Summary
Recombinant murine BID protein was used as an in vitro substrate for the CK2 holoenzyme and the catalytic CK2a subunit. The results obtained show that BID can only serve as a substrate for the catalytic CK2a 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 CK2a 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 CK2a. 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 CK2a before and after cleavage with caspase-8 showed phosphorylation of residues Thr58 and Ser76.

 
Figure 6 Mass spectrometry of phosphorylated full-length and caspase-cleaved BID.
(A) MALDI-TOF spectra of the phosphorylated full-length BID after tryptic digestion and phosphopeptide enrichment. The spectra revealed two major phosphopeptides, as indicated. Only the region of interest (m/z 1800–3000) is shown. (B) MALDI-TOF spectra of the phosphorylated caspase-8-cleaved BID after tryptic digestion and phosphopeptide enrichment. Again the spectra revealed two major phosphopeptides, as indicated. Only the region of interest (m/z 1600–2600) is shown. (C) Nanoelectrospray ionization MS/MS analysis of the phosphopeptide at m/z 2060. The peak at m/z 1030.9 corresponds to the singly phosphorylated, doubly charged tryptic peptide Ile72–Arg88. The y-fragment ions are indicated, as is the amino acid sequence of the peptide; the phosphorylated serine is indicated in bold. An asterisk denotes loss of HPO4-. (D) Magnification of the upper mass region in (C). (E) Nanoelectrospray ionization MS/MS analysis of the phosphopeptide at m/z 2344.14. The peak at m/z 1172.5 corresponds to the singly phosphorylated, doubly charged tryptic peptide Glu41–Asp59 (MH2q). The y-fragment ions are indicated, as is the amino acid sequence of the peptide; the phosphorylated threonine is indicated in bold. An asterisk denotes loss of HPO4 -. (F) Magnification of the lower mass region in (E).

Reference:
Olsen, BB. et al. “BID, an interaction partner of protein kinase CK2a”, Biol. Chem 387, p.441-449, 2006.