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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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Activation of bacterially expressed PCTAIRE-1 by mammalian cyclin Y in complex with 14-3-3(A) PCTAIRE-1 (100 ng), expressed and purified from E. coli, was incubated with COS-1 lysates (200 μg) ectopically expressing the indicated HA–cyclin Y constructs and immunoprecipitated (IP) using anti-PCTAIRE-1 (G6.1) antibody. Then, 30% of the immunoprecipitated material was immunoblotted (IB) using the indicated antibodies and 10% was assayed for kinase activity. (B) E. coli-purified PCTAIRE-1 (1 μg) was incubated with 0.5 μg of Sf21-purified His–WT cyclin Y or S326A mutant and with 1 μg of E. coli-purified 14-3-3ζ, together or in isolation and assayed for kinase activity. (C) A 100 ng amount of each of four E. coli-purified GST–PCTAIRE-1 proteins [WT (full-length), 106–476 truncation, kinase-domain (165–446) and KI (D304A) mutant] were incubated with 0.25 μg of COS-1-purified FLAG–cyclin Y and assayed for kinase activity. Immunoblotting was performed using GST-tagged PCTAIRE-1 proteins and activity assay was performed using GST-cleaved PCTAIRE-1 proteins. (D) FLAG–cyclin Y was immunoprecipitated from 50 μg of transfected COS-1 lysates using FLAG–agarose and incubated with 1 mM ARAAS*APA/14-3-3-binding phosphopeptide (* indicates phosphoserine) for 20 min at room temperature. Non-phosphopeptide ARAASAPA was used as a negative control. After washing, 100 ng of PCTAIRE-1 and 0.5 μg of 14-3-3ζ (both expressed and purified from E. coli), were then added together or in isolation and incubated with the immunoprecipitated protein for a further 20 min at room temperature. Samples were washed and 30% was immunoblotted using the indicated antibodies and 20% was assayed for PCTAIRE-1 kinase activity. Results are expressed as means± S.D. and are representative of three independent experiments.
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Figure 5: Activation of bacterially expressed PCTAIRE-1 by mammalian cyclin Y in complex with 14-3-3(A) PCTAIRE-1 (100 ng), expressed and purified from E. coli, was incubated with COS-1 lysates (200 μg) ectopically expressing the indicated HA–cyclin Y constructs and immunoprecipitated (IP) using anti-PCTAIRE-1 (G6.1) antibody. Then, 30% of the immunoprecipitated material was immunoblotted (IB) using the indicated antibodies and 10% was assayed for kinase activity. (B) E. coli-purified PCTAIRE-1 (1 μg) was incubated with 0.5 μg of Sf21-purified His–WT cyclin Y or S326A mutant and with 1 μg of E. coli-purified 14-3-3ζ, together or in isolation and assayed for kinase activity. (C) A 100 ng amount of each of four E. coli-purified GST–PCTAIRE-1 proteins [WT (full-length), 106–476 truncation, kinase-domain (165–446) and KI (D304A) mutant] were incubated with 0.25 μg of COS-1-purified FLAG–cyclin Y and assayed for kinase activity. Immunoblotting was performed using GST-tagged PCTAIRE-1 proteins and activity assay was performed using GST-cleaved PCTAIRE-1 proteins. (D) FLAG–cyclin Y was immunoprecipitated from 50 μg of transfected COS-1 lysates using FLAG–agarose and incubated with 1 mM ARAAS*APA/14-3-3-binding phosphopeptide (* indicates phosphoserine) for 20 min at room temperature. Non-phosphopeptide ARAASAPA was used as a negative control. After washing, 100 ng of PCTAIRE-1 and 0.5 μg of 14-3-3ζ (both expressed and purified from E. coli), were then added together or in isolation and incubated with the immunoprecipitated protein for a further 20 min at room temperature. Samples were washed and 30% was immunoblotted using the indicated antibodies and 20% was assayed for PCTAIRE-1 kinase activity. Results are expressed as means± S.D. and are representative of three independent experiments.

Mentions: It has been reported that bacterially expressed PCTAIRE-1 (bPCTAIRE-1) is catalytically inactive [3]. 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) [5], 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.


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)

Activation of bacterially expressed PCTAIRE-1 by mammalian cyclin Y in complex with 14-3-3(A) PCTAIRE-1 (100 ng), expressed and purified from E. coli, was incubated with COS-1 lysates (200 μg) ectopically expressing the indicated HA–cyclin Y constructs and immunoprecipitated (IP) using anti-PCTAIRE-1 (G6.1) antibody. Then, 30% of the immunoprecipitated material was immunoblotted (IB) using the indicated antibodies and 10% was assayed for kinase activity. (B) E. coli-purified PCTAIRE-1 (1 μg) was incubated with 0.5 μg of Sf21-purified His–WT cyclin Y or S326A mutant and with 1 μg of E. coli-purified 14-3-3ζ, together or in isolation and assayed for kinase activity. (C) A 100 ng amount of each of four E. coli-purified GST–PCTAIRE-1 proteins [WT (full-length), 106–476 truncation, kinase-domain (165–446) and KI (D304A) mutant] were incubated with 0.25 μg of COS-1-purified FLAG–cyclin Y and assayed for kinase activity. Immunoblotting was performed using GST-tagged PCTAIRE-1 proteins and activity assay was performed using GST-cleaved PCTAIRE-1 proteins. (D) FLAG–cyclin Y was immunoprecipitated from 50 μg of transfected COS-1 lysates using FLAG–agarose and incubated with 1 mM ARAAS*APA/14-3-3-binding phosphopeptide (* indicates phosphoserine) for 20 min at room temperature. Non-phosphopeptide ARAASAPA was used as a negative control. After washing, 100 ng of PCTAIRE-1 and 0.5 μg of 14-3-3ζ (both expressed and purified from E. coli), were then added together or in isolation and incubated with the immunoprecipitated protein for a further 20 min at room temperature. Samples were washed and 30% was immunoblotted using the indicated antibodies and 20% was assayed for PCTAIRE-1 kinase activity. Results are expressed as means± S.D. and are representative of three independent experiments.
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Figure 5: Activation of bacterially expressed PCTAIRE-1 by mammalian cyclin Y in complex with 14-3-3(A) PCTAIRE-1 (100 ng), expressed and purified from E. coli, was incubated with COS-1 lysates (200 μg) ectopically expressing the indicated HA–cyclin Y constructs and immunoprecipitated (IP) using anti-PCTAIRE-1 (G6.1) antibody. Then, 30% of the immunoprecipitated material was immunoblotted (IB) using the indicated antibodies and 10% was assayed for kinase activity. (B) E. coli-purified PCTAIRE-1 (1 μg) was incubated with 0.5 μg of Sf21-purified His–WT cyclin Y or S326A mutant and with 1 μg of E. coli-purified 14-3-3ζ, together or in isolation and assayed for kinase activity. (C) A 100 ng amount of each of four E. coli-purified GST–PCTAIRE-1 proteins [WT (full-length), 106–476 truncation, kinase-domain (165–446) and KI (D304A) mutant] were incubated with 0.25 μg of COS-1-purified FLAG–cyclin Y and assayed for kinase activity. Immunoblotting was performed using GST-tagged PCTAIRE-1 proteins and activity assay was performed using GST-cleaved PCTAIRE-1 proteins. (D) FLAG–cyclin Y was immunoprecipitated from 50 μg of transfected COS-1 lysates using FLAG–agarose and incubated with 1 mM ARAAS*APA/14-3-3-binding phosphopeptide (* indicates phosphoserine) for 20 min at room temperature. Non-phosphopeptide ARAASAPA was used as a negative control. After washing, 100 ng of PCTAIRE-1 and 0.5 μg of 14-3-3ζ (both expressed and purified from E. coli), were then added together or in isolation and incubated with the immunoprecipitated protein for a further 20 min at room temperature. Samples were washed and 30% was immunoblotted using the indicated antibodies and 20% was assayed for PCTAIRE-1 kinase activity. Results are expressed as means± S.D. and are representative of three independent experiments.
Mentions: It has been reported that bacterially expressed PCTAIRE-1 (bPCTAIRE-1) is catalytically inactive [3]. 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) [5], 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.

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