膜去极化和cAMP在胰岛细胞株HIT中活化CBPC端片段

The CBP C terminus Activated by Cyclic AMP and Membrane Depolarization in the Pancreatic lslet Cell Line HIT

在胰岛细胞株 Ｈ Ｉ Ｔ 细胞中，用瞬时转染法观察高 Ｋ＋ 导致的膜去极化与ｃ Ａ Ｍ Ｐ对 Ｃ Ｂ Ｐ Ｃ端片段转录活性的影响．发现二者均可诱导 Ｃ Ｂ Ｐ Ｃ端片段的转录活性增强，并有协同效应； Ｃ Ｂ Ｐ Ｃ端片段的突变体（ Ｓｅｒ１ ７７２ 突变为 Ａｌａ）表现相同的诱导特性，但其基本转录活性降低．说明膜去极化和 ｃ Ａ Ｍ Ｐ对 Ｃ Ｂ Ｐ Ｃ 端片段转录活性的诱导作用与 Ｐ Ｋ Ａ 磷酸化位点 Ｓｅｒ１ ７７２ 无关，而该位点的磷酸化对调节 Ｃ Ｂ Ｐ Ｃ 端片段的基本转录活性起重要作用．蛋白激酶 Ｃ 通路对 Ｃ Ｂ Ｐ Ｔｉ的转录活性无影响．

The coactivator CBP(CREB binding protein) can regulate gene transcription in pancreatic islet cells through recruitment of CBP by the cAMP response element binding protein, CREB, in response to cAMP and membrane depolarization. The C terminus of CBP ranging from residue 1 678 to residue 2 441 contains structure motifs like a consensus protein kinase A phosphorylation site at Ser 1 772 and a glutamine rich region which are well known elements of the transactivation domains of several transcription factors. In order to investigate whether the CBP C terminus conferred transcriptional activity in the insulin producing islet cell line HIT, a luciferase reporter gene under the control of five Gal4 binding sites was transiently transfected together with an expression vector for a fusion protein containing the DNA binding domain of the yeast transcription factor Gal4 and the C terminus of CBP or a CBP mutant in which the consensus PKA phosphorylation site at Ser 1 772 was destroyed.The experiments showed that high potassium induced membrane depolarization, leading to an influx of extracellular calcium, and cAMP induced transcriptional activation of the CBP C terminus. Both stimuli had a synergistic effect. The mutant of the CBP C terminus exhibited a reduced basal activity but the fold stimulation by cAMP and membrane depolarization remained unchanged. Stimulation by the phorbol ester TPA (12 O tetradecanoylphorbol 13 acetate)had no effect on CBP C terminal mediated transactivation, indicating that a PKC dependent pathway was not involved in CBP C terminal dependent transcription. These results suggest that in pancreatic islet cells transactivation of the CBP C terminus stimulated by cAMP or membrane depolarization does not depend on the phosphorylation on Ser 1 772. However, the intact consensus PKA phophorylation site is necessary to confer basal activity.