A negative feedback loop between small GTPase Rap1 and mammalian tumor suppressor homologue KrsB regulates...

Cell adhesion to the substrate influences a variety of cell behaviors and its proper regulation is essential for migration, although details of the molecular pathways regulating cell adhesion during migration are lacking. Rap1 is a small GTPase that regulates adhesion in mammalian cells, as well as in Dictyostelium discoideum social amoeba, which is an established model for studying directed cell migration. In Dictyostelium, Rap1 controls adhesion via its effects on adhesion mediator talin and Ser/Thr kinase Phg2, which inhibits myosin II function. Kinase responsive to stress B (KrsB), a homologue of mammalian tumor suppressor MST1/2 and Drosophila Hippo, also regulates cell adhesion and migration, although the molecular mechanism of KrsB action is not understood. Because KrsB has been shown to interact with active Rap1 by mass spectroscopy, we investigated the genetic interaction between Rap1 and KrsB. Cells lacking KrsB have increased adhesion to the substrate, which leads to reduced movement. Expression of constitutively active Rap1 G12V increased cell spreading and adhesion even in the absence of KrsB, suggesting that Rap1 does not require KrsB to mediate cell adhesion. In contrast, KrsB activation requires Rap1 since dominant-negative Rap1 S17N impaired KrsB phosphorylation, which has been previously shown to be necessary for KrsB activity and its function in adhesion. Even though Rap1 did not require KrsB for its function in adhesion, KrsB negatively regulates Rap1 function as seen by increased cortical localization of active Rap1 in KrsB-null cells. Consistently, Rap1 S17N completely reversed the overadhesive phenotype of KrsB-null cells. Furthermore, chemoattractant-induced activation of downstream effectors of Rap1, TalB and Phg2, was increased in the absence of KrsB. Taken together, these findings suggest that Rap1 leads to activation of KrsB, which inhibits Rap1 and its downstream targets, shutting off adhesion. The existence of a negative feedback loop between Rap1 and KrsB may contribute to the dynamic regulation of cell adhesion that is necessary for rapid amoeboid-type migration.