Screening of a library of T7 phage-displayed peptides identifies alphaC helix in 14-3-3 protein as a CBP501-binding site.
CBP501 is a chemically modified peptide composed of twelve unnatural d-amino acids, which inhibits Chk kinase and abrogates G2 arrest induced by DNA-damaging agents. Here we identified an alphaC helix in 14-3-3 protein as a CBP501-binding site using T7 phage display technology. An affinity selection of T7 phage-displayed peptide using biotinylated CBP501 identified a 14-mer peptide NSDCIISRKIEQKE. This peptide sequence showed similarity to a portion of the alphaC helix of human 14-3-3ε, suggesting that CBP501 may bind to this region. Surface plasmon resonance (SPR) and ELISA demonstrated that CBP501 interacts with 14-3-3ε specifically at the screen-guided region. An avidin-agarose bead pull-down assay showed that CBP501 also binds to other 14-3-3 isoforms in Jurkat cells. Among the other known Chk kinase inhibitors tested, CBP501 showed the strongest affinity for 14-3-3ε. Thus, we conclude that in addition to the direct inhibition of Chk kinase activity, CBP501 directly binds to cellular 14-3-3 proteins through alphaC helix.
Department of Applied Biological Sciences, Faculty of Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan.
Yuki
Y
Matsumoto
Yosuke
Y
Shindo
Yoichi
Y
Takakusagi
Kaori
K
Takakusagi
Senko
S
Tsukuda
Tomoe
T
Kusayanagi
Hitoshi
H
Sato
Takumi
T
Kawabe
Fumio
F
Sugawara
Kengo
K
Sakaguchi
D048948:14-3-3 Proteins / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D017123:Bacteriophage T7 / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002942:Circular Dichroism; D006801:Humans; D019169:Jurkat Cells; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism*; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017433:Protein Structure, Secondary; D020687:cdc25 Phosphatases / Q000378:metabolism*
D048948:14-3-3 Proteins; C517976:Cdc25C phosphatase (211-221); D010446:Peptide Fragments; D019151:Peptide Library; D010455:Peptides; D020687:cdc25 Phosphatases