Ral GTPase interacts with the N-terminal in addition to the C-terminal region of PLC-delta1.
Previously, we have shown that RalA, a calmodulin (CaM)-binding protein, binds to the C2 region in the C-terminal of PLC-delta1, and increases its enzymatic activity. Since PLC-delta1 contains a CaM-like region in its N-terminus, we have investigated if RalA can also bind to the N-terminus of PLC-delta1. Therefore, we created a GST-PLC-delta1 construct consisting of the first 294 amino acids of PLC-delta1 (GST-PLC-delta1(1-294)). In vitro binding experiments confirmed that PLC-delta1(1-294) was capable of binding directly to RalA. W-7 coupled to polyacrylamide beads bound pure PLC-delta1, demonstrating that PLC-delta1 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-delta1 were able to inhibit RalA binding to PLC-delta1(1-294). This study demonstrates that there are two binding sites for RalA in PLC-delta1 and provides further insight into the role of Ral GTPase in the regulation of PLC-delta1 function.
Grujic
Ognjen
O
Department of Oral Biology, University of Manitoba, 780 Bannatyne Avenue, Winnipeg, MB R3E0W2, Canada.
Bhullar
Rajinder P
RP
D000595:Amino Acid Sequence; D000818:Animals; D002147:Calmodulin / Q000378:metabolism; D002148:Calmodulin-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D002417:Cattle; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D054803:Phospholipase C delta / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D051381:Rats; D020725:ral GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*
D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D054803:Phospholipase C delta; D020725:ral GTP-Binding Proteins