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Title: Phosphorylation of Rab7 at serine 72 and its role in the regulation of the late endocytic pathway
Author: Wallace, M.
Awarding Body: University College London (University of London)
Current Institution: University College London (University of London)
Date of Award: 2015
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Abstract:
The small GTPase Rab7 is a key component of the endocytic pathway that controls endosomal maturation and fusion of late endosomes with lysosomes. Rab7 also regulates growth factor signaling by modulating receptor levels at the plasma membrane. While it is known how Rab7 is regulated through the GTPase cycle and interaction with its GEF and GAP, little is known about its regulation by post-translational modifications. In this study, I identified two kinases, TBK1 and IKKε, which phosphorylate Rab7 at S72 in in vitro kinase assays. Both kinases are highly homologous and are activated in the innate immune response to bacterial and viral infection. Importantly, phosphorylation of Rab7 at S72 was increased by approximately 4-fold in mouse embryonic fibroblasts following 4 h stimulation of Toll-like receptor (TLR)3 with poly(I:C), a synthetic analog of viral dsRNA. A similar increase was also seen following stimulation of TLR4 with bacterial lipopolysaccharide after 8 h stimulation. Through a pull-down and mass spectrometry analysis of differential interactors of unphosphorylated and S72-phosphorylated GST-Rab7, I found that phosphorylation of Rab7 at S72 inhibited interaction with RILP, one of its best known effectors, while, it increased interaction with the p150(Glued) subunit of the dynactin complex. The mass spectrometry analysis also identified a novel interactor, TNFR associated factor 3 (TRAF3), which is activated via the MyD88-independent pathway following stimulation of various TLRs. TRAF3 also leads to the downstream activation of TBK1 and IKKε. Using subcellular fractionation and immunofluorescence with an anti-phospho-Rab7(S72) antibody, I found that the ratio of phosphorylated to unphosphorylated Rab7 is significantly higher on the membrane compared to the cytosol. Furthermore, the membrane staining of the S72-phosphorylated Rab7 indicates that it is a subpopulation of the total Rab7 that is associated with vesicular structures. These results indicate that phosphorylation may occur on the membrane and could be a mechanism of controlling late endosome/lysosome transport and/or fusion. As the S72 site is highly conserved and located in the switch II region, which is required to bind the switch I region upon GTP binding and activation, I performed an in vitro GTP hydrolysis assay using [α-P32]GTP to determine the GTPase activity of S72-phosphorylated Rab7 compared to the unphosphorylated protein. Interestingly, there was a significant increase in GTP hydrolysis over time in the phosphorylated Rab7 compared to the unphosphorylated protein. The results of this study indicate that phosphorylation of Rab7 at S72 promotes differential interaction of Rab7 with certain effectors, which may promote its recruitment for specific functions. These results also indicate that phosphorylation may increase Rab7 turnover in vivo by stimulating the intrinsic GTPase activity.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.647250  DOI: Not available
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