A46
Determination of RhoJ signalling pathways in endothelial cells
Katarzyna Bunko, Roy Bicknell, Victoria Heath
University of Birmingham, Medical School, Institute for Biomedical Research, Division of Immunity and Infection, Birmingham, West Midlands, UK
Endothelial cells are critical for the formation of new blood vessels (angiogenesis), a process important in the pathogenesis of solid tumour. RhoJ (TCL) is an endothelial expressed protein that has been implicated in endothelial cell movement, growth and tube formation. Little is yet known about the pathways and proteins that interact with RhoJ in endothelial cells. Our aims were to identify signals which activate RhoJ in the vasculature.
RhoJ is a small Rho GTPase, which cycles between active GTP-bound and inactive GDP-bound forms. These transitions are likely to be regulated by three groups of proteins: GTPase activating proteins, guanine nucleotide exchange factors (GEFs) and guanine dissociation inhibitors. An assay was developed to assess levels of active RhoJ and was validated in human umbilical vein endothelial cells (HUVECs). This uses GST tagged Cdc42/Rac interactive-binding (CRIB) domain attached to agarose beads. CRIB domain binds only active RhoJ, and pulls it down from the whole cellular lysate. This has been used to identify stimuli which activate RhoJ, including vascular endothelial growth factor. Another assay has also been developed to identify GEFs and effecter proteins, which utilizes constitutively active or inactive RhoJ mutants.
In summary, RhoJ is a protein involved in endothelial cell biology and angiogenesis. Our work describes the development of tools to determine the signalling pathways in which RhoJ is involved.
