Schwaninger M, Blume R, Krüger M, Lux G, Oetjen E, Knepel W
Department of Biochemical Pharmacology, University of Göttingen, Federal Republic of Germany.
J Biol Chem. 1995 Apr 14;270(15):8860-6. doi: 10.1074/jbc.270.15.8860.
Gene transcription can be induced by cAMP and Ca2+ through distinct protein kinases phosphorylating the transcription factor CREB, which binds to cAMP response elements (CREs) in various genes. Induction of gene transcription by Ca2+ has been shown recently to depend on the Ca2+/calmodulin-dependent protein phosphatase calcineurin in pancreatic islet cells. This study investigates the role of calcineurin in CRE-directed gene transcription after stimulation by cAMP. Reporter fusion genes under the transcriptional control of CREs were transiently transfected into the cell line HIT. Pharmacological evidence suggests that cAMP stimulates CRE-mediated transcription through a Ca(2+)-dependent mechanism. The immunosuppressive drugs cyclosporin A and FK506 inhibited CRE-mediated transcription stimulated by cAMP. At the same concentrations they also inhibited calcineurin phosphatase activity. Reversal of calcineurin inhibition by rapamycin or overexpression of calcineurin led to disinhibition of CRE-mediated gene transcription. Immunoblots with a phosphoCREB-specific antibody showed that cyclosporin A and FK506 do not interfere with CREB phosphorylation at serine 119 stimulated with cAMP or membrane depolarization. These results indicate that in HIT cells stimulation of CRE-mediated transcription depends not only on the activity of protein kinases phosphorylating CREB but also on the Ca2+/calmodulin-dependent protein phosphatase calcineurin that is necessary for the transcriptional competence of phosphorylated CREB.
环磷酸腺苷（cAMP）和钙离子（Ca2+）可通过不同的蛋白激酶使转录因子CREB磷酸化，从而诱导基因转录，CREB可与多种基因中的环磷酸腺苷反应元件（CRE）结合。最近研究表明，在胰岛细胞中，Ca2+诱导的基因转录依赖于Ca2+/钙调蛋白依赖性蛋白磷酸酶钙调神经磷酸酶。本研究探讨了钙调神经磷酸酶在cAMP刺激后CRE介导的基因转录中的作用。将受CRE转录控制的报告基因融合体瞬时转染至细胞系HIT中。药理学证据表明，cAMP通过Ca(2+)依赖性机制刺激CRE介导的转录。免疫抑制药物环孢素A和FK506可抑制cAMP刺激的CRE介导的转录。在相同浓度下，它们也抑制钙调神经磷酸酶的磷酸酶活性。雷帕霉素逆转钙调神经磷酸酶抑制或钙调神经磷酸酶的过表达导致CRE介导的基因转录去抑制。用磷酸化CREB特异性抗体进行的免疫印迹显示，环孢素A和FK506不干扰cAMP或膜去极化刺激下丝氨酸119处的CREB磷酸化。这些结果表明，在HIT细胞中，CRE介导的转录刺激不仅取决于使CREB磷酸化的蛋白激酶的活性，还取决于Ca2+/钙调蛋白依赖性蛋白磷酸酶钙调神经磷酸酶，后者对于磷酸化CREB的转录能力是必需的。
