A small CDC25 dual-specificity tyrosine-phosphatase isoform in Arabidopsis thaliana

Abstract

The dual-specificity CDC25 phosphatases are critical positive regulators of cyclin-dependent kinases (CDKs). Even though an antagonistic Arabidopsis thaliana WEE1 kinase has been cloned and tyrosine phosphorylation of its CDKs has been demonstrated, no valid candidate for a CDC25 protein has been reported in higher plants. We identify a CDC25-related protein (Arath;CDC25) of A. thaliana, constituted by a sole catalytic domain. The protein has a tyrosine-phosphatase activity and stimulates the kinase activity of Arabidopsis CDKs. Its tertiary structure was obtained by NMR spectroscopy and confirms that Arath;CDC25 belongs structurally to the classical CDC25 superfamily with a central five-stranded beta-sheet surrounded by helices. A particular feature of the protein, however, is the presence of an additional zinc-binding loop in the C-terminal part. NMR mapping studies revealed the interaction with phosphorylated peptidic models derived from the conserved CDK loop containing the phosphothreonine-14 and phosphotyrosine-15. We conclude that despite sequence divergence, Arath;CDC25 is structurally and functionally an isoform of the CDC25 superfamily, which is conserved in yeast and in plants, including Arabidopsis and rice.

Fig. 1.

Sequence comparison of the Arath;CDC25 protein with CDC25, small CDC25-like tyrosine phosphatases, and the yeast arsenate reductase ACR2. Identical amino acids in all of the sequences are shown in white on a black background. Conserved amino acids are in bold. Secondary structure elements are indicated above the alignment for human CDC25A (PDB ID 1C25) and below for A. thaliana Arath;CDC25. The catalytic loop is indicated by the HC(X)₅R motif. CDC25ACDC25A_human, human CDC25A (accession number NP_001780); CDC25B_human, human CDC25B (NP_068658); CDC25C_human, human CDC25C (NP_073720); CDC25_pombe, Sch. pombe CDC25 (NP_013750); MIH1_cerevisiae, S. cerevisiae CDC25 (NP_013750); YGR203_cerevisiae, S. cerevisiae protein of unknown function (NP_011719); ACR2_cerevisiae, S. cerevisiae arsenate reductase (NP_015526); IBP1_pombe, Sch. pombe small CDC25-like protein (AL096796); Orysa;CDC25;1, O. sativa protein of unknown function (NP_922597); Arath;CDC25_Arabidop, this work (NP_568119).

Fig. 2.

Activation of the A. thaliana CDK kinase activity by the Arath;CDC25 protein. The Arabidopsis CDK complexes were partially purified on p10^CKS1At beads. The CDK-associated kinase activity was determined in the absence (lane 1) or presence of 4 (lane 2), 8 (lane 3), and 20 μg (lane 4) of Arath;CDC25 protein and 8 μg of Arath;CDC25 protein and 40 nM specific CDC25 inhibitor NSC95397 (lane 5). The graph illustrates the quantification of kinase activity by phosphorimager detection of the incorporation of radioactive phosphate by histone H1 normalized to the CDKA amount, control by Western blot with anti-PSTAIRE antibodies. Numbers on y axis are cpm × 10^-3.

Fig. 3.

Ribbon representation in the same orientation of the backbone of a representative conformer of the Arath;CDC25 protein (Left) and the human CDC25A (Right) (PDB entry 1C25, ref. 4). The conserved five-stranded β-sheet and α4 helices of the structures have been superposed as described in Results. The catalytic loop is indicated by the HC(X)₅R motif. The zinc ion is represented as a black sphere.

Fig. 4.

Map of the chemical-shift perturbation data on a surface and a ribbon representation in the same orientation of the Arath;CDC25 protein. Residues are colored according to the chemical-shift perturbation observed upon addition of an excess of pTyr¹⁵CDKA (Upper) or pThr¹⁴pTyr¹⁵CDKA (Lower) peptide. The color scale is as follows: yellow <0.04 ppm, orange between 0.04 and 0.1 ppm, and red >0.1 ppm combined ¹H-¹⁵N chemical-shift difference between the free and the peptide-bound form of the Arath;CDC25 protein. The zinc ion is not represented. On the left of the structure representations, overlaid sections of ¹H_N-¹⁵N-HSQC spectra acquired on Arath;CDC25 before (in red contours) and after (in blue contours) addition of 20 molar equivalent of pTyr¹⁵CDKA and pThr¹⁴pTyr¹⁵CDKA peptides.