Semester

Fall

Date of Graduation

2001

Document Type

Dissertation

Degree Type

PhD

College

School of Medicine

Department

Microbiology, Immunology, and Cell Biology

Committee Chair

Daniel Flynn.

Abstract

c-Src and c-Yes are two widely expressed and highly homologous members of the Src family of non-receptor tyrosine kinases and are involved in a diverse range of cellular signaling pathways. They are frequently activated in human cancers, and thus represent potential targets for anti-tumor drug design. The high level of homology between these two kinases includes not only sequence similarity, but also overlapping regulation and function. Despite the redundancies between c-Src and c-Yes, however, specificity in signaling has been demonstrated between the two proteins. It was hypothesized in these studies that differences in the signaling capacities of c-Src and c-Yes could be attributed to functional domain differences. In order to test this hypothesis, chimeric proteins were created in which the SH4, Unique, SH3, or SH2 domains of c-Src or the constitutively activated Src527F, were replaced by those of c-Yes, either individually or in combinations. Differences in the signaling capacities of these proteins were assessed at the levels of protein/protein interactions, downstream gene induction, and cell biology. It was demonstrated that both the SH3 and SH2 domains were capable of directing differential protein/protein interactions between c-Src and c-Yes. While no differences in substrate selection were evident, the c-Yes SH3 domain failed to efficiently bind the Src SH3 binding partner AFAP-110 and several additional tyrosine-phosphorylated binding partners. Additionally, the c-Yes SH2 domain facilitated stable complex formation between Src527F/c-Yes chimeras and an unidentified 87 kDa tyrosine-phosphorylated protein, pp87. Differences in downstream gene induction and cell biology were mediated by the amino terminal region (including the SH4 and Unique domains), as Src527F/c-Yes amino terminal chimeras failed to induce efficient upregulation of Heme Oxygenase 1 expression and were unable to induce actin filament rearrangement or changes in cell morphology upon expression in chicken embryo fibroblasts. These results occurred concomitant with a failure of these chimeras to induce activation of the PI3K/Akt pathway and inactivation of RhoA. Taken together, these results indicate that each of the non-catalytic functional domains may contribute to signaling specificity between c-Src and c-Yes. These results may contribute to the overall knowledge of biological differences between c-Src and c-Yes and how Src family kinases in general are able to achieve signaling specificity.

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