Suzuki K et al. (2015), Identification of non-Ser/Thr-Pro consensus mot...

XB-ART-53260

Sci Rep 2015 Jan 12;5:7929. doi: 10.1038/srep07929.

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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.

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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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Species referenced: Xenopus laevis
Genes referenced: cdk1 ect2 fbxo43 ikzf1 mapk1 plk1 sp1 vim yy1

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	Figure 1. 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.
	Figure 2. 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).
	Figure 3. 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.
	Figure 4. 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.
	Figure 5. 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.

References [+] :

Alexander, Spatial exclusivity combined with positive and negative selection of phosphorylation motifs is the basis for context-dependent mitotic signaling. 2011, Pubmed, Xenbase