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. 2014 Apr 8;9(4):e93394.
doi: 10.1371/journal.pone.0093394. eCollection 2014.

New classes of mind bomb-interacting proteins identified from yeast two-hybrid screens

Li-Chuan Tseng  1 , Chengjin Zhang  2 , Chun-Mei Cheng  1 , Haoying Xu  2 , Chia-Hao Hsu  3 , Yun-Jin Jiang  4
Affiliations

New classes of mind bomb-interacting proteins identified from yeast two-hybrid screens

Li-Chuan Tseng et al. PLoS One. .

Abstract

Notch signaling pathway defines an evolutionarily conserved mechanism in cell-fate determination in a broad spectrum of developmental processes through local cell interactions. mind bomb (mib) encodes an E3 ubiquitin ligase that is involved in Notch activation through Delta ubiquitylation and internalization. To further dissect the function of Mib, two yeast two-hybrid screens for zebrafish Mib/Mib2-binding proteins with different strategies have been performed. 81 putative interesting proteins were discovered and classified into six groups: ubiquitin proteasome pathway, cytoskeleton, trafficking, replication/transcription/translation factors, cell signaling and others. Confirmed by coimmunoprecipitation (Co-IP), Mib interacted with four tested proteins: ubiquitin specific protease 1 (Usp1), ubiquitin specific protease 9 (Usp9), tumor-necrosis-factor-receptor-associated factor (TRAF)-binding domain (Trabid)/zinc finger, RAN-binding domain containing 1 (Zranb1) and hypoxia-inducible factor 1, alpha subunit inhibitor (Hif1an)/factor inhibiting HIF 1 (Fih-1). Usp1, Usp9, Trabid and Fih-1 also bound to zebrafish Mib2, a Mib homolog with similar structural domains and functions. Both Mib and Mib2 can ubiquitylate Trabid and Fih-1, indicating a potential regulating role of Mib and Mib2 on Trabid and Fih-1 and, furthermore, the possible involvement of Notch signaling in hypoxia-regulated differentiation, tumorigenesis and NF-κB pathway. Finally, functions of confirmed Mib/Mib2-interacting proteins are collated, summarized and hypothesized, which depicts a regulating network beyond Notch signaling.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Structures of the bait proteins.
Domain structures of Mib and Mib2 baits. (A) Schematic structures of Mib and Mib2 proteins. (B) Domain drawings of used baits. Autoactivated baits are in gray.
Figure 2
Figure 2. Interaction of Mib and Mib2 with Usp1, Usp9 and Trabid.
Immunoprecipitation of (A) Usp1 C (349–772 aa), (B) Usp9 (full length) and (C) Trabid C (295–716 aa) by Mib and Mib2. COS7 cells were co-transfected with the expressing plasmids for HA- or Myc-tagged DUBs and FLAG-tagged Mib/Mib2. Immunoprecipitation (IP) were performed with anti-FLAG M2-Agarose and detected by Western blotting (IB) with anti-FLAG, anti-Myc or anti-HA antibody. Star indicates non-specific signals.
Figure 3
Figure 3. Interaction of Mib and Mib2 with Fih-1.
IP of Fih-1 by various Mibs and Mib2s. COS7 cells were co-transfected with pCS2-FLAG tag and pCS2-MT tag constructs. The cell lysate was incubated with anti-FLAG M2-Agarose and then subjected to IB with anti-Myc (top panel) or anti-FLAG antibody (bottom panel).
Figure 4
Figure 4. Both Mib and Mib2 promote Fih-1and Trabid ubiquitylation.
(A) Ubiquitylation of Fih-1, (B) Trabid C (295–716 aa) and (C) Trabid FL (full-length) by Mib or Mib2. COS7 cells were co-transfected with pcDNA3.1-HA-ubiquitin, pCS2-FLAG tag and pCS2-MT tag constructs. The cell lysate was incubated with anti-Myc antibody and then subjected to IB with anti-HA (top panel) or anti-Myc antibody (bottom panel).
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Grants and funding

This work was supported by the Agency of Science, Technology and Research (A*STAR, http://www.a-star.edu.sg), Singapore, the National Health Research Institutes (http://www.nhri.org.tw), Taiwan (MG-102-PP-13 and MG-102-PP-14) and grants from the National Science Council (http://web1.nsc.gov.tw), Taiwan (NSC 102-2321-B-400-018, 100-2911-I-400-002 and 100-2311-B-400-001-MY3). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.