Many genetically encoded biosensors use Förster resonance energy transfer (FRET) between fluorescent proteins to report biochemical phenomena in living cells. Most commonly, the enhanced cyan fluorescent protein (ECFP) is used as the donor fluorophore, coupled with one of several yellow fluorescent protein (YFP) variants as the acceptor. ECFP is used despite several spectroscopic disadvantages, namely a low quantum yield, a low extinction coefficient and a fluorescence lifetime that is best fit by a double exponential. To improve the characteristics of ECFP for FRET measurements, we used a site-directed mutagenesis approach to overcome these disadvantages. The resulting variant, which we named Cerulean (ECFP/S72A/Y145A/H148D), has a greatly improved quantum yield, a higher extinction coefficient and a fluorescence lifetime that is best fit by a single exponential. Cerulean is 2.5-fold brighter than ECFP and replacement of ECFP with Cerulean substantially improves the signal-to-noise ratio of a FRET-based sensor for glucokinase activation.

The serine/threonine kinase Raf-1 is an essential component of the MAPK cascade. Activation of Raf-1 by extracellular signals is initiated by association with intracellular membranes. Recruitment of Raf-1 to membranes has been reported to be mediated by direct association with Ras and by the phospholipase D product phosphatidic acid (PA). Here we report that insulin stimulation of HIRcB fibroblasts leads to accumulation of Ras, Raf-1, phosphorylated MEK, phosphorylated MAPK, and PA on endosomal membranes. Mutations that disrupt Raf-PA interactions prevented recruitment of Raf-1 to membranes, whereas disruption of Ras-Raf interactions did not affect agonistdependent translocation. Expression of a dominantnegative Ras mutant did not prevent insulin-dependent Raf-1 translocation, but inhibited phosphorylation of MAPK. Finally, the PA-binding region of Raf-1 was sufficient to target green fluorescent protein to membranes, and its overexpression blocked recruitment of Raf-1 to membranes and disrupted insulin-dependent MAPK phosphorylation. These results indicate that agonist-dependent Raf-1 translocation is primarily mediated by a direct interaction with PA and is independent of association with Ras.The MAPK 1 cascade is a signaling pathway essential for the regulation of mitogenesis by extracellular signals (1). One of the critical regulatory points in this cascade is the activation of the serine/threonine kinase Raf-1. Inactive Raf-1 exists in a large cytoplasmic complex with molecular chaperone proteins (2). Upon stimulation of cell-surface receptors, Raf-1 becomes associated with membranes and undergoes a complex series of activation steps modulated by the small GTPase Ras (3-5), 14-3-3 proteins (6 -11), and phosphorylation (12-15). Furthermore, the association of Raf-1 with membranes appears to be essential for its activation. Forced membrane recruitment through attachment of the Ras prenylation moiety to the C terminus of Raf-1 has been shown to induce kinase activation by a mechanism reportedly independent of association with Ras (16,17). It was also found that overexpression of constitutively activated Ras proteins results in the recruitment of Raf-1 to membranes (16,17). Although it was clear that Raf-1 associates with activated Ras in vivo, in vitro activation of Raf-1 by Ras has been difficult to demonstrate (18,19). Therefore, it was proposed that Ras mediates recruitment of Raf-1 to membranes (20).The recruitment of Raf-1 to membranes has also been reported to be dependent on its association with phosphatidic acid (21, 24). Disruption of agonist-dependent PLD activity either pharmacologically or by expression of catalytically inactive mutants blocks recruitment of Raf-1 to membranes and Raf-1 activation. Furthermore, addition of exogenous PA reverses the effects of PLD inhibition on Raf-1 translocation and MAPK phosphorylation. Although PA does not activate Raf-1 in vitro or in vivo, addition of exogenous PA can induce recruitment of Raf-1 to membranes in HIRcB cells or Ras-transformed cells (21). Theref...

The primary known function of phospholipase D (PLD) is to generate phosphatidic acid (PA) via the hydrolysis of phosphatidylcholine. However, the functional role of PA is not well understood. We report here evidence that links the activation of PLD by insulin and the subsequent generation of PA to the activation of the Raf-1-mitogen-activated protein kinase (MAPK) cascade. Brefeldin A (BFA), an inhibitor of the activation of ADP-ribosylation factor proteins, inhibited insulin-dependent production of PA and MAPK phosphorylation. The addition of PA reversed the inhibition of MAPK activation by BFA. Overexpression of a catalytically inactive variant of PLD2, but not PLD1, blocked insulindependent activation of PLD and phosphorylation of MAPK. Real time imaging analysis showed that insulin induced Raf-1 translocation to cell membranes by a process that was inhibited by BFA. PA addition reversed the effects of BFA on Raf-1 translocation. However, PA did not activate Raf-1 in vitro or in vivo, suggesting that the primary function of PA is to enhance the recruitment of Raf-1 to the plasma membrane where other factors may activate it. Finally, we found that the recruitment of Raf-1 to the plasma membrane was transient, but Raf-1 remained bound to endocytic vesicles.Growth factor-mediated activation of PLD 1 has been well documented and occurs in response to a broad class of mitogens, including insulin, platelet-derived growth factor, epidermal growth factor, vasopressin, and phorbol esters (1-4). Activation of PLD occurs through interaction with the small G-proteins of the ADP-ribosylation factor (ARF) (5, 6) and Rac/Rho families (7) as well as with protein kinase C (PKC) (8, 9). The relative contribution of these factors to the activation of PLD is highly dependent on the cell type and signaling model examined. For example, stimulation of Rat-1 fibroblasts overexpressing the human insulin receptor (HIRcB cells) with insulin activates PLD exclusively through the ARF pathway (10), whereas the activation of PLD by insulin in adipocytes appears to be primarily Rho-mediated (11). Activation of PLD has been implicated in a wide variety of intracellular and extracellular processes, including actin polymerization, coatomer assembly, vesicle transport, neutrophil activation, and platelet aggregation (12-16).Activated PLD catalyzes the hydrolysis of phosphatidylcholine to generate PA. However, the downstream consequences of PA generation are not well understood. Although it is clear that the principal effects of PA in some systems may be mediated by its conversion to diacylglycerol (DAG) or lysophosphatidic acid (LPA), PA may also be a potent second messenger. Several laboratories have identified putative targets for PA in growth factor signal transduction, including a protein tyrosine phosphatase (17), phospholipase C-␥ (18), and Ras-GAP (19). However, the physiological relevance of these interactions has not been established.Recently, Ghosh et al. (20) reported that PA interacts directly with the serine-threonine kinase Raf-1...

A previous study has shown an efficient, systemic transinclusion of cholesterol as a helper lipid increased the in gene expression in mice via intravenous administration of vivo transfection efficiency of LPD and more importantly, a LPD formulation composed of DOTAP liposomes, protadecrease the amount of cationic lipid required for the maximine sulfate and plasmid DNA. In this study, factors affectmal level of gene expression. Studies on the interaction ing the in vivo performance of this formulation were further between mouse serum and LPD showed that LPD became evaluated. A protocol in which liposomes were mixed with negatively charged after exposure to serum, and LPDs protamine before the addition of plasmid DNA was shown containing different helper lipids varied in the amount of to produce small condensed particles with a diameter of associated serum proteins. LPD containing DOPE was about 135 nm. These particles were stable over time and more enriched in a protein corresponding to albumin in gave a high level of gene expression in all tissues exammolecular weight. These results suggest that the mechined including lung, heart, spleen, liver and kidney with the anism of LPD-mediated intravenous gene delivery might highest level of expression in the lung. Inclusion of dioleoylbe different from that of in vitro lipofection and that serum phosphatidylethanolamine (DOPE) as a helper lipid sigprotein association might be a major factor limiting the in nificantly decreased the in vivo activity of LPD. In contrast, vivo transfection by LPD.