Identifizierung und biochemische Charakterisierung von p120ctn als Interaktionspartner der GTPase RhoC in der humanen Pankreaskarzinomzelllinie PANC-1
FacultiesFakultät für Naturwissenschaften
LicenseStandard (Fassung vom 01.10.2008)
Rho GTPases are members of the Ras superfamily of monomeric GTP-binding proteins which cycle between an active GTP-bound and an inactive GDP-bound state. In the present study RhoA and RhoC, which exhibit 92 % amino acid identity, were characterized comparatively. The aim was to identify interaction partners that bind differentially to both Rho GTPases. For the first time there was evidence of expression of the already known interaction partners of Rho GTPases RhoGDIa and RhoGDIb in the soluble fraction of EGFP-, EGFP-RhoA- and EGFP-RhoC-expressing PANC-1 cells. Coimmunoprecipitation experiments and in vitro binding studies showed no differences for the interaction of EGFP-RhoA with RhoGDIa and RhoGDIb to the interaction of EGFP-RhoC with the RhoGDIs. With further investigations the interaction of RhoA and for the first time ever of RhoC with members of the armadillo-repeat protein family p120ctn could be observed. p120ctn isoforms could be equally detected in cell lysates of EGFP-RhoA- and EGFP-RhoC-expressing PANC-1 cells. By using recombinant, bacterially expressed and purified GST-RhoA- and GST-RhoC-fusion proteins in in vitro binding experiments could be demonstrated that RhoA and RhoC interact differentially with p120ctn isoforms. With GST-RhoC there could be precipitated 40 % more of p120ctn isoforms than with GST-RhoA in cell lysates of PANC-1 cells. For the first time ever there was evidence of the interaction of p120ctn isoforms with RhoC in PANC-1 cells. In addition, some further in vitro binding experiments with GST-RhoC mutants led to the identification of the serin residue 152 of RhoC which is important for the interaction of RhoC and p120ctn isoforms. In summary, for the first time ever the present study demonstrated the interaction between the Rho GTPase RhoC and p120ctn isoforms. The different amino acid of RhoA and RhoC in position 152 has been identified to draw a significant distinction at the interaction with p120ctn.
Subject HeadingsBauchspeicheldrüsenkrebs [GND]
Pancreatic neoplasms [MeSH]
rho GTP-binding proteins [MeSH]