title: Clonal variants of PC12 pheochromocytoma cells with defects in cAMP-dependent protein kinases induce ornithine decarboxylase in response to nerve growth factor but not to adenosine agonists.
abstract: We have isolated and partially characterized three mutants of the pheochromocytoma line PC12 by using dibutyryl cyclic AMP (cAMP) as a selective agent. Each of these variants, A126-1B2, A208-4, and A208-7, was resistant to both dibutyryl cAMP and cholera toxin when cell growth was measured. In comparison to wild-type PC12 cells, each of these mutants was deficient in the ability to induce ornithine decarboxylase (ODC) in response to agents that act via a cAMP-dependent pathway. In contrast, each of these mutants induced ODC in response to nerve growth factor. To understand the nature of the mutations, the cAMP-dependent protein kinases of the wild type and of each of these mutants were studied by measuring both histone kinase activity and 8-N3-[32P]cAMP labeling. Wild-type PC12 cells contained both cAMP-dependent protein kinase type I (cAMP-PKI) and cAMP-dependent protein kinase type II (cAMP-PKII). Regulatory subunits were detected in both soluble and particulate fractions. The mutant A126-1B2 contained near wild-type PC12 levels of cAMP-PKI but greatly reduced levels of cAMP-PKII. Furthermore, when compared with wild-type PC12 cells, this cell line had an altered distribution in ion-exchange chromatography of regulatory subunits of cAMP-PKI and cAMP-PKII. The mutant A208-4 demonstrated wild-type-level binding of 8-N3-[32P]cAMP to both type I and type II regulatory subunits, but only half the wild-type level of type II catalytic activity. The mutant A208-7 had type I and type II catalytic activities equivalent to those in wild-type cells. However, the regulatory subunit of cAMP-PKI occurring in A208-7 demonstrated decreased levels of binding 8-N3-[32P]cAMP in comparison with the wild type. Furthermore, all mutants were defective in their abilities to bind 8-N3-[32P]cAMP to the type II regulatory protein in the particulate fraction. Thus, cAMP-PK was altered in each of these mutants. We conclude that both cAMP-PKI and cAMP-PKII are apparently required to induce ODC in response to increases in cAMP. Finally, since all three mutants induced ODC in response to nerve growth factor, the nerve growth factor-dependent induction of OCD was not mediated by an increase in cAMP that led to an activation of cAMP-PK. These mutants will be useful in the elucidation of the many functions controlled by cAMP and nerve growth factor.
mesh_terms: D000310:Adrenal Gland Neoplasms / Q000201:enzymology*; D000818:Animals; D001386:Azides / Q000378:metabolism; D003994:Bucladesine / Q000494:pharmacology*; D002460:Cell Line; D002772:Cholera Toxin / Q000494:pharmacology; D002999:Clone Cells; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000378:metabolism; D004790:Enzyme Induction; D014644:Genetic Variation; D007700:Kinetics; D046911:Macromolecular Substances; D051379:Mice; D009414:Nerve Growth Factors / Q000494:pharmacology*; D009955:Ornithine Decarboxylase / Q000096:biosynthesis*; D010673:Pheochromocytoma / Q000201:enzymology*; D011494:Protein Kinases / Q000235:genetics* / Q000378:metabolism
chemical_list: D001386:Azides; D046911:Macromolecular Substances; D009414:Nerve Growth Factors; C017042:8-azidoadenosine-3',5'-monophosphate; D003994:Bucladesine; D002772:Cholera Toxin; D000242:Cyclic AMP; D011494:Protein Kinases; D009955:Ornithine Decarboxylase