ABCMdb

PMID: 11331356

Button B, Reuss L, Altenberg GA
PKC-mediated stimulation of amphibian CFTR depends on a single phosphorylation consensus site. insertion of this site confers PKC sensitivity to human CFTR.
J Gen Physiol. 2001 May;117(5):457-68., [PubMed]

Sentences

No. Mutations Sentence Comment

34 ABCC7 p.His667Arg The 8-Br-cAMP-activated currents in the mutant H667R-hCFTR were very high; thus, cRNA injection was reduced to 1-2.5 ng/oocyte. Login to comment 70 ABCC7 p.Thr665Ala The following primers were used (only sense primer is shown, with mutations underlined): 5Ј-TAATAACTGAGGCCCTGAGACGATGCT-3Ј (Thr665 to Ala, T A B L E I Conductances of Xenopus Oocytes Expressing CFTR Unstimulated cAMP-stimulated 24 h 48 h 72 h Mock 2.9 Ϯ 1.7 (11) 2.7 Ϯ 1.4 (4) 2.9 Ϯ 1.6 (4) 3.2 Ϯ 2.1 (3) hCFTR 4.2 Ϯ 3.8 (35) 426 Ϯ 60 (16) 685 Ϯ 131 (13) 1,147 Ϯ 217 (6) XCFTR 3.1 Ϯ 2.1 (34) 214 Ϯ 50 (14) 1,009 Ϯ 268 (14) 2,028 Ϯ 217 (6) Xenopus laevis oocytes were injected with water (mock), hCFTR cRNA, or XCFTR cRNA, and their membrane conductances were measured at 24, 48, or 72 h after injection with and without stimulation with cAMP cocktail. Login to comment 73 ABCC7 p.Ser686Ala ABCC7 p.Ser790Ala plus addition of HaeIII site), 5Ј-GTCAAGAATAAAGCTTTTAAG- CAGG-3Ј (Ser686 to Ala, plus addition of HindIII site), 5Ј-TGGG- GATTTCGCTGAGAAAAGAAAGAG-3Ј (Ser694 to Ala, plus addition of DdeI site), and 5Ј-CAAGAAAAACTGCAGTTCG- TAAAATG-3Ј (Ser790 to Ala, plus addition of PstI site). Login to comment 74 ABCC7 p.His667Arg For construction of the H667R-hCFTR mutant, the primer used was 5Ј-AGACCTTGCGCCGTTTCTCA-3Ј (construct screened with HhaI, underlined). Login to comment 202 ABCC7 p.Ser686Ala ABCC7 p.Ser790Ala Substitution of the two conserved serine residues known to be phosphorylated (Ser686 and Ser790; Picciotto et al., 1992) with alanine residues (S686A/ S790A-XCFTR; Fig. 7, A and C) did not affect the activation by PMA. Login to comment 207 ABCC7 p.Thr665Ala Thr665 Is Essential for the Response to PKC Activation of XCFTR To investigate the roles of the unique PKC consensus sites (Thr665 and Ser694), we expressed the mutant lacking both sites (T665A/S694A-XCFTR) in Xenopus oocytes. Login to comment 214 ABCC7 p.Ser686Ala ABCC7 p.Ser790Ala (A) Representative I-V relationships from an oocyte expressing the double knockout of conserved PKC consensus phosphorylation sites (S686A/S790A-XCFTR). Login to comment 215 ABCC7 p.Thr665Ala (B) I-V plot from a cell expressing the double knockout of unique PKC consensus phosphorylation sites (T665A/S694A-XCFTR). Login to comment 221 ABCC7 p.Thr665Ala In contrast, in oocytes expressing T665A-XCFTR, PMA failed to produce full current activation (Fig. 8, B and C). Login to comment 223 ABCC7 p.Thr665Ala Thus, it is possible that the reduced activation by PMA of XCFTR mutants containing the Thr665 to Ala mutation is not absolute, but relative to the activation by PKA stimulation (i.e., the mutation increases the activation of CFTR by cAMP). Login to comment 224 ABCC7 p.Thr665Ala ABCC7 p.Ser686Ala ABCC7 p.Ser790Ala The data in Fig. 9 indicate that this is not the case because the cAMP-activated currents are not different among oocytes expressing wild-type XCFTR, S686A/ S790A-XCFTR, or T665A/S694A-XCFTR. Login to comment 228 ABCC7 p.His667Arg Since Thr665 is present in hCFTR, we decided to determine whether the formation of a PKC consensus phosphorylation site in hCFTR by substituting His with Arg at position 667 is sufficient to confer the Xenopus phenotype to hCFTR. Login to comment 229 ABCC7 p.His667Arg The mutant H667R-hCFTR was expressed in Xenopus oocytes and tested for activation by PKC stimulation. Login to comment 245 ABCC7 p.Thr665Ala The mutation Thr665 to Ala does not increase cAMP-activated XCFTR currents. Login to comment 246 ABCC7 p.Thr665Ala ABCC7 p.Ser686Ala ABCC7 p.Ser790Ala Time course of the currents after stimulation with the cAMP cocktail in oocytes injected with wild-type XCFTR, S686A/S790A-XCFTR, or T665A/S694A-XCFTR cRNAs. Login to comment 248 ABCC7 p.Thr665Ala The data show that the Thr665 to Ala mutation has no significant effect on the level of cAMP-activated currents. Login to comment 254 ABCC7 p.His667Arg Insertion of this site in the H667R-hCFTR mutant resulted in a large increase in current in response to PKC activation. Login to comment 256 ABCC7 p.His667Arg In any event, the results indicate that the PKC consensus phosphorylation site that includes Thr665 is critical for the response to PMA. If the activation of the H667R-hCFTR mutant is in fact less than that of the HXH chimera, then one would conclude also that the phosphorylation of Thr665, or its functional effect, involves other residues of the R domain of CFTR. Login to comment 274 ABCC7 p.His667Arg PMA produces a large increase in conductance in a mutant hCFTR (H667R-hCFTR) with an engineered PKC consensus phosphorylation sequence that includes Thr665. Login to comment 275 ABCC7 p.His667Arg (A) Representative I-V plot from an oocyte expressing H667R-hCFTR. Login to comment