Investigating the downstream signalling of the Rho GTPase RhoJ

Salem, Mona ORCID: 0009-0000-7091-8464 (2023). Investigating the downstream signalling of the Rho GTPase RhoJ. University of Birmingham. M.Sc.

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Abstract

RhoJ is an endothelial expressed Rho GTPase which regulates cell migration, protein trafficking and angiogenesis. Previous studies demonstrated that GTP-bound RhoJ activated MAP kinases, interacted with the GIT-PIX complex, caused a retarded electromobility shift of GIT2 and promoted focal adhesion disassembly. The aims of this study were to further characterise how RhoJ regulates MAP kinases and the GIT-PIX complex. The activity of RhoJ was manipulated in human umbilical vein endothelial cells (HUVECs) by either lentiviral transduction of constitutively active mutant form of RhoJ (daRhoJ) or transfection with RhoJ-specific si-RNA duplexes, to activate and inhibit it, respectively. Expression of daRhoJ resulted in a small increase in ERK1/2, but not JNK, phosphorylation in HUVECs cultured in complete media. In a time-course experiment, si-RNA-mediated RhoJ knockdown significantly reduced levels of phospho-ERK1/2, this was particularly evident after 15 min stimulation with VEGFA, FGF2 or complete growth media. Other groups had shown that the GIT-PIX complex interacted with and regulated the activity of MEK1 and ERK1/2, and that ERK1/2 localised to focal adhesions; we therefore aimed to test how RhoJ affected this. Experiments demonstrated that very low levels of GIT1 co-precipitated with either MEK1 or ERK1/2, and there was no evident co-localisation of ERK1/2 with vinculin, a focal adhesion protein. This low level of co-precipitation combined with some technical difficulties hindered the assessment of how RhoJ affected this interaction. This, combined with the lack of focal adhesion localisation of ERK1/2 led to our hypothesising that the GIT-PIX complex was not involved in RhoJ’s regulation of ERK1/2 in endothelial cells. One potential alternative mechanism is via p21-activated kinases (PAK), kinases known to be both downstream of RhoJ and able to regulate RAF1. The role of PAKs in the daRhoJ-mediated change in the electrophoretic mobility of GIT2 was assessed. Two PAK inhibitors were tested, and PF-3758309, but not IPA-3, abrogated GIT2’s mobility shift in daRhoJ expressing endothelial cells. This suggests that the electromobility shift of GIT2 is due to serine and threonine phosphorylation and the differential effect of the inhibitors suggests that PAK4 acts downstream of RhoJ to affect phosphorylation of GIT2. Data in this thesis further delineates the molecular pathways downstream of RhoJ and offers insight into the mechanisms by which RhoJ regulates its endothelial functions.

Type of Work:

Thesis (Masters by Research > M.Sc.)

Award Type:

Masters by Research > M.Sc.

Supervisor(s):