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

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Title:
Regulation of Ras-Associated Protein-1 By Kinase Responsive to Stress B in Dictyostelium discoideum
Creator:
Tiffany Flores
Yulia Artemenko
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Poster

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Biology
Cloning

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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 are currently generating an RFP-Rap1 expression construct. Once complete, we will examine RFP-Rap1 localization in cells with or without KrsB.
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Collected for SUNY Oswego Institutional Repository by the online self-submittal tool. Submitted by Tiffany Flores.

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SUNY Oswego Institutional Repository
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SUNY Oswego Institution
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Regulation of RasAssociated Protein1 By Kinase Responsive to Stress B in Dictyostelium discoideum Tiffany Flores, Dr. Yulia ArtemenkoDepartment of Biological Sciences, SUNY Oswego, Oswego, NY This work was supported by NSFRUI grant no. 1817378 (to Y.A.).Acknowledgements CLONING : •PCR for Rap1 yielded a lot of product and digest of both the insert and vector were successful. Gel extraction and purification also proved to work for the Rap1 insert. Two different ligation reactions were used to do transformation. Colonies from transformation were screened but none gave the expected pattern in the diagnostic digest. WESTERN BLOT: •Immunoblotting showed that the antibody against mCherry can be used to detect RFP -tagged Rap1.Future Directions•Continue cloning RFP -tagged Rap1.•Analyze the phosphorylation of Rap1 in the presence/absence of KrsB by gel electrophoresis 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 Rasassociated 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 Rap1G12Von 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 are currently generating an RFP Rap1 expression construct. Once complete, we will examine RFP Rap1 localization in cells with or without KrsB.Abstract •Dictyostelium discoideum is a social amoeba that has been used widely as a model organism.•The life stages of Dictyostelium discoideum make it uniquely suited to study migration and chemotaxis – processes that contribute to human diseases such as cancer.1( https://en.wikipedia.org/wiki/Dictyostelium_discoideum ) KrsB (Kinase Responsive to Stress B) •KrsB is a homolog of tumor suppressors MST1/2 in mammalian cells and Drosophila Hippo.1•KrsB plays a role in chemotaxis by being a negative regulator of cell adhesion and migration.1•Cells lacking KrsB have increased contact with the surface and are more difficult to detach.1 Rap1 (Ras Associated Protein 1) •Rap1 is a small GTPase that is known to regulate adhesion in mammalian cells and in Dictyostelium discoideum.2,4•In mammalian cells, Rap1 can be regulated by phosphorylation by cAMP dependent protein kinase A (PKA), and this phosphorylation negatively regulates Rap1.2•Previous studies showed that Rap1 can regulate cell adhesion without KrsB, although KrsB might modulate Rap1 function.4 HYPOTHESIS •KrsB negatively regulates Rap1 by phosphorylation, which disrupts activation of Rap1 on the membrane. Introduction ApproachTo look at the regulation of Rap1 by KrsB we plan to: 1)Look at the localization of RFP -tagged Rap1 in the presence or absence of KrsBThis requires cloning of Rap1 into an expression plasmid with an RFP gene.2)Look at the phosphorylation of Rap1 in the presence or absence of KrsBTo do this we need to examine if RFP tagged Rap1 has an electrophoretic mobility shift indicative of phosphorylation in cells with KrsB compared to without.To perform immunoblotting for RFP tagged Rap1, first we need to test whether the mCherry antibody is able to recognize the RFP tag. Methodology DiscussionReferences1.Artemenko, Y., Batsios, P., Borleis, J., Gagnon, Z., Lee, J., Rohlfs, M., Sanseau, D., Willard, S., Schleiher, M., & Devreotes, P. (2012). Tumor suppressor Hippo/MST1 kinase mediates chemotaxis by regulating spreading and adhesion. PNAS, 1363213637. doi: 10.1073/pnas.1211304109.2.Takahashi M, Dillon TJ, Liu C, Kariya Y, Wang Z, Stork PJ. Protein kinase A dependent phosphorylation of Rap1 regulates its membrane localization and cell migration. J Biol Chem . 2013;288(39):27712 27723. doi:10.1074/jbc.M113.4669043.DictyBase. Dictyostelium discoideum : Model System in Motion. http://dictybase.org/tutorial/ (accessed August 17, 2020)4.Niu , G. (2020). Genetic Interaction between Adhesion Regulators Rap1 and Kinase Responsive to Stress B in Dictyostelium discoideum. Honors Thesis, SUNY Oswego. Western Blot Testing mCherry antibody Cloning of RFP-Rap1 Figure 1. PCR of the Rap1 gene was successful and yielded intense bands on an agarose gel. Figure 2. Detection of RFP tagged constitutively active Rap1 (G12V) with an antibody against mCherry . Wild type cells expressing RFPRap1 G12V were lysed, proteins were separated by SDSPAGE and transferred to a PVDF membrane. Membrane was immunoblotted with a primary antibody against mCherry , followed by a secondary antibody conjugated with horseradish peroxidase. Signal was detected by chemiluminescence. RFP Rap1 G12V band was detected at around 40 kDa . Note that other lanes on the gel contained unrelated samples.Results