Regulation of the cystic fibrosis transmembrane conductance regulator anion channel by tyrosine phosphorylation

Abstract

The cystic fibrosis transmembrane conductance regulator (CFTR) channel is activated by PKA phosphorylation of a regulatory domain that interacts dynamically with multiple CFTR domains and with other proteins. The large number of consensus sequences for phosphorylation by PKA has naturally focused most attention on regulation by this kinase. We report here that human CFTR is also phosphorylated by the tyrosine kinases p60c-Src (proto-oncogene tyrosine-protein kinase) and the proline-rich tyrosine kinase 2 (Pyk2), and they can also cause robust activation of quiescent CFTR channels. In excised patch-clamp experiments, CFTR activity during exposure to Src or Pyk2 reached ∼80% of that stimulated by PKA. Exposure to PKA after Src or Pyk2 caused a further increase to the level induced by PKA alone, implying a common limiting step. Channels became spontaneously active when v-Src or the catalytic domain of Pyk2 was coexpressed with CFTR and were further stimulated by the tyrosine phosphatase inhibitor dephostatin. Exogenous Src also activated 15SA-CFTR, a variant that lacks 15 potential PKA sites and has little response to PKA. PKA-independent activation by tyrosine phosphorylation has implications for the mechanism of regulation by the R domain and for the physiologic functions of CFTR.

Keywords: Pyk2; R domain; Src.

Figure 1.

Phosphorylation of CFTR by Src in intact cells and in vitro. A) Representative immunoblot (IB) probed with antiphosphotyrosine antibody (upper panel) or CFTR antibody (lower panel). Lane 1 shows whole-cell lysate. In lane 2, CFTR immunoprecipitated using mAb M3A7 is recognized by the same antiphosphotyrosine antibody when v-Src and CFTR were coexpressed. No phosphotyrosine protein is detected when either v-Src (lane 3) or CFTR (lane 4) is not expressed. B) In vitro phosphorylation of CFTR by Src. The immunoblot was probed with antiphosphotyrosine antibody (upper panel), then stripped and reblotted using anti-CFTR antibody 23C5 (lower panel). Lanes marked “L” are total cell lysate controls; lanes marked “Ctl IP” are nonimmune controls for nonspecific binding of CFTR to the beads. The phosphotyrosine (phosphoTyr; indicated by arrows) and CFTR (lower blots) bands are superimposable when CFTR-expressing cells are treated with 30 U active Src for 30 min. A representative of 3 experiments for each condition is shown.

Figure 2.

Effect of Src on CFTR channel activity. A and B) Recordings obtained immediately after membrane patches were excised from an unstimulated cell into bath solution containing (A) 1 mM MgATP and 75 nM PKA or (B) 30 U/ml Src. Pipette voltage was +30 mV. Upward deflections represent channel openings. C) Single-channel i/V relationship determined in symmetric high-Cl⁻ solutions. Symbols represent means ± se of 3 experiments. D) Control experiment under same conditions as in (A) and (B) except with MgATP plus a 5 nM concentration of the PP2A inhibitor Calyculin A. E) Membrane patch excised into bath solution containing 1 mM MgATP and Src (30 U/ml). AMP-PNP (1 mM) was added subsequently as indicated.

Figure 3.

Effect of Pyk2 on CFTR channel activity. A) In vitro phosphorylation of CFTR by Pyk2. An immunoblot (IB) probed with antiphosphotyrosine antibody (upper panel), then stripped and reblotted using anti-CFTR antibody 23C5 (lower panel) is shown. The phosphotyrosine (phosphoTyr; indicated by arrows) and CFTR (lower blot) bands are superimposable when CFTR-expressing cells are incubated for 45 min with 30 U active Pyk2. A representative of 3 experiments is shown. B) Recording obtained immediately after membrane patch was excised from an unstimulated cell into bath solution containing 1 mM MgATP and 30 U/ml of the catalytic domain of Pyk2. Pipette voltage was +30 mV. Upward deflections represent channel openings. C) Single-channel i/V relationship determined in symmetric high-Cl⁻ solutions. Symbols represent means ± se of 3 experiments. D) Membrane patches excised into bath solution containing 1 mM MgATP and Pyk2 (30 U/ml). AMP-PNP (1 mM final concentration) was added as indicated.

Figure 4.

Comparison of CFTR activity stimulated by Src, Pyk2, and PKA. A) Membrane patches excised into bath solution containing 1 mM MgATP and 75 nM PKA (left trace) or 30 U/ml Src (right trace). Src (left) or PKA (right) was then added where indicated. Recording configuration is the same as in Figs. 2 and 3. B) Left histogram shows a comparison of the mean ratio of kinase-stimulated currents before and after addition of 1 mM AMP-PNP. Hatched bars represent PKA:AMP-PNP current ratios after Src or Pyk2 exposure (30 U/ml for each): Src or Pyk2 was present initially, and PKA (75 nM) was added after the maximal tyrosine kinase-induced current had been reached. Error bars show se for the number of cells indicated. ns, no significant difference. *P < 0.05 with Student’s t test. Right histogram shows a comparison of the mean ratio of the PKA or Pyk2-stimulated currents measured before and after adding 1 mM AMP-PNP; cells were bathed for 30 min in 10 µM SrcInh 1 prior to the addition of kinases.

Figure 5.

Spontaneous CFTR activity in excised patches from v-Src or knockdown-Pyk2-transfected cells. A) Representative immunoblot (IB) probed with antiphosphotyrosine (phosphoTyr; upper panel) or CFTR (lower panel) antibody. Lane 1 shows whole-cell lysate. In lane 2, CFTR immunoprecipitated using mAb M3A7 is recognized by the same antiphosphotyrosine antibody when pKH3-Pyk2 and CFTR were coexpressed. No phosphotyrosine protein was detected when either Pyk2 (lane 3) or CFTR (lane 4) was not expressed. B) Patch recording from CFTR-expressing cells transiently transfected with large T antigen and control (Ctl) plasmid lacking v-Src. C and D) CFTR activity in patches excised from cells expressing v-Src (C) or pKH3-Pyk2 (D). MgATP (1 mM) was present throughout the experiment. Dephostatin (10 µM) was added at the arrows.

Figure 6.

PKA independence of the phosphotyrosine stimulation. Recordings start immediately after excision. MgATP (1 mM) is present throughout the experiments. A) Effect of PKA on channel activity in a patch excised from a cell that is stably expressing the 15SA mutant. Left panel shows a representative recording of membrane patches excised into bath solution containing 1 mM MgATP and 75 nM PKA. Right panel shows a comparison of the PKA:AMP-PNP current ratio for wild-type (wt)- and 15SA-CFTR channels. B and C) Effect of exogenous Src (30 U/ml) (B) and Pyk2 (30 U/ml) (C) on activity of the 15SA mutant. Each recording is representative of 4 experiments.