(805.25) Kinase Responsive to Stress B negatively regulates Rap1 in Dictyostelium discoideum

Poster Board Number: A266

Tiffany Flores (State University of New York at Oswego), Kelsey Roberts (State University of New York at Oswego), Yulia Artemenko (State University of New York at Oswego)

Dynamic changes in cell adhesion to the substrate are essential for migration, although details of the pathways regulating adhesion, especially for amoeboid-type migration are lacking. Social amoeba Dictyostelium discoideum is a commonly used model organism for the study of directed migration since its movement is very similar to that of other amoeboid cells, such as neutrophils and metastatic cancer cells. Rap1 is a small GTPase that regulates adhesion in Dictyostelium cells in part via its effects on myosin II and talin. Kinase responsive to stress B (KrsB), a homolog 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. Since 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 contact with the substrate and are difficult to detach from the surface, which leads to reduced movement. Expression of constitutively active Rap1G12V increased cell adhesion, and inactive Rap1S17N reduced cell adhesion even in the absence of KrsB, suggesting that Rap1 does not require KrsB to mediate cell adhesion. However, Rap1G12V did not increase cell spreading when KrsB was overexpressed and Rap1-mediated increase in spreading was dramatically enhanced in the absence of KrsB, suggesting that KrsB might negatively regulate Rap1. In addition, chemoattractant-induced activation of Rap1, as assessed by transient cortical localization of the biosensor RalGDS, was impaired in krsB¯ cells, possibly due to increased basal activity or membrane localization of Rap1. Since mammalian Rap1 localization can be regulated by phosphorylation, KrsB might phosphorylate Rap1, thereby disrupting its activation on the membrane. Overall, our study demonstrates that KrsB negatively regulates Rap1 signaling, and we are currently investigating the molecular mechanism of this regulation.

NSF-RUI grant no. 1817378