ABCMdb

PMID: 10933805

King SA, Sorscher EJ
R-domain interactions with distal regions of CFTR lead to phosphorylation and activation.
Biochemistry. 2000 Aug 15;39(32):9868-75., 2000-08-15 [PubMed]

Sentences

No. Mutations Sentence Comment

4 ABCC7 p.Met837* A larger CFTR polypeptide that included the R-domain (M837X) also exhibited a phosphorylation-dependent mobility shift when coexpressed with ∆1-836. Login to comment 5 ABCC7 p.Met837* Moreover, coexpression of M837X and ∆1-836 led to enhanced halide permeability in living cells. Login to comment 27 ABCC7 p.Lys593* ABCC7 p.Lys593* ABCC7 p.Met837* ABCC7 p.Met837* ABCC7 p.Gly723* ABCC7 p.Gly723* 1 Abbreviations: CF, cystic fibrosis; CFTR, cystic fibrosis transmembrane conductance regulator; ABC, ATP binding cassette; NBD, nucleotide binding domain; TMD, transmembrane domain; R-domain, regulatory domain; PKA, cyclic AMP-dependent protein kinase A; His P, NBD of histidine permease; His Q and His M, TMDs of histidine permease; Mal K, NBD region of the maltose transport system; Mal F, integral membrane protein of the maltose transport system; AMP, adenosine monophosphate; ∆R-CFTR, CFTR lacking amino acids 708-835; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; DMEM, Dulbecco`s Modified Eagle`s Medium; FBS, fetal bovine serum; vTF7.3, vaccina virus encoding the T7 polymerase; MOI, multiplicity of infection; DOC, deoxycholic acid; PVDF, poly- (vinylidene difluoride); NBT, 4-nitroblue tetrazolium chloride; SPQ, 6-methoxy-N-(3-sulfopropyl)quinolonium; BCIP, 5-bromo-4-chloro-3-indolyl phosphate; -gal, -galactosidase; ∆1-836, carboxy hemi-CFTR beginning immediately after the R-domain; M837X, CFTR truncation that ends at CFTR position 836, after the R-domain; G723X, CFTR truncation ending at residue 722 within the R-domain; K593X, CFTR truncation ending immediately before the R-domain at position 592. Login to comment 52 ABCC7 p.Met837* pTM-M837X was produced by amplifying a segment of CFTR with a primer encoding nucleotides 1562-1585 (including the unique SphI restriction site) and a reverse primer containing nucleotides 2495-2529 (including a stop codon at residue 2515 followed by a StuI site). Login to comment 53 ABCC7 p.Gly723* The resulting product was inserted between the SphI and StuI sites of pTM-CFTR, to place a premature stop codon in place of CFTR amino acid 837. pTM-G723X was constructed using a similar strategy but with a stop codon in place of the glycine at residue 723. Login to comment 54 ABCC7 p.Met837* The pTM-R-domain was obtained using a PCR product to engineer a start site at methionine 596 and the stop site from pTM-M837X (leading to a vector expressing the R-domain protein, i.e., amino acids 596-837). Login to comment 111 ABCC7 p.Met837* Interactions between ∆1-836 and M837X Confer Phosphorylation of the R-Domain and ActiVation of Halide Efflux. Login to comment 112 ABCC7 p.Met837* To ascertain whether phosphorylation of the R-domain is enhanced by expression of the complete CFTR amino acid sequence, we transfected cells with both ∆1-836 and M837X (CFTR truncated at the end of the R-domain, Table 1). Login to comment 115 ABCC7 p.Met837* Immunoprecipitates from COS7 cells expressing M837X, ∆1-836, or both were studied by Western blotting, as shown in Figure 3. Login to comment 116 ABCC7 p.Met837* The M837X protein was pulled down in association with ∆1-836 (Figure 3A). Login to comment 117 ABCC7 p.Met837* The complementary experiment showed that ∆1-836 could also be co-immunoprecipitated in association with M837X (Figure 3B, lane 6). Login to comment 118 ABCC7 p.Met837* Neither M837X nor ∆1-836 bound to a control ( -gal) protein (Figure 3C). Login to comment 119 ABCC7 p.Met837* Interestingly, the M837X protein that co-immunoprecipitated with ∆1-836 was greatly enriched for an apparent higher-molecular weight conformation (Figure 3A, lane 4 with *). Login to comment 120 ABCC7 p.Met837* ABCC7 p.Met837* Direct immunoprecipitation of M837X indicated that the total level of shifted M837X protein was negligible when expressed alone (Figure 3A, lane 1). Login to comment 121 ABCC7 p.Met837* When M837X was coexpressed with ∆1-836, the higher protein band could be more readily detected, and was dramatically enriched bound to ∆1-836 (Figure 3A, lane 4). Login to comment 122 ABCC7 p.Met837* ABCC7 p.Met837* To test whether phosphorylation was responsible for the mobility shift in M837X, cells expressing M837X alone were treated with forskolin prior to lysis. Login to comment 123 ABCC7 p.Met837* Forskolin treatment reproduced the slower molecular weight migration in M837X (Figure 4A, lane 1). Login to comment 124 ABCC7 p.Met837* Alkaline phosphatase treatment of immunoprecipitated M837X protein abolished the higher-molecular weight band seen following either PKA stimulation or coexpression with ∆1-836 (Figure 4A, lanes 2 and 4). Login to comment 125 ABCC7 p.Met837* ABCC7 p.Met837* These results indicate that the expression of ∆1-836 with M837X confers a phosphorylation-dependent reduction in the mobility of M837X. Login to comment 126 ABCC7 p.Met837* Forskolin treatment did not cause phosphorylation of all the expressed R-domain or M837X. Login to comment 129 ABCC7 p.Met837* If this were so, only a subfraction of the R-domain or M837X might be situated within the cell in a position suitable for efficient phosphorylation by PKA. Login to comment 132 ABCC7 p.Met837* Since we observed that binding between M837X and ∆1-836 augments CFTR phosphorylation, we tested the functional consequences of this binding interaction. Login to comment 133 ABCC7 p.Met837* M837X, ∆1-836, or both were expressed in COS7 cells, and anion efflux was measured using the halide sensitive dye SPQ. Login to comment 134 ABCC7 p.Met837* Coexpression of M837X and ∆1-836 led to augmented cellular halide permeability (Figure 5, b), while no halide efflux above FIGURE 2: Forskolin treatment or coexpression with ∆1-836 produces an apparent higher-molecular weight R-domain protein that is sensitive to alkaline phosphatase. Login to comment 141 ABCC7 p.Met837* background was detected in cells expressing either M837X or ∆1-836 alone (Figure 5, 0 and O). Login to comment 142 ABCC7 p.Met837* Coexpression of M837X and ∆1-836 produced predominantly constitutive function of the reconstituted CFTR, similar to that reported previously for CFTR lacking a portion of the R-domain (∆R-CFTR) (15, 19). Login to comment 146 ABCC7 p.Met837* The increased halide efflux conferred by M837X and ∆1-836 could be either due to the loss of PKA-dependent regulation of CFTR [as demonstrated for ∆R-CFTR (15)] or a result of enhanced R-domain basal PKA phosphorylation. Login to comment 148 ABCC7 p.Met837* ABCC7 p.Met837* When cells coexpressing M837X and ∆1-836 were treated with Rp-8-CPT- cAMPS, a PKA antagonist, a significant decrease in the halide efflux was detected compared to that in untreated cells FIGURE 3: M837X binds ∆1-836. Login to comment 149 ABCC7 p.Met837* ABCC7 p.Met837* M837X, ∆1-836, or both were immunoprecipitated using an antibody specific to M837X, or to the C-terminal half. Login to comment 150 ABCC7 p.Met837* M837X co-immunoprecipitated with ∆1-836 (panel A, lane 4). Login to comment 151 ABCC7 p.Met837* The ∆1-836 was also detected following co-immunoprecipitation with M837X (panel B, lane 6). Login to comment 153 ABCC7 p.Met837* The asterisk denotes the shifted mobility form of M837X; see the text. The antibody used in Western blotting is defined at the right of each panel. Login to comment 154 ABCC7 p.Met837* FIGURE 4: Forskolin treatment or coexpression with ∆1-836 produces an apparent higher-molecular weight, alkaline phosphatase sensitive form of M837X. Login to comment 155 ABCC7 p.Met837* Lysates from cells expressing M837X, alone or together with ∆1-836 ("both"), were immunoprecipitated as described in the legend of Figure 3. Login to comment 157 ABCC7 p.Met837* ABCC7 p.Met837* ABCC7 p.Met837* M837X from cells treated with forskolin and M837X coexpressed with ∆1-836 resulted in an apparent higher-molecular weight M837X-CFTR protein band (*, lanes 1 and 3). Login to comment 158 ABCC7 p.Met837* The higher-molecular weight M837X protein band was eliminated by treatment with alkaline phosphatase (lanes 2 and 4). Login to comment 159 ABCC7 p.Met837* FIGURE 5: M837X and ∆1-836 coexpression produces elevated basal halide efflux. Login to comment 160 ABCC7 p.Met837* ABCC7 p.Met837* CFTR (9), M837X (0), ∆1-836 (O), or M837X and ∆1-836 (b) were expressed in COS7 cells, and halide efflux was assayed using the halide sensitive intracellular dye SPQ. Login to comment 163 ABCC7 p.Met837* Enhanced basal halide efflux following the switch to the dequench buffer was detected in cells coexpressing M837X and ∆1-836 (p < 0.001), and regulated halide efflux was seen with expression of CFTR upon forskolin stimulation (p < 0.001). Login to comment 164 ABCC7 p.Met837* No enhanced halide movement was detected when either M837X or ∆1-836 was expressed alone. Login to comment 170 ABCC7 p.Met837* ABCC7 p.Met837* These results indicate that enhanced PKA-dependent phosphorylation of M837X is responsible for the high basal halide efflux produced following coexpression of M837X and ∆1-836. Login to comment 171 ABCC7 p.Met837* The Carboxy Region of the R-Domain Is Necessary for the Phosphorylation-Dependent M837X Mobility Shift and PKA-Dependent Halide Efflux. Login to comment 172 ABCC7 p.Met837* To examine R-domain residues that are necessary for the phosphorylation of M837X elicited by ∆1-836, we tested shortened CFTR truncations missing part or all of the R-domain for functional and biochemical interactions with ∆1-836. Login to comment 173 ABCC7 p.Gly723* G723X (missing the 114 C-terminal amino acids of the R-domain, Table 1) was tested for the phosphorylation-dependent mobility shift following coexpression with ∆1-836. Login to comment 174 ABCC7 p.Met837* ABCC7 p.Gly723* ABCC7 p.Gly723* While G723X co-immunoprecipitated with ∆1-836 in a manner similar to that of M837X, this interaction did not result in a mobility shift of the G723X protein (Figure 7A, compare lanes 3 and 4). Login to comment 175 ABCC7 p.Gly723* Furthermore, no molecular weight shift of G723X could be detected when cells were treated with forskolin prior to lysis (Figure 7B, lane 4). Login to comment 176 ABCC7 p.Gly723* G723X and ∆1-836 expressed together produced constitutive (unregulated) activity (Figure 8A). Login to comment 179 ABCC7 p.Lys593* ABCC7 p.Lys593* ABCC7 p.Met837* ABCC7 p.Gly723* K593X (CFTR truncated immediately before the R-domain, Table 1) co-immunoprecipitated with ∆1-836 in a manner similar to that of M837X or G723X (data not shown), but coexpression of K593X with ∆1-836 failed to produce enhanced halide efflux (Figure 8A). Login to comment 186 ABCC7 p.Met837* Enhanced phosphorylation of the R-domain was also observed when the first half of CFTR, M837X (CFTR truncated immediately after the R-domain), was treated with forskolin or coexpressed with ∆1-836. Login to comment 188 ABCC7 p.Gly723* A shorter amino truncation (G723X) also tightly bound to ∆1-836. Login to comment 189 ABCC7 p.Met837* ABCC7 p.Met837* ABCC7 p.Met837* ABCC7 p.Gly723* In contrast to M837X, G723X failed to exhibit a reduced mobility on SDS-PAGE after either coexpression with ∆1FIGURE 6: Halide permeability produced by M837X and ∆1-836 is PKA-dependent. Login to comment 190 ABCC7 p.Met837* ABCC7 p.Met837* M837X (2) or M837X and ∆1-836 (9) were expressed in COS7 cells. Login to comment 191 ABCC7 p.Met837* Cells expressing M837X and ∆1-836 were treated with Rp-8-CPT-cAMPS (b), a PKA antagonist. Login to comment 194 ABCC7 p.Met837* Rp-8-CPT-cAMPS dramatically inhibited the halide permeability of M837X and ∆1-836 (p < 0.05). Login to comment 195 ABCC7 p.Met837* ABCC7 p.Met837* ABCC7 p.Met837* Permeability coefficients (average changes in flourescence per 20 s from 80 to 120 s) were -2.2 (M837X), -12.3 (M837X and ∆1-836), and -3.9 (M837X and ∆1-836, with Rp-8CPT-cAMPS treatment). Login to comment 197 ABCC7 p.Gly723* ABCC7 p.Gly723* FIGURE 7: G723X binds ∆1-836, but this interaction does not elicit a mobility shift of G723X. Login to comment 199 ABCC7 p.Met837* ABCC7 p.Met837* ABCC7 p.Gly723* While both G723X and M837X bind ∆1-836 (lanes 3 and 4), only the interaction of M837X with ∆1-836 produced a higher-molecular weight phosphorylated protein (*, lane 3). Login to comment 200 ABCC7 p.Met837* ABCC7 p.Gly723* (B) Cells expressing M837X or G723X were treated with forskolin prior to lysis (as described in the legend of Figure 4). Login to comment 201 ABCC7 p.Met837* ABCC7 p.Met837* M837X from cells treated with forskolin appeared as an apparent higher-molecular weight M837X-CFTR protein band (*, lane 2). Login to comment 202 ABCC7 p.Gly723* However, forskolin treament of cells expressing G723X did not result in a higher-molecular weight protein (lane 4). Login to comment 207 ABCC7 p.Met837* M837X coexpressed with ∆1-836 resulted in high-level CFTR activity (as detected by SPQ, Figure 5) in the absence of additional PKA stimulation, indicating that R-domain interactions with downstream domains can augment R-domain phosphorylation and directly regulate CFTR function. Login to comment 209 ABCC7 p.Met837* In contrast to ∆R-CFTR, the basal activity of M837X with ∆1-836 reported in this study was PKA-dependent, and could be inhibited by the PKA antagonist Rp-8-CPT-cAMPS. Login to comment 210 ABCC7 p.Met837* Therefore, while ∆R-CFTR functions independently of PKA, the high basal activity conferred by coexpression of M837X with ∆1-836 results from a different, phosphorylation-dependent mechanism. Login to comment 211 ABCC7 p.Gly723* G723X also produced high basal halide efflux when it was coexpressed with ∆1-836 (Figure 8A). Login to comment 212 ABCC7 p.Met837* However, in contrast to M837X, the activity could not be inhibited by PKA antagonists (Figure 8B), a result similar to the activity reported for ∆R-CFTR. Login to comment 213 ABCC7 p.Met837* ABCC7 p.Met837* On the basis of the observations that (1) the level of the phosphorylation-dependent higher-molecular weight M837X was dramatically increased when it was bound to ∆1-836, (2) a high basal halide permeability was produced by coexpression of M837X with ∆1-836, and (3) this activity was suppressed by PKA inhibitors, we conclude that R-domain phosphorylation and CFTR activation were promoted by R-domain interactions with downstream elements of CFTR. Login to comment 214 ABCC7 p.Met837* The increased susceptibility of the R-domain to PKA phosphorylation in the two-subunit model of CFTR (M837X and ∆1-836) may reflect greater accessibility of the R-domain to endogenous PKA, or blockade of phosphatase action. Login to comment 228 ABCC7 p.Lys593* ABCC7 p.Gly723* FIGURE 8: G723X coexpression with ∆1-836 produces enhanced halide efflux, while K593X does not. Login to comment 229 ABCC7 p.Lys593* ABCC7 p.Gly723* (A) G723X and ∆1-836 (9) or K593X and ∆1-836 (b) were expressed in COS7 cells, and halide efflux was assayed as described in the legend of Figure 5. Login to comment 230 ABCC7 p.Met837* ABCC7 p.Gly723* G723X produced high basal halide permeability when coexpressed with ∆1-836 (p < 0.01) similar to that of M837X with ∆1-836 (Figure 5). Login to comment 231 ABCC7 p.Lys593* K593X, missing the complete R-domain, failed to increase halide permeability when coexpressed with ∆1-836. Login to comment 234 ABCC7 p.Met837* ABCC7 p.Gly723* (B) M837X and ∆1-836 (0) or G723X and ∆1-836 (O) were expressed in COS7 cells and assayed for halide movement as described in the legend of Figure 6. Login to comment 235 ABCC7 p.Met837* ABCC7 p.Gly723* Rp-8-CPT- cAMPS inhibited the enhanced halide permeability of M837X and ∆1-836 (9, p < 0.05), but not that of G723X and ∆1-836 (b). Login to comment