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. 2001 Mar 15;15(6):699-709.
doi: 10.1101/gad.192601.

Mitogen-activated protein kinase phosphatase is required for genotoxic stress relief in Arabidopsis

R Ulm  1 , E Revenkova, G P di Sansebastiano, N Bechtold, J Paszkowski
Affiliations

Mitogen-activated protein kinase phosphatase is required for genotoxic stress relief in Arabidopsis

R Ulm et al. Genes Dev. .

Abstract

Genotoxic stress activates complex cellular responses allowing for the repair of DNA damage and proper cell recovery. Although plants are exposed constantly to increasing solar UV irradiation, the signaling cascades activated by genotoxic environments are largely unknown. We have identified an Arabidopsis mutant (mkp1) hypersensitive to genotoxic stress treatments (UV-C and methyl methanesulphonate) due to disruption of a gene that encodes an Arabidopsis homolog of mitogen-activated protein kinase phosphatase (AtMKP1). Growth of the mkp1 mutant under standard conditions is indistinguishable from wild type, indicating a stress-specific function of AtMKP1. MAP kinase phosphatases (MKPs), the potent inactivators of MAP kinases, are considered important regulators of MAP kinase signaling. Although biochemical data from mammalian cell cultures suggests an involvement of MKPs in cellular stress responses, there is no in vivo genetic support for this view in any multicellular organism. The genetic and biochemical data presented here imply a central role for a MAP kinase cascade in genotoxic stress signaling in plants and indicate AtMKP1 to be a crucial regulator of the MAP kinase activity in vivo, determining the outcome of the cellular reaction and the level of genotoxic resistance.

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Figures

Figure 1
Figure 1
Sensitivity of the mkp1 mutant to genotoxic stress and its complementation by ectopic expression of AtMKP1. (A) Five-day-old seedlings were transferred to liquid medium supplemented with methyl methanesulfonate (MMS) at the concentrations indicated. The picture was taken after 3 wk of further growth. (B) Root growth after UV-C irradiation of 5-day-old seedlings with 500 J/m2. Ws: Wassilewskija wild type; mkp1 mutant; line 6: mkp1 complemented by ectopic expression of AtMKP1.
Figure 2
Figure 2
Expression of the AtMKP1 gene and organization of the mkp1 locus. (A) RNA gel blot analysis of RNA isolated from 3-week-old seedlings. The blot was sequentially probed with AtMKP1 and 25S (Kiss et al. 1989) as a loading control. Ws: Wassilewskija wild type; mkp1 mutant; lines 6, 10, and 16: lines obtained by the transformation of the mkp1 mutant with the genomic fragment containing the AtMKP1 gene. (B) Structure of the AtMKP1 gene and position of the T-DNA insert. Exons are shown as gray boxes. In mkp1, the T-DNA is inserted in the second exon (T-DNA is not drawn to scale). Translational start ATG and the TAA stop codon are indicated. The depicted genomic clone was used for complementation of the mkp1 mutant phenotype in lines 6, 10, and 16.
Figure 2
Figure 2
Expression of the AtMKP1 gene and organization of the mkp1 locus. (A) RNA gel blot analysis of RNA isolated from 3-week-old seedlings. The blot was sequentially probed with AtMKP1 and 25S (Kiss et al. 1989) as a loading control. Ws: Wassilewskija wild type; mkp1 mutant; lines 6, 10, and 16: lines obtained by the transformation of the mkp1 mutant with the genomic fragment containing the AtMKP1 gene. (B) Structure of the AtMKP1 gene and position of the T-DNA insert. Exons are shown as gray boxes. In mkp1, the T-DNA is inserted in the second exon (T-DNA is not drawn to scale). Translational start ATG and the TAA stop codon are indicated. The depicted genomic clone was used for complementation of the mkp1 mutant phenotype in lines 6, 10, and 16.
Figure 3
Figure 3
Structural comparison and alignments of the AtMKP1 protein. (A) Protein sequence of AtMKP1. The extended catalytic site motif and the gelsolin homology region are underlined once and twice, respectively. (B) The structure of AtMKP1 and its homologs from maize (ZmMKP1), tomato (LeMKP1), and Medicago truncatula are shown in comparison to human (MKP-1), plant (AtDsPTP1), and yeast (MSG5) representatives of the MAP kinase phosphatase family. (C) Alignment of the dual-specificity phosphatase-specific extended catalytic site motif of AtMKP1 and proteins from plants, yeast, and mammals; Medicago truncatula EST316422, Xenopus laevis XCL100 (DDBJ/EMBL/GenBank accession no. X83742), human MKP-1 (accession no. X68277), Drosophila Puckered (PUC; accession no. AJ223360), Chlamydomonas eugametos VH-PTP13 (accession no. X77938), A. thaliana DsPTP1 (AtDsPTP1; accession no. Y18620), and Saccharomyces cerevisiae MSG5 (accession no. D17548). The position of the aspartic acid, cysteine, and serine residues implicated in the catalytic mechanism are shown in bold. (D) Alignment of the plant AtMKP1-related phosphatase-unique gelsolin homology sequences of AtMKP1, ZmMKP1, LeMKP1, and the M. truncatula EST316422 with the gelsolin family members; mouse villin (Mmvillin; accession no. M98454), mouse gelsolin (Mmgelsolin; accession no. J04953) and Drosophila flightless-I (Dmfli1; accession no. U01182). Dark shading indicates residues conserved in all entries; light shading shows amino acids identical to the AtMKP1 sequence.
Figure 4
Figure 4
MAP kinase activation by genotoxic stress treatment in Arabidopsis seedlings and AtMKP1-dependent activation level. (A) Immunoblot analysis with a p44/42 MAP kinase antibody (top) and a phospho-p44/42 MAP kinase antibody (bottom) detects phosphorylated MAP kinase orthologs after UV-C treatment (5 min after treatment with 3 kJ/m2). (B,C) Five-day-old seedlings of wild type (Ws), mkp1 mutant, and complemented line 6 were subjected to UV-C (B) or MMS (C) treatment and dose-dependent activation of a 49-kD kinase was detected in MBP in-gel kinase assays. Samples were taken after 5 min in the case of UV-C treatment and after 90 min in the case of MMS. On lower panels, protein loading controls are shown on 10% SDS-PAGE stained with Coomassie blue.
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