Integration of calcium and RAS signalling

Abstract

Calcium (Ca²⁺) is a universal second messenger that is involved in the regulation of fertilization, secretion, contraction, cell-cycle progression, cell proliferation, apoptosis, learning and memory. Intracellular, free Ca²⁺ concentrations are kept low and change dynamically due to release/re-uptake from intracellular stores, or entry/efflux across the plasma membrane. Coordinated regulation of intracellular release and extracellular influx of Ca²⁺ sets up highly complex Ca²⁺ signals in terms of amplitude, frequency, duration and spatial patterning. Ras is a small GTPase that cycles between a GTP-bound active conformation and a GDP-bound inactive conformation. Activation is dependent on the action of guanine nucleotide-exchange factors (GEFs); deactivation depends on the action of GTPase activating proteins (GAPs). Oncogenic Ras is locked in the GTP-bound form. The extent of Ras activation is dependent on the balance between GEF and GAP activity. GEFs and GAPs might be stimulated, or inhibited, to modulate Ras-GTP levels during cell stimulation. Active Ras stimulates a series of effector pathways, such as the mitogen-activates protein kinase (MAPK) cascade. The localization of Ras, GEFs, GAPs and effector proteins is a key concept for Ras-dependent signalling. Many of these events are initiated at the inner leaflet of plasma membrane, where Ras is targeted by post-translational modification. Ras guanine nucleotide-releasing factors (GRFs) are Ca²⁺-dependent Ras GEFs. Ca²⁺–calmodulin modulates Ras-GRF1 activity, and the function of this GEF seems to be particularly important in the central nervous system, in which Ca²⁺ signalling is crucial for learning and memory. Ras-GRF2 is more widely expressed and is also sensitive to changes in intracellular Ca²⁺. Evidence is accumulating that these proteins are bifunctional GEFs that also activate members of the Rho GTPase family. The Ras guanine nucleotide-releasing factors (GRPs)/CalDAG-GEFs constitute a second family of Ras GEFs. Five members of the family have been discovered that vary in their substrate specificity and mechanism of regulation. All members have some activity towards Ras GTPases (H-Ras, K-Ras and N-Ras), at least in vitro, but several also activate Rap1. Diacylglycerol (DAG) regulates all family members through a C1 domain, however they also contain EF-hands, and Ras GRP, CalDAG-GEFI and Ras GRP2 are regulated by Ca²⁺. p120 Ras GAP is known to translocate to the plasma membrane, and be potentially activated, during Ca²⁺ signalling — although the mechanisms for translocation are not entirely clear. CAPRI (Ca²⁺-promoted Ras inactivator) is a Ca²⁺-sensitive Ras GAP with little basal GAP activity in the resting cell. After Ca²⁺ elevation, CAPRI translocates to the plasma membrane through tandem C2 domains to deactivate Ras. Neuronal cells are a particularly good example of how Ca²⁺ signals are coupled in space and time to Ras-MAPK regulation, although there is still much to unravel. Non-neuronal cells are less well understood in terms of Ca²⁺-dependent Ras-MAPK signalling. The importance of frequency- and amplitude-dependent Ca²⁺ signalling for gene transcription has been shown, but is technically very difficult in terms of the Ras-MAPK cascade, or other effector pathways, to determine how complex Ca²⁺ signals influence Ras and downstream effectors. For any mechanism of cell stimulation, there is an extremely complex series of events that can lead to Ras-MAPK stimulation. The influence of Ca²⁺ occurs at many levels, such as by phosphorylation events due to PYK2, Src family members and protein kinase C. An important challenge is to put the regulation of Ca²⁺-dependent GEFs and GAPs in context with the rest of the Ras regulatory machinery during appropriate cell stimulation. The discovery of PLCɛ as a new Ras effector adds further complexity, as PLC signalling might be upstream and downstream of Ras activation. New FRET (fluorescent resonance energy transfer)-based techniques for measuring the Ras activation state in a single cell might answer the question as to whether the activation state of Ras fluctuates in concert with Ca²⁺ oscillations. If Ras is proven to oscillate during Ca²⁺-signal frequency, then this has an important impact on the interpretation of downstream effector pathway stimulation and gene transcription.