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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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Analysis of S-P motifs on human cyclin Y(A) Diagram illustrating the domain organization of human cyclin Y with a global multiple sequence alignment of amino acid sequences surrounding the S-P motifs. 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 (changes underlined). Peptide concentration was varied over the range 0–100 μM and substrate saturation curves were fitted using non-linear regression to the Michaelis–Menten model. Fitted parameters [Km (μM) and Vmax (units·mg−1)] are listed in the adjoining table. Results are expressed as means±S.D. and are representative of three independent experiments.
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Figure 1: Analysis of S-P motifs on human cyclin Y(A) Diagram illustrating the domain organization of human cyclin Y with a global multiple sequence alignment of amino acid sequences surrounding the S-P motifs. 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 (changes underlined). Peptide concentration was varied over the range 0–100 μM and substrate saturation curves were fitted using non-linear regression to the Michaelis–Menten model. Fitted parameters [Km (μM) and Vmax (units·mg−1)] are listed in the adjoining table. Results are expressed as means±S.D. and are representative of three independent experiments.

Mentions: Our recent positional scanning peptide library analysis has revealed key substrate-specificity requirements of PCTAIRE-1 [6]. We found that although an absolute requirement for a proline residue immediately C-terminal to the phospho-acceptor site (+1) and preference for a basic residue at +2 are similar to other conventional CDKs, some elements (preference for a basic residue at +4, but not at +3) were unique to PCTAIRE-1. We established a preferred consensus sequence of S-P-K/R-ϕ-K/R/H (ϕ, represents a small aliphatic amino acid) and generated an optimal substrate peptide (PCTAIRE-tide, PKSPKARKKL) for robust measurements of recombinant and endogenous PCTAIRE-1 kinase activity in vitro [6]. Analysis of the protein sequence of human cyclin Y, a cognate binding partner of PCTAIRE-1 necessary for its activation [5,6], identified two proline-directed serine residues (S-P motif; Figure 1A). One of the S-P motifs (containing Ser336 in the human sequence) is well conserved among mammals but not in lower organisms such as D. melanogaster and C. elegans, whereas the other S-P motif (containing Ser12) is conserved in humans and mouse, as well as the majority of the lower organisms inspected. We noted that residues surrounding Ser12 (SPKLRRN) of human cyclin Y resemble the preferred consensus sequence for PCTAIRE-1 (Figure 1A). Thus we hypothesized that PCTAIRE-1 phosphorylates cyclin Y and that this modification influences their interaction and hence catalytic activity of PCTAIRE-1. To test this hypothesis, we initially generated synthetic peptides encompassing residues surrounding Ser12 (termed Ser12-tide) and Ser336 (plus an additional two arginine residues to enable efficient binding of the peptide to P81 paper; termed Ser336-tide) of human cyclin Y and determined in vitro substrate kinetics using recombinant PCTAIRE-1–cyclin Y complex isolated from COS-1 cells. Ser12-tide demonstrated a comparable Km to that of PCTAIRE-tide (6.2 μM compared with 7.6 μM), but displayed an approximately 4-fold lower Vmax (Figure 1B). In contrast, no detectable activity above background was observed for Ser336-tide, possibly due to absence of a preferred basic residue at +2. In support of this, alteration of +2 (alanine) and +4 (isoleucine) amino acids to lysine and arginine respectively in Ser336-tide to more resemble PCTAIRE-tide robustly increased PCTAIRE-1 activity. However, the Km of the modified Ser336-tide was still 10-fold higher than that of Ser12- or PCTAIRE-tide (Figure 1B). Taken together, in vitro peptide analysis suggests that Ser12, but not Ser336, is potentially a candidate for PCTAIRE-1-targeted phosphorylation.


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)

Analysis of S-P motifs on human cyclin Y(A) Diagram illustrating the domain organization of human cyclin Y with a global multiple sequence alignment of amino acid sequences surrounding the S-P motifs. 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 (changes underlined). Peptide concentration was varied over the range 0–100 μM and substrate saturation curves were fitted using non-linear regression to the Michaelis–Menten model. Fitted parameters [Km (μM) and Vmax (units·mg−1)] are listed in the adjoining table. Results are expressed as means±S.D. and are representative of three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4613515&req=5

Figure 1: Analysis of S-P motifs on human cyclin Y(A) Diagram illustrating the domain organization of human cyclin Y with a global multiple sequence alignment of amino acid sequences surrounding the S-P motifs. 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 (changes underlined). Peptide concentration was varied over the range 0–100 μM and substrate saturation curves were fitted using non-linear regression to the Michaelis–Menten model. Fitted parameters [Km (μM) and Vmax (units·mg−1)] are listed in the adjoining table. Results are expressed as means±S.D. and are representative of three independent experiments.
Mentions: Our recent positional scanning peptide library analysis has revealed key substrate-specificity requirements of PCTAIRE-1 [6]. We found that although an absolute requirement for a proline residue immediately C-terminal to the phospho-acceptor site (+1) and preference for a basic residue at +2 are similar to other conventional CDKs, some elements (preference for a basic residue at +4, but not at +3) were unique to PCTAIRE-1. We established a preferred consensus sequence of S-P-K/R-ϕ-K/R/H (ϕ, represents a small aliphatic amino acid) and generated an optimal substrate peptide (PCTAIRE-tide, PKSPKARKKL) for robust measurements of recombinant and endogenous PCTAIRE-1 kinase activity in vitro [6]. Analysis of the protein sequence of human cyclin Y, a cognate binding partner of PCTAIRE-1 necessary for its activation [5,6], identified two proline-directed serine residues (S-P motif; Figure 1A). One of the S-P motifs (containing Ser336 in the human sequence) is well conserved among mammals but not in lower organisms such as D. melanogaster and C. elegans, whereas the other S-P motif (containing Ser12) is conserved in humans and mouse, as well as the majority of the lower organisms inspected. We noted that residues surrounding Ser12 (SPKLRRN) of human cyclin Y resemble the preferred consensus sequence for PCTAIRE-1 (Figure 1A). Thus we hypothesized that PCTAIRE-1 phosphorylates cyclin Y and that this modification influences their interaction and hence catalytic activity of PCTAIRE-1. To test this hypothesis, we initially generated synthetic peptides encompassing residues surrounding Ser12 (termed Ser12-tide) and Ser336 (plus an additional two arginine residues to enable efficient binding of the peptide to P81 paper; termed Ser336-tide) of human cyclin Y and determined in vitro substrate kinetics using recombinant PCTAIRE-1–cyclin Y complex isolated from COS-1 cells. Ser12-tide demonstrated a comparable Km to that of PCTAIRE-tide (6.2 μM compared with 7.6 μM), but displayed an approximately 4-fold lower Vmax (Figure 1B). In contrast, no detectable activity above background was observed for Ser336-tide, possibly due to absence of a preferred basic residue at +2. In support of this, alteration of +2 (alanine) and +4 (isoleucine) amino acids to lysine and arginine respectively in Ser336-tide to more resemble PCTAIRE-tide robustly increased PCTAIRE-1 activity. However, the Km of the modified Ser336-tide was still 10-fold higher than that of Ser12- or PCTAIRE-tide (Figure 1B). Taken together, in vitro peptide analysis suggests that Ser12, but not Ser336, is potentially a candidate for PCTAIRE-1-targeted phosphorylation.

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