scholarly journals Reversion of an S49 cell cyclic AMP-dependent protein kinase structural gene mutant occurs primarily by functional elimination of mutant gene expression.

1983 ◽  
Vol 3 (2) ◽  
pp. 250-256 ◽  
Author(s):  
T van Daalen Wetters ◽  
P Coffino
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Polyacrylamide Gel Electrophoresis ◽  
Single Amino Acid ◽  
Regulatory Subunit ◽  
Wild Type ◽  
Dibutyryl Camp ◽  
Dependent Protein Kinase ◽  
Type Function ◽  
Dependent Protein

The regulatory subunits of cyclic AMP (cAMP)-dependent protein kinase from a dibutyryl cAMP-resistant S49 mouse lymphoma cell mutant, clone U200/65.1, and its revertants were visualized by two-dimensional polyacrylamide gel electrophoresis. Clone U200/65.1 co-expressed electrophoretically distinguishable mutant and wild-type subunits (Steinberg et al., Cell 10:381-391, 1977). In all 48 clones examined, reversion of the mutant to dibutyryl cAMP sensitivity was accompanied by alterations in regulatory subunit labeling patterns. Some spontaneous (3 of 11) and N-methyl-N'-nitro-N-nitrosoguanidine-induced (2 of 11) revertants retained mutant subunits, but these were altered in charge, degree of phosphorylation, or both. The charge alterations were consistent with single amino acid substitutions, suggesting that reversion was the result of second-site mutations in the mutant regulatory subunit allele that restored wild-type function, although not wild-type structure, to the gene product. The majority of spontaneous (8 of 11) and N-methyl-N'-nitro-N-nitrosoguanidine-induced (9 of 11) revertants and all of the revertants induced by ethyl methane sulfonate (14 of 14) and ICR191 (12 of 12) displayed only wild-type subunits. Dibutyryl cAMP-resistant mutants isolated from several of these revertants displayed new mutant but not wild-type subunits, suggesting that the revertant parent expresses only a single, functional regulatory subunit allele. The mutant regulatory subunit allele can, therefore, be modified in two general ways to produce revertant phenotypes: (i) by mutations that restore its wild-type function, and (ii) by mutations that eliminate its function.

Reversion of an S49 cell cyclic AMP-dependent protein kinase structural gene mutant occurs primarily by functional elimination of mutant gene expression

1983 ◽  
Vol 3 (2) ◽  
pp. 250-256
Author(s):  
T van Daalen Wetters ◽  
P Coffino
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Polyacrylamide Gel Electrophoresis ◽  
Single Amino Acid ◽  
Regulatory Subunit ◽  
Wild Type ◽  
Dibutyryl Camp ◽  
Dependent Protein Kinase ◽  
Type Function ◽  
Dependent Protein

The regulatory subunits of cyclic AMP (cAMP)-dependent protein kinase from a dibutyryl cAMP-resistant S49 mouse lymphoma cell mutant, clone U200/65.1, and its revertants were visualized by two-dimensional polyacrylamide gel electrophoresis. Clone U200/65.1 co-expressed electrophoretically distinguishable mutant and wild-type subunits (Steinberg et al., Cell 10:381-391, 1977). In all 48 clones examined, reversion of the mutant to dibutyryl cAMP sensitivity was accompanied by alterations in regulatory subunit labeling patterns. Some spontaneous (3 of 11) and N-methyl-N'-nitro-N-nitrosoguanidine-induced (2 of 11) revertants retained mutant subunits, but these were altered in charge, degree of phosphorylation, or both. The charge alterations were consistent with single amino acid substitutions, suggesting that reversion was the result of second-site mutations in the mutant regulatory subunit allele that restored wild-type function, although not wild-type structure, to the gene product. The majority of spontaneous (8 of 11) and N-methyl-N'-nitro-N-nitrosoguanidine-induced (9 of 11) revertants and all of the revertants induced by ethyl methane sulfonate (14 of 14) and ICR191 (12 of 12) displayed only wild-type subunits. Dibutyryl cAMP-resistant mutants isolated from several of these revertants displayed new mutant but not wild-type subunits, suggesting that the revertant parent expresses only a single, functional regulatory subunit allele. The mutant regulatory subunit allele can, therefore, be modified in two general ways to produce revertant phenotypes: (i) by mutations that restore its wild-type function, and (ii) by mutations that eliminate its function.

scholarly journals The amino acid sequence of a hinge region in the regulatory subunit of bovine cardiac muscle cyclic AMP-dependent protein kinase II

FEBS Letters ◽  
1980 ◽  
Vol 114 (1) ◽  
pp. 83-88 ◽  
Author(s):  
Koji Takio ◽  
Kenneth A. Walsh ◽  
Hans Neurath ◽  
Stephen B. Smith ◽  
Edwin G. Krebs ◽  
...  
Keyword(s):  
Amino Acid ◽  
Protein Kinase ◽  
Cyclic Amp ◽  
Amino Acid Sequence ◽  
Cardiac Muscle ◽  
Hinge Region ◽  
Regulatory Subunit ◽  
Dependent Protein Kinase ◽  
Protein Kinase Ii ◽  
Dependent Protein

scholarly journals Cloning and characterization of BCY1, a locus encoding a regulatory subunit of the cyclic AMP-dependent protein kinase in Saccharomyces cerevisiae.

1987 ◽  
Vol 7 (4) ◽  
pp. 1371-1377 ◽  
Author(s):  
T Toda ◽  
S Cameron ◽  
P Sass ◽  
M Zoller ◽  
J D Scott ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Kinase ◽  
Cyclic Amp ◽  
Carbon Sources ◽  
Regulatory Subunit ◽  
Dependent Protein Kinase ◽  
Yeast Saccharomyces Cerevisiae ◽  
Regulatory Subunits ◽  
Dependent Protein

We have cloned a gene (BCY1) from the yeast Saccharomyces cerevisiae that encodes a regulatory subunit of the cyclic AMP-dependent protein kinase. The encoded protein has a structural organization similar to that of the RI and RII regulatory subunits of the mammalian cyclic AMP-dependent protein kinase. Strains of S. cerevisiae with disrupted BCY1 genes do not display a cyclic AMP-dependent protein kinase in vitro, fail to grow on many carbon sources, and are exquisitely sensitive to heat shock and starvation.

Cyclic AMP-dependent protein kinase regulates sensitivity of cells to multiple drugs

1987 ◽  
Vol 7 (9) ◽  
pp. 3098-3106
Author(s):  
I Abraham ◽  
R J Hunter ◽  
K E Sampson ◽  
S Smith ◽  
M M Gottesman ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Mammalian Cells ◽  
Multiple Drug Resistance ◽  
Regulatory Subunit ◽  
Parent Cell ◽  
Dependent Protein Kinase ◽  
Dependent Protein ◽  
Antimitotic Drug ◽  
Multiple Drugs

The isolation of mutant cell lines affecting the activity of cyclic AMP (cAMP)-dependent protein kinase (PK-A) has made it possible to determine the function of this kinase in mammalian cells. We found that both a CHO cell mutant with a defective regulatory subunit (RI) for PK-A and a transfectant cell line expressing the same mutant kinase were sensitive to multiple drugs, including puromycin, adriamycin, actinomycin D, and some antimitotic drugs. The mutant and transfectant cells, after treatment with a concentration of the antimitotic drug colcemid that had no marked effect on the wild-type parent cell, had a severely disrupted microtubule network. The phenotype of hypersensitivity to the antimitotic drug colcemid was used to select revertants of the transfectant and the original mutant. These revertants simultaneously regained normal multiple drug resistance and cAMP sensitivity, thus establishing that the characteristics of colcemid sensitivity and cAMP resistance are linked. Four revertants of the transfectant reverted because of loss or rearrangement of the transfected mutant RI gene. These revertants, as well as one revertant selected from the original mutant, had PK-A activities equal to or higher than that of the parent. In these genetic studies, in which linkage of expression of a PK-A mutation with drug sensitivity is demonstrated, it was established that the PK-A system is involved in regulating resistance of mammalian cells to multiple drugs.

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 cAMP signaling in neurons: patterns of neuronal expression and intracellular localization for a novel protein, AKAP 150, that anchors the regulatory subunit of cAMP-dependent protein kinase II beta.

1992 ◽  
Vol 3 (11) ◽  
pp. 1215-1228 ◽  
Author(s):  
S B Glantz ◽  
J A Amat ◽  
C S Rubin
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Regulatory Subunit ◽  
Mammalian Brain ◽  
Dependent Protein Kinase ◽  
Intracellular Location ◽  
Anchor Protein ◽  
Camp Dependent Protein Kinase ◽  
Protein Kinase Ii ◽  
Dependent Protein

In mammalian brain, physiological signals carried by cyclic AMP (cAMP) seem to be targeted to effector sites via the tethering of cAMP-dependent protein kinase II beta (PKAII beta) to intracellular structures. Recently characterized A kinase anchor proteins (AKAPs) are probable mediators of the sequestration of PKAII beta because they contain a high-affinity binding site for the regulatory subunit (RII beta) of the kinase and a distinct intracellular targeting domain. To establish a cellular basis for this targeting mechanism, we have employed immunocytochemistry to 1) identify the types of neurons that are enriched in AKAPs, 2) determine the primary intracellular location of the anchor protein, and 3) demonstrate that an AKAP and RII beta are coenriched and colocalized in neurons that utilize the adenylate cyclase-cyclic AMP-dependent protein kinase (PKA) signaling pathway. Antibodies directed against rat brain AKAP 150 were used to elucidate the regional, cellular and intracellular distribution of a prototypic anchor protein in the CNS. AKAP 150 is abundant in Purkinje cells and in neurons of the olfactory bulb, basal ganglia, cerebral cortex, and other forebrain regions. In contrast, little AKAP 150 is detected in neurons of the thalamus, hypothalamus, midbrain, and hindbrain. A high proportion of total AKAP 150 is concentrated in primary branches of dendrites, where it is associated with microtubules. We also discovered that the patterns of accumulation and localization of RII beta (and PKAII beta) in brain are similar to those of AKAP 150. The results suggest that bifunctional AKAP 150 tethers PKAII beta to the dendritic cytoskeleton, thereby creating a discrete target site for the reception and propagation of signals carried by cAMP.

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