BREAKPOINT CLUSTER REGION GENE PRODUCT-RELATED DOMAIN OF N-CHIMAERIN - DISCRIMINATION BETWEEN RAC-BINDING AND GTPASE-ACTIVATING RESIDUES BYMUTATIONAL ANALYSIS
S. Ahmed et al., BREAKPOINT CLUSTER REGION GENE PRODUCT-RELATED DOMAIN OF N-CHIMAERIN - DISCRIMINATION BETWEEN RAC-BINDING AND GTPASE-ACTIVATING RESIDUES BYMUTATIONAL ANALYSIS, The Journal of biological chemistry, 269(26), 1994, pp. 17642-17648
The breakpoint cluster region gene product (Bcr) is a GTPase-activatin
g protein (GAP) for members of the Rho family, Cdc42Hs, and Rac1, as i
s the brain protein n-chimaerin. At least 15 proteins have sequence id
entity to the GAP domain (150 amino acid residues) of Bcr. The widespr
ead occurrence of proteins that possess sequence identity to the Bcr-r
elated GAP domain makes it especially important to understand its stru
cture/ 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 seq
uences. Ten mutations were constructed (at least two in each of the bl
ocks of conservation), expressed as glutathione S-transferase fusion p
roteins in Escherichia coli, and purified. Seven of the mutants, inclu
ding deletions, still possessed GAP activity for Rac1. Three of the mu
tants 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 be
tween Rac1 binding and Rac1 GTPase- activating residues.