In vivo functional analysis of the daughter of sevenless protein in receptor tyrosine kinase signaling.
One mechanism used by receptor tyrosine kinases to relay a signal to different downstream effector molecules is to use adaptor proteins that provide docking sites for a variety of proteins. The daughter of sevenless (dos) gene was isolated in a genetic screen for components acting downstream of the Sevenless (Sev) receptor tyrosine kinase. Dos contains a N-terminally located PH domain and several tyrosine residues within consensus binding sites for a number of SH2 domain containing proteins. The structural features of Dos and experiments demonstrating tyrosine phosphorylation of Dos upon Sev activation suggested that Dos belongs to the family of multisite adaptor proteins that include the Insulin Receptor Substrate (IRS) proteins, Gab1, and Gab2. Here, we studied the structural requirements for Dos function in receptor tyrosine kinase mediated signaling processes by expressing mutated dos transgenes in the fly. We show that mutant Dos proteins lacking the putative binding sites for the SH2 domains of Shc, PhospholipaseC-gamma (PLC-gamma) and the regulatory subunit of Phosphoinositide 3-kinase (PI3-K) can substitute the loss of endogenous Dos function during development. In contrast, tyrosine 801, corresponding to a predicted Corkscrew (Csw) tyrosine phosphatase SH2 domain binding site, is essential for Dos function. Furthermore, we assayed whether the Pleckstrin homology (PH) domain is required for Dos function and localization. Evidence is provided that deletion or mutation of the PH domain interferes with the function but not with localization of the Dos protein. The Dos PH domain can be replaced by the Gab1 PH domain but not by a heterologous membrane anchor, suggesting a specific function of the PH domain in regulating signal transduction.
Bausenwein
B S
BS
Department of Genetics, University of Würzburg, Biozentrum, Am Hubland, D-97074, Würzburg, Germany.
Schmidt
M
M
Mielke
B
B
Raabe
T
T
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002462:Cell Membrane / Q000378:metabolism; D004330:Drosophila; D029721:Drosophila Proteins; D004742:Enhancer Elements, Genetic; D005136:Eye Proteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D005260:Female; D008297:Male; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D011401:Promoter Regions, Genetic; D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism
C100319:Dos protein, Drosophila; D029721:Drosophila Proteins; D005136:Eye Proteins; D008562:Membrane Glycoproteins; D010750:Phosphoproteins; D014443:Tyrosine; D020794:Receptor Protein-Tyrosine Kinases; C056737:sev protein, Drosophila