Ras–membrane interactions play important roles in signaling and oncogenesis. H-Ras and K-Ras have nonidentical membrane anchoring moieties that can direct them to different membrane compartments. Ras–lipid raft interactions were reported, but recent studies suggest that activated K-Ras and H-Ras are not raft resident. However, specific interactions of activated Ras proteins with nonraft sites, which may underlie functional differences and phenotypic variation between different Ras isoforms, are unexplored. Here we used lateral mobility studies by FRAP to investigate the membrane interactions of green fluorescent protein–tagged H- and K-Ras in live cells. All Ras isoforms displayed stable membrane association, moving by lateral diffusion and not by exchange with a cytoplasmic pool. The lateral diffusion rates of constitutively active K- and H-Ras increased with their expression levels in a saturable manner, suggesting dynamic association with saturable sites or domains. These sites are distinct from lipid rafts, as the activated Ras mutants are not raft resident. Moreover, they appear to be different for H- and K-Ras. However, wild-type H-Ras, the only isoform preferentially localized in rafts, displayed cholesterol-sensitive interactions with rafts that were independent of its expression level. Our findings provide a mechanism for selective signaling by different Ras isoforms.
TAZ promotes cell proliferation, development, and tumorigenesis by regulating target gene transcription. However, how TAZ orchestrates the transcriptional responses remains poorly defined. Here we demonstrate that TAZ forms nuclear condensates via liquid-liquid phase separation to compartmentalize its DNA binding co-factor TEAD4, the transcription co-activators BRD4 and MED1 and the transcription elongation factor CDK9 for activation of gene expression.TAZ, but not its paralog YAP, forms phase-separated droplets in vitro and liquid-like nuclear condensates in vivo, and this ability is negatively regulated by Hippo signaling via LATS-mediated phosphorylation and mediated by the coiled-coil domain. Deletion of the TAZ coiled-coil domain or substitution with the YAP coiled-coil domain does not affect the interaction of TAZ with its partners, but prevents its phase separation and more importantly, its ability to induce target gene expression. Thus, our study identifies a novel mechanism for the transcriptional activation by TAZ and demonstrates for the first time that pathway-specific transcription factors also engage the phase separation mechanism for efficient transcription activation.
Membrane anchorage of Ras proteins in the inner leaflet of the plasma membrane is an important factor in their signaling and oncogenic potential. Despite these important roles, the precise mode of Ras-membrane interactions is not yet understood. It is especially important to characterize these interactions at the surface of intact cells. To investigate Ras-membrane interactions in live cells, we employed studies on the lateral mobility of a constitutively active Ras isoform to characterize its membrane dynamics, and examined the effects of the Ras-displacing antagonist S-trans,trans-farnesylthiosalicylic acid (FTS) (Haklai, R., Gana-Weisz, M., Elad, G., Paz, A., Marciano, D., Egozi, Y., Ben-Baruch, G., and Kloog, Y. (1998) Biochemistry 37, 1306 -1314) on these parameters. A green fluorescent protein (GFP) was fused to the N terminus of constitutively active Ki-Ras 4B(12V) to generate GFP-Ki-Ras(12V). When stably expressed in Rat-1 cells, this protein was preferentially localized to the plasma membrane and displayed transforming activity. The lateral mobility studies demonstrated that GFP-Ki-Ras(12V) undergoes fast lateral diffusion at the plasma membrane, rather than exchange between membrane-bound and unbound states. Treatment of the cells with FTS had a biphasic effect on GFP-Ki-Ras(12V) lateral mobility. At the initial phase, the lateral diffusion rate of GFP-Ki-Ras(12V) was elevated, suggesting that it is released from some constraints on its lateral mobility. This was followed by dislodgment of the protein into the cytoplasm, and a reduction in the diffusion rate of the fraction of GFP-KiRas(12V) that remained associated with the plasma membrane. Control experiments with other S-prenyl analogs showed that these effects are specific for FTS. These results have implications for the interactions of Ki-Ras with specific membrane anchorage domains or sites.
Calcium-dependent exocytosis is regulated by a vast number of proteins. DOC2B is a synaptic protein that translocates to the plasma membrane (PM) after small elevations in intracellular calcium concentration. The aim of this study was to investigate the role of DOC2B in calcium-triggered exocytosis. Using biochemical and biophysical measurements, we demonstrate that the C2A domain of DOC2B interacts directly with the PM in a calcium-dependent manner. Using a combination of electrophysiological, morphological, and total internal reflection fluorescent measurements, we found that DOC2B acts as a priming factor and increases the number of fusioncompetent vesicles. Comparing secretion during repeated stimulation between wild-type DOC2B and a mutated DOC2B that is constantly at the PM showed that DOC2B enhances catecholamine secretion also during repeated stimulation and that DOC2B has to translocate to the PM to exert its facilitating effect, suggesting that its activity is dependent on calcium. The hypothesis that DOC2B exerts its effect at the PM was supported by the finding that DOC2B affects the fusion kinetics of single vesicles and interacts with the PM SNAREs (soluble NSF attachment receptors). We conclude that DOC2B is a calcium-dependent priming factor and its activity at the PM enables efficient expansion of the fusion pore, leading to increased catecholamine release.
Phospholipase C- (PLC) isozymes play important roles in transmembrane signaling. Their activity is regulated by heterotrimeric G proteins. The PLC 2 isozyme is unique in being stimulated also by Rho GTPases (Rac and Cdc42). However, the mechanism(s) of this stimulation are still unclear. Here, we employed fluorescence recovery after photobleaching to investigate the interaction of green fluorescent protein (GFP)-PLC 2 with the plasma membrane. For either GFP-PLC 2 or GFP-PLC 2 ⌬, a C-terminal deletion mutant lacking the region required for stimulation by G␣ q , these interactions were characterized by a mixture of exchange with a cytoplasmic pool and lateral diffusion. Constitutively active Rac2(12V) stimulated the activity of both GFP-PLC 2 and GFP-PLC 2 ⌬ in live cells, and enhanced their membrane association as evidenced by the marked reduction in their fluorescence recovery rates. Both effects required the putative N-terminal pleckstrin homology (PH) domain of PLC 2 . Importantly, Rac2(12V) dramatically increased the contribution of exchange to the fluorescence recovery of GFP-PLC 2 , but had the opposite effect on GFP-PLC 2 ⌬, where lateral diffusion became dominant. Our results demonstrate for the first time the regulation of membrane association of a PLC isozyme by a GTP-binding protein and assign a novel function to the PLC 2 C-terminal region, regulating its exchange between membrane-bound and cytosolic states.The activity of phospholipase C- (PLC) 1 enzymes that hydrolyze phosphatidylinositol 4,5-bisphosphate (PtdInsP 2 ) is stimulated with different orders of efficacy by G protein ␣ q subunits and by G protein ␥ dimers (1-3). In addition, the PLC 2 isozyme is specifically activated in vitro by the Rho GTPases Rac and Cdc42, but not by RhoA (4 -6). As for all PLC isozymes, activation by ␣ q requires the C-terminal region of PLC 2 , and mutants carrying deletions in this region, such as the mutant PLC 2 ⌬ that lacks the Phe 819 -Glu 1166 segment, are resistant to stimulation by ␣ q but are susceptible to activation by Rho GTPases and G protein ␥ subunits (3, 4, 7). Recent studies (4 -6) show that ␥ dimers and Rho GTPases activate PLC 2 by interacting with different regions of the effector enzyme. Thus, the PLC 2 catalytic subdomains X and Y are sufficient for efficient stimulation by ␥, whereas the putative pleckstrin homology (PH) domain of PLC 2 is absolutely required for stimulation by Rho GTPases (6). Among the Rho GTPases, Rac1 and Rac2 are more potent stimulators than Cdc42 (6). Evidence for a tight connection between PLC 2 and Rho GTPases in cells is provided by the chemoattractant receptor system, whose activation stimulates PLC 2 and Rac1, Rac2 and Cdc42 (8 -11). Moreover, in accord with our in vitro studies on PLC 2 activation by Rho GTPases (4, 6), a recent study conducted on myeloid-differentiated HL-60 cells demonstrated that dominant-negative Cdc42 disrupted the stimulation of inositol 1,4,5-trisphosphate formation mediated via the chemoattractant receptors whi...
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