Retinal patterning involves the specification of a diverse array of cell fates through a complex interplay of signaling pathways, using molecular factors and regulatory mechanisms that appear highly conserved. One of these is the Notch signaling pathway. It plays roles during the specification of photoreceptors such as the 'founding'R8's, the R3/4 pairs and the R7 cells. Notch mediates these effects by regulating the expression of a family of structurally related basic-helix-loop-helix (bHLH) proteins encoded by the Enhancer of Split Complex, E(spl)C. These bHLH proteins inhibit the functions of other genes/proteins whose activities, in turn, are required for specification of these neuronal cell fates. Because the inhibition by E(spl) proteins of the proneural genes is central to retinal patterning, substantial efforts have been devoted to unraveling the underlying mechanisms. Our work is focused on the mechanism by which protein kinase CK2 contributes to the regulation of E(spl) proteins via phosphorylation. Our analysis of this modification has uncovered a novel aspect of regulation, whereby phosphorylation of E(spl) appears to mediate a conformational change that is required for repression. Through these studies, CK2 is implicated as a mediator of inhibitory Notch signaling. Our studies will continue to exploit the broad potential of the Drosophila system in order to extend our characterization of the regulation of E(spl) by CK2 during retinal patterning. We now propose three aims: (1) We will analyze variants of E(spl)M8 to identify the in vivo roles of secondary phosphorylation and the mechanism by which this modification may contribute to conformational transitions. (2) We will analyze the mechanistic connection between CK2 and E(spl) using the retinal defects of Nspl, a well described and widely studied Notch mutation. (3) Using conditional (temperature sensitive) mutations in CK2, we will analyze its roles during the R3/4 and R7 cell fates and conduct screens to identify eye-specific factors that regulate repression by E(spl). Our studies are likely to be relevant to mammalian eye development because CK2 regulates, in a remarkably similar manner, the functions of mouse Hes6, a homolog of fly E(spl) proteins. Our studies will clarify the roles of CK2 and may provide insights into how misregulated cell fate specification contributes to eye defects. A successful resolution of these issues will expose a layer of complexity of Notch signaling previously unknown and unexplored.

National Institute of Health (NIH)
National Eye Institute (NEI)
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Neurogenesis and Cell Fate Study Section (NCF)
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Mariani, Andrew P
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West Virginia University
Schools of Arts and Sciences
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Bandyopadhyay, Mohna; Arbet, Scott; Bishop, Clifton P et al. (2016) Drosophila Protein Kinase CK2: Genetics, Regulatory Complexity and Emerging Roles during Development. Pharmaceuticals (Basel) 10:
Bose, Anasua; Majot, Adam T; Bidwai, Ashok P (2014) The Ser/Thr phosphatase PP2A regulatory subunit widerborst inhibits notch signaling. PLoS One 9:e101884
Eastwood, Karen; Yin, Chong; Bandyopadhyay, Mohna et al. (2011) New insights into the Orange domain of E(spl)-M8, and the roles of the C-terminal domain in autoinhibition and Groucho recruitment. Mol Cell Biochem 356:217-25
Kahali, Bhaskar; Kim, Jee-Eun; Karandikar, Umesh et al. (2010) Evidence that the C-terminal domain (CtD) autoinhibits neural repression by Drosophila E(spl)M8. Genesis 48:44-55
Kuntamalla, Pallavi P; Kunttas-Tatli, Ezgi; Karandikar, Umesh et al. (2009) Drosophila protein kinase CK2 is rendered temperature-sensitive by mutations of highly conserved residues flanking the activation segment. Mol Cell Biochem 323:49-60
Kunttas-Tatli, Ezgi; Bose, Anasua; Kahali, Bhaskar et al. (2009) Functional dissection of Timekeeper (Tik) implicates opposite roles for CK2 and PP2A during Drosophila neurogenesis. Genesis 47:647-58
Kahali, Bhaskar; Bose, Anasua; Karandikar, Umesh et al. (2009) On the mechanism underlying the divergent retinal and bristle defects of M8* (E(spl)D) in Drosophila. Genesis 47:456-68
Kahali, Bhaskar; Trott, Regina; Paroush, Ze'ev et al. (2008) Drosophila CK2 phosphorylates Hairy and regulates its activity in vivo. Biochem Biophys Res Commun 373:637-42
Bose, Anasua; Kahali, Bhaskar; Zhang, Sophia et al. (2006) Drosophila CK2 regulates lateral-inhibition during eye and bristle development. Mech Dev 123:649-64
Shaffer, Jonathan M; Karandikar, Umesh C; Bidwai, Ashok P (2006) An activity-based two-hybrid system for the selective identification of substrates of protein kinases. Anal Biochem 355:154-6

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