CONICET | Buscador de Institutos y Recursos Humanos

The cdc42 interacting protein 4 (CIP4) is an effector of cdc42 that acts in membrane deformation during endocytosis and lamellapodial protrusions. CIP4 is up-regulated during epithelial-mesenquimal transition, and is essential for the development of metastatic properties in different types of cancer cells. Previous work in our group shows the interaction of CIP4 with the PKA anchoring protein AKAP350 and that this interaction affects its subcellular localization and its pro-migratory ability. Also we demonstrated that CIP4 is a substrate of PKA.The aim of this study was to evaluate if the CIP4 pro-invasive ability is regulated throw its phosphorylation by PKA. In silico analysis revealed four potential sites for PKA phosphorylation. Using in vitro phosphorylation experiments we observe that only the CIP4T255A mutation inhibited completely the CIP4 phosphorylation. Afterwards we generated hepatocarcinome HepG2 stable cell lines expressing CIP4, CIP4T225A (non phosphorylatable mutant) or CIP4T225E (phosphomimetic mutant). The migratory capacity in wound healing assays showed an inhibition in CIP4T225A (- 43*) and an inverse effect in CIP4T225E cell line (+44*). Studies using transwell invasion assays also demonstrated that their invasive capacities greatly differ, a strong inhibition in CIP4T225A (-90%*) in contrast with an increase in CIP4T225E (+800%*) relative to control. Further experiments in xenotropic experimental metastasis assays in nude mouse revealed a similar effect in metastatic potential in the number and size of the tumors generated in the lung by the cell lines expressing the different constructs. These findings unveil a novel signaling pathway in hepatocarcinome metastasis process involving CIP4/PKA in which the phosphorylation of CIP4 in T225 residue by PKA is a crucial event in the adquisition of metastatic potential in hepatocarcime cells (*p