A phosphorylation site in the Ftz homeodomain is required for segmentation in Drosophila

grantor: University of TorontoThe 'Drosophila' homeodomain-containing protein Fushi tarazu (Ftz) is expressed sequentially in the embryo, first in alternate segments, then in specific neuroblasts and neurons in the central nervous system and finally in parts of the gut. During these different developmental stages, the protein is heavily phosphorylated on different subsets of Ser and Thr residues. This stage-specific phosphorylation suggests possible roles for signal transduction pathways in directing tissue-specific Ftz activities. The objective has been the testing of the importance of a single phosphorylation site for Ftz activity in the embryo. This was done by mapping Ftz phosphorylation sites, mutating the relevant Ser or Thr residues to Ala and Asp, and then testing the ability of the mutated proteins to rescue ' ftz' mutant embryos. My analysis has focused on T263, which is in the N-terminus of the Ftz homeodomain. This site is phosphorylated 'in vitro' by ' Drosophila' embryo extracts and by purified protein kinase A. In the embryo, mutagenesis of this site to the non-phosphorylatable residue Ala results in segmental defects. Conversely, replacement of T263 by Asp, which is also non-phosphorylatable, but which successfully mimics phosphorylated residues in a number of proteins, rescues the segmental phenotype. This suggests phosphorylation of Ftz on residue T263 is required for function in the ectoderm. In the CNS however, both T263A and T263D mutant proteins function normally, indicating that the requirement for T263 phosphorylation is stage-specific. in terms of molecular properties, mutation of T263 to Ala and Asp does not affect Ftz DNA binding activity 'in vitro', nor do the mutations affect transcriptional activity in transfected S2 cells. I conclude that T263 phosphorylation is most likely required for a homeodomain-mediated interaction with an embryonically expressed protein.Ph.D