Cyclin Y phosphorylation- and 14-3-3-binding-dependent activation of PCTAIRE-1/CDK16

Shehata, S. N., Deak, M., Morrice, N. A., Ohta, E., Hunter, R. W., Kalscheuer, V. M. and Sakamoto, K. (2015) Cyclin Y phosphorylation- and 14-3-3-binding-dependent activation of PCTAIRE-1/CDK16. Biochemical Journal, 469(3), pp. 409-420. (doi: 10.1042/bj20150486) (PMID:26205494)

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 (Ser12 and Ser336) on cyclin Y that might be catalysed by PCTAIRE-1. Although in vitro peptide analysis favoured Ser12 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 Ser336, but not Ser12, as a PCTAIRE-1-dependent phosphorylation site. Monitoring phosphorylation of Ser336 may provide a useful read-out to assess cellular activity of PCTAIRE-1 in vivo; 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 (Ser100 and Ser326) 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-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.

• Item Type: Articles
• Status: Published
• Refereed: Yes
• Glasgow Author(s) Enlighten ID: Morrice, Dr Nicholas
• Authors: Shehata, S. N., Deak, M., Morrice, N. A., Ohta, E., Hunter, R. W., Kalscheuer, V. M., and Sakamoto, K.
• College/School: College of Medical Veterinary and Life Sciences > School of Cancer Sciences
• Journal Name: Biochemical Journal
• Publisher: Portland Press Ltd.
• ISSN: 0264-6021
• ISSN (Online): 1470-8728
• Copyright Holders: Copyright © 2015 The Authors
• First Published: First published in Biochemical Journal 469(3):409-420
• Publisher Policy: Reproduced under a Creative Commons License