Calmodulin Binds RalA and RalB and Is Required for the Thrombin-induced Activation of Ral in Human Platelets

Abstract

Ral GTPases may be involved in calcium/calmodulinmediated intracellular signaling pathways. RalA and RalB are activated by calcium, and RalA binds calmodulin in vitro. It was examined whether RalA can bind calmodulin in vivo, whether RalB can bind calmodulin, and whether calmodulin is functionally involved in Ral activation. Yeast two-hybrid analyses demonstrated both Rals interact directly but differentially with calmodulin. Coimmunoprecipitation experiments determined that calmodulin and RalB form complexes in human platelets. In vitro pull-down experiments in platelets and in vitro binding assays showed endogenous Ral and calmodulin interact in a calcium-dependent manner. Truncated Ral constructs determined in vitro and in vivo that RalA has an additional calmodulin binding domain to that previously described, that although RalB binds calmodulin, its C-terminal region is involved in partially inhibiting this interaction, and that in vitro RalA and RalB have an N-terminal calciumindependent and a C-terminal calcium-dependent calmodulin binding domain. Functionally, in vitro Ral-GTP pull-down experiments determined that calmodulin is required for the thrombin-induced activation of Ral in human platelets. We propose that differential binding of calmodulin by RalA and RalB underlies possible functional differences between the two proteins and that calmodulin is involved in the regulation of the activation of Ral-GTPases.

Regulation of Phospholipase C-δ1 through Direct Interactions with the Small GTPase Ral and Calmodulin

“…We and others (29,30,39) have previously shown that Ral is an important partner in calcium and Ca 2ϩ /calmodulin mediated signal transduction pathways. Also, PLC-␦1 is highly sensitive to calcium concentrations and can be activated at concentrations as low as 10 M (40, 41).…”

“…CaM binding assays were performed using 50 l of calmodulinSepharose 4B beads or Sepharose 4B beads blocked with Tris buffer as described previously (30). To confirm the dependence on Ca 2ϩ for CaM binding to PLC-␦1, reactions were performed by incubating purified CaM with GST-PLC-␦1 overnight at 4°C diluted in commercially available Ca 2ϩ calibration buffers (Molecular Probes) exactly as described previously (35).…”

“…These proteins are also important partners in calcium and Ca 2ϩ /calmodulin (Ca 2ϩ /CaM)-mediated signal transduction pathways (29,30). We have shown that RalA and -B bind CaM in a Ca 2ϩ -dependent and -independent manner (30). Calcium-dependent CaMbinding domains in target proteins share little sequence identity but many have some common characteristics.…”

“…Ral proteins possess pivotal roles in diverse cell functions such as the control of cell proliferation (20), migration (21), filopod formation (22), differentiation (23), cytoskeletal organization (24), vesicular transport (25), exocytosis (26), and receptor endocytosis (27,28). These proteins are also important partners in calcium and Ca 2ϩ /calmodulin (Ca 2ϩ /CaM)-mediated signal transduction pathways (29,30). We have shown that RalA and -B bind CaM in a Ca 2ϩ -dependent and -independent manner (30).…”

“…These studies point to a direct role for Ral-CaM in the regulation of PLC-␦1 enzyme activity. (30). Positive clones were restreaked on selective media (lacking histidine, adenine, leucine, and tryptophan) and their DNA isolated and sequenced.…”