The ASB2¦Â Ubiquitin-interacting motif is involved in its monoubiquitination

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• 作者：Takehiro Nishiyama^a ; Sakiko Kuroda^a ; Eiko Takiguchi^a ; Takaaki Nakamura^a ; Keisuke Hashimoto^a ; Kenta Tsuzuranuki^a ; Takao Kawakami^b ; ^c ; Yasuhiko Masuho^a ; ^{masuho@rs.noda.tus.ac.jp} ; Junya Kohroki^a ; ¹
• 关键词：ASB2 ; ankyrin repeat-and SOCS box-containing protein 2 ; SOCS ; suppressor of cytokine signaling ; UIM ; Ubiquitin-interacting motif ; Ub ; ubiquitin ; polyUb- ; polyubiquitinated ; monoUb- ; monoubiquitinated ; amino acid ; aa ; UBD ; ubiquitin-binding domain ; SDS&nda
• 刊名：Biochemical and Biophysical Research Communications
• 出版年：2012
• 出版时间：13 April, 2012
• 年：2012
• 卷：420
• 期：3
• 页码：487-491
• 全文大小：421 K

文摘

ASB2 proteins are E3 ubiquitin (Ub) ligases that ubiquitinate filamins. There are two ASB2 splice variants, ASB2¦Á and ASB2¦Â. ASB2¦Â has a ubiquitin-binding motif (UIM) at the N-terminal region but ASB2¦Á does not. Here, we provide the first evidence that ASB2¦Â but not ASB2¦Á is monoubiquitinated and that this monoubiquitination involves the UIM. Myc-tagged ASB2¦Â and hemagglutinin (HA)-tagged Ub were co-expressed in HEK293 cells using the pCMV expression vector. Immunoprecipitation with an anti-Myc antibody followed by immunoblotting with anti-Myc and anti-HA antibodies showed an additional ASB2¦Â protein band that had both a Myc and a HA tag. The molecular weight of this protein was larger than that of ASB2¦Â, and the difference in molecular weight between these two proteins corresponded to the molecular weight of monoubiquitin, strongly implying that monoubiquitinated ASB2¦Â is produced in cells. ASB2¦Â with mutations in the UIM motif; either Glu¡¤Asp¡¤Glu27-29Ala¡¤Ala¡¤Ala mutations (ASB2¦Â M1) or a Ser38Ala mutation, (ASB2¦Â M2) were not monoubiquitinated, suggesting the importance of the UIM for ASB2¦Â monoubiquitination. Furthermore, an ASB2¦Â mutant that lacked a SOCS box (ASB2¦Â ¦¤C) and did not show E3 Ub ligase activity was monoubiquitinated to the same extent as the wild-type ASB2¦Â. In contrast, an ASB2¦Â mutant that lacked the UIM-containing domain (ASB2¦Â ¦¤N) was not monoubiquitinated. These results suggest that ASB2¦Â but not ASB2¦Á might be monoubiquitinated and that the ASB2¦Â UIM motif, but not its E3 Ub ligase activity, plays a pivotal role in this monoubiquitination.