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.

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.