MAST205 Competes with Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)-associated Ligand for Binding to CFTR to Regulate CFTR-mediated Fluid Transport

Ren, Aiming; Zhang, Weiqiang; Yarlagadda, Sunitha; Sinha, Chandrima; Arora, Kavisha; Moon, Chang Suk; Naren, Anjaparavanda P.

doi:10.1074/jbc.m112.432724

Cited by 15 publications

(27 citation statements)

References 43 publications

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“…Given the location of Thr1471 close to the PDZ-interacting motif that favors retention of CFTR within the cell by interacting with Golgi associated CAL (Cheng J et al JBC 2002) [39] and its competitor MAST205 [40], we argued that its phosphorylation could affect the stability of such interactions and consequently the turnover of CFTR itself. Accordingly a hCFTR mutant no longer susceptible to such a phosphorylation (T1471A) was overexpressed in BHK cells as compared to hCFTR wild type.…”

Section: Resultsmentioning

confidence: 92%

Detection of Phospho-Sites Generated by Protein Kinase CK2 in CFTR: Mechanistic Aspects of Thr1471 Phosphorylation

et al. 2013

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By mass spectrometry analysis of mouse Cystic Fibrosis Transmembrane-conductance Regulator (mCFTR) expressed in yeast we have detected 21 phosphopeptides accounting for 22 potential phospho-residues, 12 of which could be unambiguously assigned. Most are conserved in human CFTR (hCFTR) and the majority cluster in the Regulatory Domain, lying within consensus sequences for PKA, as identified in previous mammalian studies. This validates our yeast expression model. A number of phospho-residues were novel and human conserved, notably mouse Ser670, Ser723, Ser737, and Thr1467, that all lie in acidic sequences, compatible with their phosphorylation by protein kinase CK2. Thr1467 is localized in the C-terminal tail, embedded in a functionally important and very acidic sequence (EETEEE) which displays an optimal consensus for protein kinase CK2. Herein, we show that Thr1467, homologous to human Thr1471 is readily phosphorylated by CK2. Indeed a 42 amino acid peptide encompassing the C-terminal segment of human CFTR is readily phosphorylated at Thr1471 with favorable kinetics (Km 1.7 µM) by CK2 holoenzyme, but neither by its isolated catalytic subunit nor by other acidophilic Ser/Thr kinases (CK1, PLK2/3, GCK/FAM20C). Our finding that by treating CFTR expressing BHK cells with the very specific CK2 inhibitor CX4945, newly synthesized wild type CFTR (and even more its Phe508del mutant) accumulates more abundantly than in the absence of CK2 inhibitor, supports the conclusion that phosphorylation of CFTR by CK2 correlates with decreased stability of the protein.

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Section: Resultsmentioning

confidence: 92%

Detection of Phospho-Sites Generated by Protein Kinase CK2 in CFTR: Mechanistic Aspects of Thr1471 Phosphorylation

et al. 2013

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“…The protein bands were detected by chemiluminescence using ECL™ Western blotting detection reagents from GE Healthcare (Buckinghamshire, UK). We loaded 50µg of total protein per lane [20, 21]. …”

Section: 2 Materials and Methodsmentioning

confidence: 99%

“…For immunostaining, samples were incubated overnight with respective primary antibodies and then incubated with secondary antibody, Alexa Fluor® 488 phalloidin or anti-rabbit Alexa Fluor® 568 (Molecular Probes, Invitrogen) for 1 h and mounted in DAPI-containing Vectashield (Vector Labs; Burlingame, CA). Fluorescence images were taken using a NikonA1R-A1 confocal microscope system [20]. …”

Section: 2 Materials and Methodsmentioning

confidence: 99%

PKA and actin play critical roles as downstream effectors in MRP4-mediated regulation of fibroblast migration

Sinha

Ren

Arora

et al. 2015

Cellular Signalling

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Multidrug resistance protein 4 (MRP4), a member of the ATP binding cassette transporter family, functions as a plasma membrane exporter of cyclic nucleotides. Recently, we demonstrated that fibroblasts lacking the Mrp4 gene migrate faster and contain higher cyclic-nucleotide levels. Here, we show that cAMP accumulation and protein kinase A (PKA) activity are higher and polarized in Mrp4−/− fibroblasts, versus Mrp4+/+ cells. MRP4-containing macromolecular complexes isolated from these fibroblasts contained several proteins, including actin, which play important roles in cell migration. We found that actin interacts with MRP4, predominantly at the plasma membrane, and an intact actin cytoskeleton is required to restrict MRP4 to specific microdomains of the plasma membrane. Our data further indicated that the enhanced accumulation of cAMP in Mrp4−/− fibroblasts facilitates cortical actin polymerization in a PKA-dependent manner at the leading edge, which in turn increases the overall rate of cell migration to accelerate the process of wound healing. Disruption of actin polymerization or inhibition of PKA activity abolished the effect of MRP4 on cell migration. Together, our findings suggest a novel cAMP-dependent mechanism for MRP4-mediated regulation of fibroblast migration whereby PKA and actin play critical roles as downstream effectors.

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“…Proof-of concept that these signalling pathways are potential antisecretory targets has been shown by the efficacy of a small molecule phosphodiesterase (PDE) activator, which reduces cAMP and cGMP, in a closed-loop model of intestinal fluid secretion [19]. Other potential targets include modulators of membrane macromolecular complexes such as agonists of lysophosphatidic acid (LPA) receptors, which inhibit CFTR function, or the more recently identified putative modulator MAST205 [20, 21]. Agonists of the Ca 2+ sensing receptor CaSR have been shown to inhibit enterotoxin mediated fluid secretion [22] and act through a number of pathways including the enteric nervous system [23].…”

Section: Antisecretory Targets In the Intestinal Epitheliummentioning

confidence: 99%

Chloride channel-targeted therapy for secretory diarrheas

Thiagarajah¹,

Verkman²

2013

Current Opinion in Pharmacology

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Secretory diarrheas caused by bacterial and viral enterotoxins remain a significant cause of morbidity and mortality. Enterocyte Cl− channels represent an attractive class of targets for diarrhea therapy, as they are the final, rate-limiting step in enterotoxin-induced fluid secretion in the intestine. Activation of cyclic nucleotide and/or Ca2+ signalling pathways in secretory diarrheas increases the conductance of Cl− channels at the enterocyte luminal membrane, which include the cystic fibrosis transmembrane conductance regulator (CFTR) and Ca2+-activated Cl− channels (CaCCs). High-throughput screens have yielded several chemical classes of small molecule CFTR and CaCC inhibitors that show efficacy in animal models of diarrheas. Natural-product diarrhea remedies with Cl− channel inhibition activity have also been identified, with one product recently receiving FDA approval for HIV-associated diarrhea.

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MAST205 Competes with Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)-associated Ligand for Binding to CFTR to Regulate CFTR-mediated Fluid Transport

Cited by 15 publications

References 43 publications

Detection of Phospho-Sites Generated by Protein Kinase CK2 in CFTR: Mechanistic Aspects of Thr1471 Phosphorylation

Detection of Phospho-Sites Generated by Protein Kinase CK2 in CFTR: Mechanistic Aspects of Thr1471 Phosphorylation

PKA and actin play critical roles as downstream effectors in MRP4-mediated regulation of fibroblast migration

Chloride channel-targeted therapy for secretory diarrheas

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