Investigation of Calmodulin Interactions in Dictyostelium discoideum Chemotaxis

Abstract

In the model organism Dictyostelium discoideum, the purpose of calcium signaling inchemotaxis to cAMP is not well understood. The calcium-sensing protein calmodulin, highly conserved across eukaryotes, may play a role in chemotactic calcium signaling, as shown by other work with calmodulin antagonists. I investigated the role of calmodulin in chemotaxis signaling and found that the calmodulin antagonist W-7 may affect phosphorylation of the chemotactic proteins PKBA, PKBR1, and ERK1 and ERK2. Phosphorylation of these proteins begins with stimulation of the cAMP receptor 1 (cAR1), and preliminary experiments show cAR1 may co-immunoprecipitate with calmodulin, which indicates a possible interaction. To further investigate a possible cAR1-calmodulin interaction, I performed bioluminescence resonance energy transfer (BRET) experiments with the two proteins. I detected a BRET signal that, although not affected by cAMP stimulation, may indicate an interaction between cAR1 and calmodulin. For the further identification of calmodulin-binding proteins, I developed a protocol for the purification of calmodulin-binding proteins using Calmodulin-Sepharose beads in D. discoideum. Overall, my results suggest a possible interaction between the receptor cAR1 and the Ca2+ signaling protein calmodulin in D. discoideum. I have also worked towards developing a method of identifying other calmodulin-binding proteins using Calmodulin-Sepharose beads that will be useful in investigating the role of calmodulin and calcium signaling in chemotaxis.