Faculty Bibliography

Ras activation as a consequence of antigen receptor (T-cell receptor; TCR) engagement on T lymphocytes is required for T-cell development, selection and function. Lymphocyte function-associated antigen-1 (LFA-1) mediates lymphocyte adhesion, stabilization of the immune synapse and bidirectional signalling. Using a fluorescent biosensor we found that TCR activation with or without costimulation of CD28 led to activation of Ras only on the Golgi apparatus, whereas costimulation with LFA-1 induced Ras activation on both the Golgi and the plasma membrane. Ras activation on both compartments required RasGRP1, an exchange factor regulated by calcium and diacylglycerol (DAG), but phospholipase C (PLC) activity was required only for activation on the Golgi. Engagement of LFA-1 increased DAG levels at the plasma membrane by stimulating phospholipase D (PLD). PLD2 and phosphatidic acid phosphatase (PAP) were required for Ras activation on the plasma membrane. Thus, LFA-1 acts through PLD2 to reshape the pattern of Ras activation downstream of the TCR

Leukocyte-function-associated antigen-1 (LFA-1) is an integrin that is critical for T-cell adhesion and immunologic responses. As a transmembrane receptor and adhesion molecule, LFA-1 signals bidirectionally, whereby information about extracellular ligands is passed outside-in while cellular activation is transmitted inside-out to the adhesive ectodomain. Here, we review the role of small guanosine triphosphatases (GTPases) in LFA-1 signaling. Rap1, a Ras-related GTPase, appears to be central to LFA-1 function. Rap1 is regulated by receptor signaling [e.g. T-cell receptor (TCR), CD28, and cytotoxic T-lymphocyte antigen-4 (CTLA-4)] and by adapter proteins [e.g. adhesion and degranulation-promoting adapter protein (ADAP) and Src kinase-associated phosphoprotein of 55 kDa (SKAP-55)]. Inside-out signaling flows through Rap1 to regulator of adhesion and cell polarization enriched in lymphoid tissues (RAPL) and Rap1-GTP interacting adapter molecule (RIAM) that act in conjunction with the cytoskeleton on the cytosolic domain of LFA-1 to increase adhesion of the ectodomain. Outside-in signaling also relies on small GTPases such as Rho proteins. Vav-1, a guanine nucleotide exchange factor for Rho proteins, is activated as a consequence of LFA-1 engagement. Jun-activating binding protein-1 (JAB-1) and cytohesin-1 have been implicated as possible outside-in signaling intermediates. We have recently shown that Ras is also downstream of LFA-1 engagement: LFA-1 signaling through phospholipase D (PLD) to RasGRP1 was required for Ras activation on the plasma membrane following stimulation of TCR

Ras is a GTP-binding protein that plays multiple important roles in cell activation, including proliferative and inflammatory responses. Ras regulation is complex and depends upon post-translational processing, organelle-specific localization and the activation/deactivation of Ras by a number of regulatory molecules. Ras activation in T lymphocytes demonstrates unique features, including its dependence on the T cell receptor and the ability of Ras to signal from both the plasma membrane and the Golgi. Abnormalities of Ras expression, activation and signaling pathways in T lymphocytes appear to play important roles in the development of autoimmunity in general, and systemic lupus erythematosus in particular. In this manuscript, we review the basic biology of Ras in T lymphocytes, and the ways in which T lymphocyte Ras abnormalities may contribute to the development of a lupus phenotype

Several genetically encoded fluorescent biosensors for Ras family GTPases have been developed that permit spatiotemporal analysis of the activation of these signaling molecules in living cells. We describe here the use of the simplest of these probes, the Ras binding domain (RBD) of selected effectors fused with green fluorescent protein (GFP) or one of its spectral mutants. When expressed in quiescent cells, these probes are distributed homogeneously through the cytosol and nucleoplasm. On activation of their cognate GTPases on membranes, they are recruited to these compartments, and activation can be scored by redistribution of the probe. The advantage of this system is its simplicity: the probes are genetically encoded and can easily be constructed with standard cloning techniques, and the readout of activation requires only standard epifluorescence or confocal microscopy. The disadvantage of the system is that only rarely are Ras-related GTPases expressed at high enough levels to permit detection of the activation of the endogenous proteins. In general, the method requires overexpressing untagged, wild-type versions of the GTPase of interest. However, we describe a FRET-based method called bystander FRET developed to detect endogenous proteins that can be used to validate the results obtained by overexpressing Ras proteins. By use of this technique, we and others have uncovered important new features of the spatiotemporal regulation of Ras and related GTPases.

Signal transduction down the Ras/MAPK pathway, including that critical to T cell activation, proliferation, and differentiation, has been generally considered to occur at the plasma membrane. It is now clear that the plasma membrane does not represent the only platform for Ras/MAPK signaling. Moreover, the plasma membrane itself is no longer considered a uniform structure but rather a patchwork of microdomains that can compartmentalize signaling. Signaling on internal membranes was first recognized on endosomes. Genetically encoded fluorescent probes for signaling events such as GTP/GDP exchange on Ras have revealed signaling on a variety of intracellular membranes, including the Golgi apparatus. In fibroblasts, Ras is activated on the plasma membrane and Golgi with distinct kinetics. The pathway by which Golgi-associated Ras becomes activated involves PLCgamma and RasGRP1 and may also require retrograde trafficking of Ras from the plasma membrane to the Golgi as a consequence of depalmitoylation. Thus, the Ras/MAPK pathway represents a clear example of compartmentalized signaling

Proteins that terminate with a consensus sequence known as CAAX undergo a series of posttranslational modifications that include polyisoprenylation, endoproteolysis, and carboxyl methylation. These modifications render otherwise hydrophilic proteins hydrophobic at their C termini such that they associate with membranes. Whereas prenylation occurs in the cytosol, postprenylation processing is accomplished on the cytoplasmic surface of the endoplasmic reticulum and Golgi apparatus. Among the numerous CAAX proteins encoded in mammalian genomes are many signaling molecules such as monomeric GTPases, including the Ras proteins that play an important role in cancer. In the course of their processing, nascent Ras proteins traffic from their site of synthesis in the cytosol to the endomembrane and then out to the plasma membrane (PM) by at least two pathways. Recently, retrograde pathways have been discovered that deliver mature Ras from the PM back to the Golgi. The Golgi has been identified as a platform upon which Ras can signal. Thus, the subcellular trafficking of Ras proteins has the potential to increase the complexity of Ras signaling by adding a spatial dimension. The complexity of Ras trafficking also affords a wider array of potential targets for the discovery of drugs that might inhibit tumors by interfering with Ras trafficking

The discovery that Ras proteins are modified by enzymes restricted to the endoplasmic reticulum and Golgi apparatus and that, at steady state, a significant pool of Ras is localized on the Golgi has led to the hypothesis that Ras can become activated on and signal from intracellular membranes. Fluorescent probes capable of showing when and where in living cells Ras becomes activated together with studies of Ras proteins stringently tethered to intracellular membranes have confirmed this hypothesis. Thus, recent studies of Ras have contributed to the rapidly expanding field of compartmentalized signaling

K-Ras associates with the plasma membrane (PM) through farnesylation that functions in conjunction with an adjacent polybasic sequence. We show that phosphorylation by protein kinase C (PKC) of S181 within the polybasic region promotes rapid dissociation of K-Ras from the PM and association with intracellular membranes, including the outer membrane of mitochondria where phospho-K-Ras interacts with Bcl-XL. PKC agonists promote apoptosis of cells transformed with oncogenic K-Ras in a S181-dependent manner. K-Ras with a phosphomimetic residue at position 181 induces apoptosis via a pathway that requires Bcl-XL. The PKC agonist bryostatin-1 inhibited the growth in vitro and in vivo of cells transformed with oncogenic K-Ras in a S181-dependent fashion. These data demonstrate that the location and function of K-Ras are regulated directly by PKC and suggest an approach to therapy of K-Ras-dependent tumors with agents that stimulate phosphorylation of S181

In analyzing the sequence tags for mutant mouse embryonic stem (ES) cell lines in BayGenomics (a mouse gene-trapping resource), we identified a novel gene, 1-acylglycerol-3-phosphate O-acyltransferase (Agpat6), with sequence similarities to previously characterized glycerolipid acyltransferases. Agpat6's closest family member is another novel gene that we have provisionally designated Agpat8. Both Agpat6 and Agpat8 are conserved from plants, nematodes, and flies to mammals. AGPAT6, which is predicted to contain multiple membrane-spanning helices, is found exclusively within the endoplasmic reticulum (ER) in mammalian cells. To gain insights into the in vivo importance of Agpat6, we used the Agpat6 ES cell line from BayGenomics to create Agpat6-deficient (Agpat6-/-) mice. Agpat6-/- mice lacked full-length Agpat6 transcripts, as judged by northern blots. One of the most striking phenotypes of Agpat6-/- mice was a defect in lactation. Pups nursed by Agpat6-/- mothers die perinatally. Normally, Agpat6 is expressed at high levels in the mammary epithelium of breast tissue, but not in the surrounding adipose tissue. Histological studies revealed that the aveoli and ducts of Agpat6-/- lactating mammary glands were underdeveloped, and there was a dramatic decrease in the size and number of lipid droplets within mammary epithelial cells and ducts. Also, the milk from Agpat6-/- mice was markedly depleted in diacylglycerols and triacylglycerols. Thus, we identified a novel glycerolipid acyltransferase of the ER, AGPAT6, which is crucial for the production of milk fat by the mammary gland

The green fluorescent protein (GFP) of the jellyfish Aequorea victoria has revolutionized the study of protein localization and dynamics. GFP fusions permit analysis of proteins in living cells and offer distinct advantages over conventional immunofluorescence. Among these are lower background, higher resolution, robust dual color colocalization, and avoidance of fixation artifacts. In the case of Ras and Rho family proteins, GFP fusions have allowed breakthroughs in the understanding of how CAAX proteins are targeted to specific cell membranes and how signaling at different membranes can result in different cellular responses. GFP-tagged Rho proteins have also been informative in analyzing the interactions with the cytosolic chaperone, RhoGDI. The major disadvantages of studying GFP fusion proteins is that they are generally overexpressed relative to endogenous proteins, and the GFP tag can, in principle, affect protein function. Fortunately, in the case of Ras and Rho family proteins, a GFP tag at the N terminus seems to have little effect on protein targeting and function. Nevertheless, it is prudent to confirm GFP fusion protein data with the study of the endogenous protein. This chapter describes the tagging of Rho proteins with GFP and the analysis of GFP-Rho protein localization by epifluorescence and confocal microscopy. It further describes methods of analyzing endogenous Rho proteins as confirmation of data acquired using GFP-Rho fusion proteins. These techniques will be useful for anyone studying Rho protein function and are widely applicable to many cell types and signal transduction systems