Date of Award
Doctor of Philosophy (PhD)
William J. Muller
The ErbB-2/Neu receptor tyrosine kinase (RTK) plays a causal role in mammary tumourígenesis in a significant proportion of women, yet mechanistically does so in ill defined manner. The carboxy-terminus of the receptor, containing several phosphotyrosine residues, is thought to mediate transformation through interactions with cytoplasmic SH2/PTB-containing signaling molecules. To assess the role of each tyrosine phosphorylation site in cellular transformation, I created and analysed several series of phosphorylation mutants. While mutation of individual sites (Y1028, Y1144, Y1201, Y1226/7, Y1253) had little effect on neu -mediated transformation, the simultaneous mutation of each known Neu autophosphorylation site rendered the receptor transformation impaired. I assessed the role of each tyrosine phosphorylation site in cellular transformation, by restoring individual tyrosine residues to this transformation debilitated neu mutant. Reversion of any one of four mutated sites (Y1144, Y1201, Y1226/7, Y1253) restored wild-type transforming activity. These transforming "add-back" mutants displayed Ras-dependent signaling, which was further correlated with the ability of two of these "add-back" mutants to bind either the GRB2 (to Y1144) or SHC (to Y1227) adaptor molecules known to couple RTKs to Ras. Microinjection experiments suggest that Y1144 and Y1227 mediate Grb2 dependent and independent signals respectively. Additionally, several proteins were found to interact with the terminal phosphorylation site (Y1253) and using finer mutagenesis, transformation from Y1253 was correlated with the binding of an unknown 34kDa protein. By contrast, restoration of tyrosine 1028 to transforming add-back mutants suppressed the transformation. Mechanistically transformation repression correlated with a reduction in the ability to bind She and Grb2. As decreased Grb2 association was reversible by protein tyrosine phosphatase (PTP) inhibition, these data suggest that tyrosine 1028 acts to decrease transformation, in part, through the activation of PTPs. I used a RT-PCR approach to isolate PTPs expressed in the mammary gland to identify PTPs relevant in Neu signaling. One candidate, Shp-1, is overexpressed in mammary tumours and epithelial cells derived thereof and inhibits Neu-mediated transformation genetically upstream of Ras. These data argue not only that the transformation by activated neu is mediated primarily through Ras by multiple signaling proteins and is regulated in a negative manner by the Shp-1 PTP, but it also provides much insight in rational drug targets for ErbB-2-mediated human diseases.
Dankort, David L., "The genetic dissection of Neu-mediated transformation" (1999). Open Access Dissertations and Theses. Paper 2671.