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)

Although renal K(+) channels along the nephron have been explored in various animal species, little is known about the K(+) channels in human proximal tubule cells. Using the patch-clamp technique, we investigated the properties of an inwardly rectifying K(+) channel present in the surface membrane of cultured human proximal tubule cells of normal kidney origin. This channel was the most frequently observed K(+) channel in cell-attached patches, and cytoplasmic ATP was required to maintain channel activity in inside-out patches. Its single channel conductance was about 42 pS for inward currents and 7 pS for outward currents under the symmetrical K(+) condition. The ATP effect on channel activity was dose-dependently stimulatory within a range of 0.1 to 10 mM, and a nonhydrolyzable ATP analog, AMP-PNP (3 mM), had no effect on channel activity in either the presence or absence of ATP (1 mM). The channel activity observed in cell-attached patches was reduced to 30 to 50% of controls by a membrane-permeable nonspecific protein kinase inhibitor, K252a (1 microM), or a potent protein kinase A inhibitor, KT5720 (500 nM). In contrast, a membrane-permeable cAMP analog, 8Br-cAMP (100 microM), induced a twofold increase in channel activity. The addition of a catalytic subunit of protein kinase A (PKA-CS, 100 U/ml) to the bath in inside-out patches stimulated channel activity in the presence of 1 mM ATP. Furthermore, the channel activity maintained with 1 mM ATP in inside-out patches was suppressed by internal acidification and enhanced by alkalization. These results suggest that the activity of the inwardly rectifying K(+) channel in cultured human proximal tubule cells was ATP-dependent and regulated at least in part by cAMP/PKA-mediated phosphorylation processes and intracellular pH.
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PMID:An ATP-regulated and pH-sensitive inwardly rectifying K(+) channel in cultured human proximal tubule cells. 1156 89

1. The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is activated by protein kinase A (PKA) phosphorylation of its R domain and by ATP binding at its nucleotide-binding domains (NBDs). Here we investigated the functional role of a cluster of acidic residues in the amino terminal tail (N-tail) that also modulate CFTR channel gating by an unknown mechanism. 2. A disease-associated mutant that lacks one of these acidic residues (D58N CFTR) exhibited lower macroscopic currents in Xenopus oocytes and faster deactivation following washout of a cAMP -activating cocktail than wild-type CFTR. 3. In excised membrane patches D58N CFTR exhibited a two-fold reduction in single channel open probability due primarily to shortened open channel bursts. 4. Replacing this and two nearby acidic residues with alanines (D47A, E54A, D58A) also reduced channel activity, but had negligible effects on bulk PKA phosphorylation or on the ATP dependence of channel activation. 5. Conversely, the N-tail triple mutant exhibited a markedly inhibited response to AMP-PNP, a poorly hydrolysable ATP analogue that can nearly lock open the wild-type channel. The N-tail mutant had both a slower response to AMP-PNP (activation half-time of 140 +/- 20 s vs. 21 +/- 4 s for wild type) and a lower steady-state open probability following AMP-PNP addition (0.68 +/- 0.08 vs. 0.92 +/- 0.03 for wild type). 6. Introducing the N-tail mutations into K1250A CFTR, an NBD2 hydrolysis mutant that normally exhibits very long open channel bursts, destabilized the activity of this mutant as evidenced by decreased macroscopic currents and shortened open channel bursts. 7. We propose that this cluster of acidic residues modulates the stability of CFTR channel openings at a step that is downstream of ATP binding and upstream of ATP hydrolysis, probably at NBD2.
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PMID:A cluster of negative charges at the amino terminal tail of CFTR regulates ATP-dependent channel gating. 1160 Jun 81

Molecular cloning of low-voltage activated (LVA) T-type calcium channels has enabled the study of their regulation in heterologous expression systems. Here we investigate the regulation of Ca(v)3.2 alpha(1)-subunits (alpha1H) by calcium- and/or calmodulin-dependent protein kinase II (CaMKII). 293 cells stably expressing alpha1H were transiently transfected with CaMKIIgamma(C). Using the whole-cell recording configuration, we observed that elevation of pipette free Ca(2+) (1 microM) in the presence of CaM (2 microM) increases T-type channel activity selectively at negative potentials, evoking an 11 mV hyperpolarizing shift in the half-maximal potential (V(1/2)) for activation. The V(1/2) of channel inactivation is not altered by Ca(2+)/CaM. These effects reproduced modulation observed in adrenal zona glomerulosa cells. The potentiation by Ca(2+)/CaM was dependent on the co-expression of CaMKIIgamma(C) and required Ca(2+)/CaM-dependent kinase activity. Peptide (AIP) and lipophilic (KN-62) protein kinase inhibitors prevented the Ca(2+)/CaM-induced changes in channel gating without altering basal Ca(v)3.2 channel activity (27 nM free Ca(2+)) as did replacing pipette ATP with adenylyl imidodiphosphate (AMP-PNP), a non-hydrolysable analogue. CaMKII-dependent potentiation of channel opening resulted in significant increases in apparent steady-state open probability (P(o)) and sustained channel current at negative voltages. Under identical conditions, CaMKII activation did not regulate the activity of Ca(v)3.1 channels, the first cloned member (alpha1G) of the T-type Ca(2+) channel family. Our results provide the first evidence for the differential regulation of two members of the Ca(v)3 family by protein kinase activation and the first report reconstituting CaMKII-dependent regulation of any cloned Ca(2+) channel.
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PMID:Stimulation of recombinant Ca(v)3.2, T-type, Ca(2+) channel currents by CaMKIIgamma(C). 1179 Aug 4

Cell signaling pathways rely on phosphotransfer reactions that are catalyzed by protein kinases. The protein kinases themselves are typically regulated by phosphorylation and concurrent structural rearrangements, both near the catalytic site and elsewhere. Thus, physiological function requires posttranslational modification and deformable structures. A prototypical example is provided by cyclic AMP-dependent protein kinase (PKA). It is activated by phosphorylation, is inhomogeneously phosphorylated when expressed in bacteria, and exhibits a wide range of dynamic properties. Here we use (31)P nuclear magnetic resonance (NMR) spectroscopy to characterize the phosphorylation states and to estimate the flexibility of the phosphorylation sites of 2-, 3-, and 4-fold phosphorylated PKA. The phosphorylation sites Ser10, Ser139, Thr197, and Ser338 are assigned to individual NMR resonances, assisted by complexation with AMP-PNP and dephosphorylation with alkaline phosphatase. Rotational diffusion correlation times estimated from resonance line widths show progressively increasing flexibilities for phosphothreonine 197, phosphoserines 139 and 338, and disorder at phosphoserine 10, consistent with crystal structures of PKA. However, because the apparent rotational diffusion correlation time fitted for phosphothreonine 197 of the activation loop is longer than the overall PKA rotational diffusion time, microsecond to millisecond time scale conformational exchange effects involving motions of phosphothreonine 197 are probable. These may represent "open"-"closed" transitions of the uncomplexed protein in solution. These data represent direct measurements of flexibilities also associated with functional properties, such as ATP binding and membrane association, and illustrate the applicability of (31)P NMR for functional and dynamic characterization of protein kinase phosphorylation sites.
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PMID:Phosphorylation and flexibility of cyclic-AMP-dependent protein kinase (PKA) using (31)P NMR spectroscopy. 1199 91

The effect of external ATP on intracellular pH (pH(i)) was investigated using a pH imaging system in a human bronchial epithelial cell line (16HBE14o-) loaded with BCECF-AM. The steady-state pH(i) of 16HBE14o- epithelial monolayers was 7.137 +/- 0.027 (n = 46). Apical addition of ATP (10(-4) M) to epithelial monolayers induced a rapid and sustained pH(i) decrease of 0.164 +/- 0.024 pH units (n = 17; P < 0.001). The intracellular acidification was rapidly reversed upon removal of external ATP. In contrast, the non-hydrolysable ATP analogue AMP-PNP did not produce any significant change in pH(i). Inhibition of purinoreceptors by suramin did not affect the acidification induced by apical ATP. Inhibition of Na+-H+ exchange by apical Na+ removal or addition of amiloride (0.5 mM) reduced the apical ATP-induced pH(i) decrease, suggesting the involvement of a Na+-H+ exchanger or surface pH effects on the ATP-induced pH(i) response. Inhibitors of proton channels such as ZnCl2 (10(-4) M) also partially inhibited the ATP response. The pH(i) response to ATP was dependent on the external pH (pH(o)), with increasing acidification produced at lower pH(o) values. Neither the basal pH(i) nor the ATP-induced intracellular acidification was affected by thapsigargin (a Ca2+-ATPase inhibitor), chelerythrine chloride (a protein kinase C (PKC) inhibitor), RpcAMP (a protein kinase A (PKA) inhibitor) or PMA (a PKC activator). Therefore, the intracellular acidification of human bronchial epithelial cells induced by apical ATP does not involve signalling via Ca2+, PKC or PKA nor binding to a purinoreceptor. We interpret the effect of ATP to produce an intracellular acidification as a three step process: activation of H+ channels, inhibition of Na+-H+ exchange and influx of protonated ATP.
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PMID:Cellular mechanisms for apical ATP effects on intracellular pH in human bronchial epithelium. 1218 Dec 78

The small-conductance K+ channel (SK) in the apical membrane of the cortical-collecting duct (CCD) is regulated by adenosine triphosphate (ATP) and phosphorylation-dephosphorylation processes. When expressed in Xenopus oocytes, ROMK, a cloned K+ channel similar to the native SK channel, can be stimulated by phosphatidylinositol bisphosphate (PIP2), which is produced by phosphoinositide kinases from phosphatidylinositol. However, the effects of PIP2 on SK channel activity are not known. In the present study, we investigated the mechanism by which hydrolyzable ATP prevented run-down of SK channel activity in excised apical patches of principal cells from rat CCD. Channel run-down was significantly delayed by pretreatment with hydrolyzable Mg-ATP, but ATP gamma S and AMP-PNP had no effect. Addition of alkaline phosphatase also resulted in loss of channel activity. After run-down, SK channel activity rapidly increased upon addition of PIP2. Exposure of inside-out patches to phosphoinositide kinase inhibitors (LY294002, quercetin or wortmannin) decreased channel activity by 74% in the presence of Mg-ATP. PIP2 added to excised patches reactivated SK channels in the presence of these phosphoinositide kinase inhibitors. The protein kinase A inhibitor, PKI, reduced channel activity by 36% in the presence of Mg-ATP. PIP2 was also shown to modulate the inhibitory effects of extracellular and cytosolic ATP. We conclude that both ATP-dependent formation of PIP2 through membrane-bound phosphoinositide kinases and phosphorylation of SK by PKA play important roles in modulating SK channel activity.
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PMID:Hydrolyzable ATP and PIP(2) modulate the small-conductance K+ channel in apical membranes of rat cortical-collecting duct (CCD). 1240 74

The aurora kinases are a novel oncogenic family of mitotic serine/threonine kinases (S/T kinases) that are overexpressed in a number of solid tumors, including pancreas and colorectal cancer. A PSI-BLAST search [National Center for Biotechnology Information (NCBI)] with the sequence of the S/T kinase domain of human aurora1 kinase [also known as AUR1, ARK2, AIk2, AIM-1, and STK12] and human aurora2 kinase (also known as AUR2, ARK1, AIK, BTAK, and STK15) showed a high sequence similarity to the three-dimensional structures of bovine cAMP-dependent kinase [Brookhaven Protein Data Bank code 1CDK], murine cAMP-dependent kinase (1APM), and Caenorhabditis elegans twitchin kinase (1KOA). When the aurora1 or aurora2 sequence was input into the tertiary structure prediction programs THREADER and 3D-PSSM (three-dimensional position-sensitive scoring matrix), the top structural matches were 1CDK, 1APM, and 1KOA, confirming that these domains are structurally conserved. The structural models of aurora1 and aurora2 were built using 1CDK as the template structure. Molecular dynamics and docking simulations, targeting the ATP binding site of aurora2 with adenylyl imidodiphosphate (AMP-PNP), staurosporine, and six small molecular S/T kinase inhibitors, identified active-site residues that interact with these inhibitors differentially. The docked structures of the aurora2-AMP-PNP and aurora2-staurosporine complexes indicated that the adenine ring of AMP-PNP and the indolocarbazole moiety of staurosporine have similar positions and orientations and provided the basis for the docking of the other S/T kinase inhibitors. Inhibitors with isoquinoline and quinazoline moieties were recognized by aurora2 in which H-89 and 6,7-dimethoxyquinazoline compounds exhibited high binding energies compared with that of staurosporine. The calculated binding energies for the docked small-molecule inhibitors were qualitatively consistent with the IC(50) values generated using an in vitro kinase assay. The aurora2 structural model provides a rational basis for site-directed mutagenesis of the active site; design of novel H-89, staurosporine, and quinazoline analogues; and the screening of the available chemical database for the identification of other novel, small-molecular entities.
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PMID:Targeting aurora2 kinase in oncogenesis: a structural bioinformatics approach to target validation and rational drug design. 1265 23

The whole-cell patch-clamp technique was used to examine nonselective conductances in single proximal tubule cells isolated from mouse kidney. Single cells were isolated in either the presence or absence of a cocktail designed to stimulate cAMP. Patches were obtained with Na+ Ringer in the bath and Cs+ Ringer in the pipette. On initially achieving the whole-cell configuration, whole-cell currents were small. In cAMP-stimulated cells, with 5 mM ATP in the pipette solution, whole-cell currents increased with time. The activated current was linear, slightly cation-selective, did not discriminate between Na+ and K+ and was inhibited by 100 microM gadolinium. These properties are consistent with the activation of a nonselective conductance, designated G(NS). Activation of G(NS) was abolished with pipette AMP-PNP, ATP plus alkaline phosphatase or in the absence of ATP. In unstimulated cells G(NS) was activated by pipette ATP together with PKA. These data support the hypothesis that G(NS) is activated by a PKA-mediated phosphorylation event. G(NS) was also activated by a hypertonic shock. However, G(NS) does not appear to be involved in regulatory volume increase (RVI), as RVI was unaffected in the presence of the G(NS) blocker gadolinium. Instead, the ATP sensitivity of G(NS) suggests that it may be regulated by the metabolic state of the renal proximal tubule cell.
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PMID:A hypertonicity-activated nonselective conductance in single proximal tubule cells isolated from mouse kidney. 1282 Jun 64

Conformational changes are thought to play a key role in the function of active protein kinases, although little is known about how these changes relate to the mechanism of phosphorylation. Here we present four high-resolution structures of a single crystal form of Sky1p, a constitutively active serine kinase implicated in yeast RNA processing, each in a different state of nucleotide binding. By comparing the apoenzyme structure to the ADP- and ATP-bound Sky1p structures, we have revealed conformational changes caused by ATP binding or conversion from nucleotide reactant to product. Rotation of the small lobe of the kinase closes the cleft upon binding, allowing the nucleotide to interact with residues from both lobes of the kinase, although some interactions thought to be important for phosphotransfer are missing in the ATP-containing structure. In the apoenzyme, a kinase-conserved phosphate-anchoring loop is in a twisted conformation that is incompatible with ADP and ATP binding, providing a potential mechanism for facilitating ADP release in Sky1p. The nonhydrolyzable ATP analogue AMP-PNP binds in a unique mode that fails to induce lobe closure. This observation, along with comparisons between the two independent molecules in the asymmetric unit of each structure, has provided new molecular details about how the nucleotide binds and induces closure. Finally, we have used mutational analysis to establish the importance of a glycine within the linker that connects the two lobes of Sky1p.
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PMID:Nucleotide-induced conformational changes in the Saccharomyces cerevisiae SR protein kinase, Sky1p, revealed by X-ray crystallography. 1291 Dec 99

SK4/IK1 encodes an intermediate conductance, Ca2+ -activated K+ channel and fulfills a variety of physiological functions in excitable and nonexcitable cells. Although recent studies have provided evidence for the presence of SK4/IK1 channels in salivary acinar cells, the regulatory mechanisms and the physiological function of the channel remain unknown in these cells. Using molecular and electrophysiological techniques, we examined whether cytosolic ATP-dependent regulation of native SK4/IK1-like channel activity would involve endogenous cAMP-dependent protein kinase (PKA) in rat submandibular acinar (RSA) cells. Electrophysiological properties of tetraethylammonium (TEA) (10 mM)-insensitive, Ca2+ -dependent K+ currents in macropatches excised from RSA cells matched those of whole cell currents recorded from human embryonic kidney-293 cells heterologously expressing rat SK4/IK1 (rSK4/IK1) cloned from RSA cells. In outside-out macropatches, activity of native SK4/IK1-like channels, defined as a charybdotoxin (100 nM)-blockable current in the presence of TEA (10 mM) in the bathing solution, ran down unless both ATP and Mg2+ were present in the pipette solution. The nonhydrolyzable ATP analog AMP-PNP failed to support the channel activity as ATP did. The addition of Rp-cAMPS (10 microM), a PKA inhibitor, to the pipette solution containing ATP/Mg2+ induced a rundown of the Ca2+ -dependent K+ currents. Inclusion of cAMP (1 mM) into the pipette solution (1 microM free Ca2+) containing ATP/Mg2+ caused a gradual increase in the currents, the effect being pronounced for the currents induced by 0.1 microM free Ca2+. Forskolin (1 microM), an adenylyl cyclase activator, also increased the currents induced by 0.1 microM free Ca2+. In inside-out macropatches, cytosolic ATP/Mg2+ increased both the maximum current (proportional to the maximum channel activity) and Ca2+ sensitivity of current activation. Collectively, these results suggest that ATP-dependent regulation of native SK4/IK1-like channels, at least in part, is mediated by endogenous PKA in RSA cells.
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PMID:ATP-dependent regulation of SK4/IK1-like currents in rat submandibular acinar cells: possible role of cAMP-dependent protein kinase. 1460 78


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