Robinson-White Audrey, Hundley Thomas R, Shiferaw Miriam, Bertherat Jérôme, Sandrini Fabiano, Stratakis Constantine A
Section on Endocrinology and Genetics, Developmental Endocrinology Branch, NICHD, Bethesda, MD 20892, USA.
Hum Mol Genet. 2003 Jul 1;12(13):1475-84. doi: 10.1093/hmg/ddg160.
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.
卡尼综合征（CNC）由PRKAR1A失活突变引起。PRKAR1A编码环磷酸腺苷依赖性蛋白激酶（PKA）全酶的I-α型调节亚基（RIα）；RIα功能不足如何导致肿瘤发生尚不清楚。在许多细胞中，PKA抑制丝裂原活化蛋白激酶（MAPK）途径的细胞外受体激酶（ERK1/2）级联反应，从而抑制细胞增殖。我们研究了携带种系PRKAR1A突变的CNC细胞中PKA介导的对ERK1/2的抑制作用是否受到影响。携带突变的细胞在基线时以及用环磷酸腺苷刺激后的PKA活性均增强。定量信息分析表明，表达的主要PKA亚基是I型（RIα和RIβ），但突变细胞中的RIα减少。通过细胞和途径特异性刺激剂溶血磷脂酸（LPA）对ERK1/2磷酸化进行的免疫印迹分析表明，该途径以时间和浓度依赖性方式被激活，而这被一种特异性抑制剂所阻断。突变细胞的生长速率更高；福斯可林和异丙肾上腺素抑制正常细胞中LPA诱导的ERK1/2磷酸化，但不抑制突变细胞中的这种磷酸化。福斯可林抑制正常细胞中LPA诱导的细胞增殖和代谢，但刺激突变细胞中的这些参数。这些数据也在携带最常见PRKAR1A突变（c.578del TG）的垂体肿瘤细胞系以及最近显示导致PKA介导的磷酸化增强的突变PRKAR1A体外构建体中得到了重复。我们得出结论，CNC细胞中的PKA活性增加，并且福斯可林或异丙肾上腺素对其刺激会增加LPA诱导的ERK1/2磷酸化、细胞代谢和增殖。PKA介导的对该MAPK途径在CNC细胞中的抑制作用的逆转可能在这种情况下促成肿瘤发生。
