title: Protein kinase-A activity in PRKAR1A-mutant cells, and regulation of mitogen-activated protein kinases ERK1/2.
abstract: Carney complex (CNC) is caused by PRKAR1A-inactivating mutations. PRKAR1A encodes the regulatory subunit type I-alpha (RIalpha) of the cAMP-dependent kinase (PKA) holoenzyme; how RIalpha insufficiency leads to tumorigenesis remains unclear. In many cells PKA inhibits the extracellular receptor kinase (ERK1/2) cascade of the mitogen-activated protein kinase (MAPK) pathway leading to inhibition of cell proliferation. We investigated whether the PKA-mediated inhibitory effect on ERK1/2 is affected in CNC cells that carry germline PRKAR1A mutations. PKA activity both at baseline and after stimulation with cAMP was augmented in cells carrying mutations. Quantitative message analysis showed that the main PKA subunits expressed were type I (RIalpha and RIbeta) but RIalpha was decreased in mutant cells. Immunoblot assays of ERK1/2 phosphorylation by the cell- and pathway-specific stimulant lysophosphatidic acid (LPA) showed activation of this pathway in a time- and concentration-dependent manner that was prevented by a specific inhibitor. There was a greater rate of growth in mutant cells; forskolin and isoproterenol inhibited LPA-induced ERK1/2 phosphorylation in normal but not in mutant cells. Forskolin inhibited LPA-induced cell proliferation and metabolism in normal cells, but stimulated these parameters in mutant cells. These data were also replicated in a pituitary tumor cell line carrying the most common PRKAR1A mutation (c.578del TG), and an in vitro construct of mutant PRKAR1A that was recently shown to lead to augmented PKA-mediated phosphorylation. We conclude that PKA activity in CNC cells is increased and that its stimulation by forskolin or isoproterenol increases LPA-induced ERK1/2 phosphorylation, cell metabolism and proliferation. Reversal of PKA-mediated inhibition of this MAPK pathway in CNC cells may contribute to tumorigenesis in this condition.
authors.affiliation: Section on Endocrinology and Genetics, Developmental Endocrinology Branch, NICHD, Bethesda, MD 20892, USA.
authors.forename: Audrey
authors.initials: A
authors.lastname: Robinson-White
authors.forename: Thomas R
authors.initials: TR
authors.lastname: Hundley
authors.forename: Miriam
authors.initials: M
authors.lastname: Shiferaw
authors.forename: Jérôme
authors.initials: J
authors.lastname: Bertherat
authors.forename: Fabiano
authors.initials: F
authors.lastname: Sandrini
authors.forename: Constantine A
authors.initials: CA
authors.lastname: Stratakis
mesh_terms: D000818:Animals; D019556:COS Cells; D002455:Cell Division; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D054756:Cyclic AMP-Dependent Protein Kinase RIalpha Subunit; D017868:Cyclic AMP-Dependent Protein Kinases / Q000235:genetics* / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D015151:Immunoblotting; D007545:Isoproterenol / Q000494:pharmacology; D008214:Lymphocytes / Q000378:metabolism; D008246:Lysophospholipids / Q000378:metabolism; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D009154:Mutation; D010766:Phosphorylation; D012333:RNA, Messenger / Q000378:metabolism; D013997:Time Factors
chemical_list: D054756:Cyclic AMP-Dependent Protein Kinase RIalpha Subunit; D004791:Enzyme Inhibitors; D008246:Lysophospholipids; C517834:PRKAR1A protein, human; D012333:RNA, Messenger; D005576:Colforsin; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D007545:Isoproterenol