Biochemical Studies of the Mechanism of Action of the Cdc42-GTPase-activating Protein

Abstract

The small GTP-binding proteins Rac, Rho, and Cdc42 were shown to mediate a variety of signaling pathways including cytoskeletal rearrangements, cell-cycle progression, and transformation. Key to the proper function of these GTP-binding proteins is an efficient shutoff mechanism that ensures the decay of the signal. Regulatory proteins termed GAPs (GTPase-activating proteins) enhance the intrinsic GTP hydrolysis of the GTP-binding proteins, thereby ensuring signal termination. We have used site-specific mutagenesis to elucidate the limit domain for GAP activity in Cdc42-GAP, and show that in addition to the known GAP-homology domain (three conserved boxes), a C-terminal region outside that domain is also essential for GAP activity. In addition, we have replaced the conserved arginine (Arg 305 ), which was suggested by structural studies to be a key catalytic residue, with an alanine and found that the R305A Cdc42-GAP mutant has a greatly diminished catalytic capacity but is still able to bind Cdc42 with high affinity. Thus, a key catalytic role for this residue is confirmed. However, we also present evidence for the involvement of an additional residue(s), since the R305A Cdc42-GAP mutant still exhibits measurable activity. Some of this residual activity might result from a neighboring arginine, since a double mutant R305A/R306A shows a further decrease in catalytic activity.

Rational stabilization of enzymes by computational redesign of surface charge–charge interactions

“…Cdc42 is a eukaryotic protein and belongs to the family of small GTPases (24). It is a key enzyme that is involved in regulation of numerous vital cellular pathways such as gene expression, cell-cycle progression, and rearrangement of the actin cytoskeleton (25).…”

Fourier Transform Infrared Spectroscopy on the Rap·RapGAP Reaction, GTPase Activation without an Arginine Finger

“…The question arises as to whether Rap1GAP without an arginine finger still shifts charge toward the ␤-phosphate and whether the features described for the Ras⅐RasGAP system are generally applicable to other GTP-binding proteins and their respective GAPs. Here, the Rap⅐RapGAP system appeared to be particularly interesting because of the absence of both a Gln in the GTPase and an Arg residue from the GAP that are considered to be crucial in the case of the Ras (21), Rho (22,23), and Rab GTPases (24).…”

Insulin‐like growth factor‐I diminishes the activation status and expression of the small GTPase Cdc42 in articular chondrocytes

“…An increase in GTP hydrolysis, and therefore loss of [y-"PI, would be expected if IGF-I was acting to up-regulate a GAP in order to decrease Cdc42 activation. Recombinant GAP protein, GAP234, which is an efficient GAP for Cdc42 [27], serves as a positive control.…”

“…purified on glutathione-sepharose beads. and Cdc42 was isolated from GST by thrombin cleavage as previously described [27,45]. Protein concentration was determined using the Bradford method and protein purity was assessed using SDS-PAGE and Coomassie Blue staining.…”

“…Positive controls consisted of the additions of equimolar amount of CAP234. a known GTPase for Cdc42, [27] in lysis buffer and negative controls were composed of lysis buffer. …”