(F) Multiple CK2 motifs increase substrate affinity for KBTBD8

(F) Multiple CK2 motifs increase substrate affinity for KBTBD8. multisite dependency of an E3 ligase provides a powerful mechanism for switch-like cell fate transitions controlled by monoubiquitylation. account for?~90% of cases of the craniofacial disease Treacher Collins Syndrome (The Treacher Collins Syndrome Colla, 1996; Jones et al., 2008), while additional transcription factor binding sites in the promoter of drive melanoma, a cancer of neural crest origin (Hayward et al., 2017). At later stages Oxymatrine (Matrine N-oxide) of craniofacial development, CUL3 pairs up with a distinct adaptor, KLHL12, to monoubiquitylate a Oxymatrine (Matrine N-oxide) COPII vesicle coat protein and accelerate collagen secretion (Jin et al., 2012; McGourty et al., 2016), and mutations in this pathway lead to the craniofacial disorder cranio-lenticulo-sutural dysplasia (Boyadjiev et al., 2006). Together, these findings revealed critical roles of monoubiquitylation in cell differentiation and implied that tight regulation of CUL3 is essential for human development. Despite its importance for CYFIP1 neural crest specification, mechanisms that ensure accurate CUL3KBTBD8 activation and function are very poorly understood. While CUL3KBTBD8 is essential for establishing neural crest cells, it is not required for the maintenance of pluripotent stem cells (Werner et al., 2015). This suggested that CUL3KBTBD8 engages its targets at specific stages of differentiation, yet how it recognizes its substrates at the right time and place is not known. How monoubiquitylation by CUL3KBTBD8 helps TCOF1 and NOLC1 bind each other is also unclear: while monoubiquitylation often recruits effector proteins to a modified target (Dikic et al., 2009; Yau and Rape, 2016), no ubiquitin-binding domains have been identified in TCOF1, NOLC1, or their known binding partners. Indeed, rather than being organized into structural domains that engage in distinct interactions, TCOF1 and NOLC1 contain large stretches of acidic residues that are predicted to be of low structural complexity (Lee et al., 2013). How monoubiquitylation of an intrinsically disordered protein can precipitate a switch-like transition in cellular state is an open question. Here, we show Oxymatrine (Matrine N-oxide) Oxymatrine (Matrine N-oxide) that CUL3KBTBD8-dependent monoubiquitylation and neural crest specification require multisite substrate phosphorylation by CK2, a kinase whose levels gradually increase during development of the nervous system (Mestres et al., 1994). The essential CUL3KBTBD8-substrates TCOF1 and NOLC1 contain 10 or more motifs that, following their phosphorylation by CK2, can be independently recognized by a conserved surface on KBTBD8. We found that multiple CK2 motifs need to be phosphorylated in the same substrate to mediate both monoubiquitylation by CUL3KBTBD8 as well as neural crest specification. Multisite dependency allows cells to convert a gradual increase in kinase input, as seen for embryonic CK2, into decisive activation of signaling output (Gunawardena, 2005; Kapuy et al., 2009). We therefore propose that multisite dependency of CUL3KBTBD8 provides an elegant mechanism for switch-like cell fate decisions controlled by monoubiquitylation. Results CK2 kinase is required for CUL3KBTBD8-dependent neural crest specification CUL3KBTBD8 drives neural crest specification by catalyzing the monoubiquitylation of TCOF1 and NOLC1 (Werner et al., 2015), but how it selects its targets at the right time during development is not known. As substrate recognition by cullin-RING ligases often requires posttranslational modifications or co-adaptor proteins (McGourty et al., 2016; Skaar et al., 2013), we speculated that regulators of CUL3KBTBD8 could be identified as shared interactors of NOLC1 and TCOF1. We therefore affinity-purified FLAGNOLC1 and FLAGTCOF1 from human 293T embryonic kidney cells, a system that had previously allowed us to discover stem cell-related signaling pathways (Jin et al., 2012; McGourty et al., 2016; Werner et al., 2015), and analyzed the immunoprecipitates by CompPASS mass spectrometry (Huttlin et al., 2015; Sowa et al., 2009). These experiments showed that both NOLC1 and TCOF1 interacted with all subunits of the CK2 kinase (Figure 1A), which was consistent with earlier studies that found these proteins to be phosphorylated by CK2 (Jones et al., 1999; Meier and Blobel, 1992; Wise et al., 1997). We confirmed the robust interaction of NOLC1 and TCOF1 with CK2 and CK2 by affinity-purification and western blotting (Figure 1B). Open in a separate window Figure 1. CK2 kinase is required for CUL3KBTBD8-substrate binding and ubiquitylation in cells.(A) Both NOLC1 and TCOF1 associate with the CK2 kinase. FLAGNOLC1 and FLAGTCOF1 were affinity-purified from 293 T cells and specific binding partners were determined by CompPASS mass spectrometry. Total spectral counts (TSCs) of specific interactors were normalized to 1000 TSCs for each bait protein and plotted against each other. For each CUL3KBTBD8-substrate, three independent affinity-purification and mass spectrometry experiments were performed. The results of each affinity experiments were compared.