The role of protein kinase n in gastric cancer

Resumo

Extracto de Tesis 2.2 Mass spectrometry screening of effector binding to wild type and mutant RHOA As an independent and unbiased method to identify changes in the RHOA interactome caused by the recurrent RHOA mutations found in DGC (184, 185), a pulldown assay coupled with liquid chromatography/mass spectrometry (LC/MS) analysis was performed (Figure 39). Wild type RHOA (not carrying the G14V or the I90S mutations needed for the yeast two-hybrid assay, or any of the GC hotspot mutations) or the RHOA-R5Q, -G17E, - Y42C or -L57V mutants were produced in bacteria as a fusion protein with GST. These proteins were purified and used as ‘bait’ to identify binding proteins in a protein lysate obtained from mixing equal amounts of total protein extracted from 9 different diffuse gastric cancer cell lines, used here as a protein lysate representative of human gastric tumors with diffuse histology (Figure 39A). RHOA and interacting proteins were pulled down with glutation sepharose beads and binding proteins were eluted and identified using liquid chromatography/mass spectrometry analysis (LC/MS; Figure 39B). As expected, wild type RHOA was found to bind to multiple proteins that are known RHOA interactors, including PKN2, RALY, CBX1, NHP2L1 and H2AFY (supplementary Table 8). Interestingly, there are some notable absences on that such as ROCK and DIAPH1, which are among the best known RHOA effectors, but this might be due to these proteins not being expressed at significant levels in diffuse gastric cancer cell lines. Further analysis needs to be done in order to address this question. Importantly, a significant number of proteins binding to wild type RHOA but not to any of the RHOA mutants investigated were identified (supplementary Table 8). PKN2 was found to be the most abundant protein interacting with wild type RHOA but not with any of the RHOA mutants investigated (Figure 39C; supplementary Table 8). To validate these results, we tested by Western blot the levels of PKN2 in the pulldown with the different mutations of RHOA. In good agreement with the mass spectrometry results we observed robust biding of wild type RHOA to PKN2.