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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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In vitro Cdk1 kinase assays of known non-S/T-P motif sequences.(a) Non-S/T-P motif sequences of known Cdk1 substrate proteins. For each substrate protein, the phosphoacceptor Ser or Thr in its non-S/T-P motif is shown in parentheses and printed in blue in the sequence, while the Arg or Lys residue in red is a residue important for Cdk1 phosphorylation of the non-S/T-P motif, as revealed below in (c-g). Desmin, myosin II and Emi2 are from Xenopus, while vimentin and GFAP are from human. (b) GST-fused peptides (WT or a Ser/Thr → Ala mutant; for the residues of the peptides, see Methods) of the indicated substrate proteins were incubated with cyclin B1-Cdk1 and [Υ-32P]ATP, subjected to SDS-PAGE, and stained with Coomassie brilliant blue (CBB) or autoradiographed (32P). For each GST-fused peptide of each substrate protein, relative intensity of the radioactive signal (WT = 100) is shown at the bottom. (c)–(g) GST-fused peptides (WT or the indicated Ala-mutants) of vimentin (c), desmin (d), myosin II (e), GFAP (f) and Emi2 (g) were subjected to in vitro Cdk1 kinase assays and analyzed as in (b). Full scans of all autoradiographies are included in Supplementary Fig. S4.
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f1: In vitro Cdk1 kinase assays of known non-S/T-P motif sequences.(a) Non-S/T-P motif sequences of known Cdk1 substrate proteins. For each substrate protein, the phosphoacceptor Ser or Thr in its non-S/T-P motif is shown in parentheses and printed in blue in the sequence, while the Arg or Lys residue in red is a residue important for Cdk1 phosphorylation of the non-S/T-P motif, as revealed below in (c-g). Desmin, myosin II and Emi2 are from Xenopus, while vimentin and GFAP are from human. (b) GST-fused peptides (WT or a Ser/Thr → Ala mutant; for the residues of the peptides, see Methods) of the indicated substrate proteins were incubated with cyclin B1-Cdk1 and [Υ-32P]ATP, subjected to SDS-PAGE, and stained with Coomassie brilliant blue (CBB) or autoradiographed (32P). For each GST-fused peptide of each substrate protein, relative intensity of the radioactive signal (WT = 100) is shown at the bottom. (c)–(g) GST-fused peptides (WT or the indicated Ala-mutants) of vimentin (c), desmin (d), myosin II (e), GFAP (f) and Emi2 (g) were subjected to in vitro Cdk1 kinase assays and analyzed as in (b). Full scans of all autoradiographies are included in Supplementary Fig. S4.

Mentions: More than two decades ago, three intermediate filament proteins (vimentin, desmin and GFAP) and myosin II (regulatory light chain) were shown to be phosphorylated by Cdk1 on their N-terminal non-S/T-P motifs1112131425. More recently, we showed that Emi2, an inhibitor of the APC/C, is phosphorylated on a C-terminal non-S/T-P motif to be inactivated in Xenopus eggs17. To determine whether the non-S/T-P motifs in these proteins (Fig. 1a) make any consensus sequence for phosphorylation by Cdk1, we performed in vitro Cdk1 kinase assays of the respective non-S/T-P motif-spanning peptides (WT or Ala-mutants, fused to glutathione S-transferase or GST) in the presence of [Υ-32P]ATP. Initially, we confirmed that all of the phosphoacceptor Ser or Thr residues in the non-S/T-P motifs tested can be phosphorylated by Cdk1, albeit to different degrees from each other (Fig. 1b). We then examined the requirement of the individual Ser/Thr-surrounding residues of the respective non-S/T-P motifs for their phosphorylation by Cdk1. Notably, an Arg in the +3 position in both vimentin (Fig. 1c) and desmin (Fig. 1d), three Arg/Lys residues in the +2, +3 and +4 positions in myosin II (Fig. 1e), and two Arg/Lys residues in the +4 and +5 positions in Emi2 (Fig. 1g) were all more important for the phosphorylation than the other tested Ser/Thr-surrounding residues of the respective non-S/T-P motifs, while, in GFAP, only two Arg residues in the +3 and +4 positions were important for the phosphorylation (Fig. 1f). Thus, in all the proteins tested, an Arg or Lys residue(s) C-terminal to the phosphoacceptor Ser/Thr is important for Cdk1 phosphorylation of the non-S/T-P motif, although, in most cases, the majority of the other Ser/Thr-surrounding residues tested are also required to some extent. In a previous study26, an Arg in the +3 position of the synthetic vimentin non-S/T-P motif peptide, although followed by two artificially introduced Arg residues, was also shown to be important for its phosphorylation by Cdk1.


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)

In vitro Cdk1 kinase assays of known non-S/T-P motif sequences.(a) Non-S/T-P motif sequences of known Cdk1 substrate proteins. For each substrate protein, the phosphoacceptor Ser or Thr in its non-S/T-P motif is shown in parentheses and printed in blue in the sequence, while the Arg or Lys residue in red is a residue important for Cdk1 phosphorylation of the non-S/T-P motif, as revealed below in (c-g). Desmin, myosin II and Emi2 are from Xenopus, while vimentin and GFAP are from human. (b) GST-fused peptides (WT or a Ser/Thr → Ala mutant; for the residues of the peptides, see Methods) of the indicated substrate proteins were incubated with cyclin B1-Cdk1 and [Υ-32P]ATP, subjected to SDS-PAGE, and stained with Coomassie brilliant blue (CBB) or autoradiographed (32P). For each GST-fused peptide of each substrate protein, relative intensity of the radioactive signal (WT = 100) is shown at the bottom. (c)–(g) GST-fused peptides (WT or the indicated Ala-mutants) of vimentin (c), desmin (d), myosin II (e), GFAP (f) and Emi2 (g) were subjected to in vitro Cdk1 kinase assays and analyzed as in (b). Full scans of all autoradiographies are included in Supplementary Fig. S4.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: In vitro Cdk1 kinase assays of known non-S/T-P motif sequences.(a) Non-S/T-P motif sequences of known Cdk1 substrate proteins. For each substrate protein, the phosphoacceptor Ser or Thr in its non-S/T-P motif is shown in parentheses and printed in blue in the sequence, while the Arg or Lys residue in red is a residue important for Cdk1 phosphorylation of the non-S/T-P motif, as revealed below in (c-g). Desmin, myosin II and Emi2 are from Xenopus, while vimentin and GFAP are from human. (b) GST-fused peptides (WT or a Ser/Thr → Ala mutant; for the residues of the peptides, see Methods) of the indicated substrate proteins were incubated with cyclin B1-Cdk1 and [Υ-32P]ATP, subjected to SDS-PAGE, and stained with Coomassie brilliant blue (CBB) or autoradiographed (32P). For each GST-fused peptide of each substrate protein, relative intensity of the radioactive signal (WT = 100) is shown at the bottom. (c)–(g) GST-fused peptides (WT or the indicated Ala-mutants) of vimentin (c), desmin (d), myosin II (e), GFAP (f) and Emi2 (g) were subjected to in vitro Cdk1 kinase assays and analyzed as in (b). Full scans of all autoradiographies are included in Supplementary Fig. S4.
Mentions: More than two decades ago, three intermediate filament proteins (vimentin, desmin and GFAP) and myosin II (regulatory light chain) were shown to be phosphorylated by Cdk1 on their N-terminal non-S/T-P motifs1112131425. More recently, we showed that Emi2, an inhibitor of the APC/C, is phosphorylated on a C-terminal non-S/T-P motif to be inactivated in Xenopus eggs17. To determine whether the non-S/T-P motifs in these proteins (Fig. 1a) make any consensus sequence for phosphorylation by Cdk1, we performed in vitro Cdk1 kinase assays of the respective non-S/T-P motif-spanning peptides (WT or Ala-mutants, fused to glutathione S-transferase or GST) in the presence of [Υ-32P]ATP. Initially, we confirmed that all of the phosphoacceptor Ser or Thr residues in the non-S/T-P motifs tested can be phosphorylated by Cdk1, albeit to different degrees from each other (Fig. 1b). We then examined the requirement of the individual Ser/Thr-surrounding residues of the respective non-S/T-P motifs for their phosphorylation by Cdk1. Notably, an Arg in the +3 position in both vimentin (Fig. 1c) and desmin (Fig. 1d), three Arg/Lys residues in the +2, +3 and +4 positions in myosin II (Fig. 1e), and two Arg/Lys residues in the +4 and +5 positions in Emi2 (Fig. 1g) were all more important for the phosphorylation than the other tested Ser/Thr-surrounding residues of the respective non-S/T-P motifs, while, in GFAP, only two Arg residues in the +3 and +4 positions were important for the phosphorylation (Fig. 1f). Thus, in all the proteins tested, an Arg or Lys residue(s) C-terminal to the phosphoacceptor Ser/Thr is important for Cdk1 phosphorylation of the non-S/T-P motif, although, in most cases, the majority of the other Ser/Thr-surrounding residues tested are also required to some extent. In a previous study26, an Arg in the +3 position of the synthetic vimentin non-S/T-P motif peptide, although followed by two artificially introduced Arg residues, was also shown to be important for its phosphorylation by Cdk1.

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