Previously we have reported that RalA binds to the C2 domain region of PLC-δ1 in a calcium dependent manner [16]. In the present study we demonstrate that the N-terminus of PLC-δ1, comprised of the PH and EF-hand domains, contains a calcium dependent RalA binding domain. This represents a second Ral binding site within PLC-δ1 (Fig. 4C). It has been shown that the C-terminus of RalA binds CaM in a calcium dependent manner [24]. PLC-δ1 has a CaM-like structure in its N-terminal [7]. Based on this information it is reasonable to expect that the C-terminus of RalA can interact with the N-terminus of PLC-δ1. In fact, our
ASP 3026 assay results with the RalA C-terminal peptide provide evidence in support of this hypothesis. The 3D-structure of PLC-δ1 places the EF-hand domain in close proximity to the C2 domain. In fact, these two domains make direct contact [7]. Thus each of the two domains could bind one molecule of RalA, or one site could be preferred over the other. The EF-hand domain appears to be more accessible for protein binding. As reported previously, RalA increases activity of PLC-δ1 in vitro [16], possibly by targeting PLC-δ1 to the
plasma membrane where its substrate is found. PLC-δ1 is thought to be activated by increase in intracellular calcium concentration initiated by PLC-β, PLC-γ, and PLC-ε [3]. Thus it appears plausible that the interactions of Ral with PLC-δ11–294 are stronger in the presence of calcium. On the other hand, it has been suggested that membrane attachment could be a way to down regulate the enzyme [29].
