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Regulation of Ras-Associated Protein-1 By Kinase responsive to Stress B in Dictyostelium discoideum
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2021
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Quest2021_26.pdf
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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.