Rao M C, Bissonnette G B, Mahaffey T, Guggino W B, Goldstein J L
Department of Physiology and Biophysics, University of Illinois at Chicago.
Gastroenterology. 1994 Apr;106(4):890-8. doi: 10.1016/0016-5085(94)90747-1.
BACKGROUND/AIMS: Human rectal epithelium in cystic fibrosis (CF) shows impaired ion transport in response to theophylline or bethanechol, although it possesses regulatory subunits of adenosine cyclic 3',5'-monophosphate (cAMP)-dependent protein kinase (protein kinase A). Protein kinase A-specific phosphorylation of CF transmembrane conductance regulator (CFTR) in rectal tissues of control and CF volunteers was examined in this study.
CFTR was evaluated using a polyclonal antiserum (pre-NBF) raised against a peptide corresponding to residues 415-427 of CFTR. Microsomal membranes from normal and CF rectal mucosa and from T-84 cells were incubated with [gamma 32P]-adenosine triphosphate +/- protein kinase A and subjected to immunoblotting with pre-NBF and autoradiography.
Pre-NBF recognized a single band of 180 kilodaltons. Protein kinase A altered phosphorylation of this 180-kilodalton band 1.4-, 2.2- and 0.9-fold in T-84, normal, and CF rectal membranes, respectively. Catalytic activities of protein kinase A, Ca2+ calmodulin protein kinase, or protein kinase C in control and CF tissues were similar.
cAMP and Ca(2+)-signaling pathways are normal up to the kinases in CF rectal mucosa. Our results suggest differences in CFTR phosphorylation in normal and CF rectal mucosal membranes.
背景/目的：囊性纤维化（CF）患者的直肠上皮细胞对茶碱或氨甲酰甲胆碱的离子转运反应受损，尽管其具有环磷酸腺苷（cAMP）依赖性蛋白激酶（蛋白激酶A）的调节亚基。本研究检测了对照志愿者和CF志愿者直肠组织中CF跨膜传导调节因子（CFTR）的蛋白激酶A特异性磷酸化情况。
使用针对CFTR第415 - 427位残基的肽段产生的多克隆抗血清（pre - NBF）评估CFTR。将来自正常和CF直肠黏膜以及T - 84细胞的微粒体膜与[γ32P] - 三磷酸腺苷±蛋白激酶A一起孵育，然后用pre - NBF进行免疫印迹和放射自显影。
pre - NBF识别出一条180千道尔顿的单带。蛋白激酶A分别使T - 84、正常和CF直肠膜中这条180千道尔顿条带的磷酸化改变了1.4倍、2.2倍和0.9倍。对照组织和CF组织中蛋白激酶A、Ca2 + 钙调蛋白激酶或蛋白激酶C的催化活性相似。
在CF直肠黏膜中，cAMP和Ca(2 + )信号通路直至激酶都是正常的。我们的结果表明正常和CF直肠黏膜膜中CFTR磷酸化存在差异。
