Analysis of substrate specificity and cyclin Y binding of PCTAIRE-1 kinase.
PCTAIRE-1 (cyclin-dependent kinase [CDK] 16) is a highly conserved serine/threonine kinase that belongs to the CDK family of protein kinases. Little is known regarding PCTAIRE-1 regulation and function and no robust assay exists to assess PCTAIRE-1 activity mainly due to a lack of information regarding its preferred consensus motif and the lack of bona fide substrates. We used positional scanning peptide library technology and identified the substrate-specificity requirements of PCTAIRE-1 and subsequently elaborated a peptide substrate termed PCTAIRE-tide. Recombinant PCTAIRE-1 displayed vastly improved enzyme kinetics on PCTAIRE-tide compared to a widely used generic CDK substrate peptide. PCTAIRE-tide also greatly improved detection of endogenous PCTAIRE-1 activity. Similar to other CDKs, PCTAIRE-1 requires a proline residue immediately C-terminal to the phosphoacceptor site (+1) for optimal activity. PCTAIRE-1 has a unique preference for a basic residue at +4, but not at +3 position (a key characteristic for CDKs). We also demonstrate that PCTAIRE-1 binds to a novel cyclin family member, cyclin Y, which increased PCTAIRE-1 activity towards PCTAIRE-tide >100-fold. We hypothesised that cyclin Y binds and activates PCTAIRE-1 in a way similar to which cyclin A2 binds and activates CDK2. Point mutants of cyclin Y predicted to disrupt PCTAIRE-1-cyclin Y binding severely prevented complex formation and activation of PCTAIRE-1. We have identified PCTAIRE-tide as a powerful tool to study the regulation of PCTAIRE-1. Our understanding of the molecular interaction between PCTAIRE-1 and cyclin Y further facilitates future investigation of the functions of PCTAIRE-1 kinase.
MRC Protein Phosphorylation Unit, College of Life Sciences, University of Dundee, Dundee, UK.
Saifeldin N
SN
Shehata
Roger W
RW
Hunter
Eriko
E
Ohta
Mark W
MW
Peggie
Hua Jane
HJ
Lou
Frank
F
Sicheri
Elton
E
Zeqiraj
Benjamin E
BE
Turk
Kei
K
Sakamoto
D000595:Amino Acid Sequence; D001665:Binding Sites; D018844:Cyclin-Dependent Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016213:Cyclins / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D007700:Kinetics; D009154:Mutation; D019151:Peptide Library; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D014162:Transfection
C543319:CCNY protein, human; D016213:Cyclins; D019151:Peptide Library; D011994:Recombinant Proteins; D018844:Cyclin-Dependent Kinases; C089463:PCTAIRE-1 protein kinase