Ha-Ras^V12-Induced Multilayer Cellular Aggregates Is Mediated by Rac1 Activation Rather Than YAP Activation

We demonstrate that Ha-Ras^V12 overexpression induces the nuclear translocation of Hippo effector Yes-associated protein (YAP) in MDCK cells via the hippo-independent pathway at the confluent stage. Ha-Ras^V12 overexpression leads to the downregulation of Caveolin-1 (Cav1) and the disruption of junction integrity. It has been shown that the disruption of actin belt integrity causes YAP nuclear translocation in epithelial cells at high density. Therefore, we hypothesized that Ha-Ras^V12-decreased Cav1 leads to the disruption of cell junction integrity, which subsequently facilitates YAP nuclear retention. We revealed that Ha-Ras^V12 downregulated Cav1 through the ERK pathway. Furthermore, the distribution and expression of Cav1 mediated the cell junction integrity and YAP nuclear localization. This suggests that the downregulation of Cav1 induced by Ha-Ras^V12 disrupted the cell junction integrity and promoted YAP nuclear translocation. We further indicated the consequence of Ha-Ras^V12-induced YAP activation. Surprisingly, the activation of YAP is not required for Ha-Ras^V12-induced multilayer cellular aggregates. Instead, Ha-Ras^V12 triggered the ERK-Rac pathway to promote cellular aggregate formation. Moreover, the overexpression of constitutively active Rac is sufficient to trigger cellular aggregation in MDCK cells at the confluent stage. This highlights that Rac activity is essential for cellular aggregates.