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)

O2 plays a dominant role in the metabolism and viability of cells; changes in O2 supply lead to many physiological responses in the cell. Recent reports have shown that hypoxia induces the transcription of a number of genes, among them those for the glycolytic enzymes. We have investigated signalling events that may lead to enhanced activity of lactate dehydrogenase (LDH) in cultured vascular smooth muscle (VSM) cells derived from rat aorta, grown under hypoxic conditions (1% versus 20% O2). LDH was chosen because this enzyme exhibits one of the largest increases in activity among the glycolytic enzymes after hypoxic stimulation of cells. Hypoxic exposure of VSM cells for 24 h resulted in a 2-fold increase in LDH activity and in a 2.5-fold increase in intracellular cAMP levels. Agents that activate adenylate cyclase, such as forskolin, cholera toxin and 1-methyl-3-isobutylxanthine (IBMX), and thus increase cAMP production, significantly induced LDH activity. Moreover, induction of LDH activity by hypoxia was prevented in the presence of the protein kinase A inhibitor N-[2-(methyl-amino)ethyl]-5-isoquinolinsulphonamide dihydrochloride (H-8), and the cyclooxygenase inhibitor indomethacin. In contrast to the cAMP-stimulating agents, stable cGMP analogues (dibutyryl-cGMP, 8-bromo-cGMP), activators of protein kinase C [12-O-tetradecanoylphorbol-13-acetate (TPA), and 1-oleoyl-2-acetyl-glycerol (OAG), and the calcium ionophore ionomycin did not alter LDH activity in VSM cells kept at 20% O2. A dose-dependent increase in LDH activity was also observed in normoxic cells exposed to cobalt chloride (50-200 microM), indicating that a metal binding protein might be involved in this signalling cascade.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Hypoxia and cobalt stimulate lactate dehydrogenase (LDH) activity in vascular smooth muscle cells. 789 7

The regulation by endothelin-1 (ET-1) of cytosolic free calcium ion concentrations ([Ca2+]i) was investigated in single immature rat (testicular) Sertoli cells. [Ca2+]i was estimated in individual gonadal cells by digital imaging videomicroscopy using the calcium indicator dye fura-2/AM. Two concentration-dependent types of ET-1-induced [Ca2+]i signals were observed. Responses to high ET-1 concentrations (1.0-1000 nM) were characterized by a biphasic, rapid, and transient [Ca2+]i rise (spike) within 10 sec, followed by an exponential decrease toward a new steady state level (plateau phase) in 98% of responsive cells. At low concentrations of ET-1 (0.001 or 0.1 nM), the [Ca2+]i increase was slower, reaching peak values 40-100 sec after stimulation and remaining elevated for 2-3 min of observation. There was cell-cell heterogeneity in the amplitude and kinetics of the [Ca2+]i response to the same concentration of ET-1. However, there was a significant ET-1 concentration-dependent increase in the total percentage of cells responding to ET-1. Removal of extracellular Ca2+ or use of Ca2+ channel blockers (verapamil or cobalt) did not affect the ET-1-induced [Ca2+]i spike phase, but abolished the plateau phase, suggesting that ET-1 induces the mobilization of Ca2+ from internal stores, followed by calcium influx from extracellular sources. In cell population experiments, ET-1 attenuated FSH-stimulated cAMP and estradiol accumulation by Sertoli cells. These inhibitory effects were mimicked by phorbol 12-myristate 13-acetate, an activator of protein kinase-C, suggesting that ET-1 action on Sertoli cells might be linked to the protein kinase-C pathway. In conclusion, the present investigations demonstrate that ET-1 activates an intracellular signaling pathway involving [Ca2+]i in single rat Sertoli cells. The sources of the biphasic [Ca2+]i response include mobilization of Ca2+ from internal stores, followed by Ca2+ influx via verapamil- and cobalt-sensitive Ca2+ channels. Increasing ET-1 concentrations recruit an increasing number of individual Sertoli cells responding with a spike-plateau [Ca2+]i signal, thus offering a mechanism at the single cell level for the ET dose-response curve.
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PMID:Mechanisms by which endothelin-1 stimulates increased cytosolic free calcium ion concentrations in single rat Sertoli cells. 801 44

Regulation of steroidogenesis by luteinizing hormone is mediated by cAMP and calcium. We have investigated changes in cytosolic free calcium ([Ca2+]i) in Leydig cells by using Fura-2 as the fluorescent calcium indicator. Purified Leydig cells were plated on polylysine coated glass coverslips, cultured for 24 h in DMEM/F12 and loaded with Fura-2 at 37 degrees C. [Ca2+]i measurements were made fluorometrically by placing coverslips into 3 ml cuvettes with PBS+calcium. Addition of hCG increased [Ca2+]i gradually after a lag of about 2-3 min and plateaued by 5-6 min. The plateau level was sustained for at least 15 min. Absence of external Ca2+ in the medium or presence of diltiazem or nicardipine or cobalt chloride abolished the rise. Addition of BAY K 8644 or KCl caused a small but significant increase of [Ca2+]i. 8-Br-cAMP, forskolin or cholera toxin produced a gradual rise in [Ca2+]i that plateaued after 5-6 min similar to that observed with hCG. The action of hCG was inhibited by protein kinase A inhibitor (20-residues peptide) but not by protein kinase C inhibitor (staurosporine). We conclude that binding of hCG to its receptors would transmit the signal through G proteins to adenylyl cyclase to increase cAMP which would increase Ca2+ influx into cytosol across plasma membrane Ca2+ channels. Therefore, it appears that the primary action of hCG is to increase cytosolic cAMP which would then regulate [Ca2+]i as well as steroidogenesis.
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PMID:Human chorionic gonadotropin (hCG) increases cytosolic free calcium in adult rat Leydig cells. 803 93

Highly purified casein kinase II (CK II) isozymes from bovine brain gray matter (BBGM) were obtained by means of a new purification procedure consisting of one phosphocellulose and three Mono-Q steps. The phosphocellulose eluate showed two BBGM-CK II activities. The first minor component (BBGM-CK IIa) was eluted with 0.9 M NaCl and the major component was eluted at 1.1 M NaCl (BBGM-CK IIb). The protein complexes responsible for these two activities were comprised of three subunits, i.e., alpha (40 kDa), alpha' (38 kDa), and beta (28 kDa), with various subunit ratios. The two isozymes displayed the same behavior on Superose 12 fast protein liquid chromatographic gel filtration and sucrose density centrifugation. BBGM-CK IIa and b showed chromatographic and biochemical differences including differing Km for ATP and GTP and Ki for heparin and 2,3-bisphosphoglycerate. The properties of the main peak (BBGM-CK IIb) were studied in detail. The stimulatory effect of Mg2+, Mn2+, and Co2+ was highly dependent both on the nature of the substrate and on ionic type and concentration. It is surprising that with phosvitin as substrate, BBGM-CK IIb was fully active even in the absence of Mg2+ and NaCl. The inhibitory effect of heparin and the stimulatory effects of NaCl, KCl, spermine, and polylysine were highly dependent on the ionic strength, buffer type, and substrate. BBGM-CK II isozymes phosphorylated stathmine in the presence of polylysine, but the requirement for polybasic compounds was not absolute, as is the case with calmodulin and clathrin beta-light chain.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Purification and characterization of two casein kinase type II isozymes from bovine brain gray matter. 803 96

To study the cellular basis for FSH-stimulated dose-dependent graded increases in intracellular Ca2+ concentrations in populations of Sertoli cells, we investigated the effects of FSH on free Ca2+ ion concentrations ([Ca2+]i) in individual rat Sertoli cells using the Ca(2+)-sensitive dye fura-2/AM and digital fluorescent videomicroscopy. Ovine or rat FSH elicited a hormone-specific rise in [Ca2+]i within 20-140 sec, with a peak level 2.7 +/- 0.9-fold greater than the basal value (mean +/- SEM; n = 8) lasting for 4-16 min. The amplitude and kinetics of the FSH-induced [Ca2+]i signal were not dose dependent. Instead, increasing doses of FSH recruited a higher percentage of responding cells. Chelation of extracellular Ca2+ or cotreatment with verapamil or cobalt abolished FSH-induced [Ca2+]i increases. Furthermore, in the presence of extracellular Mn2+, direct evidence for FSH-mediated Ca2+ influx was obtained from the quench of fura-2 fluorescence. Induced Ca2+ increases were mimicked by forskolin or protein kinase-A type I activators [8-(6-amino-hexyl)amino-cAMP and N6-benzoyl-cAMP (N6B)]. However, the cAMP analogs, 8-bromo-cAMP, N6,2'-O-dibutyryl cAMP, or protein kinase-A type II activators (8-thiomethyl-cAMP and N6B), induced [Ca2+]i increases even in the absence of extracellular Ca2+, and the time course of the [Ca2+]i rise induced by cAMP analogs was more rapid than that induced by FSH. Similarly, the uninhibited rise in [Ca2+]i induced by FSH in pertussis toxin-pretreated Sertoli cells suggests that PT-sensitive G-proteins are not involved in the action of FSH on [Ca2+]i. In summary, we demonstrate that FSH evokes sustained [Ca2+]i increases in single Sertoli cells in a nongraded fashion and recruits increasing numbers of responding cells in a dose-dependent fashion. We also provide explicit evidence that FSH induces Ca2+ influx. Mimicry of the FSH-induced [Ca2+]i rise by certain cAMP analogs [8-(6-amino-hexyl)amino-cAMP and N6B; protein kinase-A type I activator] or forskolin suggests that Ca2+ may be part of a dual pathway of cAMP-initiated intracellular signaling.
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PMID:Cellular basis for follicle-stimulating hormone-stimulated calcium signaling in single rat Sertoli cells: possible dissociation from effects of adenosine 3',5'-monophosphate. 813 59

Phosphorylation of the peptide LRRASLG by the catalytic subunit of cAMP-dependent protein kinase was measured in the presence of various divalent metals to establish the role of electrophiles in the kinetic mechanism. Under conditions of low or high metal concentrations, the apparent second-order rate constant, kcat/Kpeptide, and the maximal rate constant, kcat, followed the trend Mg2+ > Co2+ > Mn2+. Competitive inhibition studies indicate that the former effect is not due to destabilization of the substrate complex, E.ATP.S. The effects of solvent viscosity on the steady-state kinetic parameters were interpreted according to a simple mechanism involving substrate binding, phosphotransfer, and product release steps and two metal chelation sites in the nucleotide pocket. Decreases in kcat and kcat/Kpeptide result mostly from attenuations in the dissociation rate constant for ADP and the association rate constant for the substrate, respectively. Decreases in the phosphoryl transfer rate constant have only negligible to moderate effects on these parameters. The low observed values for the association rate constant of the substrate indicate that the metals control the concentration of the productive binary form, Ea.ATP, and indirectly the accessibility of the active site. By comparison, Mg2+ is the best divalent metal catalyst because it uniformly lowers the transition state energies for all steps in the kinetic mechanism, permitting maximum flux of substrate to product. The data suggest that cAMP-dependent protein kinase uses metal ions to serve multiple roles in facilitating phosphotransfer and accelerating substrate association and product dissociation.
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PMID:Divalent metal ions influence catalysis and active-site accessibility in the cAMP-dependent protein kinase. 829 63

We have observed that soluble extracts from the extreme acidothermophilic archaebacterium Sulfolobus solfataricus contained protein phosphatase activity that was greatly stimulated by the divalent metal ions Mn2+, Mg2+, Ni2+, or Co2+. This activity apparently arose from a single enzyme since (a) stimulation by these divalent metal ions was not additive and (b) protein phosphatase activity eluted as a single peak from both a DE52 ion-exchange column and a Sephadex G-100 gel filtration column. Its apparent molecular mass was approximately 28,000 daltons. The enzyme dephosphorylated a variety of phosphoserine-containing substrates including casein, histone H2a, phosphorylase kinase, or glycogen phosphorylase. The enzyme would not dephosphorylate either histone H1 or a number of phosphotyrosine-containing compounds. It removed only half the phosphate bound to histone H2b, which is phosphorylated at two sites by the cAMP-dependent protein kinase. Protein phosphatase activity was inhibited by EDTA, Cu2+, Zn2+, NaF, inorganic phosphate, or pyrophosphate; but was unaffected by other potential activators and inhibitors such as microcystin, okadaic acid, vanadate, polyamines, or sulfhydryl modifying reagents. This enzyme represents the first protein phosphatase to be identified in any member of the third and oldest phylogenetic kingdom in nature, the archaebacteria.
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PMID:Identification of a serine/threonine-specific protein phosphatase from the archaebacterium Sulfolobus solfataricus. 838 14

Recent reports suggest that membrane-bound casein kinase I (MBCK I) activity in erythrocytes is inactivated by exogenously added phosphatidylinositol 4,5-bisphosphate (PIP2) (Bazenet et al. (1990) J. Biol. Chem. 265, 7369-7376; Brockman and Anderson (1991) J. Biol. Chem. 266, 2508-2512). Here we report that PIP2-mediated inhibition of MBCK I in erythrocytes is only observed if exogenous PIP2 and the kinase are allowed to interact in the absence of Mg2+. Prior incubation of PIP2 with 1 mM Mg2+ prevents the inactivation of MBCK I by PIP2. Other divalent cations (Ni2+, Co2+, Mn2+, Cd2+, Ca2+) and trivalent metal ions (La3+, Cr3+, Al3+) did not protect MBCK I from PIP2-mediated inactivation, indicating that the protective effect is specific for Mg2+ only. We propose a role of Mg2+ in the interaction of CK I with phosphoinositides, and that PIP2-mediated inhibition of protein kinase(s) may be a non-physiological phenomenon.
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PMID:Magnesium protects phosphatidylinositol-4,5-bisphosphate-mediated inactivation of casein kinase I in erythrocyte membrane. 839 52

Casein kinase II purified from the nuclei of Xenopus laevis oocytes as well as the recombinant alpha and beta subunits of the X. laevis CKII, produced in E. coli from the cloned cDNA genes, were tested with different divalent metal ions. The enzyme from both sources was active with either Mg2+, Mn2+, or Co2+. Optimal concentrations were 7-10 mM for Mg2+, 0.5-0.7 mM for Mn2+ and 1-2 mM for Co2+. In the presence of Mn2+ or Co2+ the enzyme used GTP more efficiently than ATP as a phosphate donor while the reverse was true in the presence of Mg2+. The apparent Km values for both nucleotide triphosphates were greatly decreased in the presence of Mn2+ as compared with Mg2+. Addition of Zn2+ (above 150 microM) to an assay containing the optimal Mg2+ ion concentration caused strong inhibition of both holoenzyme and alpha subunit. Inhibition of the holoenzyme by 400 microM Ni2+ could be reversed by high concentrations of Mg2+ but no reversal of this inhibition was observed with the alpha subunit.
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PMID:Effect of metal ions on the activity of casein kinase II from Xenopus laevis. 841 74

Mg2+ as well as Mn2+, and Co2+, which may substitute Mg2+ in the mental ion requirement of casein kinase 2 (Gatica et al., FEBS Lett: 315:173-173, 1993), have been repeatedly reported to display an optimal concentration at which activity of casein kinase 2 is maximal. As far as we know this intriguing property has always been observed with casein as substrate. This phosphoprotein is not the natural substrate of the enzyme, and it is well known that it binds divalent metal ions, which provoke the aggregation and precipitation of the protein. Since an optimal concentration of metal ion might have a regulatory role, we have examined if it is a consequence of the particular properties of casein, or it is an inherent property of the enzyme, extensive to other substrates. We have used the type II regulatory subunit of protein kinase A which is a physiological substrate of the enzyme, and the peptide RRREEETEEE as a specific substrate. No optimal concentration of Mg2+ is observed when these two substrates are used. The results explain, however, why that optimum is observed with casein. Although low concentration of Mn2+, and Co2+ render about 25% of the maximal activity found with Mg2+, they inactivate the enzyme almost fully at concentrations at which Mg2+ yield the maximal activity.
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PMID:Casein kinase 2 inactivation by Mg2+, Mn2+ and Co2+ ions. 860 6


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