Potential sites of CFTR activation by tyrosine kinases

Abstract

The CFTR chloride channel is tightly regulated by phosphorylation at multiple serine residues. Recently it has been proposed that its activity is also regulated by tyrosine kinases, however the tyrosine phosphorylation sites remain to be identified. In this study we examined 2 candidate tyrosine residues near the boundary between the first nucleotide binding domain and the R domain, a region which is important for channel function but devoid of PKA consensus sequences. Mutating tyrosines at positions 625 and 627 dramatically reduced responses to Src or Pyk2 without altering the activation by PKA, suggesting they may contribute to CFTR regulation.

Keywords: CFTR regulation; Pyk2; Src; cystic fibrosis; phosphotyrosine.

Figure 1.

Phosphorylation of CFTR by Src kinase. Blot of anto-phosphotyrosine immunoprecipitates probed with the anti-CFTR monoclonal antibody L12B4. CFTR peptides (identified by the boundary amino acid numbers indicated above each pair of lanes) co-expressed with or without v-Src and precipitated using monoclonal antibody against phosphotyrosine. Black arrows indicate the positions of CFTR phosphopeptides. The bands at ˜50 and ˜100 kDa observed in every lane indicate mono- and dimeric heavy chains of the immunoprecipitating IgG, which are recognized by the secondary antibody used for immunoblotting. Black arrows indicate CFTR fragments. A representative of 2 experiments is shown.

Figure 2.

Effect of Src and Pyk2 on activity of the CFTR double mutant YY625,627FF. (A–D): Effect of Src (A & C) and Pyk2 (B & D) on channel activity in patch excised from cell expressing wt (A&B) or YY625,627FF (C & D) CFTR. (E): Comparison of the mean ratio of the kinase- stimulated currents measured before and after addition of 1 mM AMP-PNP. Error bars show SE for number of cells indicated. *, p < 0.05; **, p < 0.01 with student t-test. The wt-CFTR results from Billet et al 2015 obtained under identical conditions are reproduced here for comparison with YY625,627FF-CFTR mutant.