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  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., et al. (2015). Cyclin Y phosphorylation- and 14-3-3-binding-dependent activation of PCTAIRE-1/CDK16. Biochemical Journal, 469(3), 409-420. doi:10.1042/BJ20150486.

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Item Permalink: http://hdl.handle.net/11858/00-001M-0000-002A-60AC-2 Version Permalink: http://hdl.handle.net/11858/00-001M-0000-002A-60AD-F
Genre: Journal Article

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Shehata, S. N., Author
Deak, M., Author
Morrice, N. A., Author
Ohta, E., Author
Hunter, R. W., Author
Kalscheuer, V. M.1, Author              
Sakamoto, K., Author
Affiliations:
1Chromosome Rearrangements and Disease (Vera Kalscheuer), Research Group Development & Disease (Head: Stefan Mundlos), Max Planck Institute for Molecular Genetics, Max Planck Society, ou_1479642              

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Free keywords: 14-3-3 Proteins/genetics/*metabolism Amino Acid Motifs Amino Acid Sequence Cyclin-Dependent Kinases/genetics/*metabolism Cyclins/chemistry/genetics/*metabolism Enzyme Activation Humans Molecular Sequence Data Phosphorylation Protein Binding Sequence Alignment 14-3-3 cyclin-dependent kinase (CDK) intellectual disability mass spectrometry protein kinase
 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 in vitro 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 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 (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-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.

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Language(s): eng - English
 Dates: 2015-06-112015-08-01
 Publication Status: Published in print
 Pages: 12
 Publishing info: -
 Table of Contents: -
 Rev. Type: -
 Identifiers: DOI: 10.1042/BJ20150486
ISSN: 1470-8728 (Electronic)0264-6021 (Print)
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Title: Biochemical Journal
Source Genre: Journal
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Publ. Info: London : Published by Portland Press on behalf of the Biochemical Society.
Pages: - Volume / Issue: 469 (3) Sequence Number: - Start / End Page: 409 - 420 Identifier: ISSN: 0264-6021
CoNE: https://pure.mpg.de/cone/journals/resource/110992357308158