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.
Highlights
Notch signaling pathway is an evolutionarily conserved signal transduction cascade in flies, worms and vertebrates
In the first screen, where the reconstitution of the functional transcription factor of LexA (DNA binding domain)-bait fusion protein and prey-Gal4 fusion protein activates the HIS3 reporter gene, 33 positive clones of 13 genes were identified by Mind bomb (Mib)-C-RF123a (783–1029 aa) from 60 million colonies, and 232 positive clones of 26 genes were identified by Mind bomb 2 (Mib2)-C-RF12 (781–999 aa) from 52 million colonies (Table 1)
The results showed that ubiquitin specific protease 1 (Usp1) C, ubiquitin specific protease 9 (Usp9) and tumor-necrosisfactor-receptor-associated factor-binding domain (Trabid) C were co-immunoprecipitated with Mib2 (Figure 2A, lane 3; Figure 2B, lane 3; Figure 2C, lane 4), but ubiquitin specific protease 5 (Usp5) was not
Summary
Notch signaling pathway is an evolutionarily conserved signal transduction cascade in flies, worms and vertebrates. Through the Notch signaling pathway, signal-sending cells transfer a lateral inhibitory signal to the adjacent signal-receiving cells to control cell fate decision during development. It plays roles in cell proliferation, cell death and self-renewal of adult stem cells [1]. Notch signaling is activated by the interaction of DSL ligands (Delta and Serrate for Drosophila and Lag-2 for C. elegans) on the surface of signal-sending cells with the Notch receptor on signalreceiving cells. NICD forms a complex with CSL transcription factors (CBF1 for human; Suppressor of Hairless for Drosophila and Lag-1 for C. elegans) to regulate the expression of downstream genes [5,6,7,8]
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