Date of Completion

6-5-2015

Embargo Period

5-27-2015

Major Advisor

Bruce J. Mayer

Associate Advisor

Daniel W. Rosenberg

Associate Advisor

Christopher D. Heinen

Associate Advisor

Stormy J. Chamberlain

Associate Advisor

Blanka Rogina

Field of Study

Biomedical Science

Degree

Doctor of Philosophy

Open Access

Open Access

Abstract

Since the discovery of the first oncogenic Crk adaptor protein, v-Crk in 1988, various studies have been conducted to understand the signaling mechanism of Crk proteins. In our previous knockdown study to investigate the importance of several CrkI SH3-binding effectors in the tumorigenesis of CrkI, we were surprised to learn that Abl kinase negatively regulates CrkI transformation in NIH3T3 cells. Because constitutively active Abl kinase, in the form of BCR-ABL, is famously known to cause chronic myelogenous leukemia (CML) in humans, we initially expected a similar tumor promoting role for Abl in CrkI transformation. However, downregulated Abl kinase (through shRNA knockdown or inhibitory drug, Imatinib) enhances the anchorage-independent growth of CrkI-transformed NIH3T3 cells. This raises our concern over the popularity of Imatinib, a potent Abl inhibitor, in various combination therapies for cancer treatment. Here, we have identified Dok1 as the Abl-phosphorylated substrate that mediates a previously unknown negative regulatory pathway for CrkI tumorigenesis. In particular, we showed that the phosphorylation of two tyrosine residues in Dok1, Y295 and Y361, are essential for limiting the anchorage independent growth of CrkI-transformed cells. Through both in vitro and in vivoSH2-interaction assays and subsequent p120RasGAP shRNA knockdown, we then confirmed p120RasGAP as the next effector for the Abl/Dok1 pathway. Although we did not find any increased Ras or Erk activation in the whole cell lysates of control, CrkI-transformed and the subsequent Abl or Dok1 knockdown cell lines, we were able to detect the dysregulation of localized Ras activation during the spreading of CrkI-overexpressing cells via live-cell imaging and a Förster resonance energy transfer (FRET) biosensor for activated Ras. In addition, our preliminary data suggest that the Abl/Dok1 pathway is most likely regulating CrkI transformation through small GTPases and its inhibition further induces Jnk activation. And finally, we demonstrated that Imatinib promotes the anchorage-independent growth of SF268, a human glioblastoma-derived cell line but more investigations are needed to understand the underlying mechanism(s).

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