Rac1b was recently identified in malignant colorectal tumors as an alternative splice variant of Rac1 containing a 19-amino acid insertion next to the switch II region. The structures of Rac1b in the GDP-and the GppNHp-bound forms, determined at a resolution of 1.75 Å, reveal that the insertion induces an open switch I conformation and a highly mobile switch II. As a consequence, Rac1b has an accelerated GEF-independent GDP/GTP exchange and an impaired GTP hydrolysis, which is restored partially by GTPase-activating proteins. Interestingly, Rac1b is able to bind the GTPasebinding domain of PAK but not full-length PAK in a GTP-dependent manner, suggesting that the insertion does not completely abolish effector interaction. The presented study provides insights into the structural and biochemical mechanism of a self-activating GTPase.

No abstract

These results suggest that PDI promotes survival after ischemic damage and that zinc-superoxide dismutase is one of the PDI molecular targets. Pharmacological modulation of this pathway might prove useful for future prevention and treatment of HF.

Rap1 is a small GTPase that is involved in signal transduction cascades. It is highly homologous to Ras but it is down-regulated by its own set of GTPase activating proteins (GAPs). To investigate the mechanism of the GTPhydrolysis reaction catalyzed by Rap1GAP, a catalytically active fragment was expressed in Escherichia coli and characterized by kinetic and mutagenesis studies. The GTPase reaction of Rap1 is stimulated 10 5 -fold by Rap1GAP and has a k cat of 6 s ؊1 at 25°C. The catalytic effect of GAPs from Ras, Rho, and Rabs depends on a crucial arginine which is inserted into the active site. However, all seven highly conserved arginines of Rap1GAP can be mutated without dramatically reducing V max of the GTP-hydrolysis reaction. We found instead two lysines whose mutations reduce catalysis 25-and 100-fold, most likely by an affinity effect. Rap1GAP does also not supply the crucial glutamine that is missing in Rap proteins at position 61. The Rap1(G12V) mutant which in Ras reduces catalysis 10 6 -fold is shown to be efficiently down-regulated by Rap1GAP. As an alternative, Rap1(F64A) is shown by kinetic and cell biological studies to be a Rap1GAP-resistant mutant. This study supports the notion of a completely different mechanism of the Rap1GAP-catalyzed GTP-hydrolysis reaction on Rap1.