Investigation of functional domains of drosophilia tribbles protein in the insulin signaling pathway
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In this dissertation, I examined the function of the Drosophila melanogaster protein Tribbles (Trbl) within the insulin/insulin-like signaling (IIS) pathway. Trbl was initially discovered as a regulator of mitosis during embryonic development and cell migration in developing egg chambers. This discovery was soon followed by the identification of mammalian Tribbles homologs, thus establishing the Tribbles (Trib) family of pseudokinases. The fly protein and mammalian Trib3 both inhibit the phosphorylation of Akt, a central regulator in the IIS pathway, and in humans a naturally-occurring single nucleotide polymorphism in Trib3 (Q84R) is associated with type 2 diabetes. In my first research project, I proposed that the functional relevance of this residue is conserved. In support of this, I observed that misexpression of a TrblR141Q mutant in the larval fat body increased phospho-dAkt, decreased circulating glucose, and increased storage metabolites. These data indicate that this conserved residue is required for the function of Trib protein in regulating IIS-mediated growth and homeostasis. Next, I studied Trbl subcellular trafficking and turnover in the larval fat body, an important site of insulin signaling in the developing fly. Akt subcellular trafficking is dynamic at the plasma membrane (PM) in response to nutritional context, and I proposed that Trbl localization in insulin-responsive cells under different dietary states might also be nutrition-dependent. To test this, I compared Trbl subcellular localization in fed and fasted larvae and observed increased Trbl protein levels and membrane accumulation of Trbl in fasted cells compared to fed, suggesting a molecular mechanism for the targeting of dAkt by Trbl. Next, I found that found that misexpression of a Trbl mutant protein with a site-directed mutation in the conserved SLE motif resulted in strong localization to the membrane, conferred stability, and enhanced binding to wild-type Trbl and dAkt. To explore the association of Trbl with the membrane, I tested the ability of Trbl to interact with bio-active lipids and identified phosphatidylserine, phosphatidylinositol-4-phosphate, and phosphatidylinositol-4,5-bisphosphate as membrane phospholipids that Trbl selectively bound. These data suggest a complex relationship between Trbl, dAkt, and phospholipids at the PM physically interacting in response to developmental and homeostatic cues.
Table of Contents
Introduction -- Drosophila Model of insulin resistance associated with the Human TRIB3 Q/R Polymorphism -- A structure-function analysis of Drosophila Tribbles reveals conserved features required for stability and subcellular distribution -- Future directions
Ph.D. (Doctor of Philosophy)