Analysis of substrate specificity and cyclin Y binding of PCTAIRE-1 kinase

Saifeldin N. Shehata, Roger W. Hunter, Eriko Ohta, Mark W. Peggie, Hua Jane Lou, Frank Sicheri, Elton Zegiraj, Benjamin E. Turk, Kei Sakamoto

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    11 Citations (Scopus)

    Abstract

    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 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. (C) 2012 Elsevier Inc. All rights reserved.

    Original languageEnglish
    Pages (from-to)2085-2094
    Number of pages10
    JournalCellular Signalling
    Volume24
    Issue number11
    DOIs
    Publication statusPublished - Nov 2012

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