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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 and inhibition of the NLS-containing non-S/T-P consensus sequence of Ect2.(a) Conservation of the NLS-containing non-S/T-P consensus sequence in Ect2 proteins from various species. (b) GST-fused peptides (WT or the indicated Ala-mutants; residues 334–363) of human Ect2 were subjected to in vitro Cdk1 kinase assays. In all constructs in this experiment, Thr342 preceding Ser345 (a) was replaced by Ala, because, reportedly, it can also be mitotically phosphorylated18, perhaps by Cdk1. RKRRR/5A, an NLS mutant. (c) Flag/His6-tagged Ect2-F constructs (WT or the indicated Ala-mutants; residues 328–388) were incubated with either interphase (I) or M phase (M) extracts (from Xenopus eggs), and then subjected to Flag-immunoprecipitation followed by λ phosphatase treatment and immunoblotting with the indicated antibodies. In the bar diagram, the levels of coimmunoprecipitated Importin β were quantitated and normalized to Ect2-F constructs; the relative values to Importin β coprecipitated with Ect2-F(WT) in interphase extracts are shown (mean ± SD, n = 4). KRR/3A, an NLS mutant. (d) Flag/His6-tagged Ect2-F constructs (WT or the indicated Asp-, Glu- or Ala-mutants) were incubated with interphase (I) extracts and analyzed as in (c) (mean ± SD, n = 4). (e) Flag/His6-tagged Ect2-F constructs (WT or the indicated Ala-mutant) were incubated with M phase extracts in the presence or absence of roscovitine (Ros; 300 μM) or RO-3306 (RO; 300 μM) and analyzed as in (c); in the bar diagram, the level of Importin β coimmunoprecipitated with Ect2-F(T342A/S345A) is set at 1.0 (mean ± SD, n = 3). Full scans of all autoradiographies and immunoblots are included in Supplementary Fig. S6.
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f5: Cdk1 phosphorylation and inhibition of the NLS-containing non-S/T-P consensus sequence of Ect2.(a) Conservation of the NLS-containing non-S/T-P consensus sequence in Ect2 proteins from various species. (b) GST-fused peptides (WT or the indicated Ala-mutants; residues 334–363) of human Ect2 were subjected to in vitro Cdk1 kinase assays. In all constructs in this experiment, Thr342 preceding Ser345 (a) was replaced by Ala, because, reportedly, it can also be mitotically phosphorylated18, perhaps by Cdk1. RKRRR/5A, an NLS mutant. (c) Flag/His6-tagged Ect2-F constructs (WT or the indicated Ala-mutants; residues 328–388) were incubated with either interphase (I) or M phase (M) extracts (from Xenopus eggs), and then subjected to Flag-immunoprecipitation followed by λ phosphatase treatment and immunoblotting with the indicated antibodies. In the bar diagram, the levels of coimmunoprecipitated Importin β were quantitated and normalized to Ect2-F constructs; the relative values to Importin β coprecipitated with Ect2-F(WT) in interphase extracts are shown (mean ± SD, n = 4). KRR/3A, an NLS mutant. (d) Flag/His6-tagged Ect2-F constructs (WT or the indicated Asp-, Glu- or Ala-mutants) were incubated with interphase (I) extracts and analyzed as in (c) (mean ± SD, n = 4). (e) Flag/His6-tagged Ect2-F constructs (WT or the indicated Ala-mutant) were incubated with M phase extracts in the presence or absence of roscovitine (Ros; 300 μM) or RO-3306 (RO; 300 μM) and analyzed as in (c); in the bar diagram, the level of Importin β coimmunoprecipitated with Ect2-F(T342A/S345A) is set at 1.0 (mean ± SD, n = 3). Full scans of all autoradiographies and immunoblots are included in Supplementary Fig. S6.

Mentions: We also examined whether Cdk1 would phosphorylate more favorable non-S/T-P consensus sequences (P)-X-S/T-X-[R/K]2–5 in any physiologically important proteins (as for myosin II; Fig. 1). According to the phosphoproteomic analysis of human cells18, mitotic phosphorylation of such preferred non-S/T-P sequences, although mediated by an unknown kinase(s), occurs in numerous proteins, of which about 20 sequences (or proteins) have four or more consecutive Arg/Lys residues after the Ser/Thr + 1 position. In at least five of these 20 sequences, which include Ect2 and Cullin 4B, the four or more consecutive Arg/Lys residues are suggested to function as a (classical) nuclear localization signal (NLS, typically consisting of four Arg/Lys residues; ref. 33)3435. Interestingly, the Cdk1 substrate Ect2, a key cytokinesis regulator23, has recently been shown to be exported from the nucleus at prophase in a Cdk1-dependent manner and, thereby, to play an essential role in mitotic cell rounding24. As phosphorylation of a Ser/Thr residue(s) near an NLS can inhibit NLS activity in certain proteins36, we suspected that Cdk1 phosphorylation of the well-conserved, NLS-containing non-S/T-P optimal sequence P-X-S-X-[R/K]5 in Ect2 (Figs 3g and 5a) might inhibit its NLS activity, thereby perhaps causing (or allowing) nuclear export of Ect2 at prophase for mitotic rounding. First, we confirmed, by in vitro kinase assays, that Ser345 in the P-X-S-X-[R/K]5 sequence of (human) Ect2 can be readily phosphorylated by Cdk1 (Fig. 5b). We also confirmed that the five consecutive Arg/Lys residues (or NLS) in the sequence are required for Cdk1 phosphorylation of Ser345 (Fig. 5b). Thus, clearly, Cdk1 can recognize and phosphorylate the NLS-containing non-S/T-P (optimal) consensus sequence P-X-S-X-[R/K]5 in Ect2. It should be noted, however, that, in these experiments, Thr342 followed by a Pro and preceding Ser345 (see Fig. 5a) was mutated to Ala, because, reportedly, it can also be mitotically phosphorylated, perhaps by Cdk1 (ref. 18; see also below).


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 and inhibition of the NLS-containing non-S/T-P consensus sequence of Ect2.(a) Conservation of the NLS-containing non-S/T-P consensus sequence in Ect2 proteins from various species. (b) GST-fused peptides (WT or the indicated Ala-mutants; residues 334–363) of human Ect2 were subjected to in vitro Cdk1 kinase assays. In all constructs in this experiment, Thr342 preceding Ser345 (a) was replaced by Ala, because, reportedly, it can also be mitotically phosphorylated18, perhaps by Cdk1. RKRRR/5A, an NLS mutant. (c) Flag/His6-tagged Ect2-F constructs (WT or the indicated Ala-mutants; residues 328–388) were incubated with either interphase (I) or M phase (M) extracts (from Xenopus eggs), and then subjected to Flag-immunoprecipitation followed by λ phosphatase treatment and immunoblotting with the indicated antibodies. In the bar diagram, the levels of coimmunoprecipitated Importin β were quantitated and normalized to Ect2-F constructs; the relative values to Importin β coprecipitated with Ect2-F(WT) in interphase extracts are shown (mean ± SD, n = 4). KRR/3A, an NLS mutant. (d) Flag/His6-tagged Ect2-F constructs (WT or the indicated Asp-, Glu- or Ala-mutants) were incubated with interphase (I) extracts and analyzed as in (c) (mean ± SD, n = 4). (e) Flag/His6-tagged Ect2-F constructs (WT or the indicated Ala-mutant) were incubated with M phase extracts in the presence or absence of roscovitine (Ros; 300 μM) or RO-3306 (RO; 300 μM) and analyzed as in (c); in the bar diagram, the level of Importin β coimmunoprecipitated with Ect2-F(T342A/S345A) is set at 1.0 (mean ± SD, n = 3). Full scans of all autoradiographies and immunoblots are included in Supplementary Fig. S6.
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Related In: Results  -  Collection

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

f5: Cdk1 phosphorylation and inhibition of the NLS-containing non-S/T-P consensus sequence of Ect2.(a) Conservation of the NLS-containing non-S/T-P consensus sequence in Ect2 proteins from various species. (b) GST-fused peptides (WT or the indicated Ala-mutants; residues 334–363) of human Ect2 were subjected to in vitro Cdk1 kinase assays. In all constructs in this experiment, Thr342 preceding Ser345 (a) was replaced by Ala, because, reportedly, it can also be mitotically phosphorylated18, perhaps by Cdk1. RKRRR/5A, an NLS mutant. (c) Flag/His6-tagged Ect2-F constructs (WT or the indicated Ala-mutants; residues 328–388) were incubated with either interphase (I) or M phase (M) extracts (from Xenopus eggs), and then subjected to Flag-immunoprecipitation followed by λ phosphatase treatment and immunoblotting with the indicated antibodies. In the bar diagram, the levels of coimmunoprecipitated Importin β were quantitated and normalized to Ect2-F constructs; the relative values to Importin β coprecipitated with Ect2-F(WT) in interphase extracts are shown (mean ± SD, n = 4). KRR/3A, an NLS mutant. (d) Flag/His6-tagged Ect2-F constructs (WT or the indicated Asp-, Glu- or Ala-mutants) were incubated with interphase (I) extracts and analyzed as in (c) (mean ± SD, n = 4). (e) Flag/His6-tagged Ect2-F constructs (WT or the indicated Ala-mutant) were incubated with M phase extracts in the presence or absence of roscovitine (Ros; 300 μM) or RO-3306 (RO; 300 μM) and analyzed as in (c); in the bar diagram, the level of Importin β coimmunoprecipitated with Ect2-F(T342A/S345A) is set at 1.0 (mean ± SD, n = 3). Full scans of all autoradiographies and immunoblots are included in Supplementary Fig. S6.
Mentions: We also examined whether Cdk1 would phosphorylate more favorable non-S/T-P consensus sequences (P)-X-S/T-X-[R/K]2–5 in any physiologically important proteins (as for myosin II; Fig. 1). According to the phosphoproteomic analysis of human cells18, mitotic phosphorylation of such preferred non-S/T-P sequences, although mediated by an unknown kinase(s), occurs in numerous proteins, of which about 20 sequences (or proteins) have four or more consecutive Arg/Lys residues after the Ser/Thr + 1 position. In at least five of these 20 sequences, which include Ect2 and Cullin 4B, the four or more consecutive Arg/Lys residues are suggested to function as a (classical) nuclear localization signal (NLS, typically consisting of four Arg/Lys residues; ref. 33)3435. Interestingly, the Cdk1 substrate Ect2, a key cytokinesis regulator23, has recently been shown to be exported from the nucleus at prophase in a Cdk1-dependent manner and, thereby, to play an essential role in mitotic cell rounding24. As phosphorylation of a Ser/Thr residue(s) near an NLS can inhibit NLS activity in certain proteins36, we suspected that Cdk1 phosphorylation of the well-conserved, NLS-containing non-S/T-P optimal sequence P-X-S-X-[R/K]5 in Ect2 (Figs 3g and 5a) might inhibit its NLS activity, thereby perhaps causing (or allowing) nuclear export of Ect2 at prophase for mitotic rounding. First, we confirmed, by in vitro kinase assays, that Ser345 in the P-X-S-X-[R/K]5 sequence of (human) Ect2 can be readily phosphorylated by Cdk1 (Fig. 5b). We also confirmed that the five consecutive Arg/Lys residues (or NLS) in the sequence are required for Cdk1 phosphorylation of Ser345 (Fig. 5b). Thus, clearly, Cdk1 can recognize and phosphorylate the NLS-containing non-S/T-P (optimal) consensus sequence P-X-S-X-[R/K]5 in Ect2. It should be noted, however, that, in these experiments, Thr342 followed by a Pro and preceding Ser345 (see Fig. 5a) was mutated to Ala, because, reportedly, it can also be mitotically phosphorylated, perhaps by Cdk1 (ref. 18; see also below).

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