Mutation-deletion analysis of a Ca(2+)-dependent phospholipid binding (CaLB) domain within p120 GAP, a GTPase-activating protein for p21 ras.
p120 GAP is a GTPase activating protein for p21 ras. It is a multidomain protein which exhibits sequence similarity with other GTPase-activating proteins, src, pleckstrin and a central portion of the protein kinase C conserved region 2 domain known as CaLB (Ca(2+)-dependent phospholipid-binding). The presence of this CaLB motif has led to the speculation that p120 GAP may be a member of a family of structurally related proteins containing a Ca(2+)-dependent membrane/lipid-binding domain. Here we have studied the in vitro Ca(2+)-dependent phospholipid-binding properties of the isolated proposed CaLB sequence in human GAP and deduce that a phospholipid-binding sequence is indeed located between amino acids 606 and 648. Binding of phosphatidylserine and phosphatidylinositol, but not phosphatidylcholine, within this sequence is Ca(2+)-dependent, with an estimated EC50 for Ca2+ of approx. 1 microM. Using deletion-mutation analysis we have further defined the minimal boundaries for this in vitro phospholipid-binding activity. p120 GAP amino acids 612-643 exhibit full phospholipid-binding activity, but further deletion of either amino acids 612-617 or amino acids 633-648 significantly decreased or abolished phospholipid binding. These studies establish that amino acids 612-643 of p120 GAP indeed constitute a functional CaLB domain and thereby imply a role for Ca2+ in the regulation of p120 GAP association with cellular (membrane) phospholipids.
Gawler
D J
DJ
Banting and Best Department of Medical Research, University of Toronto, Ontario, Canada.
Zhang
L J
LJ
Moran
M F
MF
D000595:Amino Acid Sequence; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D004252:DNA Mutational Analysis; D020690:GTPase-Activating Proteins; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D015689:Oncogene Protein p21(ras) / Q000378:metabolism*; D010743:Phospholipids / Q000378:metabolism*; D011506:Proteins / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017384:Sequence Deletion; D020703:ras GTPase-Activating Proteins
D020690:GTPase-Activating Proteins; D010743:Phospholipids; D011506:Proteins; D011993:Recombinant Fusion Proteins; D020703:ras GTPase-Activating Proteins; D015689:Oncogene Protein p21(ras); D002118:Calcium