Modulation of protein kinase CK2 activity by fragments of CFTR encompassing F508 may reflect functional links with cystic fibrosis pathogenesis.
Deletion of F508 in the first nucleotide binding domain (NBD1) of cystic fibrosis transmembrane conductance regulator protein (CFTR) is the commonest cause of cystic fibrosis (CF). Functional interactions between CFTR and CK2, a highly pleiotropic protein kinase, have been recently described which are perturbed by the F508 deletion. Here we show that both NBD1 wild type and NBD1 DeltaF508 are phosphorylated in vitro by CK2 catalytic alpha-subunit but not by CK2 holoenzyme unless polylysine is added. MS analysis reveals that, in both NBD1 wild type and DeltaF508, the phosphorylated residues are S422 and S670, while phosphorylation of S511 could not be detected. Accordingly, peptides encompassing the 500-518 sequence of CFTR are not phosphorylated by CK2; rather they inhibit CK2alpha catalytic activity in a manner which is not competitive with respect to the specific CK2 peptide substrate. In contrast, 500-518 peptides promote the phosphorylation of NBD1 by CK2 holoenzyme overcoming inhibition by the beta-subunit. Such a stimulatory efficacy of the CFTR 500-518 peptide is dramatically enhanced by deletion of F508 and is abolished by deletion of the II507 doublet. Kinetics of NBD1 phosphorylation by CK2 holoenzyme, but not by CK2alpha, display a sigmoid shape denoting a positive cooperativity which is dramatically enhanced by the addition of the DeltaF508 CFTR peptide. SPR analysis shows that NBD1 DeltaF508 interacts more tightly than NBD1 wt with the alpha-subunit of CK2 and that CFTR peptides which are able to trigger NBD1 phosphorylation by CK2 holoenzyme also perturb the interaction between the alpha- and the beta-subunits of CK2.
Pagano
Mario A
MA
Department of Biological Chemistry and CNR Institute of Neurosciences, University of Padova, viale G. Colombo 3, 35131 Padova, Italy.
Arrigoni
Giorgio
G
Marin
Oriano
O
Sarno
Stefania
S
Meggio
Flavio
F
Treharne
Kate J
KJ
Mehta
Anil
A
Pinna
Lorenzo A
LA
D000595:Amino Acid Sequence; D000818:Animals; D047390:Casein Kinase II / Q000378:metabolism*; D003550:Cystic Fibrosis / Q000378:metabolism* / Q000473:pathology; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010748:Phosphopeptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D011107:Polylysine / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D017386:Sequence Homology, Amino Acid
D010748:Phosphopeptides; C093349:cystic fibrosis transmembrane conductance regulator delta F508; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D011107:Polylysine; D047390:Casein Kinase II