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

TY - JOUR

T1 - Analysis of substrate specificity and cyclin Y binding of PCTAIRE-1 kinase

A1 - Shehata,Saifeldin N.

A1 - Hunter,Roger W.

A1 - Ohta,Eriko

A1 - Peggie,Mark W.

A1 - Lou,Hua Jane

A1 - Sicheri,Frank

A1 - Zegiraj,Elton

A1 - Turk,Benjamin E.

A1 - Sakamoto,Kei

AU - Shehata,Saifeldin N.

AU - Hunter,Roger W.

AU - Ohta,Eriko

AU - Peggie,Mark W.

AU - Lou,Hua Jane

AU - Sicheri,Frank

AU - Zegiraj,Elton

AU - Turk,Benjamin E.

AU - Sakamoto,Kei

PY - 2012/11

Y1 - 2012/11

N2 - <p>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 &gt;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.</p>

AB - <p>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 &gt;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.</p>

U2 - 10.1016/j.cellsig.2012.06.018

DO - 10.1016/j.cellsig.2012.06.018

M1 - Article

JO - Cellular Signalling

JF - Cellular Signalling

SN - 0898-6568

IS - 11

VL - 24

SP - 2085

EP - 2094

ER -