Tanner, Lewis E. S.; (2022) Characterising the effect of novel SFK phosphorylation on ADP Ribosylation Factor (ARF) state changes. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

GTP-binding proteins of the ADP-ribosylation factor (Arf) family play fundamental roles in key processes in membrane biology. Some roles are linked to disease, for example cancer progression and these are now just becoming understood. For instance, Golgi morphogenesis is mediated by Arf1 and Arf6, the most distant family member, coordinates actin rearrangements and podosome formation and is involved in endosomal recycling. Src family kinases (SFKs) also have roles in these processes. Signalling through Src, the archetypal SFK and product of a proto-oncogene, coordinates traffic in an Arf 1 dependent context at the Golgi and also coordinates actin dynamics at focal adhesions. This thesis establishes the first direct link between Src phosphorylation of two tyrosine positions in Arf6 (and a primary position in Arf1). These sites are near to one another in the tertiary structure of Arf and are positioned close to the lower part of the flexible helix and the Interswitch region both of which have roles in the switch function of Arfs. It is also shown that Arf phosphorylation has a role in mediating changes in both the Golgi and actin cytoskeleton upon challenge with active Src and that sustained Src activity drives a steady state decrease in active Arf levels. These insights have been combined with data on Src activation and Arf activation dynamics to formulate two mathematical models to capture likely features of the regulation of Arf by Src. These models recapitulate some of the observed behaviour of Arf and Src and suggests that there may exist a Golgi mechanism for the inhibition of Src during trafficking, a feature similar that believed to operate at the plasma membrane. With a view to making measurements of transitions between Arf activation states (phosphorylation and nucleotide binding status) plausible future routes to the rapid quantification of multiple Arf states are explored through the use of recombinant proteins and a novel capillary gel electrophoresis method. This established some initial separation parameters and opens the way for parameterisation of the mathematical models.

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