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

Shehata SN, Deak M, Morrice NA, Ohta E, Hunter RW, Kalscheuer VM, Sakamoto K - Biochem. J. (2015)

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.

View Article: PubMed Central - PubMed

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.

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Disease-associated human PCTAIRE-1 variants abolish cyclin Y binding and kinase activity(A) Diagram showing relative start and end positions of human PCTAIRE-1 isoform 1 and 2 (Uni Prot accession numbers in parenthese) with respect to the kinase domain (area between vertical dashed lines). Relative positions of motifs required for an active kinase (VALK, HRD and DFG motifs) are indicated by vertical unbroken lines pointing downwards. Disease-associated variants in isoform 2 that lead to truncations are shown on the right. For both isoforms, amino acid residues of the kinase domain and total length are indicated by numbers. (B) Isoform 1 of FLAG–WT PCTAIRE-1 or KI (D304A) mutant, and isoform 2 of FLAG–WT PCTAIRE-1 or disease-associated variants (R488Ter and W400VfsVPAP) were co-expressed with HA–WT cyclin Y in COS-1 cells and total lysates were immunoblotted (IB) using the indicated antibodies. Lysates were immunoprecipitated (IP) using FLAG–agarose and either assayed for kinase activity or immunoblotted using the indicated antibodies. Additional increased amounts of disease-associated variants assayed or immunoblotted after immunoprecipitation are also indicated. Results are expressed as mean±S.D. and are representative of three independent experiments.
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Figure 6: Disease-associated human PCTAIRE-1 variants abolish cyclin Y binding and kinase activity(A) Diagram showing relative start and end positions of human PCTAIRE-1 isoform 1 and 2 (Uni Prot accession numbers in parenthese) with respect to the kinase domain (area between vertical dashed lines). Relative positions of motifs required for an active kinase (VALK, HRD and DFG motifs) are indicated by vertical unbroken lines pointing downwards. Disease-associated variants in isoform 2 that lead to truncations are shown on the right. For both isoforms, amino acid residues of the kinase domain and total length are indicated by numbers. (B) Isoform 1 of FLAG–WT PCTAIRE-1 or KI (D304A) mutant, and isoform 2 of FLAG–WT PCTAIRE-1 or disease-associated variants (R488Ter and W400VfsVPAP) were co-expressed with HA–WT cyclin Y in COS-1 cells and total lysates were immunoblotted (IB) using the indicated antibodies. Lysates were immunoprecipitated (IP) using FLAG–agarose and either assayed for kinase activity or immunoblotted using the indicated antibodies. Additional increased amounts of disease-associated variants assayed or immunoblotted after immunoprecipitation are also indicated. Results are expressed as mean±S.D. and are representative of three independent experiments.

Mentions: X-linked intellectual disability (XLID) is a clinically and genetically heterogeneous disorder with >100 genes identified so far. For some of these XLID genes, only very few families with pathogenic variants have so far been reported, suggesting that mutations in these genes are very rare. In our recent study of 405 families with XLID investigated by X chromosome exome sequencing, we identified a variant in CDK16/PCTAIRE-1 in a family with four affected males who all carry the variant and their mothers are heterozygous carriers [chrX:47086041-47086043, delTG, Q00536-2 (isoform 2): p.Trp400ValfsVPAP]. This result led us to propose that CDK16/PCTAIRE-1 is a novel candidate XLID gene [19]. An additional protein truncating CDK16/PCTAIRE-1 variant, classified as pathogenic or probably pathogenic (probably affecting function) has been identified in an unrelated clinical case [chrX:47086497C>T, Q00536-2 (isoform 2): p.Arg488Ter] (http://genetics.emory.edu/egl/emvclass/emvclass.php and ClinVar database (http://www.ncbi.nlm.nih.gov/clinvar/variation/166819/). Thus we investigated whether these variants could have an effect on cyclin Y–14-3-3 binding and also on PCTAIRE-1 enzymatic activity. For this purpose, we ectopically expressed both variants of human PCTAIRE-1 (FLAG–PCTAIRE-1 Trp400ValfsVPAP and FLAG–PCTAIRE-1 Arg488Ter) and its reference WT (isoform 2) together with WT cyclin Y (Figures 6A and 6B). Isoform 2 has a 74 amino acid extension at its N-terminus compared with isoform 1 [which has been used throughout the present study (except studies shown in Figure 6) and by others]. PCTAIRE-1 isoform 2 showed modestly lower expression compared with isoform 1, but displayed relatively comparable binding to cyclin Y–14-3-3 as well as activity to that of isoform 1. We observed both pathogenic variants consistently expressed much lower than their reference WT isoform 2 (Figure 6B), suggesting that they may be less stable than WT PCTAIRE-1 isoform 1. The variants were catalytically inactive {although they still contain key features of a functional kinase such as the VALK, HRD and HFD motifs [20] (Figure 6A)} and they displayed no or marginally detectable binding to cyclin Y, even when the amount of loading was matched to that of WT (isoform 2) (Figure 6B; Supplementary Figure S3).


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

Shehata SN, Deak M, Morrice NA, Ohta E, Hunter RW, Kalscheuer VM, Sakamoto K - Biochem. J. (2015)

Disease-associated human PCTAIRE-1 variants abolish cyclin Y binding and kinase activity(A) Diagram showing relative start and end positions of human PCTAIRE-1 isoform 1 and 2 (Uni Prot accession numbers in parenthese) with respect to the kinase domain (area between vertical dashed lines). Relative positions of motifs required for an active kinase (VALK, HRD and DFG motifs) are indicated by vertical unbroken lines pointing downwards. Disease-associated variants in isoform 2 that lead to truncations are shown on the right. For both isoforms, amino acid residues of the kinase domain and total length are indicated by numbers. (B) Isoform 1 of FLAG–WT PCTAIRE-1 or KI (D304A) mutant, and isoform 2 of FLAG–WT PCTAIRE-1 or disease-associated variants (R488Ter and W400VfsVPAP) were co-expressed with HA–WT cyclin Y in COS-1 cells and total lysates were immunoblotted (IB) using the indicated antibodies. Lysates were immunoprecipitated (IP) using FLAG–agarose and either assayed for kinase activity or immunoblotted using the indicated antibodies. Additional increased amounts of disease-associated variants assayed or immunoblotted after immunoprecipitation are also indicated. Results are expressed as mean±S.D. and are representative of three independent experiments.
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Figure 6: Disease-associated human PCTAIRE-1 variants abolish cyclin Y binding and kinase activity(A) Diagram showing relative start and end positions of human PCTAIRE-1 isoform 1 and 2 (Uni Prot accession numbers in parenthese) with respect to the kinase domain (area between vertical dashed lines). Relative positions of motifs required for an active kinase (VALK, HRD and DFG motifs) are indicated by vertical unbroken lines pointing downwards. Disease-associated variants in isoform 2 that lead to truncations are shown on the right. For both isoforms, amino acid residues of the kinase domain and total length are indicated by numbers. (B) Isoform 1 of FLAG–WT PCTAIRE-1 or KI (D304A) mutant, and isoform 2 of FLAG–WT PCTAIRE-1 or disease-associated variants (R488Ter and W400VfsVPAP) were co-expressed with HA–WT cyclin Y in COS-1 cells and total lysates were immunoblotted (IB) using the indicated antibodies. Lysates were immunoprecipitated (IP) using FLAG–agarose and either assayed for kinase activity or immunoblotted using the indicated antibodies. Additional increased amounts of disease-associated variants assayed or immunoblotted after immunoprecipitation are also indicated. Results are expressed as mean±S.D. and are representative of three independent experiments.
Mentions: X-linked intellectual disability (XLID) is a clinically and genetically heterogeneous disorder with >100 genes identified so far. For some of these XLID genes, only very few families with pathogenic variants have so far been reported, suggesting that mutations in these genes are very rare. In our recent study of 405 families with XLID investigated by X chromosome exome sequencing, we identified a variant in CDK16/PCTAIRE-1 in a family with four affected males who all carry the variant and their mothers are heterozygous carriers [chrX:47086041-47086043, delTG, Q00536-2 (isoform 2): p.Trp400ValfsVPAP]. This result led us to propose that CDK16/PCTAIRE-1 is a novel candidate XLID gene [19]. An additional protein truncating CDK16/PCTAIRE-1 variant, classified as pathogenic or probably pathogenic (probably affecting function) has been identified in an unrelated clinical case [chrX:47086497C>T, Q00536-2 (isoform 2): p.Arg488Ter] (http://genetics.emory.edu/egl/emvclass/emvclass.php and ClinVar database (http://www.ncbi.nlm.nih.gov/clinvar/variation/166819/). Thus we investigated whether these variants could have an effect on cyclin Y–14-3-3 binding and also on PCTAIRE-1 enzymatic activity. For this purpose, we ectopically expressed both variants of human PCTAIRE-1 (FLAG–PCTAIRE-1 Trp400ValfsVPAP and FLAG–PCTAIRE-1 Arg488Ter) and its reference WT (isoform 2) together with WT cyclin Y (Figures 6A and 6B). Isoform 2 has a 74 amino acid extension at its N-terminus compared with isoform 1 [which has been used throughout the present study (except studies shown in Figure 6) and by others]. PCTAIRE-1 isoform 2 showed modestly lower expression compared with isoform 1, but displayed relatively comparable binding to cyclin Y–14-3-3 as well as activity to that of isoform 1. We observed both pathogenic variants consistently expressed much lower than their reference WT isoform 2 (Figure 6B), suggesting that they may be less stable than WT PCTAIRE-1 isoform 1. The variants were catalytically inactive {although they still contain key features of a functional kinase such as the VALK, HRD and HFD motifs [20] (Figure 6A)} and they displayed no or marginally detectable binding to cyclin Y, even when the amount of loading was matched to that of WT (isoform 2) (Figure 6B; Supplementary Figure S3).

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.

View Article: PubMed Central - PubMed

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
Related in: MedlinePlus