| Summary: | 碩士 === 國立成功大學 === 藥理學研究所 === 88 === Eps8 is a common substrate of receptor and noreceptor tyrosine kinase implicated in the control of the cell proliferation. Constitutive tyrosine phosphorylation of Eps8 was detected in many tumor cell lines, indicating that Eps8 might be part of pathway preferentially selected in tumor growth. The SH3 domain of Eps8 has been shown to interact with several signaling proteins including Shb 、Shc、 RN-tre、 E3B1. Previous studies have indicated that Eps8 can exist as a dimer in vivo, and that dimerization requires an intact SH3 domain. In order to further study the interaction between Eps8 SH3 domain and the interaction between the Eps8 SH3 and its binding proteins, we utilized site-directed mutagenesis to generate the defective SH3 domain mutant. We mutated the tryptophan residue at position 566 of Eps8 to lysine (W566K mutant). This residue is highly conserved in various SH3 domains and has been shown to be required for binding to the proline rich sequence. In addition, we generated GST-fusion protein containing either the wild type or the defective mutant to examine its binding ability in vitro. Form the lysates of the v-Src transformed cells, we observed GST-Eps8SH3 domain interacted with high amount of 68kDa proteins and low amount of 97kDa proteins, and GST-Eps8W566K loss its binding activity to 97kDa and 68 kDa protein. Furthermore, we found that the interaction beteween Eps8SH3 and p97Eps8 or p68Eps8 was not affected by the mutation of W566 to K566. Our results suggested that different amino acids of SH3 domain is required for the formation of SH3-SH3 interwind dimer.
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