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: It has been reported that bacterially expressed PCTAIRE-1 (bPCTAIRE-1) is catalytically inactive . In addition, we have observed that co-incubation with bacterially expressed cyclin Y does not activate bPCTAIRE-1 in a cell-free assay (results not shown). This is unsurprising considering that our results suggest an absolute requirement for cyclin Y phosphorylation (and potentially 14-3-3 binding) for efficient binding/activation of PCTAIRE-1. This suggests that bPCTAIRE-1 may be activated by the cyclin Y–14-3-3 complex isolated from eukaryotic cells. We therefore incubated bPCTAIRE-1 with or without COS-1 lysates ectopically expressing WT or phospho-deficient/14-3-3-binding-deficient mutants (S100A or S326A) of cyclin Y, immunoprecipitated using anti-PCTAIRE-1 antibody and assessed binding and kinase activity (Figure 5A). As expected, bPCTAIRE-1 on its own was inactive. In contrast, WT cyclin Y (in complex with 14-3-3), but not S100A or S326A, bound and robustly activated bPCTAIRE-1. To rule out the possibility that the observed activity was due to the presence of a contaminating kinase in the purified HA–WT cyclin Y preparation, we also incubated the WT cyclin Y with KI (D304A) bPCTAIRE-1. This confirmed that the observed activity is intrinsic to bPCTAIRE-1. We also performed a similar experiment using Sf21-derived cyclin Y preparations (Figure 4D) and again observed that WT, but not S326A, activated bPCTAIRE-1 but only in the presence of additional recombinant 14-3-3 (of low abundance in Sf21-derived cyclin Y) suggesting that the formation of a ternary complex of PCTAIRE-1–cyclin-Y–14-3-3 is essential for kinase activity (Figure 5B). In line with a previous report (using a preparation of PCTAIRE-1 from mammalian cells) , partially truncated forms (Δ1–105 and Δ477–496/106–476), but not the kinase domain fragment (165–446) of bPCTAIRE-1, could be activated when co-incubated with COS-1 cell-derived WT cyclin Y preparation (Figure 5C), indicating the importance/requirement of regions N- and C-terminal to/flanking the kinase domain for cyclin Y-mediated PCTAIRE-1 activation. To further demonstrate that 14-3-3 is necessary for cyclin Y binding and activation of bPCTAIRE-1, we measured cyclin Y binding and activation of bPCTAIRE-1 after addition of the 14-3-3-binding phosphopeptide ARAAS*APA (where S* denotes phosphoserine). Non-phosphopeptide (ARAASAPA) was used as a negative control (Figure 5D). FLAG–WT cyclin Y was immunoprecipitated from COS-1 cells followed by incubation with the 14-3-3-binding phosphopeptide/ARAAS*APA. After washing of unbound/dissociated proteins, along with excess phosphopeptide, bPCTAIRE-1 and 14-3-3 were added/incubated together or in isolation with the immunoprecipitate, washed and then analysed by immunoblotting and kinase activity assay. We observed that the 14-3-3-binding phosphopeptide/ARAAS*APA severely diminished cyclin Y interaction with bPCTAIRE-1 and abolished bPCTAIRE-1 activity, which was restored when 14-3-3 proteins were exogenously added. In contrast, control peptide (non-phosphopeptide/ARAASAPA) did not affect cyclin Y binding to bPCTAIRE-1 or the consequent bPCTAIRE-1 activation.
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.