Calmodulin binding to the small GTPase Ral requires isoprenylated Ral

Ral, a member of the Ras-p21 superfamily of small GTPases, has been shown to require the calcium-signaling protein calmodulin (CaM) for activation. In the present work, we investigated the properties of the Ral-CaM interaction. Using CaM affinity binding assay with lysates from mammalian cells overexpressing various Ral mutants, we found that RalB(V23, ΔCAAX) lacking the C-terminal isoprenylation region bound significantly less efficiently to CaM. Binding of other mutants containing critical amino acid changes in the nucleotide or substrate binding regions (residues 23, 28, and 49) was not affected. In addition, all mutants bound significantly better in the presence of calcium versus the calcium chelator EGTA. Using in vitro transcription-translation in the presence of geranylgeranyl pyrophosphate, we demonstrate enhanced Ral binding to CaM. Inhibition of isoprenylation in cells in culture with lovastatin resulted in decreased binding of CaM to Ral. The present results show that post-translational isoprenylation of Ral is important in Ral-CaM interaction.