Abstract
Copines make up a family of calcium-dependent, phospholipid-binding proteins found in numerous eukaryotic organisms. Copine proteins consist of two C2 domains at the N-terminus followed by an A domain similar to the von Willebrand A domain found in integrins. We are studying copine protein function in the model organism, Dictyostelium discoideum, which has six copine genes, cpnA-cpnF. Previous research showed that cells lacking the cpnA gene exhibited a cytokinesis defect, a contractile vacuole defect, and developmental defects. To provide insight into the role of CpnA in these cellular processes, we used column chromatography and immunoprecipitation to isolate proteins that bind to CpnA. These proteins were identified by mass spectrometry. One of the proteins identified was actin. Purified CpnA was shown to bind to actin filaments in a calcium-dependent manner in vitro. cpnA− cells exhibited defects in three actin-based processes: chemotaxis, cell polarity, and adhesion. These results suggest that CpnA plays a role in chemotaxis and adhesion and may do so by interacting with actin filaments.
Highlights
Copines make up a family of calcium-dependent phospholipid-binding proteins found in many eukaryotic organisms [1,2]
We found that CpnA binds to actin filaments in a calcium-dependent manner in vitro
These results indicate that cells lacking cpnA are defective in their actin polymerization in their actin polymerization response to cAMP
Summary
Copines make up a family of calcium-dependent phospholipid-binding proteins found in many eukaryotic organisms [1,2]. Copine proteins contain two C2 domains in the N-terminal half of the protein followed by an A domain similar to the von Willebrand A (VWA) domain in the C-terminal half of the protein [1]. The C2 domain is a calcium-dependent phospholipid-binding motif originally identified in protein kinase C. Most proteins containing a single C2 domain are involved in signaling pathways, while most proteins that have multiple C2 domains are involved in membrane trafficking [3]. The VWA domain is named from the von Willebrand Factor, a plasma and extracellular matrix protein. VWA domains have been studied in integrins and several extracellular matrix proteins, and appear to function as protein-binding domains [4]. The exact function of copines is not known, a growing body of evidence suggests that copines may mediate an array of cellular processes by conferring calcium regulation to various signaling pathways [5,6,7,8]
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