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. 2010 Dec 7;107(49):20974-9.
doi: 10.1073/pnas.1007225107. Epub 2010 Nov 15.

Oxidation-induced intramolecular disulfide bond inactivates mitogen-activated protein kinase kinase 6 by inhibiting ATP binding

Yarui Diao  1 , Wei Liu, Catherine C L Wong, Xi Wang, Kaman Lee, Po-yan Cheung, Lifeng Pan, Tao Xu, Jiahuai Han, John R Yates 3rd, Mingjie Zhang, Zhenguo Wu
Affiliations

Oxidation-induced intramolecular disulfide bond inactivates mitogen-activated protein kinase kinase 6 by inhibiting ATP binding

Yarui Diao et al. Proc Natl Acad Sci U S A. .

Abstract

Mitogen-activated protein kinase kinase 6 (MKK6) is a member of the mitogen-activated protein kinase (MAPK) kinase (MAP2K) subfamily that specifically phosphorylates and activates the p38 MAPKs. Based on both biochemical and cellular assays, we found that MKK6 was extremely sensitive to oxidation: It was inactivated by oxidation and its kinase activity was fully restored upon treatment with a reducing agent. Detailed mechanistic studies showed that cysteines 109 and 196, two of the six cysteines in MKK6, formed an intramolecular disulfide bond upon oxidation that inactivated MKK6 by inhibiting its ATP binding. This mechanism is distinct from that seen in other redox-sensitive kinases. The two cysteines involved in intramolecular disulfide formation are conserved in all seven members of the MAP2K family. Consistently, we confirmed that other MAP2Ks were also sensitive to oxidation. Our work reveals that MKK6 and other MAP2Ks are a distinct class of cellular redox sensors.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
The kinase activity of MKK6 was sensitive to oxidation both in vitro and in cells. (A) The bacterially expressed human MKK6 with (lanes 1 and 2) or without (lanes 3 and 4) a Trx tag was purified and extensively dialyzed in the absence of any reducing agent. The purified MKK6 was subjected to in vitro kinase assays using GST-p38(KM) as a substrate in the presence or absence of 1 mM DTT. The phosphorylated p38 was detected by autoradiography. (B) The purified MKK6 with or without a Trx tag was electrophoresed on a SDS-polyacrylamide gel and visualized by Coomassie blue staining. (C and D) HeLa cells were either left untreated or treated with increasing doses of H2O2 as indicated. In C, cells were harvested after 5 min of H2O2 treatment. In D, cells were pretreated with H2O2 for 5 min followed by stimulation with 10 ng/mL TNFα for 10 min. (E) Serum-starved RAW264.7 cells were either left untreated or treated with 1 μM PMA for 30 min. The same amount of the endogenous MKK6 from C–E was immunoprecipitated from whole cell extracts (WCE) and subjected to in vitro kinase assays with GST-p38(KM) as a substrate. The phosphorylated p38 (p-p38) was revealed by Western blot analysis. Ten percent of total WCE used in the immunoprecipitation experiments in C–E was analyzed by Western blotting to reveal the amount of MKK6 as well as p-p38 and total p38 (D) in each sample.
Fig. 2.
Fig. 2.
Identification of cysteines involved in intramolecular disulfide bond formation in MKK6 oxidized in vitro. (A) Similar amount of purified WT MKK6, MKK6-4C (4C), MKK6-4C/C109S (4C/C109S), MKK6-4C/C196S (4C/C196S), and MKK6-6C (6C) were subjected to in vitro kinase assays with GST-p38(KM) as a substrate with or without 1 mM DTT (Upper). The phosphorylated p38 (p-p38) was revealed by Western blot analysis. The amount and purity of MKK6 proteins used in the above kinase assays were shown on a Coomassie blue-stained nonreducing gel (Lower). (B) Full MS spectra of the [M + 2H]2+ (m/z 1,769.84) and [M + 2H]3+ (m/z 1,180.23) ions of the disulfide-linked TVDC109PFTVTFYGALFR and MC196DFGISGYLVDSVAK.
Fig. 3.
Fig. 3.
Cys109 and Cys196 were involved in disulfide bond formation in MKK6 oxidized in mammalian cells. (A and B) HeLa cells were transiently transfected with plasmids encoding either the WT HA-MKK6 or HA-MKK6-4C (4C). Twenty-four hours after transfection, cells were either mock treated or treated with 200 μM H2O2 for 10 min before harvest. Cellular proteins were either labeled with 100 μM BIAM alone (A), or double labeled with IAA and BIAM (B). (C) Serum-starved RAW264.7 cells were either mock treated or treated with 1 µM PMA for 30 min. Cells were lysed in the buffer containing BIAM. Similar amount of HA-MKK6 from A and B or the endogenous MKK6 from C was then immunoprecipitated from WCE and the biotin-conjugated MKK6 (b-MKK6) was detected by HRP-streptavidin. Ten percent of total WCE used in the immunoprecipitation experiments in A–C was analyzed by Western blotting to reveal the levels of either the transfected or the endogenous MKK6. (D and E) MKK3/6 double knockout cells were transfected with either an empty vector or expression vectors encoding either the WT HA-MKK6, HA-MKK6-4C (4C), or HA-MKK6-6C (6C). Twenty-four hours after transfection, cells were either mock treated or treated with increasing does of H2O2 for 5 min, followed by treatment with sorbitol (0.4 M) for 10 min. Fifty micrograms of WCE were analyzed by Western blotting.
Fig. 4.
Fig. 4.
The oxidized MKK6 was defective in ATP binding. (A) The CD spectra for both reduced and oxidized MKK6 were shown. W/O, without. (B) GST (lanes 1 and 3) or GST-p38α (lanes 2 and 4) immobilized on glutathione-Sepharose beads were incubated with purified MKK6 with or without 1 mM DTT in a GST pull-down assay. The bound MKK6 was detected by Western blot analysis. (C) Equilibrium fluorescence anisotropy was measured with 30 nM BODIPY FL-AMPPNP and increasing amount of MKK6 in a binding buffer with or without 1 mM DTT. The binding curve was fitted with the MicroCal Origin software package.
Fig. 5.
Fig. 5.
The kinase activity of JNKK1 and MKK1 was also sensitive to oxidation. (A) The amino acid sequences (partial) of seven human MAP2Ks were aligned using the Clustal W program. Cysteines corresponding to Cys109 and Cys196 of MKK6 were highlighted in yellow. (B) The bacterially expressed JNKK1 (Left) and MKK1 (Right) were incubated with GST-JNK1 and GST-ERK2, respectively, in the in vitro kinase assays with or without 1 mM DTT. The phosphorylated JNK1 and ERK2 were detected by autoradiography.
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