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

---

Query: EC:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The apical membrane of intestinal epithelial cells harbors a unique isozyme of cGMP-dependent protein kinase (cGK type II) which acts as a key regulator of ion transport systems, including the cystic fibrosis transmembrane conductance regulator (CFTR)-chloride channel. To explore the mechanism of cGK II membrane-anchoring, recombinant cGK II was expressed stably in HEK 293 cells or transiently in COS-1 cells. In both cell lines, cGK II was found predominantly in the particulate fraction. Immunoprecipitation of solubilized cGK II did not reveal any other tightly associated proteins, suggesting a membrane binding motif within cGK II itself. The primary structure of cGK II is devoid of hydrophobic transmembrane domains; cGK II does, however, contain a penultimate glycine, a potential acceptor for a myristoyl moiety. Metabolic labeling showed that cGK II was indeed able to incorporate [3H]myristate. Moreover, incubation of cGK II-expressing 293 cells with the myristoylation inhibitor 2-hydroxymyristic acid (1 mM) significantly increased the proportion of cGK II in the cytosol from 10 +/- 5 to 35 +/- 4%. Furthermore, a nonmyristoylated cGK II Gly2 --> Ala mutant was localized predominantly in the cytosol after transient expression in COS-1 cells. The absence of the myristoyl group did not affect the specific enzyme activity or the Ka for cGMP and only slightly enhanced the thermal stability of cGK II. These results indicate that N-terminal myristoylation fulfills a crucial role in directing cGK II to the membrane.
...
PMID:N-terminal myristoylation is required for membrane localization of cGMP-dependent protein kinase type II. 863 33

Earlier studies (Hawkins, C., Xu, A., and Narayanan, N. (1994) J. Biol. Chem. 269, 31198-31206) have suggested that the Vmax of Ca2+ uptake is enhanced up to 2-fold through phosphorylation of Ser38 in the cardiac Ca2+-ATPase (SERCA2a) by calmodulin-dependent protein kinase (CaM kinase). It is difficult, however, to determine whether stimulation is caused by phosphorylation of the Ca2+-ATPase or by phosphorylation of phospholamban in cardiac microsomes. We have expressed SERCA2a in HEK-293 cells in the presence or absence of phospholamban and measured the effects on Ca2+ uptake activity of phosphorylation of microsomal proteins by CaM kinase or protein kinase A (PKA). We found no effect on the Vmax of Ca2+ uptake following phosphorylation by CaM kinase or PKA in either the presence or absence of phospholamban. The K0.5 for Ca2+ dependence of Ca2+ transport, however, was shifted following phosphorylation by either CaM kinase or PKA in those microsomes containing both SERCA2a and phospholamban, but not in those expressing only SERCA2a. Thus, we cannot confirm earlier reports of stimulation of SERCA2a activity by CaM kinase II phosphorylation of Ser38. Our studies, however, emphasize the need for adequate controls for measurement of Vmax.
...
PMID:The vmax of the Ca2+-ATPase of cardiac sarcoplasmic reticulum (SERCA2a) is not altered by Ca2+/calmodulin-dependent phosphorylation or by interaction with phospholamban. 866 32

We have characterized the phosphorylation of the glutamate receptor subunit GluR1, using biochemical and electrophysiological techniques. GluR1 is phosphorylated on multiple sites that are all located on the C-terminus of the protein. Cyclic AMP-dependent protein kinase specifically phosphorylates SER-845 of GluR1 in transfected HEK cells and in neurons in culture. Phosphorylation of this residue results in a 40% potentiation of the peak current through GluR1 homomeric channels. In addition, protein kinase C specifically phosphorylates Ser-831 of GluR1 in HEK-293 cells and in cultured neurons. These results are consistent with the recently proposed transmembrane topology models of glutamate receptors, in which the C-terminus is intracellular. In addition, the modulation of GluR1 by PKA phosphorylation of Ser-845 suggests that phosphorylation of this residue may underlie the PKA-induced potentiation of AMPA receptors in neurons.
...
PMID:Characterization of multiple phosphorylation sites on the AMPA receptor GluR1 subunit. 866 94

UMR106-06 cells predominantly express the C1a isoform of the rat calcitonin (CT) receptor (CTR). We have compared the homologous regulation of the C1a CTR endogenously expressed in UMR106-06 cells with the cloned C1a CTR in transfected HEK 293 cells, in which expression is driven by a heterologous promoter. It was found that treatment of both cell lines with either salmon CT or human CT reduced the density of cell surface CTR in a dose- and time-dependent manner. However, the magnitude of the response was greater in UMR106-06 cells, and salmon CT was more potent than human CT in both cell lines. Recovery from down-regulation was rapid in transfected cells (< 2 h), but was comparatively delayed in UMR106-06 cells, where less than 70% of receptor-binding capacity had returned by 24 h. In both cell lines, treatment with either agonist increased the basal activity of CT-sensitive adenylate cyclase and caused a time-dependent reduction in the responsiveness of adenylate cyclase to a second challenge with CT. Reduced responsiveness occurred under conditions of minimal loss of CTR from the cell surface, consistent with an uncoupling of the receptor from the signal transduction apparatus. Recovery of CT-sensitive adenylate cyclase was complete in transfected cells by 24 h, but was delayed in UMR106-06 cells, paralleling the slow recovery of receptor binding. CT-induced down-regulation of the CTR was not mimicked by receptor-independent activation of protein kinase A or protein kinase C. However, treatment of cells for 24 h, but not for 4 h, with phorbol ester caused a partial loss of CTR binding in UMR106-06 cells and resulted in an approximately 200% increase in CTR binding in transfected HEK 293 cells. CTR messenger RNA levels, as assessed by reverse transcription-PCR, were not changed by any of the above treatments. These results suggest that CT-induced receptor down-regulation and modulation of the ability of CT to activate adenylate cyclase are inherent properties of the receptor, as they can be recapitulated in an otherwise CTR-naive cell line, in which receptor expression is driven by a heterologous gene promoter. Moreover, and in contrast with CTR regulation in osteoclasts, activation of protein kinase A is insufficient for ligand-induced regulation of the CTR in these nonosteoclastic cell lines, and receptor regulation does not appear to involve altered messenger RNA levels.
...
PMID:Homologous regulation of the rat C1a calcitonin receptor (CTR) in nonosteoclastic cells is independent of CTR messenger ribonucleic acid changes and cyclic adenosine 3',5'-monophosphate-dependent protein kinase activation. 889 20

Parathyroid hormone (PTH) regulates calcium metabolism through a specific G protein-coupled, seven-transmembrane helix-containing receptor. This receptor also binds and is activated by PTH-related protein (PTHrP). The human (h) PTH/PTHrP receptor is a membrane glycoprotein with an apparent molecular weight of approximately 85000 which contains four putative N-glycosylation sites. To elucidate the functional role of receptor glycosylation, if any, we studied hormone binding and signal transduction in human embryonic kidney cells transfected with hPTH/PTHrP receptor (HEK-293/C-21). These cells stably express 300000-400000 receptors per cell. Inhibition of N-glycosylation with an optimized concentration of tunicamycin yielded completely nonglycosylated hPTH/PTHrP receptor (approximately 60 kDa). This receptor form is fully functional; it maintains nanomolar binding affinity for PTH- and PTHrP-derived agonists and antagonists. PTH and PTHrP agonists stimulate cyclic AMP accumulation and increases in cytosolic calcium levels. In addition, the highly potent benzophenone (pBz2)-containing PTH-derived radioligand [Nle8,18,Lys13(epsilon-pBz2),L-2-Nal23,Tyr34 3-125I)]bPTH(1-34)NH2 can photoaffinity cross-link specifically to the nonglycosylated receptor. The molecular weight (approximately 60000) of the band representing the photo-cross-linked, nonglycosylated receptor (obtained from the tunicamycin-treated HEK-293/C-21 cells) was similar to that of the deglycosylated photo-cross-linked receptor (obtained by enzymatic treatment with Endoglycosidase-F/N-glycosidase-F). Our findings indicate that glycosylation of the hPTH/PTHrP receptor is not essential for its effective expression on the plasma membrane or for the binding of ligands known to interact with the native receptor. The nonglycosylated hPTH/PTHrP receptor remains fully functional with regard to both of its known signal transduction pathways: cAMP-protein kinase A and phospholipase C-cytosolic calcium.
...
PMID:Role of glycosylation in expression and function of the human parathyroid hormone/parathyroid hormone-related protein receptor. 896 54

The oxime derivative 2,3-butanedione monoxime (BDM) is used as an inorganic phosphatase to probe the phosphorylation state of many cellular proteins including the L-type calcium channel in various tissues. We used BDM further to shed light on the controversy surrounding direct phosphorylation of the L-type Ca2+ channel. We employed a recombinant system that utilizes HEK 293 cells expressing wild type and mutant human heart calcium channels. BDM reversibly reduced the calcium channel current induced by expression of the wild type channel in a concentration-dependent manner with an apparent IC50 value of 15.3 mM. Deletion of part of the carboxyl terminus of the alpha 1 subunit, which contains one putative protein kinase A site, or mutating all of the protein kinase A consensus sites of the pore forming subunit, did not significantly change the apparent IC50 value or alter in any other way the blocking effect of BDM on the expressed currents. Our data suggest that BDM produces reversible modifications of the cardiac calcium channel protein leading to an expected reduction in the amplitude of the expressed currents, but the site of action must be different from that of the consensus sites for protein kinase A dependent phosphorylation.
...
PMID:Inhibition of cloned human L-type cardiac calcium channels by 2,3-butanedione monoxime does not require PKA-dependent phosphorylation sites. 901 46

Mitogen-activated protein kinase kinase (MEK) is a dual-specificity protein kinase that is located primarily in the cellular cytosol, both prior to and upon mitogenic stimulation. The existence of a nuclear export signal in the N-terminal domain of MEK [Fukuda, M., Gotoh, I., Gotoh, Y. & Nishida, E. (1996) J. Biol. Chem. 271, 20024-20028] suggests that there are circumstances under which MEK enters the nucleus and must be exported. Using mutants of MEK, we show that the deletion of the nuclear export signal sequence from constitutively active MEK caused constitutive localization of MEK in the nucleus of COS7 and HEK-293T cells. However, when the same region was deleted from a catalytically inactive MEK, cytoplasmic localization was observed in resting cells, which turned nuclear upon stimulation. Confocal microscopy of COS7 cells expressing the above mutants showed localization of the active MEK in the nuclear envelope and also in the cell periphery. The differences in cellular localization between the wild-type and mutant MEKs are not due to severe changes in specificity because the recombinant, constitutively active MEK that lacked its N-terminal region exhibited the same substrate specificity as the wild-type MEK, both in vitro and in intact cells. Taken together, our results indicate that upon mitogenic stimulation, MEK, like extracellular signal responsive kinase and p90(RSK), is massively translocated to the nucleus. Rapid export from the nucleus, which is mediated by the nuclear export signal, is probably the cause for the cytoplasmic distribution observed with wild-type MEK.
...
PMID:Nuclear translocation of mitogen-activated protein kinase kinase (MEK1) in response to mitogenic stimulation. 910 48

Long-term potentiation (LTP), a cellular model of learning and memory, requires calcium-dependent protein kinases. Induction of LTP increased the phosphorus-32 labeling of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors (AMPA-Rs), which mediate rapid excitatory synaptic transmission. This AMPA-R phosphorylation appeared to be catalyzed by Ca2+- and calmodulin-dependent protein kinase II (CaM-KII): (i) it correlated with the activation and autophosphorylation of CaM-KII, (ii) it was blocked by the CaM-KII inhibitor KN-62, and (iii) its phosphorus-32 peptide map was the same as that of GluR1 coexpressed with activated CaM-KII in HEK-293 cells. This covalent modulation of AMPA-Rs in LTP provides a postsynaptic molecular mechanism for synaptic plasticity.
...
PMID:Regulatory phosphorylation of AMPA-type glutamate receptors by CaM-KII during long-term potentiation. 922 9

Previous studies indicated that partial agonists cause less desensitization of the beta2-adrenergic receptor (betaAR) than full agonists; however, the molecular basis for this in intact cells has not been investigated. In the present work, we have determined the rates of desensitization, internalization, and phosphorylation caused by a series of betaAR agonists displaying a 95-fold range of coupling efficiencies. These studies were performed with HEK-293 cells overexpressing the betaAR with hemagglutinin and 6-histidine epitopes introduced into the N and C termini, respectively. This modified betaAR behaved identically to the wild type receptor with regard to agonist Kd, coupling efficiency, and desensitization. The coupling efficiencies for betaAR agonist activation of adenylyl cyclase relative to epinephrine (100%) were 42% for fenoterol, 4.9% for albuterol, 2.5% for dobutamine, and 1.1% for ephedrine. At concentrations of these agonists yielding >90% receptor occupancy, the rate and extent (0-30 min) of agonist-induced desensitization of betaAR activation of adenylyl cyclase followed the same order as coupling efficiency, i.e. epinephrine >/= fenoterol > albuterol > dobutamine > ephedrine. The rate of internalization of the betaAR with respect to these agonists also followed the same order as the desensitization and exhibited a slight lag. Like internalization and desensitization, betaAR phosphorylation exhibited a dependence on agonist strength. The two strongest agonists, epinephrine and fenoterol, provoked 11-13-fold increases in the level of betaAR phosphorylation after just 1 min, whereas the weak agonists dobutamine and ephedrine caused only 3-4-fold increases, similar to levels induced by cAMP-dependent protein kinase activation with forskolin. With longer treatment times, the level of betaAR phosphorylation declined with strong agonists, but it progressively increased with the weaker partial agonists, such that after 30 min the -fold elevation with epinephrine (6.2 +/- 0.82) was not appreciably different from ephedrine (5.0 +/- 0.96) and significantly less than that caused by albuterol (10.4 +/- 1.7). In summary, our results demonstrate an excellent proportionality between the agonist strength and agonist-induced desensitization, internalization, and the rapid initial phase of phosphorylation. The data support the hypothesis that increasing agonist-coupling efficiency primarily affects desensitization by increasing the rate of betaARK phosphorylation of the betaAR.
...
PMID:beta2-adrenergic receptor desensitization, internalization, and phosphorylation in response to full and partial agonists. 929 36

1. We have used non-stationary variance analysis to examine the single channel conductance and the probability of channel opening at the peak of the homomeric GluR6 response (Po,peak) to 100-200 ms application (10-90% exchange time, 0.3 ms) of glutamate onto excised membrane patches from transiently transfected human embryonic kidney cells (HEK 293). 2. Our determinations of both Po,peak and single channel conductance of simulated current responses are insensitive to system filtering, response rise time, desensitization rate and measured variation in our drug perfusion speed. Isolation of stochastic current fluctuations using the local mean response waveform minimizes problems associated with modest rundown of response amplitude during the experiment. 3. The slope conductance calculated from the weighted mean unitary currents for the channels activated in response to glutamate application is 16 pS. Chord conductance between-40 and -80 mV is independent of agonist concentration. Conversion of the codon for glutamine621 to arginine (Q621R) by RNA editing reduces conductance by more than 35-fold to less than 0.4 pS without changing response time course, desensitization, or Po,peak. 4. Po,peak is high at saturating glutamate concentrations (0.65 +/- 0.23; mean +/- S.D.) and varies with agonist concentrations. The half-maximally effective glutamate concentration (EC50) determined for Po,peak (0.2 mM; Hill slope = 0.6) is similar to that determined for the macroscopic peak current amplitude (0.5 mM; Hill slope = 1.0) in response to rapid agonist application. 5. Inclusion of the purified catalytic subunit of cAMP-dependent protein kinase A (PKA) in the patch pipette increases Po,peak to 0.85 +/- 0.12 and co-transfection of cells with a cDNA encoding the catalytic subunit of PKA (C alpha-PKA) increases Po,peak to 0.94 +/- 0.09. 6. Inclusion of purified calcineurin plus its coactivators 200 nM Ca2+ and calmodulin in the patch pipette decreases Po,peak to 0.48 +/- 0.10. The calcineurin-stimulated decrease of Po,peak in cells co-transfected with C alpha-PKA is blocked by 800 nM deltamethrin, a calcineurin inhibitor. Calmodulin, 200 nM Ca2+ and deltamethrin have no effect on Po,peak in the absence of calcineurin. As predicted from its effects on Po,peak, inclusion of calcineurin in the patch pipette accelerates the run-down of whole cell GluR6 responses in cells co-transfected with C alpha-PKA. 7. The effects of both calcineurin and PKA on Po,peak for GluR6 receptors in excised patches occur without any detectable changes to response time course, desensitization, or chord conductance. 8. We conclude that the binding of glutamate to homomeric GluR6 receptors is associated with a high probability of channel opening, which is under the control of two signalling systems that are known to be co-localized at the neuronal membrane: PKA (Po,peak near 1.0) and calcineurin (Po,peak near 0.5).
...
PMID:Control of rat GluR6 glutamate receptor open probability by protein kinase A and calcineurin. 937 2


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>

---