Regulation of ras exchange factors and cellular localization of ras activation by lipid messengers in T cells

Abstract

The Ras-MAPK signaling pathway is highly conserved throughout evolution and is activated downstream of a wide range of receptor stimuli. Ras guanine nucleotide exchange factors (RasGEFs) catalyze GTP loading of Ras and play a pivotal role in regulating receptor-ligand induced Ras activity. In T cells, three families of functionally important RasGEFs are expressed: RasGRF, RasGRP, and Son of Sevenless (SOS)-family GEFs. Early on it was recognized that Ras activation is critical for T cell development and that the RasGEFs play an important role herein. More recent work has revealed that nuances in Ras activation appear to significantly impact T cell development and selection. These nuances include distinct biochemical patterns of analog versus digital Ras activation, differences in cellular localization of Ras activation, and intricate interplays between the RasGEFs during distinct T cell developmental stages as revealed by various new mouse models. In many instances, the exact nature of these nuances in Ras activation or how these may result from fine-tuning of the RasGEFs is not understood. One large group of biomolecules critically involved in the control of RasGEFs functions are lipid second messengers. Multiple, yet distinct lipid products are generated following T cell receptor (TCR) stimulation and bind to different domains in the RasGRP and SOS RasGEFs to facilitate the activation of the membrane-anchored Ras GTPases. In this review we highlight how different lipid-based elements are generated by various enzymes downstream of the TCR and other receptors and how these dynamic and interrelated lipid products may fine-tune Ras activation by RasGEFs in developing T cells.

Keywords: LAT; P38; Ras; RasGRP; SOS; T cell; lipids; signaling.

Figure 1

Regulation of Ras family proteins. The Ras GTPases cycle between GDP-bound inactive and GTP-bound active forms. Activation of Ras is regulated by the balance of opposing actions of two classes of Ras regulatory enzymes. Guanine nucleotide exchange factors (GEFs) promote GTP-bound Ras state by enhancing exchange of GDP with GTP. GTPase activating proteins (GAPs) enhance slow rate of intrinsic Ras GTPase activity, promoting the inactive GDP-bound state of Ras.

Figure 2

Structural domain organization of three families of RasGEFs expressed in T cells. Cartoon highlighting the general protein domains in the three families of RasGEFs: SOS, RasGRP, and RasGRF. Cdc25, Cdc25 homology domain; DH, Dbl homology domain; HF, N-terminal histone-like fold; PH, Pleckstrin homology domain; PR, C-terminal PR domain; REM, Ras exchange motif; EF, Ca²⁺-binding EF hand; C1, DAG-binding C1 domain; CC-IQ, coiled coil – ilimaquinone domain. Protein size is drawn to approximate scale based on SOS1, RasGRP1, and RasGRF1 (53).

Figure 3

Multiple membrane-derived signals determine the RasGEF activity of SOS. (A) Model of inactive SOS. In the inactive state, SOS’s DH-PH domains obscure the allosteric Ras-binding pocket. Without engagement of the allosteric pocket by Ras·GTP, SOS only shows low reactivity for Ras·GDP at the catalytic binding site. The HF docks itself to a helical linker region (not depicted) between PH and REM domains, further stabilizing auto-inhibited state of SOS. The protein structure of the C-terminal proline-rich domain has not been determined to date. (B) Model of allosteric activation of SOS. Ras·GTP binding to the allosteric site enhances SOS exchange activity by increasing Ras-binding affinity for the catalytic pocket, establishing a positive feedback mechanism. Other SOS domains are omitted for simplicity. (C) Model of full activation of SOS. Full activation of SOS requires the integration of multiple membrane-derived signals. Grb2-mediated membrane recruitment of SOS to phosphorylated LAT is thought to be one of the initial membrane recruitment mechanisms. Membrane phospholipids such as PIP₂ and PA interact with HF and PH domains, and these interactions further relieve auto-inhibition state of SOS, allowing efficient access of Ras to both the allosteric and catalytic sites.

Figure 4

Activation of RasGRP. (A) Depiction of RasGRP with its protein domains. RasGRPs must be controlled to prevent spurious Ras activation but the exact mechanism of auto-inhibition is unknown. Roles for various domains C-terminal of the Cdc25 domain to limit membrane recruitment of RasGRP have been proposed. (B) DAG-regulated membrane recruitment of RasGRP. Receptor-induced generation of diacylglycerol (DAG) results in efficient membrane recruitment on RasGRP1, RasGRP3, and RasGRP4 where these RasGEFs can encounter Ras·GDP to activate it to Ras·GTP. RasGRP1 and RasGRP3 are known to be phosphorylated on a conserved threonine residue at the very start of the Cdc25 domain, which enhances their catalytic activity through an unknown mechanism. RasGRP2 does not efficiently bind DAG and must have a different membrane-recruiting mechanism. (C) Other regulatory mechanisms for RasGRP. Amino acid sequence homologies predict that RasGRPs lack and allosteric Ras-binding pocket as the one observed for SOS. RasGRP proteins contain EF hands, structure that can often bind calcium. Calcium has been implicated in the recruitment of RasGRP1 to the membrane but nuances appear to exist in different cell types. It is not known if other lipid moieties such as PIP₂ can regulate the activity or residence time of RasGRP1 at the membrane.

Figure 5

Model of synergy between RasGRP and SOS in TCR signaling. TCR stimulation is connected to activation of RasGRP via tyrosine phosphorylation of the adapter molecule LAT and activation of PLCγ1, that metabolizes PIP₂ into IP₃ and DAG to trigger two second messenger pathways; Ca²⁺ and DAG. Activated RasGRP can enhance the full activation of SOS by providing Ras·GTP, allosterically activating SOS. In principle, the TCR-LAT-PLCγ1 pathway can also indirectly facilitate SOS activation via DAG; DGK metabolizes DAG and converts it to PA, which is a possible target for the HF and/or PH domains in SOS.

Figure 6

Differential activation of RasGEF determines the quantity and quality of Ras-ERK output. Left: full activation of the ERK response requires activation of both RasGRP and SOS and can lead to bimodal (digital) ERK activation patterns. In this mode of signaling, RasGRP activation temporally precedes activation of SOS and provides initial Ras·GTP that primes full activation of SOS. Middle: in the absence of SOS, there is substantial Ras-ERK activation mediated by RasGRP alone, but the ERK activation patterns are analog and therefore differs both quantitatively and qualitatively from ERK signal generated by two RasGEFs in synergy. Right: in lymphocytes, RasGRP plays a dominant role in connecting TCR-Ras-ERK pathway. SOS alone has difficulty to prime its own allosteric activation, which results in a high threshold for Ras-ERK activation.

Figure 7

An adapter function for SOS in oligomeric LAT clusters? Grb2-SOS complexes can serve as a cytosolic linkers and aggregate multiple LAT molecules and LAT signalosome-constituent proteins together. This SOS-containing complex may facilitate activation of other, non-canonical Ras-ERK signal transduction pathways such as activation of the MAPK p38, perhaps through a Vav-Rac·GTP connection. We found that regulation of p38 is independent of any enzymatic function of SOS, further strengthening the notion that SOS can signal as an adapter to non-canonical pathways in lymphocytes.