CONICET | Buscador de Institutos y Recursos Humanos

The cdc42 interacting protein 4 (CIP4) is an effector of cdc42 that acts in membranedeformation during endocytosis and lamellapodial protrusions. CIP4 is up-regulatedduring epithelial-mesenquimal transition, and is essential for the development ofmetastatic properties in different types of cancer cells. Previous work in our groupshows the interaction of CIP4 with the PKA anchoring protein AKAP350 and thatthis interaction affects its subcellular localization and its pro-migratory ability. Alsowe demonstrated that CIP4 is a substrate of PKA. The aim of this study was toevaluate if the CIP4 pro-invasive ability is regulated throw its phosphorylation byPKA. In silico analysis revealed four potential sites for PKA phosphorylation. Usingin vitro phosphorylation experiments we observe that only the CIP4T255A mutationinhibited completely the CIP4 phosphorylation. Afterwards we generatedhepatocarcinoma HepG2 stable cell lines expressing CIP4, CIP4T225A (nonphosphorylatable mutant) or CIP4T225E (phosphomimetic mutant). The migratorycapacity in wound healing assays showed an inhibition in CIP4T225A (- 43*) andan inverse effect in CIP4T225E cell line (+44*). Studies using transwell invasionassays also demonstrated that their invasive capacities greatly differ, a stronginhibition in CIP4T225A (-90%*) in contrast with an increase in CIP4T225E(+800%*) relative to control. Further experiments in xenotropic experimentalmetastasis assays in nude mouse revealed a similar effect in metastatic potential inthe number and size of the tumors generated in the lung by the cell lines expressingthe different constructs. These findings unveil a novel signaling pathway inhepatocarcinoma metastasis process involving CIP4/PKA in which thephosphorylation of CIP4 in T225 residue by PKA is a crucial event in theadquisition of metastatic potential in hepatocarcima cells (*p