Role of multi-site phosphorylation in regulation of E(spl) M8 activity during Drosophila neurogenesis

Notch signaling is an evolutionarily conserved pathway that regulates cell-fate determination at various stages of development in invertebrate and vertebrate species. The Notch pathway mediates juxtacrine signaling through a process termed as lateral inhibition by which two equipotent cells adopt distinct cell fates. The functions of Notch have been exceptionally well analyzed during neurogenesis in Drosophila, particularly during eye and mechanosensory bristle morphogenesis. At the onset of neurogenesis, proneural bHLH activators encoded by atonal (ato) or the achaete-scute complex (ASC) is expressed in groups of cells, called proneural clusters (PNCs) in the eye and the bristle tissue respectively. From each PNC, however, only a single cell becomes the neural progenitor, the sensory organ precursor (SOP). This selection is mediated through E(spl) repressors, a group of evolutionary conserved bHLH repressors and the terminal effectors of the Notch pathway. Accumulating evidence suggest that repression of Ato/ASC by E(spl) occurs in a phosphorylation dependent manner. Protein kinase CK2 phosphorylates E(spl)-M8, which then undergoes a conformational change and antagonizes the proneural proteins. The work described in this dissertation aims to provide a more detailed understanding of the mechanism of neural repression by M8. The work described in Chapter 2 provides in vivo evidence that multi-site phosphorylation of the C-terminal domain (CtD) of M8 may be involved in regulation of repressor activity. Using phospho-mimetic variants of M8 we have shown that both CK2 and MAPK motif in the CtD may be modulated for the protein to exhibit neural repression. Multisite phosphorylation may act as a \u27switch\u27 controlling the activity and onset of repression by M8 during neurogenesis. The studies in Chapter 3 demonstrate a direct role of P-domain, a conserved phosphorylation domain of CtD, in autoinhibition model of M8. The deletion variant studies provide strong evidence that the P-domain provides critical autoinhibitory contact with HLH and/or Orange domain to regulate repression of Ato/ASC. This study provides a fundamental reinterpretation of the mechanism by which truncated protein, M8* encoded by m8 allele E(spl)D, that lacks the autoinhibitory domain, elicits precocious Ato antagonism thus interfering with first phase of Notch signaling and perturbing eye development. Our studies also implicate P-domain as a target of Slmb, a subunit of E3 ubiquitin ligase that may give rise to \u27phosphodegron\u27 necessary for proteasomal degradation and rapid clearance of M8. Together, the studies described in this dissertation provide a mechanism of M8 regulation by posttranslational regulation to elicit the effects of inhibitory Notch signaling during lateral inhibition