Regulation of Ras-Associated Protein-1 By Kinase responsive to Stress B in Dictyostelium discoideum

Abstract

Dictyostelium discoideum is a social amoeba that is commonly used as a model organism for studying chemotaxis, which is a directed migration along a chemical gradient, due to its similarities to human neutrophils and metastatic cancer cells. There are multiple pathways involved in regulating migration. In particular, kinase responsive to stress B (KrsB), a homolog of mammalian tumor suppressor MST1/2 and Drosophila Hippo, is a negative regulator of cell adhesion and migration in D. discoideum. However, little is known about the molecular mechanism of KrsB action. Another regulator of adhesion is small GTPase Ras-associated protein 1 (Rap1), which acts by affecting talin and myosin II. In mammalian cells Rap1 can be phosphorylated, which leads to its inhibition. We hypothesized that KrsB might negatively regulate Rap1 by phosphorylation, thereby disrupting the activation of Rap1 on the membrane. To determine if KrsB phosphorylates Rap1 we will perform immunoblotting for Rap1 in cells with or without KrsB and look for a shift in the electrophoretic mobility as an indicator of phosphorylation. In this study, we were able to detect RFP-tagged constitutively active Rap1G12V on an immunoblot using an antibody against mCherry. We will now continue to conduct immunoblotting to detect mobility shifts of phosphorylated Rap1. To be able to track Rap1 localization, we successfully generated an RFP-Rap1 expression construct. We will examine RFP-Rap1 localization in cells with or without KrsB.