golgi region. Next, LLC-PK1 cells were stably transfected with AQP2 cDNA in which the PKA phosphorylation site, Ser256, was replaced with alanine (S256A). S256A-AQP2 was not phosphorylated in vitro by PKA, and S256A-AQP2 was mainly localized to intracellular vesicles in the basal condition, similar to wild-type AQP2. However, after stimulation with vasopressin or forskolin, the cellular distribution of S256A-AQP2 remained unchanged. In addition, the usual vasopressin-induced increase in endocytosis seen in AQP2-transfected cells was not observed in S256A-AQP2-transfected cells. These results demonstrate that the Ser256 PKA phosphorylation site is possibly involved in the vasopressin-induced trafficking of AQP2 from intracellular vesicles to the plasma membrane and in the subsequent stimulation of endocytosis.
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PMID:Protein kinase A phosphorylation is involved in regulated exocytosis of aquaporin-2 in transfected LLC-PK1 cells. 922 44

Protein kinase D (PKD) is a serine/threonine protein kinase that is activated by phorbol esters via protein kinase C in intact cells. To assess the physiological significance of this putative pathway, we examined the regulation of PKD in living cells by mitogenic regulatory peptides and by platelet-derived growth factors (PDGF). Our results demonstrate that bombesin rapidly induces PKD activation in Swiss 3T3 cells, as shown by autophosphorylation and syntide-2 phosphorylation assays. Maximum PKD activation (14-fold above base-line levels) was obtained 90 s after bombesin stimulation. Bombesin also induced PKD activation in Rat-1 cells stably transfected with the bombesin/gastrin releasing peptide (GRP) receptor and in COS-7 cells transiently co-transfected with PKD and bombesin/GRP receptor expression constructs. No inducible kinase activity was demonstrated when COS-7 cells were transfected with a kinase-deficient PKD mutant. Bombesin-mediated PKD activation was prevented by treatment of Swiss 3T3 cells with the protein kinase C inhibitors GF 1092030X and Ro 31-8220. In contrast, these compounds did not inhibit PKD activity when added directly in vitro. Vasopressin, endothelin, and bradykinin also activated PKD in Swiss 3T3 cells through a PKC-dependent pathway. Platelet-derived growth factor-stimulated PKD activation in Swiss 3T3 cells and in porcine aortic endothelial cells stably transfected with PDGF-beta receptors. Treatment with GF 1092030X or Ro 31-8220 inhibited PKD activation induced by PDGF. Thus, our results indicate that PKD is activated by multiple signaling peptides through a protein kinase C-dependent signal transduction pathway in a variety of cell types.
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PMID:Bombesin, vasopressin, endothelin, bradykinin, and platelet-derived growth factor rapidly activate protein kinase D through a protein kinase C-dependent signal transduction pathway. 929 46

Ca2+ functions as an intracellular signal to transfer hormonal messages to different cellular compartments, including mitochondria, where it activates intramitochondrial Ca2(+)-dependent enzymes. However, excessive mitochondrial Ca2+ uptake can promote the mitochondrial permeability transition (MPT), a process known to be associated with cell injury. The factors controlling mitochondrial Ca2+ uptake and release in intact cells are poorly understood. In this paper, we investigate mitochondrial Ca2+ accumulation in intact hepatocytes in response to the elevation of cytosolic Ca2+ levels ([Ca2+]c) induced either by a hormonal stimulus (vasopressin), or by thapsigargin, an inhibitor of the endoplasmic reticulum Ca2+ pump. After stimulation, cells were rapidly permeabilized for the determination of the mitochondrial Ca2+ content (Ca2+(m)) and to analyze the susceptibility of the mitochondria to undergo the MPT. Despite very similar levels of [Ca2+]c elevation, vasopressin and thapsigargin had markedly different effects on mitochondrial Ca2+ accumulation. Vasopressin caused a rapid (< 90 sec), but modest (< 2 fold) increase in Ca2+(m) that was not further increased during prolonged incubations, despite a sustained [Ca2+]c elevation. By contrast, thapsigargin induced a net Ca2+ accumulation in mitochondria that continued for up to 30 min and reached Ca2+(m) levels 10-20 fold over basal. Accumulation of mitochondrial Ca2+ was accompanied by a markedly increased susceptibility to undergo the MPT. Both mitochondrial Ca2+ accumulation and MPT activation were modulated by treatment of the cells with inhibitors of protein kinases and phosphatases. The results indicate that net mitochondrial Ca2+ uptake in response to hormonal stimulation is regulated by processes that depend on protein kinase activation. These controls are inoperative when the cytosol is flooded by Ca2+ through artificial means, enabling mitochondria to function as a Ca2+ sink under these conditions.
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PMID:Hormonal stimulation, mitochondrial Ca2+ accumulation, and the control of the mitochondrial permeability transition in intact hepatocytes. 930 83

cAMP and Ca2+ acted together with the acute phase cytokine interleukin-1beta (IL-1beta) to inhibit hepatocyte DNA replication. At sub-basal activity of cAMP-dependent protein kinase (PKA), neither IL-1beta nor the Ca2+-elevating hormone vasopressin affected hepatocyte proliferation. Basal level of PKA activity permitted IL-1beta action. Increased PKA activity also permitted vasopressin action and sensitized further towards IL-1beta, which acted at 10-50 pM concentrations. Vasopressin acted via Ca2+/calmodulin-dependent protein kinase II (CaMKII), and its action was mimicked by the serine/threonine phosphatase inhibitor microcystin, which activates CaMKII. Inhibitors (KN93 and KT5926) of CaMKII selectively counteracted the effects of vasopressin and microcystin on hepatocyte proliferation at concentrations similar to those required to inhibit CaMKII in vitro. Two-dimensional gel electrophoresis of 32P-prelabeled hepatocytes revealed a common set of proteins phosphorylated in response to vasopressin and microcystin. Their phosphorylation was counteracted by CaMKII inhibitor (KT5926). Phosphorylation of the CaMKII substrate phenylalanine hydroxylase (PAH; EC 1.14.16.1) was used as an endogenous marker of CaMKII activation. It was found that treatment of the cells with vasopressin or microcystin increased the phosphorylation of PAH, and that the vasopressin-induced PAH phosphorylation was inhibited by KT5926. In conclusion, the Ca2+-elevating hormone vasopressin potentiated the antiproliferative effects of cAMP and IL-1beta through CaMKII activation.
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PMID:Synergistic antiproliferative actions of cyclic adenosine 3',5'-monophosphate, interleukin-1beta, and activators of Ca2+/calmodulin-dependent protein kinase in primary hepatocytes. 932 53

Vasopressin is one of several small neuropeptides that are reported to be autocrine growth factors for small cell carcinoma of the lung (SCCL). It has been assumed that this peptide exercises its mitogenic influences through the vasopressin V1a receptor, and we have previously demonstrated that this receptor is expressed by classical and variant SCCL. Activation of the vasopressin V1a receptor produces changes in phospholipases C, D, and A2, in protein kinase C, and in Ca2+ mobilization. This study demonstrates that SCCL cells express not only vasopressin V1a receptors but also mRNAs and proteins representing normal V1b receptors and V2 receptors. They were also shown to express mRNA for a human form of the putative receptor rabbit vasopressin-activated calcium-mobilizing receptor (VACM-1). Additionally, SCCL tumor cells were found to express mRNA and protein representing a possible nonfunctional, shortened, "diabetic" form of the vasopressin V2 receptor that is the product of incomplete posttranscriptional splicing. At least four of these five vasopressin receptors were produced by cell lines exemplifying classical and variant forms of SCCL. No differences in the sequences for the V1 receptors between classical and variant SCCL were found. However, although the nature and expression of both vasopressin V1 receptors and human VACM are apparently unaffected by dedifferentiation in SCCL, only the abnormal (and probably nonfunctional) form of the V2 receptor could be demonstrated in variant cell line NCI H82. Functional engagement of vasopressin V2 receptors is reported to produce rises in cAMP and activation of protein kinase A, whereas stimulation of V1b receptors is believed to produce similar changes to those produced by V1a receptors, i.e., activation of phospholipases and of protein kinase C. Stimulation of VACM receptors raises intracellular free Ca2+ through currently unknown but phosphoinositide-independent mechanisms. The presence of all known vasopressin receptors that are, together, potentially capable of inducing several different transduction cascades in small cell tumor cells suggests that this peptide serves a multifaceted role in tumor physiology.
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PMID:Expression of all known vasopressin receptor subtypes by small cell tumors implies a multifaceted role for this neuropeptide. 958 26


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