Abstract
Mitogen-activated protein kinases (MAPKs) are important mediators that integrate signaling from upstream pathways in response to various environmental cues. In order to control appropriate gene expression through phosphorylation of transcription factors, the activity of MAPKs must be tightly regulated by the actions coordinated between protein kinases and phosphatases. In this study, we explore the underlying mechanism through which the oxidative stress-activated c-Jun N-terminal kinases (JNKs), members of MAPKs, are regulated by dual specificity phosphatases (DUSPs). DUSPs are a group of enzymes that belong to the superfamily of protein-tyrosine phosphatases. They are able to recognize phospho-Ser/Thr and phospho-Tyr residues in substrates. Using quantitative real time PCR, we found that stimulation of human embryonic kidney 293T cells with H(2)O(2) or xanthine/xanthine oxidase led to inducible expression of multiple DUSPs. We used RNA interference to characterize the functional role of these DUSPs and found rapid and transient induction of DUSP1 and DUSP10 to be essential for determining the appropriate magnitude of JNK activation in response to oxidative stress. The transcription factor ATF2, which is phosphorylated and activated by JNK, is a critical mediator for inducible expression of DUSP1 and DUSP10 in this signaling pathway. We further demonstrated that DUSP4 and DUSP16, both showing significant late phase induction, dephosphorylate JNK effectively, causing the down-regulation of the signaling cascade. Thus, this study provides new insights into the role of several DUSPs that coordinate with each other to control the magnitude and duration of JNK activity in response to oxidative stress.
Highlights
Modules is composed of a highly conserved cascade of three kinases, in which mitogen-activated protein kinases (MAPKs)3 are the primary component [1]
De Novo mRNA and Protein Synthesis Are Involved in Downregulating Oxidative Stress-induced Jun N-terminal kinases (JNKs) Signaling in 293T Cells—In this study, we wanted to identify endogenous dual specificity phosphatases (DUSPs) involved in the regulation of MAPK signaling in response to oxidative stress
A recent comprehensive survey demonstrated that there are 107 protein-tyrosine phosphatases (PTPs) genes in the human genome [14]. Of these PTP genes, 81 that is consistent with the known function of these phospha- genes encode active forms of phosphatases [14], comparable with tases in controlling JNK activation at an early stage in the sig- 85 genes encoding catalytically active forms of protein-tyrosine naling response to oxidative stress
Summary
Cell Culture and Transient Transfection with Small Interfering RNA—Human embryonic kidney 293T cells were routinely maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and 1% glutamine. For transient transfection with small interfering RNA (siRNA) oligonucleotides, cells were plated in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum for 16 h. Cells were continuously incubated in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum for 8 h, followed by serum deprivation for an additional 16 h prior to stimulation with H2O2 or X/XO. The cDNA was synthesized from total RNA with Transcriptor reverse transcriptase (Roche Applied Science) using oligo(dT) primers according to the manufacturer’s instructions. The primer sequences for the transcription factors p53, c-Jun, and ATF2 as well as a housekeeping gene, hydroxymethylbilane synthase (HMBS), were as follows: p53 forward, 5Ј-ATGTGTAACAGTTCCTGC-3Ј; p53 reverse, 5Ј-GCTCGCTTAGTGCTCC-3Ј; 28396 JOURNAL OF BIOLOGICAL CHEMISTRY.
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