Rectal epithelial expression of protein kinase A phosphorylation of cystic fibrosis transmembrane conductance regulator

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. Methods: 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 [γ³²P]-adenosine triphosphate ± protein kinase A and subjected to immunoblotting with pre-NBF and autoradiography. Results: 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. Conclusions: cAMP and Ca²⁺-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.