ABCC7 p.Ser422Asp
| Predicted by SNAP2: | A: N (87%), C: N (87%), D: N (72%), E: N (82%), F: N (66%), G: N (82%), H: N (82%), I: N (72%), K: N (87%), L: N (78%), M: N (78%), N: N (87%), P: N (87%), Q: N (93%), R: N (82%), T: N (93%), V: N (78%), W: N (61%), Y: N (72%), |
| Predicted by PROVEAN: | A: N, C: N, D: N, E: N, F: N, G: N, H: N, I: N, K: N, L: N, M: N, N: N, P: N, Q: N, R: N, T: N, V: N, W: N, Y: N, |
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[hide] PIKfyve upregulates CFTR activity. Biochem Biophys Res Commun. 2009 Dec 18;390(3):952-7. Epub 2009 Oct 21. Gehring EM, Lam RS, Siraskar G, Koutsouki E, Seebohm G, Ureche ON, Ureche L, Baltaev R, Tavare JM, Lang F
PIKfyve upregulates CFTR activity.
Biochem Biophys Res Commun. 2009 Dec 18;390(3):952-7. Epub 2009 Oct 21., 2009-12-18 [PMID:19852935]
Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-activated Cl(-) channel critically important in Cl(-) secreting epithelia. Mutations in the CFTR gene, such as (DeltaF508)CFTR leads to cystic fibrosis, a severe disease with defective Cl(-) secretion. CFTR is stimulated by the serum and glucocorticoid-inducible kinase SGK1. The SGK1 dependent regulation of several carriers and channels involves the phosphatidylinositol-3-phosphate-5-kinase PIKfyve, which similarly mediates the regulation of glucose carriers by PKB/Akt. The present study was thus performed to elucidate whether PKB/Akt and PIKfyve are regulators of CFTR. To this end CFTR or (DeltaF508)CFTR were expressed in Xenopus oocytes alone or together with PKB, PIKfyve or the SGK1/PKB resistant mutant (S318A)PIKfyve, and the current generated by cAMP upregulation with 10muM forskolin+1mM IBMX determined utilizing dual electrode voltage clamp. As a result, forskolin/IBMX treatment triggered a current (I(cAMP)) in CFTR-expressing Xenopus oocytes, but not in oocytes expressing (DeltaF508)CFTR. Coexpression of PKB/Akt and PIKfyve, but not of (S318A)PIKfyve, stimulated I(cAMP) in CFTR-expressing ( approximately 2- to 3-fold) but not in (DeltaF508)CFTR-expressing or water injected Xenopus oocytes. Immunohistochemistry revealed that the coexpression of PIKfyve, but not of (S318A)PIKfyve, enhanced the CFTR protein abundance but not the (DeltaF508)CFTR protein abundance in CFTR or (DeltaF508)CFTR-expressing oocytes. The present observations reveal a novel powerful regulator of intact but not of defective CFTR.
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No. Sentence Comment
141 water CFTR CFTR PIKfyve CFTR S318APIKfyve ΔF508CFTR PIKfyve C FTR +H 2 O C FTR +H 2 O C FTR +PIKfyve C FTR + S318A PIKfyve C FTR +PIKfyve C FTR + S422D SG K1 ~170 KDa ~170 KDa A B Fig. 4.
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ABCC7 p.Ser422Asp 19852935:141:152
status: NEW[hide] CFTR mutations altering CFTR fragmentation. Biochem J. 2013 Jan 1;449(1):295-305. doi: 10.1042/BJ20121240. Tosoni K, Stobbart M, Cassidy DM, Venerando A, Pagano MA, Luz S, Amaral MD, Kunzelmann K, Pinna LA, Farinha CM, Mehta A
CFTR mutations altering CFTR fragmentation.
Biochem J. 2013 Jan 1;449(1):295-305. doi: 10.1042/BJ20121240., [PMID:23067305]
Abstract [show]
Most CF (cystic fibrosis) results from deletion of a phenylalanine (F508) in the CFTR {CF transmembrane-conductance regulator; ABCC7 [ABC (ATP-binding cassette) sub-family C member 7]} which causes ER (endoplasmic reticulum) degradation of the mutant. Using stably CFTR-expressing BHK (baby-hamster kidney) cell lines we demonstrated that wild-type CTFR and the F508delCFTR mutant are cleaved into differently sized N- and C-terminal-bearing fragments, with each hemi-CFTR carrying its nearest NBD (nucleotide-binding domain), reflecting differential cleavage through the central CFTR R-domain. Similar NBD1-bearing fragments are present in the natively expressing HBE (human bronchial epithelial) cell line. We also observe multiple smaller fragments of different sizes in BHK cells, particularly after F508del mutation (ladder pattern). Trapping wild-type CFTR in the ER did not generate a F508del fragmentation fingerprint. Fragments change their size/pattern again post-mutation at sites involved in CFTR's in vitro interaction with the pleiotropic protein kinase CK2 (S511A in NBD1). The F508del and S511A mutations generate different fragmentation fingerprints that are each unlike the wild-type; yet, both mutants generate new N-terminal-bearing CFTR fragments that are not observed with other CK2-related mutations (S511D, S422A/D and T1471A/D). We conclude that the F508delCFTR mutant is not degraded completely and there exists a relationship between CFTR's fragmentation fingerprint and the CFTR sequence through putative CK2-interactive sites that lie near F508.
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No. Sentence Comment
44 Cell culture, lysis, protein solubilization and Western blotting The cell culture methods to create the stable CFTR-expressing cell lines (WT, F, WT S422A, WT S422D, WT S511A, WT S511D, WT T1471A and WT T1471D) and their culture protocols have been described recently [25].
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ABCC7 p.Ser422Asp 23067305:44:160
status: NEW191 Compared with each other S422D- and S422A-CFTR revealed no gross differences in fragmentation (Figures 7A-7D, compare lanes 3 and 4).
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ABCC7 p.Ser422Asp 23067305:191:25
status: NEW[hide] Activation of 3-phosphoinositide-dependent kinase ... J Biol Chem. 2014 Dec 26;289(52):35953-68. doi: 10.1074/jbc.M114.598649. Epub 2014 Nov 10. Caohuy H, Yang Q, Eudy Y, Ha TA, Xu AE, Glover M, Frizzell RA, Jozwik C, Pollard HB
Activation of 3-phosphoinositide-dependent kinase 1 (PDK1) and serum- and glucocorticoid-induced protein kinase 1 (SGK1) by short-chain sphingolipid C4-ceramide rescues the trafficking defect of DeltaF508-cystic fibrosis transmembrane conductance regulator (DeltaF508-CFTR).
J Biol Chem. 2014 Dec 26;289(52):35953-68. doi: 10.1074/jbc.M114.598649. Epub 2014 Nov 10., [PMID:25384981]
Abstract [show]
Cystic fibrosis (CF) is due to a folding defect in the CF transmembrane conductance regulator (CFTR) protein. The most common mutation, DeltaF508, prevents CFTR from trafficking to the apical plasma membrane. Here we show that activation of the PDK1/SGK1 signaling pathway with C4-ceramide (C4-CER), a non-toxic small molecule, functionally corrects the trafficking defect in both cultured CF cells and primary epithelial cell explants from CF patients. The mechanism of C4-CER action involves a series of mutual autophosphorylation and phosphorylation events between PDK1 and SGK1. Detailed mechanistic studies indicate that C4-CER initially induces autophosphorylation of SGK1 at Ser(422). SGK1[Ser(P)(422)] and C4-CER coincidently bind PDK1 and permit PDK1 to autophosphorylate at Ser(241). Then PDK1[Ser(P)(241)] phosphorylates SGK1[Ser(P)(422)] at Thr(256) to generate fully activated SGK1[Ser(422), Thr(P)(256)]. SGK1[Ser(P)(422),Thr(P)(256)] phosphorylates and inactivates the E3 ubiquitin ligase Nedd4-2. DeltaF508-CFTR is thus free to traffic to the plasma membrane. Importantly, C4-CER-mediated activation of both PDK1 and SGK1 is independent of the PI3K/Akt/mammalian target of rapamycin signaling pathway. Physiologically, C4-CER significantly increases maturation and stability of DeltaF508-CFTR (t(1/2) approximately 10 h), enhances cAMP-activated chloride secretion, and suppresses hypersecretion of interleukin-8 (IL-8). We suggest that candidate drugs for CF directed against the PDK1/SGK1 signaling pathway, such as C4-CER, provide a novel therapeutic strategy for a life-limiting disorder that affects one child, on average, each day.
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No. Sentence Comment
82 In Vitro Phosphorylation of CFTR-Wild-type and èc;F508-CFTR immunoprecipitates from untreated repaired and CFPAC-1 cell lysates, respectively, were washed with phosphorylation buffer (50 mM Tris, pH 7.5, 0.1 mM EGTA, 0.1% 2-mercaptoethanol) and then resuspended in 30 òe;l of the same buffer containing 100 ng of purified recombinant human S422D-SGK1 mutant protein (Millipore) and 1afb; Mg2af9; -ATP mixture (Millipore).
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ABCC7 p.Ser422Asp 25384981:82:348
status: NEW336 The in vitro kinase reaction mixture consists of active PDK1, mutant S422D-SGK1, and substrate CFTR that was immunoprecipitated from either untreated CFPAC-1 or repaired CFPAC-1 pLJ6 cells.
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ABCC7 p.Ser422Asp 25384981:336:69
status: NEW415 Phosphorylation of immunoprecipitated CFTR or phosphorylation at Thr256 of S422D-SGK1 in the whole reaction mixture(WRM)wasdetectedbyWesternblotting.D,effectofC4-CERonCFTRubiquitination.ThecelllysatespreparedasdescribedinBweresubjectedtoCFTR immunoprecipitation, followed by Western blot analysis for total CFTR expression or CFTR ubiquitination.
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ABCC7 p.Ser422Asp 25384981:415:75
status: NEW425 Furthermore, the importance of active SGK kinase for the addition of CFTR to the plasma membrane in Xenopus oocytes (50) is consistent with our observation that PDK1-activated S422D-SGK1 phosphorylates both wild-type and mutant CFTR.
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ABCC7 p.Ser422Asp 25384981:425:176
status: NEW