Ankyrin Repeat and SOCS Box 3 (ASB3) Mediates Ubiquitination and Degradation of Tumor Necrosis Factor Receptor II

Abstract

Ankyrin repeat and SOCS box (ASB) family members have a C-terminal SOCS box and an N-terminal ankyrin-related sequence of variable repeats belonging to the SOCS superfamily. While SH2-domain-bearing SOCS proteins are mainly involved in the negative feedback regulation of the protein tyrosine kinase-STAT pathway in response to a variety of cytokines, the roles of ASB family members remain largely unknown. To investigate ASB functions, we screened for ASB3-interacting factors by using antibody array technology and identified tumor necrosis factor receptor II (TNF-R2) as an ASB3 binding target. ASB3 expression and activities are required for (i) TNF-R2 ubiquitination both in vivo and in vitro, (ii) TNF-R2 proteolysis via the proteasome pathway, and (iii) the inhibition of TNF-R2-mediated Jun N-terminal protein kinase (JNK) activation. While the ankyrin repeats of ASB3 interact with the C-terminal 37 amino acids of TNF-R2, the SOCS box of ASB3 is responsible for recruiting the E3 ubiquitin ligase adaptors Elongins-B/C, leading to TNF-R2 ubiquitination on multiple lysine residues within its C-terminal region. Downregulation of ASB3 expression by a small interfering RNA inhibited TNF-R2 degradation and potentiated TNF-R2-mediated cytotoxicity. The data presented here implicate ASB3 as a negative regulator of TNF-R2-mediated cellular responses to TNF-α by direct targeting of TNF-R2 for ubiquitination and proteasome-mediated degradation.

ACKNOWLEDGMENTS

We thank J. Singer and W. Min for discussions and for reading the manuscript. We thank F. K. Chan for the 4E3 cell line, Z. Q. Pan for HA-ubiquitin, J. W. Conaway for Elongin-C, and D. V. Goeddel for TRAF2 and TNF-R2 expression constructs.

This work was supported by an RO1 grant (CA-82549) from the National Institutes of Health (NIH) (to Y.E.C.) and by a COBRE grant from the NIH to Brown University. A.S.C. was supported by an NIH predoctoral training grant (GM-07601).