Rlim, an E3 ubiquitin ligase, influences the stability of Stathmin protein in human osteosarcoma cells

Highlights

• We identify that Rlim could interact with Stathmin.

• Rlim induces ubiquitination of Stathmin and promotes degradation of Stathmin.

• Rlim is involved in the negative regulation of Stathmin protein level.

• Rlim-Stathmin interaction can turn over malignant phenotype in osteosarcomas cells.

Abstract

Stathmin is an oncoprotein and is expressed at high levels in a wide variety of human malignancies, which plays important roles in maintenance of malignant phenotypes. The regulation of Stathmin gene overexpression has been wildly explored, but the exact mechanism still needs to be elucidated. It is believed that regulation of an oncogene protein abundance through post-translational modifications is essential for maintenance of malignant phenotypes. Here we identified the Rlim, a Ring H2 zinc finger protein with intrinsic ubiquitin ligase activity, as a Stathmin-interacting protein that could increase Stathmin turnover through binding with this targeted protein and then induce its degradation by proteasome in a ubiquitin-dependent manner. Inhibition of endogenous Rlim expression by siRNA could increase the level of Stathmin protein, which further led to cell proliferation and cell cycle changes in human osteosarcoma cell lines. On the other hand, forced overexpression of Rlim could decrease the level of Stathmin protein. These results demonstrate that Rlim is involved in the negative regulation of Stathmin protein level through physical interaction and ubiquitin-mediated proteolysis. Hence, Rlim is a novel regulator of Stathmin protein in a ubiquitin-dependent manner, and represents a new pathway for malignant phenotype turnover by modulating the level of Stathmin protein in human osteosarcomas.

Introduction

Stathmin (STMN1), a 19 kDa cytosolic protein and also known as Op18, is a major phosphoprotein that plays crucial roles in the control of cell division and proliferation by regulating the dynamics of the microtubules [1], [2], [3]. Stathmin is also an oncoprotein and is expressed at high levels in a wide variety of human malignancies including osteosarcoma, lung cancer, uterine cervix cancer, bladder cancer, and breast cancer, which demonstrates that the overexpression of Stathmin may play an important role in maintenance of malignant phenotypes in malignant tumors [4], [5], [6], [7], [8]. Our previous studies on Stathmin have shown that Stathmin overexpression is associated with cellular proliferation, and inhibition of Stathmin expression can abolish the transformed phenotypes in malignant cells [9], [10], [11]. In addition, inhibition of Stathmin expression in malignant tumor cells interferes with their proliferation through the cell cycle arrest and abrogates their transformed phenotypes [12], [13], [14]. Thus, Stathmin provides an attractive molecular target for the treatment of malignant tumors.

In a search for proteins capable of interacting with Stathmin, we identified Rlim as a candidate of such proteins. Rlim (RING finger LIM domain-binding protein), encoded by the Rnf12 gene and located around 500 kb telomeric to the Xist gene on the X chromosome [15], [16], is originally identified as an E3 ubiquitin ligase. Rlim acts as a negative coregulator for LIM homeodomain transcription factors by mediating the ubiquitination and subsequent degradation of LIM cofactors LDB1 and LDB2 [17], [18], [19]. It has been reported that Rlim also plays a role in telomere length-mediated growth suppression by mediating the ubiquitination and degradation of TRF1, and acts as an activator of random inactivation of X chromosome in the embryo by targeting ZFP42 for degradation [20], [21]. Furthermore, study has shown that Rlim also enhances transcriptional activation of endogenous estrogen receptor alpha (ERalpha) target genes [22], [23]. Since signaling pathways controlled by ERalpha are profoundly implicated in mammary oncogenesis, Rlim may play an important role during the development of human cancers.

However, whether and how Rlim affects Stathmin protein levels and further influences the Stathmin functional roles during carcinogenesis have not been determined so far. In this study, we found that Rlim increased Stathmin turnover by binding it for degradation through the proteasome in a ubiquitin-dependent manner. Depletion of endogenous Rlim expression by siRNA stabilized Stathmin protein, whereas overexpression of Rlim promoted degradation of Stathmin and further impaired cell growth. In sum, these results demonstrated that Rlim was a critical negative regulator of Stathmin protein abundance and represented a new target for cancer therapy.