Cyclin Y phosphorylation- and 14-3-3-binding-dependent activation of PCTAIRE-1/CDK16.
Bottom Line: Recombinant WT cyclin Y, but not a S100A/S326A mutant, prepared in COS-1 cells co-purified with 14-3-3 and was able to activate bacterially expressed recombinant PCTAIRE-1 in cell-free assays.Finally, we observed that recently identified PCTAIRE-1 variants found in patients with intellectual disability were unable to interact with cyclin Y, and were inactive enzymes.Collectively, the present work has revealed a new mechanistic insight into activation of PCTAIRE-1, which is mediated through interaction with the phosphorylated form of cyclin Y in complex with 14-3-3.
Affiliation: Nestlé Institute of Health Sciences SA, EPFL Innovation Park, bâtiment G, 1015 Lausanne, Switzerland School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne (EPFL), 1015 Lausanne, Switzerland.Show MeSH
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Mentions: Our recent positional scanning peptide library analysis has revealed key substrate-specificity requirements of PCTAIRE-1 . We found that although an absolute requirement for a proline residue immediately C-terminal to the phospho-acceptor site (+1) and preference for a basic residue at +2 are similar to other conventional CDKs, some elements (preference for a basic residue at +4, but not at +3) were unique to PCTAIRE-1. We established a preferred consensus sequence of S-P-K/R-ϕ-K/R/H (ϕ, represents a small aliphatic amino acid) and generated an optimal substrate peptide (PCTAIRE-tide, PKSPKARKKL) for robust measurements of recombinant and endogenous PCTAIRE-1 kinase activity in vitro . Analysis of the protein sequence of human cyclin Y, a cognate binding partner of PCTAIRE-1 necessary for its activation [5,6], identified two proline-directed serine residues (S-P motif; Figure 1A). One of the S-P motifs (containing Ser336 in the human sequence) is well conserved among mammals but not in lower organisms such as D. melanogaster and C. elegans, whereas the other S-P motif (containing Ser12) is conserved in humans and mouse, as well as the majority of the lower organisms inspected. We noted that residues surrounding Ser12 (SPKLRRN) of human cyclin Y resemble the preferred consensus sequence for PCTAIRE-1 (Figure 1A). Thus we hypothesized that PCTAIRE-1 phosphorylates cyclin Y and that this modification influences their interaction and hence catalytic activity of PCTAIRE-1. To test this hypothesis, we initially generated synthetic peptides encompassing residues surrounding Ser12 (termed Ser12-tide) and Ser336 (plus an additional two arginine residues to enable efficient binding of the peptide to P81 paper; termed Ser336-tide) of human cyclin Y and determined in vitro substrate kinetics using recombinant PCTAIRE-1–cyclin Y complex isolated from COS-1 cells. Ser12-tide demonstrated a comparable Km to that of PCTAIRE-tide (6.2 μM compared with 7.6 μM), but displayed an approximately 4-fold lower Vmax (Figure 1B). In contrast, no detectable activity above background was observed for Ser336-tide, possibly due to absence of a preferred basic residue at +2. In support of this, alteration of +2 (alanine) and +4 (isoleucine) amino acids to lysine and arginine respectively in Ser336-tide to more resemble PCTAIRE-tide robustly increased PCTAIRE-1 activity. However, the Km of the modified Ser336-tide was still 10-fold higher than that of Ser12- or PCTAIRE-tide (Figure 1B). Taken together, in vitro peptide analysis suggests that Ser12, but not Ser336, is potentially a candidate for PCTAIRE-1-targeted phosphorylation.
Affiliation: Nestlé Institute of Health Sciences SA, EPFL Innovation Park, bâtiment G, 1015 Lausanne, Switzerland School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne (EPFL), 1015 Lausanne, Switzerland.