Concerted regulation of cell dynamics by BNIP-2 and Cdc42GAP homology/Sec14p-like,proline-rich,and GTPase-activating protein domains of a novel Rho GTPase-activating protein,BPGAP1

RhoA, Cdc42, and Rac1 are small GTPases that regulate cytoskeletal reorganization leading to changes in cell morphology and cell motility. Their signaling pathways are activated by guanine nucleotide exchange factors and inactivated by GTPase-activating proteins (GAPs). We have identified a novel RhoGAP, BPGAP1 (for BNIP-2 and Cdc42GAP Homology (BCH) domain-containing, Proline-rich and Cdc42GAP-like protein subtype-1), that is ubiquitously expressed and shares 54% sequence identity to Cdc42GAP/p50RhoGAP. BP-GAP1 selectively enhanced RhoA GTPase activity in vivo although it also interacted strongly with Cdc42 and Rac1. "Pull-down" and co-immunoprecipitation studies indicated that it formed homophilic or heterophilic complexes with other BCH domain-containing proteins. Fluorescence studies of epitope-tagged BPGAP1 revealed that it induced pseudopodia and increased migration of MCF7 cells. Formation of pseudopodia required its BCH and GAP domains but not the proline-rich region, and was differentially inhibited by coexpression of the constitutively active mutant of RhoA, or dominant negative mutants of Cdc42 and Rac1. However, the mutant without the proline-rich region failed to confer any increase in cell migration despite the induction of pseudopodia. Our findings provide evidence that cell morphology changes and migration are coordinated via multiple domains in BPGAP1 and present a novel mode of regulation for cell dynamics by a RhoGAP protein.     

Sec14 family of lipid transfer proteins in yeasts

The hydrophobicity of lipids prevents their free movement across the cytoplasm. To achieve highly heterogeneous and precisely regulated lipid distribution in different cellular membranes, lipids are transported by lipid transfer proteins (LTPs) in addition to their transport by vesicles. Sec14 family is one of the most extensively studied groups of LTPs. Here we provide an overview of Sec14 family of LTPs in the most studied yeast Saccharomyces cerevisiae as well as in other selected non-Saccharomyces yeasts—Schizosaccharomyces pombe, Kluyveromyces lactis, Candida albicans, Candida glabrata, Cryptococcus neoformans, and Yarrowia lipolytica. Discussed are specificities of Sec14-domain LTPs in various yeasts, their mode of action, subcellular localization, and physiological function. In addition, quite few Sec14 family LTPs are target of antifungal drugs, serve as modifiers of drug resistance or influence virulence of pathologic yeasts. Thus, they represent an important object of study from the perspective of human health.     

Secretion of the mammalian Sec14p-like phosphoinositide-binding p45 protein

Protein-lipid interactions are important for protein targeting, signal transduction, lipid transport, and the maintenance of cellular compartments and membranes. Specific lipid-binding protein domains, such as PH, FYVE, PX, PHD, C2 and SEC14 homology domains, mediate interactions between proteins and specific phospholipids. We recently cloned a 45-kDa protein from rat olfactory epithelium, which is homologous to the yeast Sec14p phosphatidylinositol (PtdIns) transfer protein and we report here that this protein binds to PtdIns(3,4,5)P3 and far weaker to less phosphorylated derivatives of PtdIns. Expression of the p45 protein in COS-1 cells resulted in accumulation of the protein in secretory vesicles and in the extracellular space. The secreted material contained PtdIns(3,4,5)P3. Our findings are the first report of a Sec14p-like protein involved in transport out of a cell and, to the best of our knowledge, inositol-containing phospholipids have not previously been detected in the extracellular space. Our findings suggest that p45 and phosphoinositides may participate in the formation of the protective mucus on nasal epithelium.     

Sec14 related proteins in yeast

Lipid transport between membranes of eukaryotic organisms represents an essential aspect of organelle biogenesis. This transport must be strictly selective and directional to assure specific lipid composition of individual membranes. Despite the intensive research effort in the last few years, our understanding of how lipids are sorted and moved within cells is still rather limited. Evidence indicates that at least some of the mechanisms generating and maintaining non-random distribution of lipids in cells are linked to the action of phosphatidylinositol transfer proteins (PITPs). The major PITP in yeast Saccharomyces cerevisiae, Sec14p, is essential in promoting Golgi secretory function by modulating of its membrane lipid composition. This review focuses on a group of five yeast proteins that share significant sequence homology with Sec14p. Based on this sequence identity, they were termed Sfh (Sec fourteen homologue) proteins. It is a diverse group of proteins with distinct subcellular localizations and varied physiological functions related to lipid metabolism, phosphoinositide mediated signaling and membrane trafficking.     

Electron microscopic imaging of Ras signaling domains

Ras isoform-specific signaling from the plasma membrane appears to be regulated by interactions with distinct functional microdomains. We have developed protocols allowing the generation of 2-D spatial maps describing cell surface microdomain distributions. The combined electron microscopic (EM)-statistics approach provides nanometer scale resolution allowing both inner and outer leaflet domains to be visualized and cross-correlated with each other or with a protein of interest. In particular, the technique has allowed the interaction of Ras isoforms with signaling microdomains and proteins regulating these compartments to be screened. By allowing detailed monitoring of cell surface organization and compartmentalization, the approach has widespread potential for studies of plasma membrane-dependent cell biology, including regulated signaling and membrane trafficking.     

Eph receptor-ephrin bidirectional signals that target Ras and Rho proteins

The ability of cells to respond to their surrounding environment and relay signals to the cell interior is essential for numerous processes during the development and maintenance of tissues. Eph receptors and their membrane-bound ligands, the ephrins, are unique in the receptor tyrosine kinase family in that their signaling is bidirectional, through both the receptor and the ligand. Eph receptors and ephrins are essential for a variety of biological processes, and play a particularly important role in regulating cell shape and cell movement. Recent data have linked Eph receptor-ephrin signaling complexes to the Ras and Rho families of small molecular weight GTPases and also to heterotrimeric G proteins. Understanding the signaling networks involved is an important step to understand the molecular basis for normal and defective cell-cell communication through Eph receptors and ephrins.     

Dystrophin-glycoprotein complex and Ras and Rho GTPase signaling are altered in muscle atrophy

The dystrophin-glycoprotein complex (DGC)is a sarcolemmal complex whose defects cause muscular dystrophies. Thenormal function of this complex is not clear. We have proposed thatthis is a signal transduction complex, signaling normal interactionswith matrix laminin, and that the response is normal growth andhomeostasis. If so, the complex and its signaling should be altered inother physiological states such as atrophy. The amount of some of the DGC proteins, including dystrophin, -dystroglycan, and-sarcoglycan, is reduced significantly in rat skeletal muscleatrophy induced by tenotomy. Furthermore, H-Ras, RhoA, and Cdc42decrease in expression levels and activities in muscle atrophy. Whenthe small GTPases were assayed after laminin or -dystroglycandepletion, H-Ras, Rac1, and Cdc42 activities were reduced, suggesting aphysical linkage between the DGC and the GTPases. Dominant-negativeCdc42, introduced with a retroviral vector, resulted in fibers thatappeared atrophic. These data support a putative role for the DGC intransduction of mechanical signals in muscle.

     

Monitoring spatio-temporal regulation of Ras and Rho GTPase with GFP-based FRET probes

GFP-based fluorescence resonance energy transfer (FRET) probes that visualize local activity-changes of Ras and Rho GTPases in living cells are now available for examining the spatio-temporal regulation of these proteins. This article describes principles and strategies to develop intramolecular FRET probes for Ras- and Rho-family GTPases. The procedure for characterizing candidate probes, and image acquisition and processing are also explained. An optimal FRET probe should have (i) a wide dynamic range (which means a high sensitivity), (ii) a high fluorescence intensity, (iii) target specificity, and (iv) a minimal perturbation to endogenous signaling cascades. Although an improvement of FRET probes should be executed in a trial-and-error manner, practical tips for optimization are provided here. In addition, we illustrate some applications of FRET probes for neuronal cells, which are composed of diverse subcellular compartments with different functions; thus, tools to decipher the dynamics of GTPase activity in each compartment have long been desired.     

Role of the Sec14-like domain of Dbl family exchange factors in the regulation of Rho family GTPases in different subcellular sites

Mechanisms underlying subcellular region-specific regulation of Rho family GTPases through Dbl family guanine nucleotide exchange factors (GEFs) remain totally unknown. Here we show that the Sec14-like domain, which lies in the N-terminus of the Dbl family GEFs Dbl and Ost, directs the subcellular localization of these GEFs and also their substrate Cdc42. When coexpressed with Cdc42 in human adenocarcinoma HeLa cells, Dbl-I and Ost-I, which lack the Sec14-like domain, translocated Cdc42 to the plasma membrane, where Dbl-I or Ost-I was colocalized. In marked contrast, Dbl-II and Ost-II, which contain the Sec14-like domain, were colocalized with Cdc42 in endomembrane compartments. Furthermore, ruffle membrane formation upon epidermal growth factor treatment was mediated by Dbl-I or Ost-I, but neither Dbl-II nor Ost-II, supporting a notion that GEFs with or without the Sec14-like domain are linked to different upstream signals. By employing a novel method to detect the active GTP-bound form of Cdc42 in situ, we demonstrate that Dbl-I and Ost-I, but neither Dbl-II nor Ost-II, indeed activate colocalized Cdc42.     

Cloning of novel human SEC14p-like proteins: ligand binding and functional properties

We describe the cloning and expression of two novel genes highly similar to the tocopherol-associated protein (hTAP/SEC14L2/SPF). Immunoprecipitation of the three recombinant hTAPs and extraction of their associated lipid-soluble molecules indicates that they bind not just tocopherols, but also phosphatidylinositol, phosphatidylcholine, and phosphatidylglycerol. Ligand competition analysis by isoelectric point mobility shift assay indicates that phosphatidylcholine, tocopherols, and tocopheryl-succinate compete with phosphatidylinositol binding to hTAPs. To investigate a possible function of hTAPs on enzymes involved in phospholipids metabolism, the activity of recombinant phosphatidylinositol 3-kinase (PI3Kgamma/p110gamma) was tested. Recombinant hTAPs reduce in vitro the activity of the recombinant catalytic subunit of PI3Kgamma and stimulate it in the presence of alpha-tocopherol up to 5-fold. Immunoprecipitation of hTAP1 from cells results in co-precipitation of PI3-kinase activity, indicating a physical contact between the two proteins at a cellular level. In summary, hTAPs may modulate, in a tocopherol-sensitive manner, phosphatidylinositol-3-kinase, a central enzyme in signal transduction, cell proliferation, and apoptosis. It is possible that other phosphatidylinositol- and phosphatidylcholine-dependent signaling pathways are modulated by hTAPs and tocopherols, possibly by transporting and presenting these ligands to the corresponding enzymes.     

Imaging metabolism of phosphatidylinositol 4,5-bisphosphate in T-cell GM1-enriched domains containing Ras proteins

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) and Ras proteins are involved in signalling pathways originating at the plasma membrane. The localisation and metabolism of PI(4,5)P(2) was studied in Jurkat T cells using fluorescence microscopic imaging with EGFP-tagged and antibody probes. Software was developed to objectively quantitate colocalisation and was used to show that plasma membrane PI(4,5)P(2) was enriched in lipid raft-containing patches of GM1 ganglioside, formed by crosslinking cholera toxin B-subunit (CT-B). The PI(4,5)P(2) metabolites phosphatidylinositol 3,4,5-trisphosphate and diacylglycerol appeared in plasma membrane CT-B-GM1 patches upon induction of signalling. Transferrin receptor and the CD45 tyrosine phosphatase did not colocalise with CT-B-GM1 patches, whereas the tyrosine kinase Lck, the scaffolding protein LAT, and endogenous Ras proteins did partially colocalise with CT-B-GM1 patches as did transfected EGFP-K-Ras(4B) and EGFP-H-Ras. The results demonstrate that T-cell PI(4,5)P(2) metabolism is occurring in GM1-enriched domains and that Ras proteins are present in these domains in vivo.     

Characterization of three human sec14p-like proteins: alpha-tocopherol transport activity and expression pattern in tissues

Three closely related human sec14p-like proteins (hTAP1, 2, and 3, or SEC14L2, 3, and 4, respectively) have been described. These proteins may participate in intracellular lipid transport (phospholipids, squalene, tocopherol analogues and derivatives) or influence regulatory lipid-dependent events. Here, we show that the three recombinant hTAP proteins associate with the Golgi apparatus and mitochondria, and enhance the in vitro transport of radioactively labeled alpha-tocopherol to mitochondria in the same order of magnitude as the human alpha-tocopherol transfer protein (alpha-TTP). hTAP1 and hTAP2 are expressed in several cell lines, whereas the expression level of hTAP3 is low. Expression of hTAP1 is induced in human umbilical cord blood-derived mast cells upon differentiation by interleukin 4. In tissues, the three hTAPs are detectable ubiquitously at low level; pronounced and localized expression is found for hTAP2 and hTAP3 in the perinuclear region in cerebellum, lung, liver and adrenal gland. hTAP3 is well expressed in the epithelial duct cells of several glands, in ovary in endothelial cells of small arteries as well as in granulosa and thecal cells, and in testis in Leydig cells. Thus, the three hTAPs may mediate lipid uptake, secretion, presentation, and sub-cellular localization in a tissue-specific manner, possibly using organelle- and enzyme-specific docking sites.     

Rho proteins of plants--functional cycle and regulation of cytoskeletal dynamics

Rho-related ROP proteins are molecular switches that essentially regulate a wide variety of processes. Of central interest is their influence on the plant cytoskeleton by which they affect vital processes like cell division, growth, morphogenesis, and pathogen defense. ROPs switch between GTP- and GDP-bound conformations by strictly regulated nucleotide exchange and GTP-hydrolysis, and only the active GTP-form interacts with downstream effectors to ultimately provoke a biological response. However, the mode of action of the engaged regulators and effectors as well as their upstream and downstream interaction partners have long been largely unknown. As opposed to analogous systems in animals and fungi, plants use specific GTPase activating proteins (RopGAPs) with a unique domain composition and novel guanine nucleotide exchange factors (RopGEFs) with a probable link to cell surface receptors. Moreover, plants comprise novel effector molecules and adapters connecting ROPs to mostly unknown downstream targets on the route to the cytoskeleton. This review aims to summarize recent knowledge on the molecular mechanisms and reaction cascades involved in ROP dependent cytoskeletal rearrangements, addressing the structure and function of the unusual RopGAPs, RopGEFs and effectors, and the upstream and downstream pathways linking ROPs to cell receptor-like kinases, actin filaments, and microtubules.     

Assembly of Trp1 in a signaling complex associated with caveolin-scaffolding lipid raft domains

Trp1 has been proposed as a component of the store-operated Ca(2+) entry (SOC) channel. However, neither the molecular mechanism of SOC nor the role of Trp in this process is yet understood. We have examined possible molecular interactions involved in the regulation of SOC and Trp1 and report here for the first time that Trp1 is assembled in signaling complex associated with caveolin-scaffolding lipid raft domains. Endogenous hTrp1 and caveolin-1 were present in low density fractions of Triton X-100-extracted human submandibular gland cell membranes. Depletion of plasma membrane cholesterol increased Triton X-100 solubility of Trp1 and inhibited carbachol-stimulated Ca(2+) signaling. Importantly, thapsigargin stimulated Ca(2+) influx, but not internal Ca(2+) release, and inositol 1,4,5-triphosphate (IP(3))-stimulated I(soc) were also attenuated. Furthermore, both anti-Trp1 and anti-caveolin-1 antibodies co-immunoprecipitated hTrp1, caveolin-1, Galpha(q/11), and IP(3) receptor-type 3 (IP(3)R3). These results demonstrate that caveolar microdomains provide a scaffold for (i) assembly of key Ca(2+) signaling proteins into a complex and (ii) coordination of the molecular interactions leading to the activation of SOC. Importantly, we have shown that Trp1 is also localized in this microdomain where it interacts with one or more components of this complex, including IP(3)R3. This finding is potentially important in elucidating the physiological function of Trp.