BID, an interaction partner of protein kinase CK2alpha.
Recombinant murine BID protein was used as an in vitro substrate for the CK2 holoenzyme and the catalytic CK2alpha subunit. The results obtained show that BID can only serve as a substrate for the catalytic CK2alpha subunit. Phosphorylation of BID using the CK2 holoenzyme was only possible in the presence of polylysine, supporting the notion that BID behaves similarly to calmodulin. Co-immunoprecipitation of BID and CK2 subunits revealed that BID is preferentially associated with the CK2alpha subunit. Enzyme kinetic analyses yielded a Km value for BID that is a level of magnitude lower than that measured for casein and the synthetic peptide, suggesting more specific and tight binding of BID to CK2alpha. In contrast are the Vmax values observed, with a significantly higher phosphorylation rate measured for casein and the synthetic peptide than for BID. When BID was phosphorylated by polylysine-stimulated CK2 holoenzyme prior to caspase-8 cleavage, the formation of tC-BID was reduced in comparison to treatment with caspase-8 in the absence of protein kinase. Mass spectrometric analysis of BID phosphorylated by CK2alpha before and after cleavage with caspase-8 showed phosphorylation of residues Thr58 and Ser76.
Olsen
Birgitte B
BB
Institute for Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark.
Petersen
Jørgen
J
Issinger
Olaf-Georg
OG
D000595:Amino Acid Sequence; D000818:Animals; D051023:BH3 Interacting Domain Death Agonist Protein / Q000378:metabolism*; D047390:Casein Kinase II / Q000378:metabolism*; D053181:Caspase 8; D020169:Caspases / Q000378:metabolism / Q000494:pharmacology; D020134:Catalytic Domain; D047468:Immunoprecipitation; D007700:Kinetics; D013058:Mass Spectrometry; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D010766:Phosphorylation; D021122:Protein Subunits / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012694:Serine / Q000378:metabolism; D013912:Threonine / Q000378:metabolism
D051023:BH3 Interacting Domain Death Agonist Protein; D021122:Protein Subunits; D011994:Recombinant Proteins; D013912:Threonine; D012694:Serine; D047390:Casein Kinase II; C505526:Casp8 protein, mouse; D053181:Caspase 8; D020169:Caspases