Identification of motifs in the tribbles kinase required for substrate interactions
type: CreativeWork
description: The gene tribbles encodes an atypical kinase that is evolutionarily conserved with important roles in regulation of cell signaling. In Drosophila melanogaster Tribbles (Trbl) directs the proteasomal degradation of the C/EBP transcription factor Slbo (slow border cells) to regulate cell migration in the fly ovary. Trbl's structure is homologous to serine/threonine kinases with N-terminal and C-terminal structures that flank a central kinase-like domain, which includes a conserved catalytic DLK motif needed in normal kinases to mediate phosphotransfer during kinase-substrate interactions. Genetic misexpression assays in the fly eye and wing were used to confirm Trbl suppression of Slbo phenotypes and to determine the essential nature of the conserved DLK motif. In addition to this kinase motif, Trbl has C-terminal MEK1 and COP1 binding sites, raising the possibility that Trbl is a docking kinase with motifs that impart specificity to bind and degrade multiple pathway components. To test the ability of Trbl to directly bind Slbo while avoiding the problem of Slbo degradation that ensues from this interaction, a yeast two hybrid system was established to measure interaction strength by growth on selective media and in a quantitative reporter gene assay. Using site-directed mutagenesis, Trbl mutants were constructed based on these conserved motifs and used in the yeast two hybrid assay to test their ability to interact with Slbo, a known substrate. It was found that Trbl in the prey vector interacts robustly with Slbo in the bait plasmid, and this interaction is dependent on both the kinase DLK motif and the MEK1 binding domain. Because some kinases form dimers/multimers in vivo to regulate their activity, Trbl-Trbl interactions were tested by yeast two hybrid. It was found that Trbl interacts with itself but with a lower strength than its interaction with Slbo and this interaction depends on the kinase DLK motif as well. An additional Trbl mutant was created which was b…
abstract: The gene tribbles encodes an atypical kinase that is evolutionarily conserved with important roles in regulation of cell signaling. In Drosophila melanogaster Tribbles (Trbl) directs the proteasomal degradation of the C/EBP transcription factor Slbo (slow border cells) to regulate cell migration in the fly ovary. Trbl's structure is homologous to serine/threonine kinases with N-terminal and C-terminal structures that flank a central kinase-like domain, which includes a conserved catalytic DLK motif needed in normal kinases to mediate phosphotransfer during kinase-substrate interactions. Genetic misexpression assays in the fly eye and wing were used to confirm Trbl suppression of Slbo phenotypes and to determine the essential nature of the conserved DLK motif. In addition to this kinase motif, Trbl has C-terminal MEK1 and COP1 binding sites, raising the possibility that Trbl is a docking kinase with motifs that impart specificity to bind and degrade multiple pathway components. To test the ability of Trbl to directly bind Slbo while avoiding the problem of Slbo degradation that ensues from this interaction, a yeast two hybrid system was established to measure interaction strength by growth on selective media and in a quantitative reporter gene assay. Using site-directed mutagenesis, Trbl mutants were constructed based on these conserved motifs and used in the yeast two hybrid assay to test their ability to interact with Slbo, a known substrate. It was found that Trbl in the prey vector interacts robustly with Slbo in the bait plasmid, and this interaction is dependent on both the kinase DLK motif and the MEK1 binding domain. Because some kinases form dimers/multimers in vivo to regulate their activity, Trbl-Trbl interactions were tested by yeast two hybrid. It was found that Trbl interacts with itself but with a lower strength than its interaction with Slbo and this interaction depends on the kinase DLK motif as well. An additional Trbl mutant was created which was b…
author: Pence, Laramie Elise
date published: 2013
date modified: 2026-07-24T05:17:10Z
provider: University of Missouri - Kansas City
publisher: Networked Digital Library of Theses and Dissertations
type: WebPage
source organization: University of Missouri--Kansas City

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property ID: Handle
value: http://hdl.handle.net/10355/35460