Function and subcellular location of Ro52β

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Abstract

Autoantigen Ro52α was recently identified as an E3 ubiquitin ligase. Its splicing variant Ro52β, which lacks a leucine zipper, has not been characterized yet. We therefore characterized Ro52β in contrast to Ro52α. Our biochemical assays revealed that both Ro52α and Ro52β function as E3 ubiquitin ligases and self-ubiquitinate in cooperation with UbcH5B in vitro. In addition, both Ro52α and Ro52β are ubiquitinated when overexpressed with ubiquitin in HEK293T cells, suggesting that both function as E3 ligases and self-ubiquitinate in vivo. However, cytological studies revealed that Ro52α mainly localizes to the cytoplasmic rod-like structures, whereas Ro52β diffusely localizes to both the cytoplasm and the nucleus. Since the leucine zipper plays a role in the homodimerization and heterodimerization of Ro52α, the dimerization might be required for the localization of Ro52α to the rod-like structures. On the basis of these results, Ro52α and Ro52β appear to ubiquitinate their particular substrates at different locations.

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Materials and methods

Cell culture. Human embryonic kidney HEK293 cells and HEK293T cells (American Type Culture Collection, Manassas, VA) were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum and antibiotics.

Antibodies. Mouse anti-FLAG antibody (M5) was purchased from Sigma Chemical Company (St. Louis, MO). Mouse anti-RH antibody (specific for the amino acid sequences RGSHHHH and GGSHHHH) was purchased from QIAGEN (Santa Clara, CA). Mouse anti-HA antibody (16B12) was purchased

Ro52β structurally lacks the leucine zipper

To compare human Ro52α and Ro52β, we first summarized their domain structure, which was previously reported [2]. As shown in Fig. 1, Ro52α possesses a RING finger and a B box at the N-terminal region. This RING-finger motif is required for the activity of E3 ubiquitin ligase [3]. In the central domain, Ro52α possesses two coiled coils, the second of which is a leucine zipper encoded in exon 4. Recently, Ro52α was shown to form homodimers through its leucine zipper [11]. At the C-terminus, Ro52α

Acknowledgment

This work was supported by National Institutes of Health Grant R01 DK56298 (to T.K.).

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