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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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Identification of non-S/T-P consensus sequences for Cdk1.Data are taken from Fig. 2b (mean ± SD, n = 4) for comparison of the effects of the position of a single Arg/Lys residue (a) or two to five consecutive Arg/Lys residues (b)-(e), the number of (consecutive) Arg/Lys residues (f), the presence or absence of a -2 Pro (g), and the identity of the phosphoacceptor as Ser or Thr (h), on Cdk1 phosphorylation of the non-S/T-P sequences. In (g) and (h), two non-S/T-P or S/T-P sequences to be compared directly are vertically lined. In each panel, the phosphorylation level of XXSPXXXXX is also shown for comparison and is set at 1.0; note, however, that its scales are different from each other between (a), (b) and (c), (d)–(f) and (g) and (h). *P < 0.05; **P < 0.01.
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f3: Identification of non-S/T-P consensus sequences for Cdk1.Data are taken from Fig. 2b (mean ± SD, n = 4) for comparison of the effects of the position of a single Arg/Lys residue (a) or two to five consecutive Arg/Lys residues (b)-(e), the number of (consecutive) Arg/Lys residues (f), the presence or absence of a -2 Pro (g), and the identity of the phosphoacceptor as Ser or Thr (h), on Cdk1 phosphorylation of the non-S/T-P sequences. In (g) and (h), two non-S/T-P or S/T-P sequences to be compared directly are vertically lined. In each panel, the phosphorylation level of XXSPXXXXX is also shown for comparison and is set at 1.0; note, however, that its scales are different from each other between (a), (b) and (c), (d)–(f) and (g) and (h). *P < 0.05; **P < 0.01.

Mentions: From the data in Fig. 2b, we compared Cdk1 phosphorylations of the various Arg/Lys-containing non-S/T-P motifs by classifying them into several categories. When a single Arg/Lys was present in either of the five positions after the phosphoacceptor Ser, S-X-X-R/K, containing an Arg/Lys in the +3 position, was significantly most strongly phosphorylated, albeit to a much lesser extent than the conventional minimal consensus sequence S-P (Fig. 3a), suggesting that S-X-X-R/K may be the non-S/T-P minimal consensus sequence for Cdk1. As for two and three (consecutive) Arg/Lys residues, S-X-[R/K]2 and S-X-[R/K]3 were significantly much more strongly phosphorylated than the other [R/K]2- and [R/K]3-containing sequences, respectively, albeit still to a lesser extent than the conventional minimal consensus S-P (Fig. 3b, c), but much more strongly than S-X-X-R/K (see Figs 2b and 3a). These results indicate that, when the +1 position is not an Arg/Lys, a +2 Arg/Lys, but not a +4 or +5 Arg/Lys, greatly enhances the phosphorylation based on the +3 Arg/Lys. Furthermore, as for four and five consecutive Arg/Lys residues, S-X-[R/K]4 was a much better substrate than S-[R/K]4, as expected, and was somewhat better than even the conventional minimal sequence S-P (Fig. 3d), while S-X-[R/K]5 was much better than both S-[R/K]5 and S-P, being phosphorylated to the same extent as the conventional S/T-P optimal sequence S-P-X-R/K (Fig. 3e). These results and the data summarized in Fig. 3f clearly show that, when present consecutively (on a +2/+3 Arg/Lys-background), either of the Arg/Lys residues in the +4, +5 and +6 positions has a positive and additive effect (about 3-fold each, under the present experimental conditions) on the +2/+3 Arg/Lys-based phosphorylation, whereas an Arg/Lys in the +1 position has a negative (less than 0.5-fold) effect (compare SXRR with SRRR, SXRRR with SRRRR, and SXRRRR with SRRRRR in Fig. 3f).


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)

Identification of non-S/T-P consensus sequences for Cdk1.Data are taken from Fig. 2b (mean ± SD, n = 4) for comparison of the effects of the position of a single Arg/Lys residue (a) or two to five consecutive Arg/Lys residues (b)-(e), the number of (consecutive) Arg/Lys residues (f), the presence or absence of a -2 Pro (g), and the identity of the phosphoacceptor as Ser or Thr (h), on Cdk1 phosphorylation of the non-S/T-P sequences. In (g) and (h), two non-S/T-P or S/T-P sequences to be compared directly are vertically lined. In each panel, the phosphorylation level of XXSPXXXXX is also shown for comparison and is set at 1.0; note, however, that its scales are different from each other between (a), (b) and (c), (d)–(f) and (g) and (h). *P < 0.05; **P < 0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: Identification of non-S/T-P consensus sequences for Cdk1.Data are taken from Fig. 2b (mean ± SD, n = 4) for comparison of the effects of the position of a single Arg/Lys residue (a) or two to five consecutive Arg/Lys residues (b)-(e), the number of (consecutive) Arg/Lys residues (f), the presence or absence of a -2 Pro (g), and the identity of the phosphoacceptor as Ser or Thr (h), on Cdk1 phosphorylation of the non-S/T-P sequences. In (g) and (h), two non-S/T-P or S/T-P sequences to be compared directly are vertically lined. In each panel, the phosphorylation level of XXSPXXXXX is also shown for comparison and is set at 1.0; note, however, that its scales are different from each other between (a), (b) and (c), (d)–(f) and (g) and (h). *P < 0.05; **P < 0.01.
Mentions: From the data in Fig. 2b, we compared Cdk1 phosphorylations of the various Arg/Lys-containing non-S/T-P motifs by classifying them into several categories. When a single Arg/Lys was present in either of the five positions after the phosphoacceptor Ser, S-X-X-R/K, containing an Arg/Lys in the +3 position, was significantly most strongly phosphorylated, albeit to a much lesser extent than the conventional minimal consensus sequence S-P (Fig. 3a), suggesting that S-X-X-R/K may be the non-S/T-P minimal consensus sequence for Cdk1. As for two and three (consecutive) Arg/Lys residues, S-X-[R/K]2 and S-X-[R/K]3 were significantly much more strongly phosphorylated than the other [R/K]2- and [R/K]3-containing sequences, respectively, albeit still to a lesser extent than the conventional minimal consensus S-P (Fig. 3b, c), but much more strongly than S-X-X-R/K (see Figs 2b and 3a). These results indicate that, when the +1 position is not an Arg/Lys, a +2 Arg/Lys, but not a +4 or +5 Arg/Lys, greatly enhances the phosphorylation based on the +3 Arg/Lys. Furthermore, as for four and five consecutive Arg/Lys residues, S-X-[R/K]4 was a much better substrate than S-[R/K]4, as expected, and was somewhat better than even the conventional minimal sequence S-P (Fig. 3d), while S-X-[R/K]5 was much better than both S-[R/K]5 and S-P, being phosphorylated to the same extent as the conventional S/T-P optimal sequence S-P-X-R/K (Fig. 3e). These results and the data summarized in Fig. 3f clearly show that, when present consecutively (on a +2/+3 Arg/Lys-background), either of the Arg/Lys residues in the +4, +5 and +6 positions has a positive and additive effect (about 3-fold each, under the present experimental conditions) on the +2/+3 Arg/Lys-based phosphorylation, whereas an Arg/Lys in the +1 position has a negative (less than 0.5-fold) effect (compare SXRR with SRRR, SXRRR with SRRRR, and SXRRRR with SRRRRR in Fig. 3f).

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