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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 phosphorylation of non-S/T-P and S/T-P motif sequences using Arg/Lys-scanning oriented peptide libraries.(a) Schematic representation of Arg/Lys-scanning oriented peptide libraries. See the lower part of the panel for explanation of X0, X1, X2, X3 and R′. Slashes indicate “or”. (b) Each peptide library used (shown only for the -2 to the +6 positions) was incubated with [Υ-32P]ATP and either ERK2 or Cdk1, and analyzed as described in the text and Methods. The radioactive signal of each library (32P) was quantitated and its value is shown just above the signal and in the bar diagram, with the value of XXSPXXXXX being set at 1.0 (mean ± SD, n = 3 for ERK2 and N = 4 for Cdk1). R corresponds to R′ (a mixture of Arg and Lys) in (a), while X is either X0, X1, X2 or X3 in (a), depending on its corresponding positions in (a).
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f2: In vitro phosphorylation of non-S/T-P and S/T-P motif sequences using Arg/Lys-scanning oriented peptide libraries.(a) Schematic representation of Arg/Lys-scanning oriented peptide libraries. See the lower part of the panel for explanation of X0, X1, X2, X3 and R′. Slashes indicate “or”. (b) Each peptide library used (shown only for the -2 to the +6 positions) was incubated with [Υ-32P]ATP and either ERK2 or Cdk1, and analyzed as described in the text and Methods. The radioactive signal of each library (32P) was quantitated and its value is shown just above the signal and in the bar diagram, with the value of XXSPXXXXX being set at 1.0 (mean ± SD, n = 3 for ERK2 and N = 4 for Cdk1). R corresponds to R′ (a mixture of Arg and Lys) in (a), while X is either X0, X1, X2 or X3 in (a), depending on its corresponding positions in (a).

Mentions: The above results indicated that one or more (consecutive) Arg/Lys residues in the +2 to +5 positions are important for Cdk1 phosphorylation of the known non-S/T-P motifs. To test more systematically for the position and number of C-terminal (consecutive) Arg/Lys residues that would be important for such phosphorylation, we used such Arg/Lys-scanning oriented peptide libraries as summarized in Fig. 2a. Each library contained an N-terminal biotin tag, a central Ser or Thr as the phosphoacceptor, and a second fixed amino acid(s) (Ala, Pro, or an equimolar mixture (R′) of Arg and Lys) in an appropriate position(s), particularly after the phosphoacceptor site; in all other positions (within the 13-amino acid window) was present a degenerate equimolar mixture of 17 (X0), 16 (X1), 15 (X2) or 14 (X3) amino acids, which are defined respectively in Fig. 2a. The actual (truncated) sequences of the peptide libraries used are shown in Fig. 2b, in which R corresponds to R′ (a mixture of Arg and Lys) in Fig. 2a, while X is either X0, X1, X2 or X3 in Fig. 2a, depending on its corresponding positions in Fig. 2a. These peptide libraries were incubated individually with cyclin B-Cdk1 and [Υ-32P] ATP in solution, transferred to an avidin-coated membrane, washed and imaged with a phosphoimager.


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 phosphorylation of non-S/T-P and S/T-P motif sequences using Arg/Lys-scanning oriented peptide libraries.(a) Schematic representation of Arg/Lys-scanning oriented peptide libraries. See the lower part of the panel for explanation of X0, X1, X2, X3 and R′. Slashes indicate “or”. (b) Each peptide library used (shown only for the -2 to the +6 positions) was incubated with [Υ-32P]ATP and either ERK2 or Cdk1, and analyzed as described in the text and Methods. The radioactive signal of each library (32P) was quantitated and its value is shown just above the signal and in the bar diagram, with the value of XXSPXXXXX being set at 1.0 (mean ± SD, n = 3 for ERK2 and N = 4 for Cdk1). R corresponds to R′ (a mixture of Arg and Lys) in (a), while X is either X0, X1, X2 or X3 in (a), depending on its corresponding positions in (a).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: In vitro phosphorylation of non-S/T-P and S/T-P motif sequences using Arg/Lys-scanning oriented peptide libraries.(a) Schematic representation of Arg/Lys-scanning oriented peptide libraries. See the lower part of the panel for explanation of X0, X1, X2, X3 and R′. Slashes indicate “or”. (b) Each peptide library used (shown only for the -2 to the +6 positions) was incubated with [Υ-32P]ATP and either ERK2 or Cdk1, and analyzed as described in the text and Methods. The radioactive signal of each library (32P) was quantitated and its value is shown just above the signal and in the bar diagram, with the value of XXSPXXXXX being set at 1.0 (mean ± SD, n = 3 for ERK2 and N = 4 for Cdk1). R corresponds to R′ (a mixture of Arg and Lys) in (a), while X is either X0, X1, X2 or X3 in (a), depending on its corresponding positions in (a).
Mentions: The above results indicated that one or more (consecutive) Arg/Lys residues in the +2 to +5 positions are important for Cdk1 phosphorylation of the known non-S/T-P motifs. To test more systematically for the position and number of C-terminal (consecutive) Arg/Lys residues that would be important for such phosphorylation, we used such Arg/Lys-scanning oriented peptide libraries as summarized in Fig. 2a. Each library contained an N-terminal biotin tag, a central Ser or Thr as the phosphoacceptor, and a second fixed amino acid(s) (Ala, Pro, or an equimolar mixture (R′) of Arg and Lys) in an appropriate position(s), particularly after the phosphoacceptor site; in all other positions (within the 13-amino acid window) was present a degenerate equimolar mixture of 17 (X0), 16 (X1), 15 (X2) or 14 (X3) amino acids, which are defined respectively in Fig. 2a. The actual (truncated) sequences of the peptide libraries used are shown in Fig. 2b, in which R corresponds to R′ (a mixture of Arg and Lys) in Fig. 2a, while X is either X0, X1, X2 or X3 in Fig. 2a, depending on its corresponding positions in Fig. 2a. These peptide libraries were incubated individually with cyclin B-Cdk1 and [Υ-32P] ATP in solution, transferred to an avidin-coated membrane, washed and imaged with a phosphoimager.

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