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

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 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.

PCTAIRE Kinase 3/Cyclin-dependent Kinase 18 Is Activated through Association with Cyclin A and/or Phosphorylation by Protein Kinase A

“…to PCTK1 (24). This Lys-Glu pair is also found in other cyclins and is likely to be a conserved structural motif in cyclin-CDK complexes.…”

“…PCTK1, a member of the PCTK subfamily, is activated by membrane-associated cyclin Y (10,24). We hypothesized that PCTK3 was also activated by cyclin family members.…”

Atypical cyclins: the extended family portrait

“…These two groups of cyclins show significant conservation within the CBD region near residues K257 and E286 of CCNB1 (indicated by red dots in Fig. 1a), which were described to be critical for CDK binding in the six CDK/cyclin complexes that have known crystallographic models [30]. Indeed, mutation of this lysine in the CBDs of cyclin D1 and cyclin Y impairs the interaction with their respective CDK partners [30,31].…”

“…1a), which were described to be critical for CDK binding in the six CDK/cyclin complexes that have known crystallographic models [30]. Indeed, mutation of this lysine in the CBDs of cyclin D1 and cyclin Y impairs the interaction with their respective CDK partners [30,31]. A close look at this region reveals that there are also other well-conserved residues, of which there is less experimental evidence (Fig.…”

Large-scale profiling of protein kinases for cellular signaling studies by mass spectrometry and other techniques

“…The positional scanning peptide library approach was applied for characterization of the substrate preference of Arabidopsis abscisic acid-activated kinase OST1 [55], mTORC1 [34], cyclindependent kinase 16 (PCTAIRE-1) [56], non-receptor tyrosine kinase BMX [57], and p90 ribosomal S6 kinase [58]. Mok et al profiled the yeast kinome using a large-scale position scanning peptide library [59].…”