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Identification of non-Ser/Thr-Pro consensus motifs for Cdk1 and their roles in mitotic regulation of C2H2 zinc finger proteins and Ect2.

Suzuki K, Sako K, Akiyama K, Isoda M, Senoo C, Nakajo N, Sagata N - Sci Rep (2015)

Bottom Line: Second, using Arg/Lys-scanning oriented peptide libraries, we demonstrate that Cdk1 phosphorylates a minimal sequence S/T-X-X-R/K and more favorable sequences (P)-X-S/T-X-[R/K](2-5) as its non-S/T-P consensus motifs.Third, on the basis of these results, we find that highly conserved linkers (typically, T-G-E-K-P) of C2H2 zinc finger proteins and a nuclear localization signal-containing sequence (matching P-X-S-X-[R/K]5) of the cytokinesis regulator Ect2 are inhibitorily phosphorylated by Cdk1, well accounting for the known mitotic regulation and function of the respective proteins.We suggest that non-S/T-P Cdk1 consensus motifs identified here may function to regulate many other proteins during mitosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Graduate School of Sciences, Kyushu University, Hakozaki 6-10-1, Fukuoka 812-8581, Japan.

ABSTRACT
The cyclin B-dependent protein kinase Cdk1 is a master regulator of mitosis and phosphorylates numerous proteins on the minimal consensus motif Ser/Thr-Pro (S/T-P). At least in several proteins, however, not well-defined motifs lacking a Pro in the +1 position, referred herein to as non-S/T-P motifs, have been shown to be phosphorylated by Cdk1. Here we show that non-S/T-P motifs in fact form consensus sequences for Cdk1 and probably play roles in mitotic regulation of physiologically important proteins. First, we show, by in vitro kinase assays, that previously identified non-S/T-P motifs all harbour one or more C-terminal Arg/Lys residues essential for their phosphorylation by Cdk1. Second, using Arg/Lys-scanning oriented peptide libraries, we demonstrate that Cdk1 phosphorylates a minimal sequence S/T-X-X-R/K and more favorable sequences (P)-X-S/T-X-[R/K](2-5) as its non-S/T-P consensus motifs. Third, on the basis of these results, we find that highly conserved linkers (typically, T-G-E-K-P) of C2H2 zinc finger proteins and a nuclear localization signal-containing sequence (matching P-X-S-X-[R/K]5) of the cytokinesis regulator Ect2 are inhibitorily phosphorylated by Cdk1, well accounting for the known mitotic regulation and function of the respective proteins. We suggest that non-S/T-P Cdk1 consensus motifs identified here may function to regulate many other proteins during mitosis.

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Cdk1 phosphorylation of the linkers of C2H2 zinc finger proteins.(a) Linker sequences of (human) Ikaros, Sp1 and YY1. For each linker, the phosphoacceptor Ser or Thr is shown in parentheses and printed in blue in the sequence, while the Arg/Lys in the +3 position in red. (b) GST-fused linker-spanning peptides (WT or a Ser/Thr → Ala mutant; for the residues of the peptides, see Methods) of the indicated zinc finger proteins were subjected to in vitro kinase assays by using [Υ-32P]ATP and either Cdk1, Plk1 or Aurora B. For each linker peptide of each protein, relative intensity of the radioactive signal (WT = 100) is shown at the bottom. (c)–(e) In vitro Cdk1 kinase assays of the linker-spanning peptides (WT or the indicated Ala-mutants) of Ikaros (c), Sp1 (d) and YY1 (e). (f) Flag/His6-tagged YY1 protein (WT or T348A) was incubated with either interphase (I) or M phase (M) extracts (from Xenopus eggs) in the presence or absence of roscovitine (Ros; 300 μM), BI2536 (BI; 15 μM) or ZM447439 (ZM; 30 μM), and then subjected to Flag-immunoprecipitation followed by immunoblotting with the indicated antibodies. In input, the arrowhead shows Flag/His6-tagged YY1(WT). Full scans of all autoradiographies and immunoblots are included in Supplementary Fig. S5.
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f4: Cdk1 phosphorylation of the linkers of C2H2 zinc finger proteins.(a) Linker sequences of (human) Ikaros, Sp1 and YY1. For each linker, the phosphoacceptor Ser or Thr is shown in parentheses and printed in blue in the sequence, while the Arg/Lys in the +3 position in red. (b) GST-fused linker-spanning peptides (WT or a Ser/Thr → Ala mutant; for the residues of the peptides, see Methods) of the indicated zinc finger proteins were subjected to in vitro kinase assays by using [Υ-32P]ATP and either Cdk1, Plk1 or Aurora B. For each linker peptide of each protein, relative intensity of the radioactive signal (WT = 100) is shown at the bottom. (c)–(e) In vitro Cdk1 kinase assays of the linker-spanning peptides (WT or the indicated Ala-mutants) of Ikaros (c), Sp1 (d) and YY1 (e). (f) Flag/His6-tagged YY1 protein (WT or T348A) was incubated with either interphase (I) or M phase (M) extracts (from Xenopus eggs) in the presence or absence of roscovitine (Ros; 300 μM), BI2536 (BI; 15 μM) or ZM447439 (ZM; 30 μM), and then subjected to Flag-immunoprecipitation followed by immunoblotting with the indicated antibodies. In input, the arrowhead shows Flag/His6-tagged YY1(WT). Full scans of all autoradiographies and immunoblots are included in Supplementary Fig. S5.

Mentions: Having identified non-S/T-P consensus sequences for Cdk1, we next addressed whether the minimal consensus sequence S/T-X-X-R/K would occur in any physiologically important proteins. While such a sequence can be phosphorylated by Cdk1 at least in several proteins, including Wee1 (ref. 15) and Boi1 (ref. 16) as well as vimentin and desmin (Fig. 1), a large number of similar sequences (S-A/G-X-R/K) have been shown to be phosphorylated by an unknown kinase(s) in human mitotic cells18. Specifically, short, highly-conserved linkers (matching or resembling T-G-E-K-P) of C2H2 zinc finger proteins, which include such proteins as Ikaros, Sp1 and YY1 and represent the largest family of transcription factors19, have been shown to be mitotically phosphorylated by an unknown kinase(s), thereby causing inactivation of the zinc fingers during mitosis202122. We therefore investigated whether the (multiple) linkers of Ikaros, Sp1 and YY1 (Fig. 4a) would be phosphorylated by Cdk1. For this, first we performed in vitro kinase assays of the respective linker-spanning peptides (WTs or Ser/Thr → Ala mutants, fused to GST), using [Υ-32P] ATP and either Cdk1 or other mitotic kinases, Plk1 and Aurora B, which are localized at kinetochores (potentially with C2H2 transcription factors) in prometaphase and metaphase210. Importantly, all of the phosphoacceptor Ser or Thr residues (seven in total) in the linkers of Ikaros/Sp1/YY1, except Thr378 in YY1, were phosphorylated by Cdk1, mostly if not very strongly, whereas none of them and only Ser196 in Ikaros were phosphorylated by Plk1 and Aurora B, respectively (Fig. 4b). Somewhat interestingly, Plk1 was able to (weakly) phosphorylate most of the Ikaros/Sp1/YY1 peptides at some other sites than their linker phosphoacceptors (Fig. 4b), although usually its activity requires priming phosphorylation of the substrates31. Thus, among the mitotic kinases tested, only Cdk1 can phosphorylate nearly all linkers of Ikaros/Sp1/YY1. Then we also asked whether an Arg/Lys in the +3 position of the respective linkers would be important for their phosphorylation by Cdk1. All the (seven) Arg/Lys residues in the linkers of Ikaros/Sp1/YY1, except Arg381 in YY1, were required for Cdk1 phosphorylation of the respective linkers (Fig. 4c–e), similar to, and nearly to the same extent as, their respective phosphoacceptor Ser/Thr residues (Fig. 4b–e). Furthermore, in the linker-spanning peptide (containing S196/K199) of Ikaros, Lys199 was most important, among the seven Ser196-surrounding residues tested, for Ser196 phosphorylation by Cdk1 (Supplementary Fig. S1a). Thus, in nearly all cases, the S/T-X-X-R/K motif (typically, T-G-E-K) in the linker can apparently serve as a Cdk1 phosphorylation site in vitro.


Identification of non-Ser/Thr-Pro consensus motifs for Cdk1 and their roles in mitotic regulation of C2H2 zinc finger proteins and Ect2.

Suzuki K, Sako K, Akiyama K, Isoda M, Senoo C, Nakajo N, Sagata N - Sci Rep (2015)

Cdk1 phosphorylation of the linkers of C2H2 zinc finger proteins.(a) Linker sequences of (human) Ikaros, Sp1 and YY1. For each linker, the phosphoacceptor Ser or Thr is shown in parentheses and printed in blue in the sequence, while the Arg/Lys in the +3 position in red. (b) GST-fused linker-spanning peptides (WT or a Ser/Thr → Ala mutant; for the residues of the peptides, see Methods) of the indicated zinc finger proteins were subjected to in vitro kinase assays by using [Υ-32P]ATP and either Cdk1, Plk1 or Aurora B. For each linker peptide of each protein, relative intensity of the radioactive signal (WT = 100) is shown at the bottom. (c)–(e) In vitro Cdk1 kinase assays of the linker-spanning peptides (WT or the indicated Ala-mutants) of Ikaros (c), Sp1 (d) and YY1 (e). (f) Flag/His6-tagged YY1 protein (WT or T348A) was incubated with either interphase (I) or M phase (M) extracts (from Xenopus eggs) in the presence or absence of roscovitine (Ros; 300 μM), BI2536 (BI; 15 μM) or ZM447439 (ZM; 30 μM), and then subjected to Flag-immunoprecipitation followed by immunoblotting with the indicated antibodies. In input, the arrowhead shows Flag/His6-tagged YY1(WT). Full scans of all autoradiographies and immunoblots are included in Supplementary Fig. S5.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4300507&req=5

f4: Cdk1 phosphorylation of the linkers of C2H2 zinc finger proteins.(a) Linker sequences of (human) Ikaros, Sp1 and YY1. For each linker, the phosphoacceptor Ser or Thr is shown in parentheses and printed in blue in the sequence, while the Arg/Lys in the +3 position in red. (b) GST-fused linker-spanning peptides (WT or a Ser/Thr → Ala mutant; for the residues of the peptides, see Methods) of the indicated zinc finger proteins were subjected to in vitro kinase assays by using [Υ-32P]ATP and either Cdk1, Plk1 or Aurora B. For each linker peptide of each protein, relative intensity of the radioactive signal (WT = 100) is shown at the bottom. (c)–(e) In vitro Cdk1 kinase assays of the linker-spanning peptides (WT or the indicated Ala-mutants) of Ikaros (c), Sp1 (d) and YY1 (e). (f) Flag/His6-tagged YY1 protein (WT or T348A) was incubated with either interphase (I) or M phase (M) extracts (from Xenopus eggs) in the presence or absence of roscovitine (Ros; 300 μM), BI2536 (BI; 15 μM) or ZM447439 (ZM; 30 μM), and then subjected to Flag-immunoprecipitation followed by immunoblotting with the indicated antibodies. In input, the arrowhead shows Flag/His6-tagged YY1(WT). Full scans of all autoradiographies and immunoblots are included in Supplementary Fig. S5.
Mentions: Having identified non-S/T-P consensus sequences for Cdk1, we next addressed whether the minimal consensus sequence S/T-X-X-R/K would occur in any physiologically important proteins. While such a sequence can be phosphorylated by Cdk1 at least in several proteins, including Wee1 (ref. 15) and Boi1 (ref. 16) as well as vimentin and desmin (Fig. 1), a large number of similar sequences (S-A/G-X-R/K) have been shown to be phosphorylated by an unknown kinase(s) in human mitotic cells18. Specifically, short, highly-conserved linkers (matching or resembling T-G-E-K-P) of C2H2 zinc finger proteins, which include such proteins as Ikaros, Sp1 and YY1 and represent the largest family of transcription factors19, have been shown to be mitotically phosphorylated by an unknown kinase(s), thereby causing inactivation of the zinc fingers during mitosis202122. We therefore investigated whether the (multiple) linkers of Ikaros, Sp1 and YY1 (Fig. 4a) would be phosphorylated by Cdk1. For this, first we performed in vitro kinase assays of the respective linker-spanning peptides (WTs or Ser/Thr → Ala mutants, fused to GST), using [Υ-32P] ATP and either Cdk1 or other mitotic kinases, Plk1 and Aurora B, which are localized at kinetochores (potentially with C2H2 transcription factors) in prometaphase and metaphase210. Importantly, all of the phosphoacceptor Ser or Thr residues (seven in total) in the linkers of Ikaros/Sp1/YY1, except Thr378 in YY1, were phosphorylated by Cdk1, mostly if not very strongly, whereas none of them and only Ser196 in Ikaros were phosphorylated by Plk1 and Aurora B, respectively (Fig. 4b). Somewhat interestingly, Plk1 was able to (weakly) phosphorylate most of the Ikaros/Sp1/YY1 peptides at some other sites than their linker phosphoacceptors (Fig. 4b), although usually its activity requires priming phosphorylation of the substrates31. Thus, among the mitotic kinases tested, only Cdk1 can phosphorylate nearly all linkers of Ikaros/Sp1/YY1. Then we also asked whether an Arg/Lys in the +3 position of the respective linkers would be important for their phosphorylation by Cdk1. All the (seven) Arg/Lys residues in the linkers of Ikaros/Sp1/YY1, except Arg381 in YY1, were required for Cdk1 phosphorylation of the respective linkers (Fig. 4c–e), similar to, and nearly to the same extent as, their respective phosphoacceptor Ser/Thr residues (Fig. 4b–e). Furthermore, in the linker-spanning peptide (containing S196/K199) of Ikaros, Lys199 was most important, among the seven Ser196-surrounding residues tested, for Ser196 phosphorylation by Cdk1 (Supplementary Fig. S1a). Thus, in nearly all cases, the S/T-X-X-R/K motif (typically, T-G-E-K) in the linker can apparently serve as a Cdk1 phosphorylation site in vitro.

Bottom Line: Second, using Arg/Lys-scanning oriented peptide libraries, we demonstrate that Cdk1 phosphorylates a minimal sequence S/T-X-X-R/K and more favorable sequences (P)-X-S/T-X-[R/K](2-5) as its non-S/T-P consensus motifs.Third, on the basis of these results, we find that highly conserved linkers (typically, T-G-E-K-P) of C2H2 zinc finger proteins and a nuclear localization signal-containing sequence (matching P-X-S-X-[R/K]5) of the cytokinesis regulator Ect2 are inhibitorily phosphorylated by Cdk1, well accounting for the known mitotic regulation and function of the respective proteins.We suggest that non-S/T-P Cdk1 consensus motifs identified here may function to regulate many other proteins during mitosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Graduate School of Sciences, Kyushu University, Hakozaki 6-10-1, Fukuoka 812-8581, Japan.

ABSTRACT
The cyclin B-dependent protein kinase Cdk1 is a master regulator of mitosis and phosphorylates numerous proteins on the minimal consensus motif Ser/Thr-Pro (S/T-P). At least in several proteins, however, not well-defined motifs lacking a Pro in the +1 position, referred herein to as non-S/T-P motifs, have been shown to be phosphorylated by Cdk1. Here we show that non-S/T-P motifs in fact form consensus sequences for Cdk1 and probably play roles in mitotic regulation of physiologically important proteins. First, we show, by in vitro kinase assays, that previously identified non-S/T-P motifs all harbour one or more C-terminal Arg/Lys residues essential for their phosphorylation by Cdk1. Second, using Arg/Lys-scanning oriented peptide libraries, we demonstrate that Cdk1 phosphorylates a minimal sequence S/T-X-X-R/K and more favorable sequences (P)-X-S/T-X-[R/K](2-5) as its non-S/T-P consensus motifs. Third, on the basis of these results, we find that highly conserved linkers (typically, T-G-E-K-P) of C2H2 zinc finger proteins and a nuclear localization signal-containing sequence (matching P-X-S-X-[R/K]5) of the cytokinesis regulator Ect2 are inhibitorily phosphorylated by Cdk1, well accounting for the known mitotic regulation and function of the respective proteins. We suggest that non-S/T-P Cdk1 consensus motifs identified here may function to regulate many other proteins during mitosis.

Show MeSH
Related in: MedlinePlus