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Author

Richard Chan

Date of Award

6-2004

Degree Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

Department

Biology

Supervisor

Professor William J. Muller

Abstract

The ErbB2 receptor tyrosine kinase plays important roles in both mammalian development and in oncogenesis. Signal transduction mediated by the receptor is dependent on the catalytic activity of its tyrosine kinase domain and the phosphorylation of discrete tyrosine residues within the receptor. To perform structure-function studies in vivo and to evaluate the function of tyrosine phosphorylation, an allelic series of erbB2 cDNA knock-in animals were generated such that the erbB2 cDNA was under the transcriptional control of the endogenous promoter. Mice homozygous for the kinasedead erbB2 allele died at midgestation and displayed the same spectrum of embryonic defects observed in erbB2 deficient animals. In contrast, expression of ErbB2 receptors harboring a single tyrosine site mutation in the carboxy-terminus of the receptor resulted in viable animals. However, expression of ErbB2 receptors de-coupled from the Shc signaling pathway (Y 1226/7F) lead to subtle defects in the development of cutaneous sensory nerves. The cDNA knock-in alleles were inadvertently hypomorphic expressing a minimum threshold level of ErbB2 (~1 0% of wild type) required for survival. Indeed, a further 2-fold reduction in ErbB2 levels in hemizygous knock-in animals resulted in perinatal lethality due to defective innervation of the diaphragm leading to acute respiratory distress. Interestingly, this hypomorphic phenotype was genetically rescued when the ErbB2-Y1028F mutant receptor was expressed in animals using a comparable knock-in strategy. Subsequent molecular analyses revealed that the YI028F allele expressed higher levels of ErbB2, due to the decreased turnover rate of the receptor following removal ofY1028. This downregulation was independent of c-Cbl association and ubiquitylation of the receptor. Instead, ErbB2-Yl144 and Y122617 can associate with c-Cbl but the receptor remains refractory to c-Cbl mediated ubiquitylation. Additionally, ErbB2 is monoubiquitylated through an unidentified mechanism mediated by the Y1253 phosphorylation site. Taken together, these results provide important novel insight into the function and regulation of the ErbB2 receptor.

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