Ras-ERK MAPK Cascade Regulates GATA3 Stability and Th2 Differentiation through Ubiquitin-Proteasome Pathway

Masakatsu Yamashita,Ryo Shinnakasu,Hikari Asou,Motoko Kimura,Akihiro Hasegawa,Kahoko Hashimoto,Naoya Hatano,Masato Ogata,Toshinori Nakayama

doi:10.1074/jbc.m502333200

Masakatsu Yamashita, Ryo Shinnakasu + Show 7 more

Open Access

https://doi.org/10.1074/jbc.m502333200

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Abstract

Differentiation of naive CD4 T cells into Th2 cells requires protein expression of GATA3. Interleukin-4 induces STAT6 activation and subsequent GATA3 transcription. Little is known, however, on how T cell receptor-mediated signaling regulates GATA3 and Th2 cell differentiation. Here we demonstrated that T cell receptor-mediated activation of the Ras-ERK MAPK cascade stabilizes GATA3 protein in developing Th2 cells through the inhibition of the ubiquitin-proteasome pathway. Mdm2 was associated with GATA3 and induced ubiquitination on GATA3, suggesting its role as a ubiquitin-protein isopeptide ligase for GATA3 ubiquitination. Thus, the Ras-ERK MAPK cascade controls GATA3 protein stability by a post-transcriptional mechanism and facilitates GATA3-mediated chromatin remodeling at Th2 cytokine gene loci leading to successful Th2 cell differentiation.

Highlights

Differentiation of naive CD4 T cells into Th2 cells requires protein expression of GATA3
The Ras-ERK MAPK Cascade Controls Histone Hyperacetylation of the Th2Ϫ Cytokine Gene Loci—We reported that Th2 cell differentiation and certain Th2 responses are dependent on the extent of activation of the Ras-ERK MAPK cascade [11, 13]
We provide evidence indicating that TCRmediated activation of the Ras-ERK MAPK cascade controls GATA3 protein stability through the ubiquitin-proteasome pathway

Summary

The abbreviations used are

IFN␥, interferon-␥; dn, dominant-negative; ERK, extracellular signal-regulated kinase; Erk sem., an active form of ERK2; KO, knock out (deficient); Tg, transgenic; Ub, ubiquitination; WT, wild type; TCR, T cell receptor; STAT, signal transducer and activator of transcription; IL, interleukin; PMA, phorbol 12-myristate 13-acetate; mAb, monoclonal antibody; ChIP, chromatin immunoprecipitation; MAPK, mitogen-activated protein kinase; MEK, MAPK/ ERK kinase; siRNA, small interfering RNA; CHX, cycloheximide; GFP, green fluorescent protein; EGFP, enhanced GFP; hNGFR, human nerve growth factor receptor p75; FCS, fetal calf serum; E3, ubiquitin-protein isopeptide ligase. Th2 cell differentiation is accompanied by chromatin remodeling of the Th2 cytokine (IL-4/IL-5/IL-13) gene loci, e.g. hyperacetylation of histones H3 and H4 [23,24,25]. The GATA3-response element appears to play a crucial role for GATA3-mediated targeting and downstream spreading of core histone hyperacetylation within the IL-13 and IL-4 gene loci in developing CD4ϩ Th2 and CD8ϩ Tc2 cells [23, 27]. A well known example of the ubiquitin-dependent regulation in the immune system is the proteasome-dependent processing of peptides in antigen-presenting cells [33]. We investigated the molecular targets of the Ras-ERK MAPK cascade that control chromatin remodeling of the Th2 cytokine gene loci and subsequent Th2 cell differentiation, and we found that the Ras-ERK MAPK cascade controls the stability of the GATA3 protein through the ubiquitin-proteasome pathway. We demonstrated that the ubiquitination of GATA3 by Mdm is dependent on a ring finger domain

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION