Human Molecular Genetics, 2003, Vol. 12, No. 13 1475-1484
DOI: 10.1093/hmg/ddg160
© 2003 Oxford University Press
Protein kinase-A activity in PRKAR1A-mutant cells, and regulation of mitogen-activated protein kinases ERK1/2
1Section on Endocrinology and Genetics, Developmental Endocrinology Branch, NICHD, Bethesda, MD 20892, USA, 2LMI, NHLBI, National Institutes of Health, Bethesda, MD 20892, USA and 3Département d'Endocrinologie, Institut Cochin, INSERM U576, CNRS UMR 8104, IFR116, Université-Paris V, Paris, France
Received February 5, 2003; Revised April 17, 2003; Accepted April 28, 2003
Carney complex (CNC) is caused by PRKAR1A-inactivating mutations. PRKAR1A encodes the regulatory subunit type I-
(RI) of the cAMP-dependent kinase (PKA) holoenzyme; how RI 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 (RI and RIß) but RI 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.
* To whom correspondence should be addressed at: Section on Endocrinology and Genetics, DEB, NICHD, NIH, Building 10, Room 10N262, 10 Center Dr. MSC1862, Bethesda, MD 20892-1862, USA. Tel: +1 3014964686; Fax: +1 3014020574; Email: stratakc{at}mail.nih.gov
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