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 gene defective in cystic fibrosis encodes a Cl- channel named CFTR, which belongs to the family of transport proteins identified by their cytoplasmic domains that bind and hydrolyse ATP. CFTR channels require phosphorylation by protein kinase A at one or more serine residues in the large central regulatory domain before they will open. Severl findings argue that hydrolysis of ATP at the N-terminal nucleotide binding domain is the rate-limiting step for opening a phosphorylated CFTR channel. Although AMP-PNP the non-hydrolysable, but close structural, analog of ATP fails to open phosphorylated CFTR channels, once a channel has been opened, AMP-PNP can bind tightly to the channel and "lock" it into the open conformation for several minutes. This tight binding of AMP-PNP presumably occurs at CFTR's C-terminal nucleotide binding domain. Because it structurally resembles AMP-PNP, ATP must also bind tightly there, which suggests that hydrolysis of that ATP normally prompts channel closing. That conclusion is supported by the finding that free [Mg2+] level controls the rate of CFTR channel closure. A normal closed-open-closed gating cycle of a CFTR channel thus seems to involve hydrolysis of one ATP molecule to open it, and hydrolysis of a second ATP to close it. Stabilization of an active state by tight binding of a nucleotide, and termination of that state by hydrolysis of the nucleotide, are characteristics reminiscent of G proteins. Indeed, CFTR's nucleotide binding domains share with G proteins not only this functional similarity, but also some sequence homology, at least in certain highly conserved motifs.
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PMID:ATP hydrolysis cycles and the gating of CFTR Cl- channels. 978 67

1. The degree of cell-to-cell coupling between ventricular myocytes of neonatal rats appeared well preserved when studied in the perforated version of the patch clamp technique or, in double whole-cell conditions, when ATP was present in the patch pipette solution. In contrast, when ATP was omitted, the amplitude of junctional current rapidly declined (rundown). 2. To examine the mechanism(s) of ATP action, an 'internal perfusion technique' was adapted to dual patch clamp conditions, and reintroduction of ATP partially reversed the rundown of junctional channels. 3. Cell-to-cell communication was not preserved by a non-hydrolysable ATP analogue (5'-adenylimidodiphosphate, AMP-PNP), indicating that the effect most probably did not involve direct interaction of ATP with the channel-forming proteins. 4. An ATP analogue supporting protein phosphorylation but not active transport processes (adenosine 5'-O-(3-thiotriphosphate), ATPgammaS) maintained normal intercellular communication, suggesting that the effect was due to kinase activity rather than to altered intracellular Ca2+. 5. A broad spectrum inhibitor of endogenous serine/threonine protein kinases (H7) reversibly reduced the intercellular coupling. A non-specific exogenous protein phosphatase (alkaline phosphatase) mimicked the effects of ATP deprivation. The non-specific inhibition of endogenous protein phosphatases resulted in the preservation of substantial cell-to-cell communication in ATP-free conditions. 6. The activity of gap junctional channels appears to require both the presence of ATP and protein kinase activity to counteract the tonic activity of endogenous phosphatase(s).
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PMID:ATP counteracts the rundown of gap junctional channels of rat ventricular myocytes by promoting protein phosphorylation. 1008 44

The CFTR chloride channel is regulated by phosphorylation by protein kinases, especially PKA, and by nucleotides interacting with the two nucleotide binding domains, NBD-A and NBD-B. Giant excised inside-out membrane patches from Xenopus oocytes expressing human epithelial cystic fibrosis transmembrane conductance regulator (CFTR) were tested for their chloride conductance in response to the application of PKA and nucleotides. Rapid changes in the concentration of ATP, its nonhydrolyzable analogue adenylylimidodiphosphate (AMP-PNP), its photolabile derivative ATP-P3-[1-(2-nitrophenyl)ethyl]ester, or ADP led to changes in chloride conductance with characteristic time constants, which reflected interaction of CFTR with these nucleotides. The conductance changes of strongly phosphorylated channels were slower than those of partially phosphorylated CFTR. AMP-PNP decelerated relaxations of conductance increase and decay, whereas ATP-P3-[1-(2-nitrophenyl)ethyl]ester only decelerated the conductance increase upon ATP addition. ADP decelerated the conductance increase upon ATP addition and accelerated the conductance decay upon ATP withdrawal. The results present the first direct evidence that AMP-PNP binds to two sites on the CFTR. The effects of ADP also suggest two different binding sites because of the two different modes of inhibition observed: it competes with ATP for binding (to NBD-A) on the closed channel, but it also binds to channels opened by ATP, which might either reflect binding to NBD-A (i.e., product inhibition in the hydrolysis cycle) or allosteric binding to NBD-B, which accelerates the hydrolysis cycle at NBD-A.
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PMID:Dual effects of ADP and adenylylimidodiphosphate on CFTR channel kinetics show binding to two different nucleotide binding sites. 1039 91

Cardiac sarcoplasmic reticulum (SR) contains an endogenous phosphorylation system that under specific conditions phosphorylates two proteins with apparent molecular masses of 150 and 130 kDa. The conditions for their phosphorylation are as for the skeletal muscle sarcalumenin and the histidine-rich Ca2+ binding protein (HCP) with respect to: (i) Ca2+ and high concentrations of NaF are required; (ii) phosphorylation is obtained with no added Mg2+ and shows a similar time course and ATP concentration dependence; (iii) inhibition by similar concentrations of La3+; (iv) phosphorylation is obtained with [gamma-32P]GTP; (v) ryanodine binding is inhibited parallel to the phosphorylation of the two proteins. The endogenous kinase is identified as casein kinase II (CK II) based on its ability to use GTP as effectively as ATP, and its inhibition by La3+. The association of CK II with the cardiac SR, even after EGTA extraction at alkaline pH, is demonstrated using antibodies against CK II. The cardiac 130 kDa protein is identified as sarcalumenin based on its partial amino acid sequence and its blue staining with Stains-All. Cardiac sarcalumenin is different from the skeletal muscle protein based on electrophoretic mobilities, immunological analysis, peptide and phosphopeptide maps, as well as amino acid sequencing. Preincubation of SR with NaF and ATP, but not with NaF and AMP-PNP caused strong inhibition of ryanodine binding. This is due to decrease in Ca2+- and ryanodine-binding affinities of the ryanodine receptor (RyR) by about 6.6 and 18-fold, respectively. These results suggest that cardiac sarcalumenin is an isoform of the skeletal muscle protein. An endogenous CK II can phosphorylate sarcalumenin, and in parallel to its phosphorylation the properties of the ryanodine receptor are modified.
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PMID:Cardiac sarcalumenin: phosphorylation, comparison with the skeletal muscle sarcalumenin and modulation of ryanodine receptor. 1039 59

Previously, we have shown that the soluble form of brain glutamic acid decarboxylase (GAD) is inhibited by ATP through protein phosphorylation and is activated by calcineurin-mediated protein dephosphorylation (Bao, J., Cheung, W. Y., and Wu, J. Y. (1995) J. Biol. Chem. 270, 6464-6467). Here we report that the membrane-associated form of GAD (MGAD) is greatly activated by ATP, whereas adenosine 5'-[beta,gamma-imido]triphosphate (AMP-PNP), a non-hydrolyzable ATP analog, has no effect on MGAD activity. ATP activation of MGAD is abolished by conditions that disrupt the proton gradient of synaptic vesicles, e.g. the presence of vesicular proton pump inhibitor, bafilomycin A1, the protonophore carbonyl cyanide m-chorophenylhydrazone or the ionophore gramicidin, indicating that the synaptic vesicle proton gradient is essential in ATP activation of MGAD. Furthermore, direct incorporation of (32)P from [gamma-(32)P]ATP into MGAD has been demonstrated. In addition, MGAD (presumably GAD65, since it is recognized by specific monoclonal antibody, GAD6, as well as specific anti-GAD65) has been reported to be associated with synaptic vesicles. Based on these results, a model linking gamma-aminobutyric acid (GABA) synthesis by MGAD to GABA packaging into synaptic vesicles by proton gradient-mediated GABA transport is presented. Activation of MGAD by phosphorylation appears to be mediated by a vesicular protein kinase that is controlled by the vesicular proton gradient.
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PMID:Role of synaptic vesicle proton gradient and protein phosphorylation on ATP-mediated activation of membrane-associated brain glutamate decarboxylase. 1044 15

To test the role of nucleotide-binding fold (NBF) 2 and its interaction with the regulatory (R) domain in the function of the cystic fibrosis transmembrane conductance regulator (CFTR) channel, we used three deletion mutants of CFTR: DeltaR(708-835), DeltaNBF2(1185-1349) and DeltaR-DeltaNBF2. In lipid bilayers, DeltaNBF2 channel activity is ATP- and cAMP-dependent protein kinase (PKA)-dependent, but unlike wild-type (wt) CFTR, it displays a reduced activity and insensitivity to 5'-adenylylimidodiphosphate (AMP-PNP). Both DeltaR and DeltaR-DeltaNBF2 channels are PKA-independent, but DeltaR activity is reduced whereas DeltaR-DeltaNBF2 activity is increased. Deletion of NBF2 from CFTR affects protein trafficking and channel gating kinetics. The data suggest that NBF2 could have inhibitory and stimulatory roles in CFTR activity by interaction with NBF1 directly or indirectly via the R domain.
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PMID:Function of the second nucleotide-binding fold in the CFTR chloride channel. 1051 14

We determined the effect of nucleotides and protein kinase A (PKA) on the Ca(2+)-dependent gating of the cloned intermediate conductance, Ca(2+)-dependent K(+) channel, hIK1. In Xenopus oocytes, during two-electrode voltage-clamp, forskolin plus isobutylmethylxanthine induced a Ca(2+)-dependent increase in hIK1 activity. In excised inside-out patches, addition of ATP induced a Ca(2+)-dependent increase in hIK1 activity (NP(o)). In contrast, neither nonhydrolyzable (AMP-PNP, AMP-PCP) nor hydrolyzable ATP analogs (GTP, CTP, UTP, and ITP) activated hIK1. The ATP-dependent activation of hIK1 required Mg(2+) and was reversed by either exogenous alkaline phosphatase or the PKA inhibitor PKI(5-24). The Ca(2+) dependence of hIK1 activation was best fit with a stimulatory constant (K(s)) of 350 nM and a Hill coefficient (n) of 2.3. ATP increased NP(o) at [Ca(2+)] >100 nM while having no effect on K(s) or n. Mutation of the single PKA consensus phosphorylation site at serine 334 to alanine (S334A) had no effect on the PKA-dependent activation during either two-electrode voltage-clamp or in excised inside-out patches. When expressed in HEK293 cells, ATP activated hIK1 in a Mg(2+)-dependent fashion, being reversed by alkaline phosphatase. Neither PKI(5-24) nor CaMKII(281-309) or PKC(19-31) affected the ATP-dependent activation. Northern blot analysis revealed hIK1 expression in the T84 colonic cell line. Endogenous hIK1 was activated by ATP in a Mg(2+)- and PKI(5-24)-dependent fashion and was reversed by alkaline phosphatase, whereas CaMKII(281-309) and PKC(19-31) had no effect on the ATP-dependent activation. The Ca(2+)-dependent activation (K(s) and n) was unaffected by ATP. In conclusion, hIK1 is activated by a membrane delimited PKA when endogenously expressed. Although the oocyte expression system recapitulates this regulation, expression in HEK293 cells does not. The effect of PKA on hIK1 gating is Ca(2+)-dependent and occurs via an increase in NP(o) without an effect on either Ca(2+) affinity or apparent cooperativity.
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PMID:Kinase-dependent regulation of the intermediate conductance, calcium-dependent potassium channel, hIK1. 1061 55

Using the patch-clamp technique, we have identified an intermediate conductance Ca(2+)-activated K(+) channel from bullfrog (Rana catesbeiana) erythrocytes and have investigated the regulation of channel activity by cytosolic ATP. The channel was highly selective for K(+) over Na(+), gave a linear I-V relationship with symmetrical 117.5 mM K(+) solutions and had a single-channel conductance of 60 pS. Channel activity was dependent on Ca(2+) concentration (K(1/2) = 600 nM) but voltage-independent. These basic characteristics are similar to those of human and frog erythrocyte Ca(2+)-activated K(+) (Gardos) channels previously reported. However, cytoplasmic application of ATP reduced channel activity with block exhibiting a novel bell-shaped concentration dependence. The channel was inhibited most by approximately 10 microM ATP (P(0) reduced to 5% of control) but less blocked by lower and higher concentrations of ATP. Moreover, the novel type of ATP block did not require Mg(2+), was independent of PKA or PKC, and was mimicked by a nonhydrolyzable ATP analog, AMP-PNP. This suggests that ATP exerts its effect by direct binding to sites on the channel or associated regulatory proteins, but not by phosphorylation of either of these components.
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PMID:A novel type of ATP block on a Ca(2+)-activated K(+) channel from bullfrog erythrocytes. 1086 55

The properties of the inward-rectifying Cl- conductance in rat choroid plexus epithelial cells were investigated to allow comparisons to be made with ClC-2. All experiments were performed using the whole-cell configuration of the patch-clamp method. The conductance was transiently activated using an electrode solution which contained 375 nM catalytic subunit of protein kinase A (PKA). PKA failed to activate the conductance, however, when cells were pre-incubated with phorbol esters, which activate protein kinase C [1 microM phorbol 12-myristate 13-acetate (PMA) and 1 microM phorbol 12,13-dibutyrate (PDBu)]. Sustained activation of the conductance by PKA was observed in Ca2+-free conditions (5 mM BAPTA in the electrode solution), or when 100 nM calphostin C, a PKC inhibitor, was added to the electrode solution. The inward-rectifying Cl- conductance in choroid plexus is therefore similar to ClC-2 in that it is inhibited by PKC. The inward-rectifying conductance was blocked when Cd2+ (30 and 300 microM) and Zn2+ (1, 30 and 300 microM) were added to the bath solution. ClC-2 channels are also blocked by Zn2+ and Cd2+. The magnitude of the inward conductance was dependent on the concentration of ATP in the electrode solution. The conductance was not observed when ATP in the electrode was replaced with non-hydrolysable ATP analogues [adenosine 5'-O-(3-thiotriphosphate) (ATP[gamma-S]) and 5'-adenylylimidodiphosphate (AMP-PNP)), but it was supported by UTP and GTP. These data contrast with those of previous studies in which ClC-2 channels were activated in the absence of ATP. In conclusion, the inward-rectifying Cl- channel in rat choroid plexus shares some properties with ClC-2 (inhibition by PKC and block by divalent cations), but differs in that it depends on intracellular ATP.
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PMID:Properties of the inward-rectifying Cl- channel in rat choroid plexus: regulation by intracellular messengers and inhibition by divalent cations. 1104 61

After phosphorylation by protein kinase A, gating of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is regulated by the interaction of ATP with its nucleotide binding domains (NBDs). Models of this gating regulation have proposed that ATP hydrolysis at NBD1 and NBD2 may drive channel opening and closing, respectively (reviewed in Nagel, G. (1999) Biochim. Biophys. Acta 1461, 263-274). However, as yet there has been little biochemical confirmation of the predictions of these models. We have employed photoaffinity labeling with 8-azido-ATP, which supports channel gating as effectively as ATP to evaluate interactions with each NBD in intact membrane-bound CFTR. Mutagenesis of Walker A lysine residues crucial for azido-ATP hydrolysis to generate the azido-ADP that is trapped by vanadate indicated a greater role of NBD1 than NBD2. Separation of the domains by limited trypsin digestion and enrichment by immunoprecipitation confirmed greater and more stable nucleotide trapping at NBD1. This asymmetry of the two domains in interactions with nucleotides was reflected most emphatically in the response to the nonhydrolyzable ATP analogue, 5'-adenylyl-beta,gamma-imidodiphosphate (AMP-PNP), which in the gating models was proposed to bind with high affinity to NBD2 causing inhibition of ATP hydrolysis there postulated to drive channel closing. Instead we found a strong competitive inhibition of nucleotide hydrolysis and trapping at NBD1 and a simultaneous enhancement at NBD2. This argues strongly that AMP-PNP does not inhibit ATP hydrolysis at NBD2 and thereby questions the relevance of hydrolysis at that domain to channel closing.
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PMID:Differential interactions of nucleotides at the two nucleotide binding domains of the cystic fibrosis transmembrane conductance regulator. 1127 83


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