Altered cyclic AMP‐dependent protein kinase activity in a mutant adrenocortical tumor cell line

Gutmann, N S; Rae, Patricia A.; Schimmer, Bernard P.

doi:10.1002/jcp.1040970320

Cited by 45 publications

(17 citation statements)

References 30 publications

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“…Similar findings have been reported in neuroblastoma (27), Y1 adrenocortical (14), macrophage-like (25), and Chinese hamster ovary (28) cell lines. Only one Bt2cAMP-resistant S49 variant that does not affect cA-PK has been characterized (30).…”

supporting

confidence: 87%

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

Wetters¹,

Coffino

1982

Mol. Cell. Biol.

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Dibutyryl adenosine 3',5'-phosphate (Bt2cAMP)-sensitive (Bt2cAMP1) 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 i0-5. The mutagens ethyl methane sulfonate (EMS), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), and ICR191 increased the reversion frequency. In all cases, reversion to Bt2cAMP sensitivity was associated with restoration of wildtype 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 lability. 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.

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supporting

confidence: 87%

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

Wetters¹,

Coffino

1982

Mol. Cell. Biol.

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“…We observed that an increase in cAMP content (and activation of PKA (7,23)) triggers the rapid (Յ1 min) and selective dephosphorylation of tyrosine residues on paxillin in Y1 cells. The dephosphorylation reaction precedes depletion of paxillin from focal adhesions, disassembly of adhesion plaques, dissolution of stress fibers, and rearrangement of the actin cytoskeleton.…”

Section: Camp Regulates Paxillin Dephosphorylation and Cell Shapementioning

confidence: 81%

Regulation of Cytoskeleton Organization and Paxillin Dephosphorylation by cAMP

Han¹,

Rubin²

1996

Journal of Biological Chemistry

103

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Cyclic AMP induces corticosteroid production, differential gene transcription, and cell cycle arrest in adrenal cortex-derived Y1 cells. These responses follow a cAMP-controlled transformation in Y1 cell morphology: the conversion of flat epithelial cells into rounded, highly refractile cells with short processes. Little is known about effector proteins and mechanisms that link activated protein kinase A to the alteration in cell shape. We now report that cAMP causes rapid ( 1 min) and selective tyrosine dephosphorylation of paxillin, a focal adhesion protein. Paxillin is maximally dephosphorylated before other physiological effects of cAMP are detected in Y1 cells. Dephosphopaxillin translocates from focal adhesions to the cytoplasm as stress fibers vanish and F-actin accumulates in membrane ruffles and cytoplasmic aggregates. Remnants of focal adhesion complexes dissociate from the cell cortex and coalesce into large structures that contain aggregated F-actin. Pervanadate, an inhibitor of protein-tyrosine phosphatases, abrogates all effects of cAMP. Conversely, genistein-sensitive protein-tyrosine kinase activity is essential for establishing epithelial morphology and reversing effects of cAMP in Y1 cells. Thus, cAMP/protein kinase A (PKA) actions are initially targeted to focal adhesions and cortical actin cytoskeleton; paxillin is an early and unexpected downstream target in a PKA-mediated signaling pathway, and protein-tyrosine phosphatase activity provides an essential link between PKA activation and the control of cell shape.The second messenger cAMP and its effector, protein kinase A (PKA), 1 mediate the regulation of many processes including glycogenolysis, ion transport, gene transcription, and cell proliferation and differentiation (1-4). Increased intracellular levels of cAMP also cause a reorganization of the actin cytoskeleton in REF52 fibroblasts (5) and a marked alteration in the shape of epithelial cells (6). Hormone-induced changes in cell shape often precede alterations in cell physiology and/or differentiation. An example is provided by murine Y1 cells. The cells have a flat epithelioid morphology and contain receptors for adrenocorticotropic hormone (ACTH) that activate adenylate cyclase (7). Increases in cAMP content elicit a rapid morphological transition that yields rounded refractile cells with short processes (6 -8). This cell shape transition is completed before other responses to cAMP are detected. Slower responses are: synthesis and secretion of corticosteroids, induction of cytochrome P-450 steroid hydroxylase genes, and withdrawal of Y1 cells from the cell cycle (7-10). The cited responses to cAMP produce a phenotype in Y1 cells that mimics normal secretory cells of the zona fasciculata of the adrenal cortex. Furthermore, the change in shape of Y1 cells appears to be required for the subsequent appearance of this differentiated phenotype (11).The abrupt rounding of ACTH-treated Y1 cells apparently reflects a reorganization of the actin cytoskeleton (7,8,11). Although this morpholog...

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“…The basis for this divergence, however, is not clear. The defects in the mutant Kin-i and clones likely are in the type I regulatory subunit of the cyclic AMP-dependent protein kinase (23,36). The natures of the mutations in the other Kin clones have not been explored.…”

Section: Discussionmentioning

confidence: 99%

“…The Y6 cell line is a spontaneous variant of the original Y1 cell line (25). Y1(Kin) and Y1(Cyc) mutants were obtained after mutagenesis with ethyl methanesulfonate or N-methyl-N'-nitro-N-nitrosoguanidine, followed by selection in 0.8 mM 8-bromoadenosine 3',5'-monophosphate (8BrcAMP) as described (22)(23)(24). Routine aspects of cell culture have been described elsewhere (26).…”

mentioning

confidence: 99%

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Regulation of ornithine decarboxylase activity by corticotropin in adrenocortical tumor cell clones: roles of cyclic AMP and cyclic AMP-dependent protein kinase.

Kudlow¹,

Rae²,

Gutmann³

et al. 1980

Proc. Natl. Acad. Sci. U.S.A.

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In Y1 adrenocortical tumor cells, corticotropin (ACTH), cyclic AMP, and 8-bromoadenosine 3',5'-monophosphate (8BrcAMP) stimulated ornithine decarboxylase activity (L-ornithine carboxy-lyase, EC 4.1.1.17) and steroidogenesis. The concentrations required for half-maximal activation of ornithine decarboxylase were 60 pM for ACTH and 1 mM for 8BrcAMP; the concentrations required for half-maximal activation of steroidogenesis were 50 pM for ACTH and 0.2 mM for 8BrcAMP. Ornithine decarboxylase activity increased 1.5 hr after the addition of these agents, reached a maximum between 4 and 6 hr, and then declined. Mutant clones with impaired ACTH-responsive adenylate cyclase systems [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] did not respond to ACTH with increased ornithine decarboxylase activity, but they responded normally to added cyclic AMP. These results indicate that adenylate cyclase and cyclic AMP are necessary for the stimulation of ornithine decarboxylase activity by ACTH. In a series of Y1(Kin) mutants with altered cyclic AMP-dependent protein kinase activities (ATP:protein phosphotransferase, EC 2.7.1.37), the effects of ACTH on ornithine decarboxylase also were attenuated. These findings suggest that cyclic AMP-dependent protein kinase also plays a necessary role in the stimulation of ornithine decarboxylase activity by ACTH. The effects of ACTH on ornithine decarboxylase in the Kin mutants, however, were quantitatively different from the effects on steroidogenesis and did not closely reflect the degree of defect in cyclic AMP-dependent protein kinase activity. These differences suggest that the pathways of ACTH action leading to stimulation of steroidogenesis and ornithine decarboxylase activity diverge subsequent to activation of the protein kinase.

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Altered cyclic AMP‐dependent protein kinase activity in a mutant adrenocortical tumor cell line

Cited by 45 publications

References 30 publications

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

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

Regulation of Cytoskeleton Organization and Paxillin Dephosphorylation by cAMP

Regulation of ornithine decarboxylase activity by corticotropin in adrenocortical tumor cell clones: roles of cyclic AMP and cyclic AMP-dependent protein kinase.

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