ABCMdb

ABCC7 p.Phe833Leu

Predicted by SNAP2:	A: D (71%), C: D (75%), D: D (85%), E: D (80%), G: D (80%), H: D (66%), I: D (66%), K: D (80%), L: D (53%), M: D (80%), N: D (71%), P: D (80%), Q: D (71%), R: D (75%), S: D (75%), T: D (71%), V: D (66%), W: D (80%), Y: D (59%),
Predicted by PROVEAN:	A: N, C: N, D: N, E: N, G: N, H: N, I: N, K: N, L: N, M: N, N: N, P: D, Q: N, R: N, S: N, T: N, V: N, W: N, Y: N,

[switch to compact view]
Comments [show]

None has been submitted yet.

Publications
[hide] NMR spectroscopy to study the dynamics and interac... Methods Mol Biol. 2011;741:377-403. Kanelis V, Chong PA, Forman-Kay JD
NMR spectroscopy to study the dynamics and interactions of CFTR.
Methods Mol Biol. 2011;741:377-403., [PMID:21594798]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) is a multi-domain membrane chloride channel whose activity is regulated by ATP at two nucleotide-binding domains (NBD1 and NBD2) and by phosphorylation of the regulatory (R) region. The NBDs and the R region have functionally relevant motions that are critical for channel gating. Nuclear magnetic resonance (NMR) spectroscopy is a highly useful technique for obtaining information on the structure and interactions of CFTR and is extremely powerful for probing dynamics. NMR approaches for studying CFTR are reviewed, using our previous NBD1 and the R region results to provide examples. These NMR data are yielding insights into the dynamic properties and interactions that facilitate normal CFTR regulation as well as pathological effects of mutations, including the most common disease mutant, deletion of F508 in NBD1.

Comments [show]

None has been submitted yet.

Sentences [show]
No. Sentence Comment
127 As a disordered protein having a high fraction of charged residues, the R region was more easily amenable to solution NMR studies, once a better behaved construct containing the polymorphism F833L was identified (20). Login to comment

[hide] Phosphorylation-dependent 14-3-3 protein interacti... Mol Biol Cell. 2012 Mar;23(6):996-1009. Epub 2012 Jan 25. Liang X, Da Paula AC, Bozoky Z, Zhang H, Bertrand CA, Peters KW, Forman-Kay JD, Frizzell RA
Phosphorylation-dependent 14-3-3 protein interactions regulate CFTR biogenesis.
Mol Biol Cell. 2012 Mar;23(6):996-1009. Epub 2012 Jan 25., [PMID:22278744]

Abstract [show]
Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP/protein kinase A (PKA)-regulated chloride channel whose phosphorylation controls anion secretion across epithelial cell apical membranes. We examined the hypothesis that cAMP/PKA stimulation regulates CFTR biogenesis posttranslationally, based on predicted 14-3-3 binding motifs within CFTR and forskolin-induced CFTR expression. The 14-3-3beta, gamma, and epsilon isoforms were expressed in airway cells and interacted with CFTR in coimmunoprecipitation assays. Forskolin stimulation (15 min) increased 14-3-3beta and epsilon binding to immature and mature CFTR (bands B and C), and 14-3-3 overexpression increased CFTR bands B and C and cell surface band C. In pulse-chase experiments, 14-3-3beta increased the synthesis of immature CFTR, reduced its degradation rate, and increased conversion of immature to mature CFTR. Conversely, 14-3-3beta knockdown decreased CFTR B and C bands (70 and 55%) and elicited parallel reductions in cell surface CFTR and forskolin-stimulated anion efflux. In vitro, 14-3-3beta interacted with the CFTR regulatory region, and by nuclear magnetic resonance analysis, this interaction occurred at known PKA phosphorylated sites. In coimmunoprecipitation assays, forskolin stimulated the CFTR/14-3-3beta interaction while reducing CFTR's interaction with coat protein complex 1 (COP1). Thus 14-3-3 binding to phosphorylated CFTR augments its biogenesis by reducing retrograde retrieval of CFTR to the endoplasmic reticulum. This mechanism permits cAMP/PKA stimulation to make more CFTR available for anion secretion.

Comments [show]

None has been submitted yet.

Sentences [show]
No. Sentence Comment
261 The human CFTR R region (654- 838; F833L) was expressed from a pPROEX HTb vector (Invitrogen) with an N-terminal hexahistidine tag and purified as described previously (Baker et al., 2007). Login to comment
262 (Note that the F833L polymorphism yields an R region that is significantly more soluble.) Login to comment

[hide] Regulatory R region of the CFTR chloride channel i... Proc Natl Acad Sci U S A. 2013 Nov 19;110(47):E4427-36. doi: 10.1073/pnas.1315104110. Epub 2013 Nov 4. Bozoky Z, Krzeminski M, Muhandiram R, Birtley JR, Al-Zahrani A, Thomas PJ, Frizzell RA, Ford RC, Forman-Kay JD
Regulatory R region of the CFTR chloride channel is a dynamic integrator of phospho-dependent intra- and intermolecular interactions.
Proc Natl Acad Sci U S A. 2013 Nov 19;110(47):E4427-36. doi: 10.1073/pnas.1315104110. Epub 2013 Nov 4., [PMID:24191035]

Abstract [show]
Intrinsically disordered proteins play crucial roles in regulatory processes and often function as protein interaction hubs. Here, we present a detailed characterization of a full-length disordered hub protein region involved in multiple dynamic complexes. We performed NMR, CD, and fluorescence binding studies on the nonphosphorylated and highly PKA-phosphorylated human cystic fibrosis transmembrane conductance regulator (CFTR) regulatory region, a approximately 200-residue disordered segment involved in phosphorylation-dependent regulation of channel trafficking and gating. Our data provide evidence for dynamic, phosphorylation-dependent, multisite interactions of various segments of the regulatory region for its intra- and intermolecular partners, including the CFTR nucleotide binding domains 1 and 2, a 42-residue peptide from the C terminus of CFTR, the SLC26A3 sulphate transporter and antisigma factor antagonist (STAS) domain, and 14-3-3beta. Because of its large number of binding partners, multivalent binding of individually weak sites facilitates rapid exchange between free and bound states to allow the regulatory region to engage with different partners and generate a graded or rheostat-like response to phosphorylation. Our results enrich the understanding of how disordered binding segments interact with multiple targets. We present structural models consistent with our data that illustrate this dynamic aspect of phospho-regulation of CFTR by the disordered regulatory region.

Comments [show]

None has been submitted yet.

Sentences [show]
No. Sentence Comment
237 The human CFTR R region (aa 654-838; F833L) was prepared as described in the work by Baker et al. (23), and the F833L polymorphism was used to optimize solubility. Login to comment