Phosphorylation of FOXP3 by LCK Downregulates MMP9 Expression and Represses Cell Invasion

Kumiko Nakahira,Akihiro Morita,Nam-Soon Kim,Itaru Yanagihara,Laszlo Buday

doi:10.1371/journal.pone.0077099

Abstract

Forkhead Box P3 (FOXP3) is a member of the forkhead/winged helix family of the transcription factors and plays an important role not only as a master gene in T-regulatory cells, but also as a tumor suppressor. In this study, we identified lymphocyte-specific protein tyrosine kinase (LCK), which correlates with cancer malignancy, as a binding partner of FOXP3. FOXP3 downregulated LCK-induced MMP9, SKP2, and VEGF-A expression. We observed that LCK phosphorylated Tyr-342 of FOXP3 by immunoprecipitation and in vitro kinase assay, and the replacement of Tyr-342 with phenylalanine (Y342F) abolished the ability to suppress MMP9 expression. Although FOXP3 decreased the invasive ability induced by LCK in MCF-7 cells, Y342F mutation in FOXP3 diminished this suppressive effect. Thus we demonstrate for the first time that LCK upregulates FOXP3 by tyrosine phosphorylation, resulting in decreased MMP9, SKP2, and VEGF-A expression, and suppressed cellular invasion. We consider that further clarification of transcriptional mechanism of FOXP3 may facilitate the development of novel therapeutic approaches to suppress cancer malignancy.

Highlights

Forkhead box transcriptional factor families are involved in the network of post-translational modifications, including phosphorylation and protein–protein interactions, which provide an integrated cellular response to changes in the physiological status [1,2,3]
Forkhead box P3 (FOXP3) acts as a transcriptional repressor of oncogenes, HER-2/ErbB2 [10] and S-phase kinase-associated protein 2 (SKP2) [15], and FOXP3-regulated microRNAs suppress special AT-rich sequence-binding protein 1 [16], whereas deletions of FOXP3 exons extinguish those suppressive function in a breast cancer cell line [10]
We confirmed that MCF-7 cells expressed four FOXP3 isoforms lacking exon 3, 3-4, 3 and 8, and 3-4 and 8, supporting the idea that FOXP3 acts as a tumor suppressor

Summary

Introduction

Forkhead box transcriptional factor families are involved in the network of post-translational modifications, including phosphorylation and protein–protein interactions, which provide an integrated cellular response to changes in the physiological status [1,2,3]. FOXP3 interacts with other transcription factors, including a nuclear factor of activated T cells [7,8], a nuclear factor kappa-B (NFκB) [8], and an acute myeloid leukemia 1 [9], and blocks their ability to induce endogenous target gene expression, such as interleukin 2, interleukin 4, and interferon-gamma. Recent reports reveal that FOXP3 expression appears widespread in normal epithelia and aberrant in various solid tumors including breast cancer [10], ovarian cancer [11], prostate cancer [12], and pancreatic carcinoma [13] and cells lines of colon cancer [14]. Tumor suppression by FOXP3 has been investigated by many researchers, regulatory proteins that functionally modify FOXP3 are still unknown

Methods

Results

Conclusion