Event Title
In Vivo Biotinylation and Streptavidin Pull down of Fzd9bto Evaluate the Role of Ubiquitination of the Intracellular Domains of Fzd9b in Wnt9a Signal Specificity
Location
Loosemore Auditorium
Description
PURPOSE: Numerous developmental processes, including stem cell amplification, are dependent on Wnt signaling. Wnt signaling between one of the (19+) Wnt ligands and (10+) Frizzled (Fzd) receptors was previously thought to be mostly nonspecific. However, our lab has established that Wnt9a signals specifically through Fzd9b to amplify hematopoietic stem cells in the aorta during development. Mechanistically, we have also shown that phosphorylation of the Fzd9b intracellular tail by the epidermal growth factor receptor (EGFR) is required for signaling specificity. Yet, the other mechanisms driving specificity of this signal are poorly understood and may rely in part on ubiquitin modification of the Fzd9b receptor. PROCEDURES: We have devised a method for in vitro biotinylation of Fzd9b, which enables Fzd9b purification with streptavidin beads. Optimization of the pulldown methodology allows the ubiquitination status of Fzd9b to be determined through western blot. OUTCOME: We have identified 4 lysine (K) residues on the third intracellular loop of Fzd9b, which may act as ubiquitination sites. IMPACT: Mutation of these residues to arginine (R), which cannot be ubiquitinated has impacts on signaling. Furthermore, an unbiased proteomics screen for proteins interacting with Fzd9b uncovered several ubiquitin modifying enzymes, suggesting that Fzd9b has a change in ubiquitination status in response to Wnt9a. Fzd9b is a seven-pass transmembrane receptor, and these are inherently difficult to isolate from the membrane for immunoprecipitation.
In Vivo Biotinylation and Streptavidin Pull down of Fzd9bto Evaluate the Role of Ubiquitination of the Intracellular Domains of Fzd9b in Wnt9a Signal Specificity
Loosemore Auditorium
PURPOSE: Numerous developmental processes, including stem cell amplification, are dependent on Wnt signaling. Wnt signaling between one of the (19+) Wnt ligands and (10+) Frizzled (Fzd) receptors was previously thought to be mostly nonspecific. However, our lab has established that Wnt9a signals specifically through Fzd9b to amplify hematopoietic stem cells in the aorta during development. Mechanistically, we have also shown that phosphorylation of the Fzd9b intracellular tail by the epidermal growth factor receptor (EGFR) is required for signaling specificity. Yet, the other mechanisms driving specificity of this signal are poorly understood and may rely in part on ubiquitin modification of the Fzd9b receptor. PROCEDURES: We have devised a method for in vitro biotinylation of Fzd9b, which enables Fzd9b purification with streptavidin beads. Optimization of the pulldown methodology allows the ubiquitination status of Fzd9b to be determined through western blot. OUTCOME: We have identified 4 lysine (K) residues on the third intracellular loop of Fzd9b, which may act as ubiquitination sites. IMPACT: Mutation of these residues to arginine (R), which cannot be ubiquitinated has impacts on signaling. Furthermore, an unbiased proteomics screen for proteins interacting with Fzd9b uncovered several ubiquitin modifying enzymes, suggesting that Fzd9b has a change in ubiquitination status in response to Wnt9a. Fzd9b is a seven-pass transmembrane receptor, and these are inherently difficult to isolate from the membrane for immunoprecipitation.