Breakpoint cluster region gene product-related domain of n-chimaerin. Discrimination between Rac-binding and GTPase-activating residues by mutational analysis.
The breakpoint cluster region gene product (Bcr) is a GTPase-activating protein (GAP) for members of the Rho family, Cdc42Hs, and Rac1, as is the brain protein n-chimaerin. At least 15 proteins have sequence identity to the GAP domain (150 amino acid residues) of Bcr. The widespread occurrence of proteins that possess sequence identity to the Bcr-related GAP domain makes it especially important to understand its structure/function relationships. Amino acid sequence alignment of these proteins reveals three blocks of conservation in the GAP domain. Here, we present a mutational analysis of this domain using n-chimaerin sequences. Ten mutations were constructed (at least two in each of the blocks of conservation), expressed as glutathione S-transferase fusion proteins in Escherichia coli, and purified. Seven of the mutants, including deletions, still possessed GAP activity for Rac1. Three of the mutants had no Rac1-GAP activity but were still able to bind Rac1. IC50 values obtained from competition experiments suggest that n-chimaerin and the mutants with no GAP activity bound Rac1 with similar apparent binding constants. Thus, this mutant analysis allows discrimination between Rac1-binding and Rac1 GTPase- activating residues.
Ahmed
S
S
Institute of Neurology, London, United Kingdom.
Lee
J
J
Wen
L P
LP
Zhao
Z
Z
Ho
J
J
Best
A
A
Kozma
R
R
Lim
L
L
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D020848:Chimerin 1; D004247:DNA; D004926:Escherichia coli; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D020690:GTPase-Activating Proteins; D008969:Molecular Sequence Data; D005810:Multigene Family; D016297:Mutagenesis, Site-Directed; D009419:Nerve Tissue Proteins / Q000235:genetics*; D015513:Oncogene Proteins / Q000235:genetics; D011505:Protein-Tyrosine Kinases; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D011518:Proto-Oncogene Proteins; D051562:Proto-Oncogene Proteins c-bcr; D016415:Sequence Alignment; D020764:cdc42 GTP-Binding Protein; D020744:rac GTP-Binding Proteins
D020848:Chimerin 1; D020690:GTPase-Activating Proteins; D009419:Nerve Tissue Proteins; D015513:Oncogene Proteins; D011506:Proteins; D011518:Proto-Oncogene Proteins; D004247:DNA; D011505:Protein-Tyrosine Kinases; D051562:Proto-Oncogene Proteins c-bcr; D019204:GTP-Binding Proteins; D020764:cdc42 GTP-Binding Protein; D020744:rac GTP-Binding Proteins