A phosphorylation site in the ftz homeodomain is required for activity.
The 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. Here we show that one of the Ftz phosphorylation sites, T263 in the N-terminus of the Ftz homeodomain, is phosphorylated in vitro by Drosophila embryo extracts and protein kinase A. In the embryo, mutagenesis of this site to the non-phosphorylatable residue Ala resulted in loss of ftz-dependent segments. Conversely, substitution of T263 with Asp, which is also non-phosphorylatable, but which successfully mimics phosphorylated residues in a number of proteins, rescued the mutant phenotype. This suggests that T263 is in the phosphorylated state when functioning normally in vivo. We also demonstrate that the T263 substitutions of Ala and Asp do not affect Ftz DNA-binding activity in vitro, nor do they affect stability or transcriptional activity in transfected S2 cells. This suggests that T263 phosphorylation is most likely required for a homeodomain-mediated interaction with an embryonically expressed protein.
Dong
J
J
Banting and Best Department of Medical Research, University of Toronto, C.H.Best Institute, Toronto, Ontario, Canada M5G 1L6.
Hung
L H
LH
Strome
R
R
Krause
H M
HM
D000409:Alanine / Q000235:genetics / Q000378:metabolism; D000818:Animals; D030801:Animals, Genetically Modified; D001224:Aspartic Acid / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D002460:Cell Line; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004247:DNA / Q000378:metabolism; D004330:Drosophila / Q000196:embryology / Q000378:metabolism; D029721:Drosophila Proteins; D051717:Fushi Tarazu Transcription Factors; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism*; D006706:Homeostasis; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011518:Proto-Oncogene Proteins / Q000235:genetics; D013329:Structure-Activity Relationship; D013912:Threonine / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics; D014158:Transcription, Genetic; D051155:Wnt1 Protein
D029721:Drosophila Proteins; C063459:En protein, Drosophila; D051717:Fushi Tarazu Transcription Factors; D018398:Homeodomain Proteins; D011518:Proto-Oncogene Proteins; D014157:Transcription Factors; D051155:Wnt1 Protein; C497169:ftz protein, Drosophila; C074622:wg protein, Drosophila; D013912:Threonine; D001224:Aspartic Acid; D004247:DNA; D017868:Cyclic AMP-Dependent Protein Kinases; D000409:Alanine