Abstract
PCTAIRE-1 [also known as cyclin-dependent kinase 16 (CDK16)] is implicated in various physiological processes such as neurite outgrowth and vesicle trafficking; however, its molecular regulation and downstream targets are largely unknown. Cyclin Y has recently been identified as a key interacting/activating cyclin for PCTAIRE-1; however, the molecular mechanism by which it activates PCTAIRE-1 is undefined. In the present study, we initially performed protein sequence analysis and identified two candidate phosphorylation sites (Ser(12) and Ser(336)) on cyclin Y that might be catalysed by PCTAIRE-1. Although invitro peptide analysis favoured Ser(12) as the candidate phosphorylation site, immunoblot analysis of cell lysates that had been transfected with wild-type (WT) or kinase-inactive (KI) PCTAIRE-1 together with WT or phospho-deficient mutants of cyclin Y suggested Ser(336), but not Ser(12), as a PCTAIRE-1-dependent phosphorylation site. Monitoring phosphorylation of Ser(336) may provide a useful read-out to assess cellular activity of PCTAIRE-1 invivo; however, a phospho-deficient S336A mutant displayed normal interaction with PCTAIRE-1. Unbiased mass spectrometry and targeted mutagenesis analysis of cyclin Y identified key phosphorylation sites (Ser(100) and Ser(326)) required for 14-3-3 binding. 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-1in 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.
Highlights
The human cyclin-dependent kinase 16 (CDK16) gene, which encodes the protein kinase PCTAIRE-1, maps to the X chromosome (Xp11.3) whose defects and copynumber variants have been linked to various diseases, including intellectual disability and related disorders [1]
Uni Prot accession numbers are indicated in parentheses. (B) The in vitro phosphotransferase activity of FLAG–PCTAIRE-1–HA–cyclin Y complex was determined using peptides derived from residues surrounding Ser12 and Ser336 on human cyclin Y, as well as a derivative of the Ser336 peptide based on the PCTAIRE-tide sequence
We demonstrate in the present study that phosphorylation of cyclin Y (Ser100 and/or Ser326) and cyclin Y-like-1 protein (Ser122 and/or Ser344, equivalent to Ser100 and Ser326 of cyclin Y respectively) promotes binding to 14-3-3 proteins in which both events are necessary for binding to, and activation of, PCTAIRE-1
Summary
The human cyclin-dependent kinase 16 (CDK16) gene, which encodes the protein kinase PCTAIRE-1 ( known as CDK16), maps to the X chromosome (Xp11.3) whose defects and copynumber variants have been linked to various diseases, including intellectual disability and related disorders [1]. The original work implicating that cyclin Y might be a potential partner for PCTAIRE-1 came from a large-scale yeast two-hybrid screen in Drosophila melanogaster, where Eip63E (ecdysoneinduced protein encoded by a gene at chromosomal position 63E), most similar to the mammalian PFTAIRE-1/CDK14, interacted with a novel cyclin-like protein, coded as CG14939. Ou et al [16] have demonstrated that the Caenorhabditis elegans homologue of Abbreviations: bPCTAIRE-1, bacterially expressed PCTAIRE-1; CDK, cyclin-dependent kinase; Eip63E, ecdysone-induced protein encoded by a gene at chromosomal position 63E; HA, haemagglutinin; HRP, horseradish peroxidase; KI, kinase-inactive; MKL, megakaryoblastic leukemia; TBST, tris buffered saline with Tween-20; TEAB, triethylammonium bicarbonate; WT, wild-type; XLID, X-linked intellectual disability
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