Rectal epithelial expression of protein kinase A phosphorylation of cystic fibrosis transmembrane conductance regulator.
OBJECTIVE
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 [gamma 32P]-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 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.
Rao
M C
MC
Department of Physiology and Biophysics, University of Illinois at Chicago.
Bissonnette
G B
GB
Mahaffey
T
T
Guggino
W B
WB
Goldstein
J L
JL
D000328:Adult; D015153:Blotting, Western; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D003550:Cystic Fibrosis / Q000378:metabolism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007413:Intestinal Mucosa / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D012007:Rectum / Q000378:metabolism*; D012016:Reference Values
C505032:CFTR protein, human; D008565:Membrane Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D011494:Protein Kinases; D017868:Cyclic AMP-Dependent Protein Kinases