Gilfillan, Iain;
(1998)
Cell cycle analysis of lck and fyn.
Doctoral thesis (Ph.D), UCL (University College London).
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Abstract
The aim of this Ph.D was to investigate cell cycle-dependent roles of the src-family members, p56lck and p59fyn, in the T cell line, Jurkat. The SH3 and SH2 domains were analysed with respect to substrate binding and specificity, in cells blocked in S phase and mitosis. Cellular substrates were isolated by affinity precipitation using p56lck and p59fyn domains expressed as GST fusion proteins. Although quantitative differences in substrate binding were seen between the SH2 and SH3 domains, no cell cycle-dependent protein interactions were detected. To identify these constitutively interacting proteins, Coomassie Blue stained proteins were excised from the acrylamide gel and subjected to peptide sequencing. This revealed the following interacting proteins for the fynSH3 domain; vimentin, hnRNPK, p54nrb, Sam68 and BF-1/HFK1. FynSH2-binding proteins included HSP70, nucleolin, KU-70kDa subunit and KU-84kDa subunit. These interactions, excluding hnRNPK and Sam68, are novel. Sequence from a 40kDa peptide associating with the fynSH2 domain did not correspond to any known protein. Sam68 was shown to associate with the SH3 and SH3SH2 domains of p56lck and p59fyn and with the SH2 domain of p59fyn. An immunologically related 57kDa protein associated with the SH3 and SH2 domains of p59fyn in asynchronous and mitotic cells but with the SH2 domain of p56lck in asynchronous cells only. A 57/58kDa doublet was found in association with the SH2 and SH3 domains of p59fyn in mitotic cells with the mitotic band exhibiting a retarded mobility. The association of Sam68 and the 57kD protein with fynSH3 was inhibited by poly-proline. Sam68 association was constitutive and independent of anti-CD3 stimulation or position in the cell cycle. Evidence is also reported, suggesting the presence of a family of Sams in a range of cell types.
| Type: | Thesis (Doctoral) |
|---|---|
| Qualification: | Ph.D |
| Title: | Cell cycle analysis of lck and fyn |
| Open access status: | An open access version is available from UCL Discovery |
| Language: | English |
| Additional information: | Thesis digitised by ProQuest. |
| Keywords: | Biological sciences; Cell cycle |
| URI: | https://discovery.ucl.ac.uk/id/eprint/10102165 |
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