Ral GTPase interacts with the N-terminal in addition to the C-terminal region of PLC-δ1

Abstract

Previously, we have shown that RalA, a calmodulin (CaM)-binding protein, binds to the C2 region in the C-terminal of PLC-δ1, and increases its enzymatic activity. Since PLC-δ1 contains a CaM-like region in its N-terminus, we have investigated if RalA can also bind to the N-terminus of PLC-δ1. Therefore, we created a GST-PLC-δ1 construct consisting of the first 294 amino acids of PLC-δ1 (GST-PLC-δ1 1–294). In vitro binding experiments confirmed that PLC-δ1 1–294 was capable of binding directly to RalA. W-7 coupled to polyacrylamide beads bound pure PLC-δ1, demonstrating that PLC-δ1 contains a CaM-like region. Competition assays with W-7, peptides representing RalA and the newly identified RalB CaM-binding regions, or the IQ peptide from PLC-δ1 were able to inhibit RalA binding to PLC-δ1 1–294. This study demonstrates that there are two binding sites for RalA in PLC-δ1 and provides further insight into the role of Ral GTPase in the regulation of PLC-δ1 function.