IDENTIFICATION OF PROTEIN-KINASE-A PHOSPHORYLATION SITES ON NBD1-DOMAINS AND R-DOMAINS OF CFTR USING ELECTROSPRAY MASS-SPECTROMETRY WITH SELECTIVE PHOSPHATE ION MONITORING

HPLC-electrospray mass spectrometry was used to identify the phosphory lated sites on a bacterially expressed cystic fibrosis transmembrane c onductance regulator (CFTR) fragment containing the first nucleotide b inding domain (NBD1) and the regulatory domain (R). Tryptic digests of NBD1-R (CFTR residues 404-830) were analyzed after protein kinase A ( PKA) treatment for all possible peptides and phosphopeptides (a total of 118 species) containing Ser residues within ''high-probability'' PK A consensus sequences: R-R/K-X-S/T, R-X-X-S/T, and R-X-S/T. Three crit eria were used to assign phosphorylated sites: (1) an 80-Da increase i n the predicted average molecular weight of the tryptic peptides; (2) coelution with the PO3- ion induced by stepped energy collision, and ( 3) the relative elution positions of the phosphorylated and unmodified peptides. Ser residues within the eight dibasic sites in the NBD1 and R domains (positions 422, 660, 700, 712, 737, 768; 795, and 813) were phosphorylated, a pattern similar to that observed for full-length CF TR. The serine at position 753, which in CFTR is phosphorylated in viv o, was not phosphorylated. The remaining potential PKA sites, Ser(489) , Ser(519), Ser(557), Ser(670), and Thr(788), were not phosphorylated. The ''low-probability'' PKA sites (those not containing an Arg residu e) were not phosphorylated. The results suggest that isolated domains of CFTR developed useful models for investigating the biochemical and structural effects of phosphorylation within CFTR. The mass spectromet ry approach in this study should prove useful for defining phosphoryla tion sites of CFTR in vitro and in vivo.