Abstract
Insulin-like growth factor (IGF) is a potent mitogen that activates the IGF receptor (IGFR)/insulin receptor substrate (IRS) axis, thus stimulating growth in normal cells and uncontrolled cell proliferation in cancer. Posttranslational modifications of IRS such as ubiquitination tightly control IGF signaling, and we previously identified IRS-1 as a potential substrate for the E3 ubiquitin ligase TRAF4 using an unbiased screen. Here we provide evidence that TRAF4-mediated ubiquitination of IRS-1 is physiologically relevant and crucial for IGF signal transduction. Through site-directed mutagenesis we found that TRAF4 promotes an atypical K29-linked ubiquitination at the C-terminal end of IRS-1. Its depletion abolishes AKT and ERK phosphorylation downstream of IGF-1 and inhibits breast cancer cell proliferation. Overexpression of TRAF4 enhances IGF1-induced IGFR–IRS-1 interaction, IRS-1 tyrosine phosphorylation, and downstream effector protein activation, whereas mutation of IRS-1 ubiquitination sites completely abolishes these effects. Altogether, our studies demonstrate that nonproteolytic ubiquitination of IRS-1 is a key step in conveying IGF-1 stimulation from IGFR to IRS-1.
Highlights
There are six members of the insulin receptor substrate (IRS) family
We found that IRS-1 was among the top candidates whose ubiquitination levels significantly increased upon TRAF4 overexpression
Immunoprecipitation using an anti-myc antibody identified a direct association between IRS-1 and TRAF4 compared with cells expressing only FLAG-IRS-1 as a control (Fig. 1A)
Summary
The E3 ligase TRAF4 promotes IGF signaling by mediating atypical ubiquitination of IRS-1. Wenjuan Yu1,‡, Ramesh Singh2,‡, Zhao Wang1,2 , Bert W. O’Malley, and Ping Yi2,* From the 1Department of Biochemistry and Molecular Biology, 2Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas, USA
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