IFIBYNE   05513
INSTITUTO DE FISIOLOGIA, BIOLOGIA MOLECULAR Y NEUROCIENCIAS
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Visualization of the activation/deactivation cycle of RhoC in inflammatory breast cancer
Autor/es:
ELIZABETH KENNEDY; ALEJANDRA C VENTURA; ZHIFEN WU; HECTOR GARCIA; SHWETHA MADDUR; SOFIA D MERAJVER
Reunión:
Conferencia; 2010 AACR Annual Meeting; 2010
Resumen:
A key event in the development of Inflammatory Breast Cancer, a particularly aggressive, metastatic form of breast cancer, isthe over-expression of the GTPase protein RhoC. RhoC plays a role in regulating cell shape, attachment and motility andover-expression is likely associated with tumor proliferation. RhoC is activated into a GTP-bound state by the regulatoryproteins GEFs (guanine nucleotide exchange factor proteins) and is deactivated to a GDP-bound state by GAPs (GTPaseactivating proteins). Stimulation of IBC cells with LPA (lysophophatidic acid), an activator of the cycle, showed a transientRhoC-GTP increase, peaking at 2 minutes and then diminishing until 20 minutes. It has been suggested that LPA can activateboth GEF and GAP proteins, and that a delay in the GAP protein activation could explain this RhoC-GTP behavior. BecauseRhoC activity occurs at the plasma membrane, the translocation of GEF and GAP proteins was studied as an indication ofRhoC interaction. Immunofluorescent stains for the three key proteins_RhoC, GAP, and GEF_were performed on an IBCcell line before and after stimulation with LPA and observed using confocal microscopy. Preliminary images revealed aco-localization of three proteins RhoC, p190B (a GAP protein), and PDZ (a GEF protein) in the cytosol, forming a gradientof high to low protein concentration from the nuclear membrane to the plasma membrane. Further analysis with ImageJsoftware showed slight differences in protein concentration at the two membranes upon stimulation with LPA. RhoC proteinlevels increased at both membranes, PDZ quantities increased slightly at the plasma membrane, and the p190B levelsdecreased at both membranes as a result of stimulation. This data suggests that after 5 minutes, LPA has a positive regulatoryeffect on the GEF protein and a negative one on the GAP protein at the plasma membrane. In addition, co-localization of theproteins RhoC and p190B was analyzed at different time points. The data indicates a decrease in co-localization after LPAstimulation that is slightly recovered 20 minutes after treatment. A possible explanation for these results is that 5 minutesafter stimulation, RhoC undergoes translocation to the nuclear and plasma membranes, and is not followed by the GAPprotein until later times. As of now, these conclusions are preliminary. These results will be verified using a new RhoCantibody, and additional co-localization analysis.