Studying the spatial and temporal regulation of Ras GTPase-activating proteins.

Two classes of proteins govern Ras activation. Guanine-nucleotide exchange factors (Ras GEFs) catalyze the activation of Ras by inducing the dissociation of GDP to allow association of the more abundant GTP, whereas GTPase-activating proteins (Ras GAPs), bind to the GTP-bound form and, by enhancing the intrinsic GTPase activity, catalyze Ras inactivation. A wide range of Ras GEFs and Ras GAPs have been identified from the various genome projects, and in a few instances, the mechanisms by which signals originating from activated receptors converge on specific GEFs and GAPs have been mapped. However, for most Ras GEFs and GAPs we have a poor understanding of their regulation. Here we focus on describing methods used to study the regulation of the GAP1 family of Ras GAPs. In particular, we emphasize how by combining biochemical, molecular, and imaging techniques, one can determine some of the complex array of mechanisms that have evolved to modulate the spatial and temporal dynamics of Ras regulation through these various Ras GAPs. By combining biochemical, molecular, and imaging techniques, we describe the visualization of the diverse and dynamic mechanisms through which stimulation of cell surface receptors leads to the regulation of these proteins. Thus, although each member of the GAP1 family performs the same basic biological function, that is, they function as Ras GAPs, each is designed to respond and decode signals from distinct second messenger pathways.

GAP1 family members constitute bifunctional Ras and Rap GTPase-activating proteins.

GAP1(IP4BP) is a member of the GAP1 family of Ras GTPase-activating proteins (Ras GAPs) that includes GAP1(m), CAPRI, and RASAL. Composed of a central Ras GAP domain, surrounded by amino-terminal C(2) domains and a carboxyl-terminal pleckstrin homology/Bruton's tyrosine kinase domain, GAP1(IP4BP) has previously been shown to possess an unexpected GAP activity on the Ras-related protein Rap, besides the predicted Ras GAP activity (Cullen, P. J., Hsuan, J. J., Truong, O., Letcher, A. J., Jackson, T. R., Dawson, A. P., and Irvine, R. F. (1995) Nature 376, 527-530). Here we have shown that GAP1(IP4BP) is indeed an efficient Ras/Rap GAP, having K(m)s of 213 and 42 microm and estimated k(cat)s of 48 and 16 s(-1) for Ras and Rap, respectively. For this dual activity, regions outside the Ras GAP domain are required, as the isolated domain (residues 291-569) retains a pronounced Ras GAP activity yet has very low activity toward Rap. Interestingly, mutagenesis of the Ras GAP arginine finger, and surrounding residues important in Ras binding, inhibit both Ras and Rap GAP activity of GAP1(IP4BP). Although the precise details by which GAP1(IP4BP) can function as a Rap GAP remain to be determined, these data are consistent with Rap associating with GAP1(IP4BP) through the Ras-binding site within the Ras GAP domain. Finally, we have established that such dual Ras/Rap GAP activity is not restricted to GAP1(IP4BP). Although GAP1(m) appears to constitute a specific Ras GAP, CAPRI and RASAL display dual activity. For CAPRI, its Rap GAP activity is modulated upon its Ca(2+)-induced association with the plasma membrane.

Engineering the phosphoinositide-binding profile of a class I pleckstrin homology domain.

Pleckstrin homology (PH) domains are protein modules that bind with varying degrees of affinity and specificity membrane phosphoinositides. Previously we have shown that although the PH domains of the Ras GTPase-activating proteins GAP1m and GAP1IP4BP are 63% identical at the amino acid level they possess distinct phosphoinositide-binding profiles. The GAP1m PH domain binds phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3), whereas the domain from GAP1IP4BP binds PtdIns(3,4,5)P3 and phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) equally well. These phosphoinositide specificities are translated into distinct subcellular localizations. GAP1m is cytosolic and undergoes a rapid PtdIns(3,4,5)P3-dependent association with the plasma membrane following growth factor stimulation. In contrast, GAP1IP4BP is constitutively associated, in a PtdIns(4,5)P2-dependent manner, with the plasma membrane (Cozier, G. E., Lockyer, P. J., Reynolds, J. S., Kupzig, S., Bottomley, J. R., Millard, T., Banting, G., and Cullen, P. J. (2000) J. Biol. Chem. 275, 28261-28268). In the present study, we have used molecular modeling to identify residues in the GAP1IP4BP PH domain predicted to be required for high affinity binding to PtdIns(4,5)P2. This has allowed the isolation of a mutant, GAP1IP4BP-(K591T), which while retaining high affinity for PtdIns(3,4,5)P3 has a 6-fold reduction in its affinity for PtdIns(4,5)P2. Importantly, GAP1IP4BP-(K591T) is predominantly localized to the cytosol and undergoes a PtdIns(3,4,5)P3-dependent association with the plasma membrane following growth factor stimulation. We have therefore engineered the phosphoinositide-binding profile of the GAP1IP4BP PH domain, thereby emphasizing that subtle changes in PH domain structure can have a pronounced effect on phosphoinositide binding and the subcellular localization of GAP1IP4BP.

Identification of a Ras GTPase-activating protein regulated by receptor-mediated Ca2+ oscillations.

Receptor-mediated increases in the concentration of intracellular free calcium ([Ca2+]i) are responsible for controlling a plethora of physiological processes including gene expression, secretion, contraction, proliferation, neural signalling, and learning. Increases in [Ca2+]i often occur as repetitive Ca2+ spikes or oscillations. Induced by electrical or receptor stimuli, these repetitive Ca2+ spikes increase their frequency with the amplitude of the receptor stimuli, a phenomenon that appears critical for the induction of selective cellular functions. Here we report the characterisation of RASAL, a Ras GTPase-activating protein that senses the frequency of repetitive Ca2+ spikes by undergoing synchronous oscillatory associations with the plasma membrane. Importantly, we show that only during periods of plasma membrane association does RASAL inactivate Ras signalling. Thus, RASAL senses the frequency of complex Ca2+ signals, decoding them through a regulation of the activation state of Ras. Our data provide a hitherto unrecognised link between complex Ca2+ signals and the regulation of Ras.