New Classes of Mind Bomb-Interacting Proteins Identified from Yeast Two-Hybrid Screens

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.

The E3 ubiquitin ligase Mib1 regulates Plk4 and centriole biogenesis

“…Recent proteomics and yeast two-hybrid studies have hinted at a centrosome-associated function for the E3 ubiquitin ligase Mib1 (Akimov et al, 2011;Jakobsen et al, 2011;Tseng et al, 2014). In line with these results, we found that Mib1 colocalizes with PCM-1 (Fig.…”

“…Such conditions are known to occur in breast cancer as well as other tumor types (Chng et al, 2008;Macmillan et al, 2001;Mason et al, 2014;van de Vijver et al, 2002). Specifically, we propose that SCF b-TrCP is the major E3 ligase controlling steady-state Plk4 levels under physiological conditions (Cunha-Ferreira et al, 2013; Cunha-Ferreira et al, , and the centriole maker Cep135 (blue).…”

“…One important mechanism for controlling Plk4 abundance is based on the ability of Plk4 to dimerize and trans-autophosphorylate, which results in the generation of a phosphodegron for recognition by Skp, Cullin, F-box containing complex coanatining b-TrCP (SCF b-TrCP ), an E3 ligase, and subsequent degradation of Plk4 by the proteasome (Cunha-Ferreira et al, 2013;Cunha-Ferreira et al, 2009;Guderian et al, 2010;Holland et al, 2012;Holland et al, 2010;Klebba et al, 2013;Sillibourne et al, 2010). Other regulatory mechanisms are likely to exist, and a full understanding of Plk4 regulation is important, particularly in view of the frequent deregulation of this kinase in human cancers (Chng et al, 2008;Macmillan et al, 2001;Mason et al, 2014;van de Vijver et al, 2002) and the impact of Plk4 mutations on brain development and body growth (Martin et al, 2014).…”

Sequential Elution Interactome Analysis of the Mind Bomb 1 Ubiquitin Ligase Reveals a Novel Role in Dendritic Spine Outgrowth

“…In our previous study (12), Choe et al presented evidence for Mib1 interaction with several other kinases in addition to proteins involved in membrane trafficking, the UPS, the cytoskeleton, and cell adhesion. These categories of Mib1 protein interaction were reiterated and elaborated upon by a yeast two-hybrid screen, revealing 81 putative binding partners for Mib1 and Mib2 (19). The present study reports an even larger list of potential Mib1 binding partners from adult rat brain, further expanding the Mib1 interaction network, reinforcing several pathways mentioned above, and introducing more components.…”

“…Mib1 was found to mediate the degradation of survival motor neuron 1 (SMN1), which contributes to spinal muscular atrophy (17). Mib1 was reported to be essential for Wnt3A activation of beta-catenin signaling through the receptor RYK (18), and a recent yeast two-hybrid screen indicated that Mib1 interacts with 81 candidate proteins beyond the canonical Notch pathway (19). The ongoing identification of new Mib1 interaction partners and functions underscores the need to characterize the Mib1 interactome en masse with high confidence.…”