Calmodulin binds RalA and RalB and is required for the thrombin-induced activation of Ral in human platelets.
Ral GTPases may be involved in calcium/calmodulin-mediated 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 calcium-independent 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.
Clough
Richard R
RR
Department of Oral Biology, University of Manitoba, Winnipeg, Manitoba R3E 0W2, Canada.
Sidhu
Ranjinder S
RS
Bhullar
Rajinder P
RP
D001792:Blood Platelets / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism*; D004789:Enzyme Activation; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D014176:Protein Biosynthesis; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013917:Thrombin / Q000378:metabolism*; D014158:Transcription, Genetic; D020798:Two-Hybrid System Techniques; D029867:Xenopus Proteins; D020725:ral GTP-Binding Proteins / Q000378:metabolism*
D002147:Calmodulin; C119391:RalB protein, Xenopus; D011993:Recombinant Fusion Proteins; D029867:Xenopus Proteins; D005982:Glutathione Transferase; D013917:Thrombin; D020558:GTP Phosphohydrolases; C492198:RALA protein, human; D020725:ral GTP-Binding Proteins; D002118:Calcium