The Ubiquitin-like Protein MNSFβ Regulates ERK-MAPK Cascade

Seiji Yamaguchi,Morihiko Nakamura

doi:10.1074/jbc.m509907200

Abstract

MNSFbeta is a ubiquitously expressed member of the ubiquitin-like family that has been implicated in various biological functions. Previous studies have demonstrated that MNSFbeta covalently binds to intracellular proapoptotic protein Bcl-G in mitogen-activated murine T cells. In this study, we further investigated the intracellular mechanism of action of MNSFbeta in macrophage cell line, Raw 264.7 cells. We present evidence that MNSFbeta.Bcl-G complex associates with ERKs in non-stimulated Raw 264.7. We found that MNSFbeta.Bcl-G directly bound to ERKs and inhibited ERK activation by MEK1. In Raw 264.7 cells treated with MNSFbeta small interfering RNA (siRNA) lipopolysaccharide (LPS)-induced ERK1/2 activation was enhanced and LPS-induced JNK and p38 activation was unaffected. SiRNA-mediated knockdown of MNSFbeta increased tumor necrosis factor alpha (TNFalpha) expression at mRNA and protein levels in LPS-stimulated Raw 264.7 cells. Finally, we found that transfection with MNSFbeta expression construct resulted in a significant inhibition of LPS-induced ERK activation and TNFalpha production. Co-transfection experiments with MNSFbeta and Bcl-G greatly enhanced this inhibition. Collectively, these findings indicate that MNSFbeta might be implicated in the macrophage response to LPS.

Highlights

Monoclonal nonspecific suppressor factor (MNSF), a lymphokine produced by murine T cell hybridoma, possesses pleiotrophic anti
We have shown that MNSF␤ covalently conjugates to acceptor proteins and forms MNSF␤ adducts including 33.5-kDa protein in concanavalin A- and IFN␥-stimulated D.10 cells [18]
MNSF␤ Complex Is Associated with ERKs—It has been reported that ubiquitin-like protein, ISG15, covalently binds to ERK1 [8]

Summary

Introduction

Monoclonal nonspecific suppressor factor (MNSF), a lymphokine produced by murine T cell hybridoma, possesses pleiotrophic anti-. We observed that MNSF␤ siRNA increased ERK activation and TNF␣ production by LPS-stimulated Raw 264.7 cells. In Raw macrophages as well as D.10 cells, anti-MNSF␤ antibody recognized several bands including a band of 33.5-kDa protein (Fig. 1A). Activated GST-MEK1 was incubated with unphosphorylated GST-ERK2 in the presence or absence of MNSF␤1⁄7Bcl-G as described under “Experimental Procedures.” The reaction mixture was immunoblotted with using anti-phospho-ERK1/2 antibody.

Results

Conclusion