Abstract
The interleukin (IL)-6-type cytokines play major roles in a variety of biological processes by signaling through a common receptor subunit, glycoprotein (gp) 130. We performed yeast two-hybrid screening to identify new binding partners of the activated gp130 and the associated Janus kinases. LMO4, a LIM domain-containing protein that belongs to a family of oncogenes, was identified in this assay. Further studies show that LMO4 associates with gp130 and Janus kinase1 in several mammalian cell types. It also interacts with protein-tyrosine phosphatase 2 (SHP2) and suppressor of cytokine signaling 3 (SOCS3). The binding domains involved in these interactions were mapped, and the interactions were shown to be in a direct manner by in vitro binding assays. It is likely that LMO4 exists in the gp130 complex. The cellular localization of LMO4 was detected primarily in the nucleus with a substantial amount also detected in the cytoplasm in several cell types. The effect of LMO4 in IL-6 signaling was subsequently examined. Overexpression of LMO4 enhanced the transcriptional activity and target gene expression of Stat 3 (signal transducers and activators of transcription 3). Consistent with this, silencing LMO4 expression in stable cell lines expressing the small interfering RNA of LMO4 decreased Stat3 activity. Furthermore, the half-life of gp130 was shortened, and the production of acute phase proteins induced by IL-6 was reduced. Together, our data reveal a positive regulatory role of LMO4 in IL-6 signaling, possibly by acting as a scaffold for stabilization of the gp130 complex. These studies may open up a link between the oncogenic effect of LMO proteins and their regulatory role in cytokine signaling in general.
Highlights
The interleukin (IL1)-6-type cytokines belong to a cytokine family that plays major roles in hematopoiesis, immune re
We found that LMO4 coimmunoprecipitated with gp130-JH1 fusion protein as well as gp130 or JH1 alone but not with the vector control (Fig. 1A), indicating LMO4 may interact with both gp130 and Jak2
We further examined whether LMO4 interacted with the fulllength Jak2 as well as Jak1, because gp130 binds to Jak1, Jak2, and Tyk2, Jak1 activity is predominant in IL-6 signaling [26, 27]
Summary
The interleukin (IL1)-6-type cytokines belong to a cytokine family that plays major roles in hematopoiesis, immune re-. We used the human hepatoma cell line HepG2 that expresses gp130 and Jak1 in relatively high levels to overexpress HA-tagged LMO4 to observe its interaction with the endogenous gp130 and Jak1 under either unstimulated conditions or stimulated with IL-6.
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