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Functional analysis of the Rac-binding protein POSH

Bishop, Anne Louise; (2001) Functional analysis of the Rac-binding protein POSH. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Abstract

The scaffold protein POSH (Plenty of SH3s) was discovered to be an effector for the GTPase Rac using the yeast-two-hybrid system. Rac regulates numerous cellular processes including actin reorganisation and the control of gene transcription (JNK and p38 MAP kinase pathways and NF-κB and SRF transcription factors). It was previously shown that POSH, like Rac, is able to activate both JNK and NF-κB. Unlike Rac, POSH was also shown to induce apoptosis when over-expressed. The induction of apoptosis by POSH provided the starting point for the functional analysis of POSH described in this thesis. Data is presented here showing that the Rac-binding domain of POSH contributes to apoptosis, by inhibiting Rac, and that activated Rac rescues POSH-expressing cells by providing a survival signal that is independent of binding to POSH. The whole of the N-terminus of POSH (containing two SH3 domains and a RING finger) was also found to be required, in addition to the Rac-binding domain, for POSH-induced apoptosis. Investigation of the binding partners for POSH N-terminus revealed that the major binding partner was dynamin, a GTPase required for endocytosis. The two N-terminal SH3 domains of POSH were sufficient and necessary for binding of POSH to dynamin and POSH constructs containing this region inhibited endocytosis, suggesting both a physical and a functional link between POSH and dynamin. The RING finger of POSH was essential for apoptosis and increased the potency of POSH inhibition of endocytosis. Initial data suggests that POSH, like many other RING finger-containing proteins, is itself ubiquitinated and may be involved in the ubiquitination of other, as yet unknown, protein substrates. Overall, the data presented here suggests the existence of a novel POSH-dependent link between Rac activity and endocytosis (or another less well characterised dynamin-dependent process), which is regulated by ubiquitin-dependent protein degradation.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: Functional analysis of the Rac-binding protein POSH
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by ProQuest.
Keywords: Biological sciences; Apoptosis
URI: https://discovery.ucl.ac.uk/id/eprint/10102230
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