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Abstract
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 GTP-hydrolysis 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 degrees 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.
Highlights
Rap is a small guanine nucleotide-binding protein implied in signal transduction processes
Just as Ras, it cycles between a GDP-bound inactive and a GTP-bound active form, and this switching is regulated by specific guanine nucleotide exchange factors (GEFs)1 and GTPase activating proteins (GAPs)
Biochemical experiments have demonstrated that the Cdc25 domain is fully capable for catalyzing nucleotide exchange [20], and the three-dimensional structure of the Ras-SOS complex has shown that the Ras exchanger motif domain is stabilizing the Cdc25 domain [21]
Summary
Rap is a small guanine nucleotide-binding protein implied in signal transduction processes (reviewed in Ref. 1). The GTPase reaction of Rap1 is stimulated 105-fold by Rap1GAP and has a kcat of 6 s؊1 at 25 °C. The Rap1(G12V) mutant which in Ras reduces catalysis 106-fold is shown to be efficiently down-regulated by Rap1GAP.
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