Poster Presenter

Interaction Between the Ral Gtp-Binding protein and Phospholipase c Delta1
Rajinder Pal Bhullar
Canada

Ras p21 related or small guanine nucleotide binding proteins have been implicated in the regulation of a variety of cellular processes including: cell growth and cell movement, exocytosis and other physiological functions. This often requires an interaction between the GTP-binding protein and cellular proteins. Small GTP-binding proteins have been shown to bind and regulate phosphoinositide-specific phospholipase C (PLC). Ral is a small GTP-binding protein that has been shown to interact with cellular proteins such as, calmodulin and RIP1. We used the yeast two hybrid system to identify new interacting proteins for Ral. We have shown that Ral interacts with PLC-δ1, an enzyme that plays a crucial role in regulating intracellular calcium concentration through its role in the generation of inositol 1,4,5-trisphosphate, a universal calcium mobilizing agent. Previously, we have shown that RalA 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. We created a GST-PLC-δ1 construct consisting of the first 294 amino acids of PLC-δ1 (GST-PLC-δ11-294). In vitro binding experiments confirmed that PLC-δ11-294 was capable of binding directly to RalA and PLC-δ1. 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-δ11-294. 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.