Expression, purification and characterization of recombinant human choline acetyltransferase: phosphorylation of the enzyme regulates catalytic activity

2000 ◽  
Vol 349 (1) ◽  
pp. 141-151 ◽  
Author(s):  
Tomas DOBRANSKY ◽  
Wanda L. DAVIS ◽  
Gong-Hua XIAO ◽  
R. Jane RYLETT
Keyword(s):  
Enzyme Activity ◽  
Catalytic Activity ◽  
Protein Kinase ◽  
Choline Acetyltransferase ◽  
Insect Cells ◽  
Two Dimensional ◽  
Cam Kinase ◽  
Wild Type ◽  
Dependent Protein Kinase ◽  
Dependent Protein

Choline acetyltransferase synthesizes acetylcholine in cholinergic neurons and, in humans, may be produced in 82- and 69-kDa forms. In this study, recombinant choline acetyltransferase from baculovirus and bacterial expression systems was used to identify protein isoforms by two-dimensional SDS/PAGE and as substrate for protein kinases. Whereas hexa-histidine-tagged 82- and 69-kDa enzymes did not resolve as individual isoforms on two-dimensional gels, separation of wild-type choline acetyltransferase expressed in insect cells revealed at least nine isoforms for the 69-kDa enzyme and at least six isoforms for the 82-kDa enzyme. Non-phosphorylated wild-type choline acetyltransferase expressed in Escherichia coli yielded six (69 kDa) and four isoforms (82 kDa) respectively. Immunofluorescent labelling of insect cells expressing enzyme showed differential subcellular localization with the 69-kDa enzyme localized adjacent to plasma membrane and the 82-kDa enzyme being cytoplasmic at 24 h. By 64 h, the 69-kDa form was in cytoplasm and the 82-kDa form was only present in nucleus. Studies in vitro showed that recombinant 69-kDa enzyme was a substrate for protein kinase C (PKC), casein kinase II (CK2) and α-calcium/calmodulin-dependent protein kinase II (α-CaM kinase), but not for cAMP-dependent protein kinase (PKA); phosphorylation by PKC and CK2 enhanced enzyme activity. The 82-kDa enzyme was a substrate for PKC and CK2 but not for PKA or α-CaM kinase, with only PKC yielding increased enzyme activity. Dephosphorylation of both forms of enzyme by alkaline phosphatase decreased enzymic activity. These studies are of functional significance as they report for the first time that phosphorylation enhances choline acetyltransferase catalytic activity.

scholarly journals Ischemia-Induced Neuronal Damage: A Role for Calcium/Calmodulin-Dependent Protein Kinase II

1996 ◽  
Vol 16 (1) ◽  
pp. 1-6 ◽  
Author(s):  
M. Neal Waxham ◽  
James C. Grotta ◽  
Alcino J. Silva ◽  
Roger Strong ◽  
Jaroslaw Aronowski
Keyword(s):  
Protein Kinase ◽  
Neuronal Damage ◽  
Alpha Subunit ◽  
Cam Kinase ◽  
Wild Type ◽  
Dependent Protein Kinase ◽  
Knock Out ◽  
Calcium Calmodulin Dependent ◽  
Protein Kinase Ii ◽  
Dependent Protein

Calcium/calmodulin-dependent protein kinase II (CaM-kinase) is a central enzyme in regulating neuronal processes. Imbalances in the activity and distribution of this enzyme have been reported following in vivo ischemia, and sustained decreases in activity correlate with subsequent neuronal death. In this report, mice that had been rendered deficient in the alpha subunit of CaM-kinase using gene knock-out technology were utilized to determine whether this enzyme is causally related to ischemic damage. Using a focal model of cerebral ischemia, we showed that homozygous knock-out mice lacking the alpha subunit exhibited an infarct volume almost twice that of wild-type litter mates. Heterozygous mice exhibited slightly less damage following ischemia than did homozygous mice, but infarct volumes remained significantly larger than those of wild-type litter mates. We conclude that reduced amounts of the alpha subunit of CaM-kinase predisposes neurons to increased damage following ischemia and that any perturbation that decreases the amount or activity of the enzyme will produce enhanced susceptibility to neuronal damage.

Differential effects of phospholamban and Ca2+/calmodulin-dependent kinase II on [Ca2+]i transients in cardiac myocytes at physiological stimulation frequencies

2008 ◽  
Vol 294 (5) ◽  
pp. H2352-H2362 ◽  
Author(s):  
Andreas A. Werdich ◽  
Eduardo A. Lima ◽  
Igor Dzhura ◽  
Madhu V. Singh ◽  
Jingdong Li ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Sarcoplasmic Reticulum ◽  
Cardiac Myocytes ◽  
Wild Type ◽  
Dependent Protein Kinase ◽  
Frequency Dependent ◽  
Physiological Range ◽  
Dependent Protein ◽  
Isolated Cardiac Myocytes

In cardiac myocytes, the activity of the Ca2+/calmodulin-dependent protein kinase II (CaMKII) is hypothesized to regulate Ca2+ release from and Ca2+ uptake into the sarcoplasmic reticulum via the phosphorylation of the ryanodine receptor 2 and phospholamban (PLN), respectively. We tested the role of CaMKII and PLN on the frequency adaptation of cytosolic Ca2+ concentration ([Ca2+]i) transients in nearly 500 isolated cardiac myocytes from transgenic mice chronically expressing a specific CaMKII inhibitor, interbred into wild-type or PLN null backgrounds under physiologically relevant pacing conditions (frequencies from 0.2 to 10 Hz and at 37°C). When compared with that of mice lacking PLN only, the combined chronic CaMKII inhibition and PLN ablation decreased the maximum Ca2+ release rate by more than 50% at 10 Hz. Although PLN ablation increased the rate of Ca2+ uptake at all frequencies, its combination with CaMKII inhibition did not prevent a frequency-dependent reduction of the amplitude and the duration of the [Ca2+]i transient. High stimulation frequencies in the physiological range diminished the effects of PLN ablation on the decay time constant and on the maximum decay rate of the [Ca2+]i transient, indicating that the PLN-mediated feedback on [Ca2+]i removal is limited by high stimulation frequencies. Taken together, our results suggest that in isolated mouse ventricular cardiac myocytes, the combined chronic CaMKII inhibition and PLN ablation slowed Ca2+ release at physiological frequencies: the frequency-dependent decay of the amplitude and shortening of the [Ca2+]i transient occurs independent of chronic CaMKII inhibition and PLN ablation, and the PLN-mediated regulation of Ca2+ uptake is diminished at higher stimulation frequencies within the physiological range.

Mutation of the Gs protein alpha subunit NH2 terminus relieves an attenuator function, resulting in constitutive adenylyl cyclase stimulation

1990 ◽  
Vol 10 (6) ◽  
pp. 2931-2940
Author(s):  
S Osawa ◽  
L E Heasley ◽  
N Dhanasekaran ◽  
S K Gupta ◽  
C W Woon ◽  
...  
Keyword(s):  
Amino Acids ◽  
Protein Kinase ◽  
G Proteins ◽  
Fold Increase ◽  
Alpha Subunit ◽  
Wild Type ◽  
Dependent Protein Kinase ◽  
Dependent Protein ◽  
Gs Protein ◽  
Camp Levels

G-proteins couple hormonal activation of receptors to the regulation of specific enzymes and ion channels. Gs and Gi are G-proteins which regulate the stimulation and inhibition, respectively, of adenylyl cyclase. We have constructed two chimeric cDNAs in which different lengths of the alpha subunit of Gs (alpha s) have been replaced with the corresponding sequence of the Gi alpha subunit (alpha i2). One chimera, referred to as alpha i(54)/s' replaces the NH2-terminal 61 amino acids of alpha s with the first 54 residues of alpha i. Within this sequence there are 7 residues unique to alpha s, and 16 of the remaining 54 amino acids are nonhomologous between alpha i and alpha s. The second chimera, referred to as alpha i/s(Bam), replaces the first 234 amino acids of alpha s with the corresponding 212 residues of alpha i. Transient expression of alpha i(54)/s in COS-1 cells resulted in an 18- to 20-fold increase in cyclic AMP (cAMP) levels, whereas expression of either alpha i/s(Bam) or the wild-type alpha s polypeptide resulted in only a 5- to 6-fold increase in cellular cAMP levels. COS-1 cells transfected with alpha i showed a small decrease in cAMP levels. Stable expression of the chimeric alpha i(54)/s polypeptide in Chinese hamster ovary (CHO) cells constitutively increased both cAMP synthesis and cAMP-dependent protein kinase activity. CHO clones expressing transfected alpha i/s(Bam) or the wild-type alpha s and alpha i cDNAs exhibited cAMP levels and cAMP-dependent protein kinase activities similar to those in control CHO cells. Therefore, the alpha i(54)/s chimera behaves as a constitutively active alpha s polypeptide, whereas the alpha i/s(Bam) polypeptide is regulated similarly to wild-type alpha s. Expression in cyc-S49 cells, which lack expression of wild-type alpha s, confirmed that the alpha i(54)/s polypeptide is a highly active alpha s molecule whose robust activity is independent of any change in intrinsic GTPase activity. The difference in phenotypes observed upon expression of alpha i(54)/s or alpha i/s(Bam) indicates that the NH2-terminal moieties of alpha s and alpha i function as attenuators of the effector enzyme activator domain which is within the COOH-terminal half of the alpha subunit. Mutation at the NH2 terminus of alpha s relieves the attenuator control of the Gs protein and results in a dominant active G-protein mutant.

Revertants of an S49 cell mutant that expresses altered cyclic AMP-dependent protein kinase

1982 ◽  
Vol 2 (10) ◽  
pp. 1229-1237
Author(s):  
T van Daalen Wetters ◽  
P Coffino
Keyword(s):  
Protein Kinase ◽  
Regulatory Subunit ◽  
Mutational Event ◽  
Wild Type ◽  
Cell Mutant ◽  
Dependent Protein Kinase ◽  
Cell Extracts ◽  
Counter Selection ◽  
Dependent Protein ◽  
Gene Lesion

Dibutyryl adenosine 3',5'-phosphate (Bt2cAMP)-sensitive (Bt2cAMPS) revertants were isolated from a resistant S49 cell mutant carrying a structural gene lesion in the regulatory subunit of cAMP-dependent protein kinase (cA-PK). This was accomplished with a counter-selection in which, first, Bt2cAMP was used to reversibly arrest revertants, and then a sequence of treatments with bromodeoxyuridine, 33258 Hoechst dye, and white light was used to kill cycling mutant cells. Reversion rates in nonmutagenized cultures could not be accurately measured, but spontaneous revertants do occur and with frequencies of less than 10(-7) to 10(-5). The mutagens ethyl methane sulfonate (EMS), N-methyl-N'-nitro-N-nitro-soguanidine (MNNG), and ICR191 increased the reversion frequency. In all cases, reversion to Bt2cAMP sensitivity was associated with restoration of wild-type levels and apparent activation constant for cAMP of cA-PK. MNNG induced revertants whose cell extracts contained cA-PK activity distinguishable from that of wild type by thermal liability. EMS did not. The counter-selection effectively isolates rare phenotypes and is therefore a useful tool in further somatic genetic experiments. The association of reversion with alterations in cA-PK function supports all previous data from this and other laboratories implicating cA-PK as the intracellular mediator of cAMP effects. Reversion is probably the result of a mutational event. Induction of reversion by ICR191 suggests the existence of a novel mechanism for generating revertants in somatic cells.

scholarly journals Cyclic AMP-dependent protein kinase in rat mammary tissue: expression of catalytic and regulatory subunits throughout pregnancy and lactation

1994 ◽  
Vol 301 (3) ◽  
pp. 807-812 ◽  
Author(s):  
R A Gardner ◽  
M T Travers ◽  
M C Barber ◽  
W R Miller ◽  
R A Clegg
Keyword(s):  
Catalytic Activity ◽  
Protein Kinase ◽  
Mammary Development ◽  
Mammary Tissue ◽  
Early Lactation ◽  
Dependent Protein Kinase ◽  
Regulatory Subunits ◽  
C Subunit ◽  
Pregnancy And Lactation ◽  
Dependent Protein

‘Expressed’ and ‘total’ activities of cyclic AMP-dependent protein kinase (PK-A) were measured in extracts of rat mammary tissue sampled throughout pregnancy and lactation. Expression of the genes encoding the catalytic subunit (C-subunit) isoforms C alpha and C beta was examined by Northern blotting, as a function of mammary development, to determine relative levels of their respective mRNAs. The content of C-subunit protein (all isoforms) was estimated immunochemically and related to levels of C-subunit catalytic activity and of mRNAs. It was found that C-subunit isoform mRNAs are expressed co-ordinately during mammary development and that a marked decline in expression, per cell, at around parturition is paralleled by a fall in ‘total’ PK-A activity. The ‘expressed’ activity of PK-A activity underwent characteristic changes throughout pregnancy and lactation, reaching a peak late in pregnancy. The PK-A activity ratio reached a peak in early lactation. C-subunit protein mass closely parallel ‘total’ PK-A activity throughout pregnancy and lactation, thereby demonstrating the constancy of C-subunit specific catalytic activity during these developmental events. Regulatory subunits (R-subunits) were probed with the photoaffinity label 8-azido-[32P]cAMP. The abundance of R-II as a proportion of total R-subunit increased throughout pregnancy and lactation, and quantitative analysis of the photoaffinity labelling suggested inconstancy in the ratio of R:C subunits, with highest values occurring in late pregnancy/early lactation.

Expression of Wild-type and Mutant Subunits of the cAMP-dependent Protein Kinase

1988 ◽  
Vol 53 (0) ◽  
pp. 111-119 ◽  
Author(s):  
G.S. McKnight ◽  
G.G. Cadd ◽  
C.H. Clegg ◽  
A.D. Otten ◽  
L.A. Correll
Keyword(s):  
Protein Kinase ◽  
Wild Type ◽  
Dependent Protein Kinase ◽  
Camp Dependent Protein Kinase ◽  
Dependent Protein

Beta-adrenoceptor metabolism in wild-type, Gs, and protein kinase A-variant S49 cells

1990 ◽  
Vol 259 (1) ◽  
pp. C41-C46 ◽  
Author(s):  
J. R. Jasper ◽  
H. J. Motulsky ◽  
L. C. Mahan ◽  
P. A. Insel
Keyword(s):  
Protein Kinase ◽  
Adrenergic Receptors ◽  
Protein Kinase Activity ◽  
Wild Type ◽  
Beta Adrenergic Receptors ◽  
Dependent Protein Kinase ◽  
Beta Adrenergic ◽  
Camp Dependent Protein Kinase ◽  
Dependent Protein ◽  
Gs Alpha

To determine the role of the stimulatory guanine nucleotide-binding protein, Gs, and adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase in the basal metabolism of beta-adrenergic receptors in S49 lymphoma cells, we measured the return of receptor number and function after irreversible blockade of receptors. After inactivation of receptors with the irreversible ligand N8-(bromoacetyl)-N'-[3-(4-indolyoxy)-2-hydroxypropyl]-(2)-1,8-diam ino-p- methane (BIM), beta-adrenergic receptors (defined as [125I]iodocyanopindolol binding sites) reappeared in a biphasic manner, the faster phase having a half-time (t 1/2) of 3-8 h (approximately 50% of the sites) and the slower phase greater than 40 h. Although the slow phase is not readily explained, recovery of binding sites during the first 10 h matched recovery of receptor function after BIM treatment (as measured by stimulation of cAMP accumulation) and recovery of receptor sites after downregulation induced by the agonist isoproterenol. Thus quantifying receptor recovery during the first 10 h after BIM treatment appears to be a reasonable method for examining basal receptor metabolism in S49 cells. Measured in this way, metabolism of beta-adrenergic receptors is very similar in wild-type S49 and the following variant clones: cyc- (absent Gs alpha), UNC and H21a (defective Gs alpha), and kin- (lacking cAMP-dependent protein kinase activity). Although previous data have demonstrated that agonist-promoted downregulation of beta-adrenergic receptors requires functional receptor-Gs coupling, the current data suggest that neither Gs nor cAMP-dependent protein kinase activity plays an important role in the regulation of basal metabolism of beta-adrenergic receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

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