Detection of phospho-sites generated by protein kinase CK2 in CFTR

mechanistic aspects of Thr1471 phosphorylation

Andrea Venerando, Cinzia Franchin, Natasha Cant, Giorgio Cozza, Mario A. Pagano, Kendra Tosoni, Ateeq Al-Zahrani, Giorgio Arrigoni, Robert C. Ford, Anil Mehta, Lorenzo A. Pinna

    Research output: Contribution to journalArticle

    23 Citations (Scopus)

    Abstract

    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.
    Original languageEnglish
    Article number e74232
    JournalPLoS ONE
    Volume8
    Issue number9
    DOIs
    Publication statusPublished - 2013

    Fingerprint

    Casein Kinase II
    Phosphorylation
    protein kinases
    Yeast
    phosphorylation
    Phosphopeptides
    Cystic Fibrosis Transmembrane Conductance Regulator
    Holoenzymes
    Mass spectrometry
    Phosphotransferases
    Yeasts
    yeasts
    Amino Acids
    Kinetics
    Protein Stability
    consensus sequence
    cystic fibrosis
    mice
    Consensus Sequence
    protein subunits

    Cite this

    Venerando, A., Franchin, C., Cant, N., Cozza, G., Pagano, M. A., Tosoni, K., ... Pinna, L. A. (2013). Detection of phospho-sites generated by protein kinase CK2 in CFTR: mechanistic aspects of Thr1471 phosphorylation. PLoS ONE, 8(9), [ e74232]. https://doi.org/10.1371/journal.pone.0074232
    Venerando, Andrea ; Franchin, Cinzia ; Cant, Natasha ; Cozza, Giorgio ; Pagano, Mario A. ; Tosoni, Kendra ; Al-Zahrani, Ateeq ; Arrigoni, Giorgio ; Ford, Robert C. ; Mehta, Anil ; Pinna, Lorenzo A. / Detection of phospho-sites generated by protein kinase CK2 in CFTR : mechanistic aspects of Thr1471 phosphorylation. In: PLoS ONE. 2013 ; Vol. 8, No. 9.
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    abstract = "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.",
    author = "Andrea Venerando and Cinzia Franchin and Natasha Cant and Giorgio Cozza and Pagano, {Mario A.} and Kendra Tosoni and Ateeq Al-Zahrani and Giorgio Arrigoni and Ford, {Robert C.} and Anil Mehta and Pinna, {Lorenzo A.}",
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    Venerando, A, Franchin, C, Cant, N, Cozza, G, Pagano, MA, Tosoni, K, Al-Zahrani, A, Arrigoni, G, Ford, RC, Mehta, A & Pinna, LA 2013, 'Detection of phospho-sites generated by protein kinase CK2 in CFTR: mechanistic aspects of Thr1471 phosphorylation', PLoS ONE, vol. 8, no. 9, e74232. https://doi.org/10.1371/journal.pone.0074232

    Detection of phospho-sites generated by protein kinase CK2 in CFTR : mechanistic aspects of Thr1471 phosphorylation. / Venerando, Andrea; Franchin, Cinzia; Cant, Natasha; Cozza, Giorgio; Pagano, Mario A.; Tosoni, Kendra; Al-Zahrani, Ateeq; Arrigoni, Giorgio; Ford, Robert C.; Mehta, Anil; Pinna, Lorenzo A.

    In: PLoS ONE, Vol. 8, No. 9, e74232, 2013.

    Research output: Contribution to journalArticle

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    T1 - Detection of phospho-sites generated by protein kinase CK2 in CFTR

    T2 - mechanistic aspects of Thr1471 phosphorylation

    AU - Venerando, Andrea

    AU - Franchin, Cinzia

    AU - Cant, Natasha

    AU - Cozza, Giorgio

    AU - Pagano, Mario A.

    AU - Tosoni, Kendra

    AU - Al-Zahrani, Ateeq

    AU - Arrigoni, Giorgio

    AU - Ford, Robert C.

    AU - Mehta, Anil

    AU - Pinna, Lorenzo A.

    PY - 2013

    Y1 - 2013

    N2 - 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.

    AB - 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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