Date of Award
Doctor of Philosophy (PhD)
The v-Crk oncogene product, while possessing no intrinsic tyrosine kinase activity, causes an elevation in levels of cellular phosphotyrosine as it transforms chicken embryo fibroblasts (CEF). Because of this similarity to v-Src transformation and EGF receptor (EGFR) stimulation, signalling pathways that might be activated in v-Crk transformed cells were investigated by analogy to pathways utilized by v-Src and the activated EGFR , and a search for serine/threonine kinases activated in v-Crk transformed CEF was conducted. Mapk was identified as one kinase, the activity of which is constitutively elevated in v-Crk transformed CEF. Mek activity was also somewhat elevated. However, the Shc proteins, which probably mediate activation of the Ras pathway in v-Src transformed and EGF-stimulated cells, were not tyrosine phosphorylated, indicating that the activation of Mapk was probably mediated by a different mechanism. v-Crk transformed NIH-3T3 cell lines were established in order to examine the effects of blocking the Ras pathway in v-Crk transformed cells. In contrast to previous studies done in mammalian cells, v-Crk caused morphological transformation and promoted anchorage-independent growth of the transfected NIH-3T3 cells. In addition, elevated levels of tyrosine phosphorylation were observed on the characteristic p70 and p130. Expression of dominant negative Ras in the v-Crk NIH-3T3 caused morphological reversion of these cells, as well as an inhibition of colony formation. in soft agar, indicating a requirement for Ras function in v-Crk transformation. However, no decrease in tyrosine phosphorylation of cellular proteins was observed upon dominant negative Ras expression. A delay in serum stimulation of Mapk activity in oncogene transformed cells was also observed. This delay was shown to correlate with increase in metabolic rate, rather than degree of morphological transformation. A possible mechanism involving a delay in Shc phosphorylation is proposed.
Greulich, Heidi, "A Role for RAS in V-CRK Transformation" (1995). Student Theses and Dissertations. 336.