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Mutation-deletion analysis of a Ca(2+)-dependent phospholipid binding (CaLB) domain within p120 GAP, a
D J Gawler1, L J Zhang, M F Moran
1Banting and Best Department of Medical Research, University of Toronto, Ontario, Canada.
Abstract:
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.
Insights
p120 GTPase-activating protein (GAP) contains a Ca(2+)-dependent phospholipid-binding (CaLB) domain. This domain, located at amino acids 612-643, binds specific phospholipids in a calcium-dependent manner, suggesting a regulatory role.
Area of Science:
- Molecular Biology
- Cell Signaling
- Protein Biochemistry
Background:
- p120 GTPase-activating protein (GAP) is a multidomain protein involved in p21 ras signaling.
- Sequence similarity suggests p120 GAP may possess a Ca(2+)-dependent phospholipid-binding (CaLB) domain, similar to other related proteins.
- The CaLB motif's presence implies a potential role for calcium in regulating p120 GAP's interaction with cellular membranes.
Purpose of the Study:
- To investigate the in vitro Ca(2+)-dependent phospholipid-binding properties of the proposed CaLB sequence in human p120 GAP.
- To identify the specific amino acid boundaries of the functional CaLB domain.
- To elucidate the role of calcium in the regulation of p120 GAP's membrane association.
Main Methods:
- In vitro binding assays using isolated CaLB sequences of human p120 GAP.
- Deletion-mutation analysis to define the minimal functional boundaries of the CaLB domain.
- Assessment of binding affinity for various phospholipids (phosphatidylserine, phosphatidylinositol, phosphatidylcholine) in a Ca(2+)-dependent manner.
Main Results:
- A Ca(2+)-dependent phospholipid-binding sequence was identified in human p120 GAP between amino acids 606 and 648.
- The minimal functional CaLB domain was mapped to amino acids 612-643, exhibiting full phospholipid-binding activity.
- Binding of phosphatidylserine and phosphatidylinositol was Ca(2+)-dependent (EC50 ≈ 1 µM), while phosphatidylcholine binding was not observed.
Conclusions:
- Amino acids 612-643 of p120 GAP constitute a functional CaLB domain.
- Calcium ions play a significant role in regulating the association of p120 GAP with cellular phospholipids.
- These findings suggest a novel mechanism for calcium-mediated regulation of p120 GAP activity and localization.