Investigating the role played by protein-lipid and protein-protein interactions in the membrane association of the p120GAP CaLB domain.
The GTPase activating protein, p120GAP, contains an amino acid sequence motif called the Ca2+-dependent lipid binding domain (CaLB) which mediates a protein-protein interaction between p120GAP and annexin VI and also binds to negatively charged phospholipids. Because membrane association of p120GAP is important for the regulation of p21 Ras activity, we have studied the roles played by Ca2+, phospholipids and annexin VI in the membrane association of p120GAP. Here we demonstrate that a truncated CaLB domain GST fusion protein (GSTGAP618-632), lacking the ability to bind to phospholipids, is able to bind to rat fibroblast membranes in a Ca2+- and concentration-dependent manner. In addition, this fusion protein also binds to annexin VI in an amino acid sequence specific but Ca2+ independent manner. Also, when bound to annexin VI in the presence of Ca2+, this fusion protein has the ability to co-bind to phosphatidylserine vesicles. Thus, annexin VI may simultaneously mediate an interaction with p120GAP and also an interaction with membrane phospholipids. This may in part explain the mechanism by which p120GAP associates with membranes in response to Ca2+ elevation and suggests the potential importance of annexin VI in the regulation of p21 Ras and the role CaLB domains may play in the specific recognition of cellular membranes.
Chow
A
A
Department of Pharmacology, University of Leeds, United Kingdom.
Davis
A J
AJ
Gawler
D J
DJ
D000595:Amino Acid Sequence; D000818:Animals; D017317:Annexin A6 / Q000378:metabolism*; D001665:Binding Sites; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D020690:GTPase-Activating Proteins; D006801:Humans; D008969:Molecular Sequence Data; D010743:Phospholipids / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D020703:ras GTPase-Activating Proteins
D017317:Annexin A6; D020690:GTPase-Activating Proteins; D010743:Phospholipids; D011506:Proteins; D011993:Recombinant Fusion Proteins; D020703:ras GTPase-Activating Proteins