Extracellular Signal-regulated Kinase 1/2-mediated Phosphorylation of JunD and FosB Is Required for Okadaic Acid-induced Activator Protein 1 Activation

Sabine F Rosenberger,Joanne S Finch,Ashok Gupta,G Tim Bowden

doi:10.1074/jbc.274.2.1124

Abstract

Previously, we reported that in papilloma-producing 308 mouse keratinocytes, the tumor promoter okadaic acid, a serine-threonine phosphatase inhibitor, increased binding of activator protein 1 (AP-1) to a consensus 12-O-tetradecanoylphorbol-13-acetate-responsive element (Rosenberger, S. F., and Bowden, G. T. (1996) Oncogene 12, 2301-2308). In this study, we investigated the correlation between AP-1 DNA binding and transactivation and examined molecular mechanisms involved in this process. Using a luciferase reporter driven by region -74 to +63 of the human collagenase gene, we demonstrated induction of AP-1-mediated transcription following okadaic acid treatment. By performing in vitro kinase assays, we found elevated activities of extracellular signal-regulated kinase (ERK) 1/2, c-Jun N-terminal kinase, and p38 mitogen-activated protein kinase. The ERK-1/2-specific inhibitor PD 98059 completely abrogated okadaic acid-induced AP-1 transactivation without altering AP-1 expression, DNA binding, or complex composition. Phosphorylation analyses indicated that inhibition of ERK-1/2 decreased okadaic acid-elevated phosphorylation of JunD and FosB. To further examine the role of JunD and FosB in okadaic acid-induced AP-1 transactivation, we generated fusion proteins of the DNA-binding domain of the yeast transcription factor Gal4 and the transactivation domain of either JunD or FosB. Cotransfection experiments of these constructs with a Gal4-luciferase reporter demonstrated that both JunD and FosB are required for okadaic acid-induced transcription. Treatment with PD 98059 reduced JunD/FosB-dependent transactivation, suggesting that ERK-1/2-mediated phosphorylation is a critical component in this process.

Highlights

Ulating biological processes, such as proliferation, differentiation, and cell death, which are involved in expansion of “initiated” cells and tumor development
Okadaic Acid Increases activator protein 1 (AP-1) Transactivation—Previously, we reported that in papilloma-producing 308 mouse keratinocytes, okadaic acid increases AP-1 binding to a consensus 12O-tetradecanoylphorbol-13-acetate-responsive element [17]
To analyze whether this okadaic acid-induced increase in AP-1 DNA binding activity results in increased AP-1 transactivation, we transiently transfected 308 cells with a luciferase reporter construct driven by region Ϫ74 to ϩ63 of the human collagenase gene containing a tetradecanoylphorbol13-acetate-responsive element (TRE)

Summary

Introduction

Ulating biological processes, such as proliferation, differentiation, and cell death, which are involved in expansion of “initiated” cells and tumor development. Several lines of evidence indicate the transcription factor complex AP-1 1 as a critical component in epidermal tumor development in mouse skin. In this system, elevated AP-1 activity has been correlated with increased neoplastic transformation. AP-1 activity is regulated at various levels, including transcriptional and post-transcriptional mechanisms leading to increased AP-1 expression and post-translational modifications, such as phosphorylation and oxidation/reduction, altering DNA binding affinity and transactivation potential. Mitogen-activated protein kinases (MAPKs) have been discussed as important mediators of signal transduction regulating gene expression, and activation of these enzymes has been implicated in altered phosphorylation of several transcription factors, including Elk-1 [18], c-Jun [19], and ATF-2 [20]. JNKs/stress-activated protein kinases and p38 MAP kinases are considered part of stress response pathways mediating inhibition of proliferation or cell death [22]

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