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At least 235 records · Page 13Linked to original sources
PubMed2006

A KSR/CNK complex mediated by HYP, a novel SAM domain-containing protein, regulates RAS-dependent RAF activation in Drosophila.

RAF is a critical effector of the small GTPase RAS in normal and malignant cells. Despite intense scrutiny, the mechanism regulating RAF activation remains partially understood. Here, we show that the scaffold KSR (kinase suppressor of RAS), a RAF homolog known to assemble RAF/MEK/ERK complexes, induces RAF activation in Drosophila by a mechanism mediated by its kinase-like domain, but which is independent of its scaffolding property or putative kinase activity. Interestingly, we found that KSR is recruited to RAF prior to signal activation by the RAF-binding protein CNK (connector enhancer of KSR) in association with a novel SAM (sterile alpha motif) domain-containing protein, named Hyphen (HYP). Moreover, our data suggest that the interaction of KSR to CNK/HYP stimulates the RAS-dependent RAF-activating property of KSR. Together, these findings identify a novel protein complex that controls RAF activation and suggest that KSR does not only act as a scaffold for the MAPK (mitogen-activated protein kinase) module, but may also function as a RAF activator. By analogy to catalytically impaired, but conformationally active B-RAF oncogenic mutants, we discuss the possibility that KSR represents a natural allosteric inducer of RAF catalytic function.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Three regions within ActA promote Arp2/3 complex-mediated actin nucleation and Listeria monocytogenes motility.

The Listeria monocytogenes ActA protein induces actin-based motility by enhancing the actin nucleating activity of the host Arp2/3 complex. Using systematic truncation analysis, we identified a 136-residue NH(2)-terminal fragment that was fully active in stimulating nucleation in vitro. Further deletion analysis demonstrated that this fragment contains three regions, which are important for nucleation and share functional and/or limited sequence similarity with host WASP family proteins: an acidic stretch, an actin monomer-binding region, and a cofilin homology sequence. To determine the contribution of each region to actin-based motility, we compared the biochemical activities of ActA derivatives with the phenotypes of corresponding mutant bacteria in cells. The acidic stretch functions to increase the efficiency of actin nucleation, the rate and frequency of motility, and the effectiveness of cell-cell spread. The monomer-binding region is required for actin nucleation in vitro, but not for actin polymerization or motility in infected cells, suggesting that redundant mechanisms may exist to recruit monomer in host cytosol. The cofilin homology sequence is critical for stimulating actin nucleation with the Arp2/3 complex in vitro, and is essential for actin polymerization and motility in cells. These data demonstrate that each region contributes to actin-based motility, and that the cofilin homology sequence plays a principal role in activation of the Arp2/3 complex, and is an essential determinant of L. monocytogenes pathogenesis.

Actin-Related Protein 2↗
PubMed2001

The conserved npl4 protein complex mediates proteasome-dependent membrane-bound transcription factor activation.

Proteolytic activation of membrane-bound transcription factors has emerged as an important mechanism for the regulation of gene expression. Two membrane-bound transcription factors regulated in this manner are the Saccharomyces cerevisiae proteins Mga2p and Spt23p, which direct transcription of the Delta9-fatty acid desaturase gene OLE1. We now show that a membrane-associated complex containing the highly conserved Npl4p, Ufd1p, and Cdc48p proteins mediates the proteasome-regulated cleavage of Mga2p and Spt23p. Mutations in NPL4, UFD1, and CDC48 cause a block in Mga2p and Spt23p processing, with concomitant loss of OLE1 expression. Taken together, our data indicate that the Npl4 complex may serve to target the proteasome to the ubiquitinated endoplasmic reticulum membrane-bound proteins Mga2p and Spt23p. Given the recent finding that NPL4 is allelic to the ERAD gene HRD4, we further propose that this NPL4 function extends to all endoplasmic reticulum-membrane-associated targets of the proteasome.

Adaptor Proteins, Vesicular Transport↗
PubMed2001

Downregulation of the tissue-specific transcription factor Fork head by Broad-Complex mediates a stage-specific hormone response.

Drosophila development is coordinated by pulses of the steroid hormone 20-hydroxyecdysone (20E). During metamorphosis, the 20E-inducible Broad-Complex (BR-C) gene plays a key role in the genetic hierarchies that transduce the hormone signal, being required for the destruction of larval tissues and numerous aspects of adult development. Most of the known BR-C target genes, including the salivary gland secretion protein (Sgs) genes, are terminal differentiation genes that are thought to be directly regulated by BR-C-encoded transcription factors. Here, we show that repression of Sgs expression is indirectly controlled by the BR-C through transcriptional down-regulation of fork head, a tissue-specific gene that plays a central role in salivary gland development and is required for Sgs expression. Our results demonstrate that integration of a tissue-specific regulatory gene into a 20E-controlled genetic hierarchy provides a mechanism for hormonal repression. Furthermore, they suggest that the BR-C is placed at a different position within the 20E-controlled hierarchies than previously assumed, and that at least part of its pleiotropic functions are mediated by tissue-specific regulators.

Animals↗
PubMed1999

A novel clathrin adaptor complex mediates basolateral targeting in polarized epithelial cells.

Although polarized epithelial cells are well known to maintain distinct apical and basolateral plasma membrane domains, the mechanisms responsible for targeting membrane proteins to the apical or basolateral surfaces have remained elusive. We have identified a novel form of the AP-1 clathrin adaptor complex that contains as one of its subunits mu1B, an epithelial cell-specific homolog of the ubiquitously expressed mu1A. LLC-PK1 kidney epithelial cells do not express mu1B and missort many basolateral proteins to the apical surface. Stable expression of mu1B selectively restored basolateral targeting, improved the overall organization of LLC-PK1 monolayers, and had no effect on apical targeting. We conclude that basolateral sorting is mediated by an epithelial cell-specific version of the AP-1 complex containing mu1B.

Adaptor Protein Complex 1↗
PubMed2003

RETRACTED: The glycoprotein Ib-IX-V complex mediates localization of factor XI to lipid rafts on the platelet membrane.

Factor XI binds to activated platelets where it is efficiently activated by thrombin. The factor XI receptor is the platelet membrane glycoprotein (GP) Ib-IX-V complex (Baglia, F. A., Badellino, K. O., Li, C. Q., Lopez, J. A., and Walsh, P. N. (2002) J. Biol. Chem. 277, 1662-1668), a significant fraction of which exists within lipid rafts on stimulated platelets (Shrimpton, C. N., Borthakur, G., Larrucea, S., Cruz, M. A., Dong, J. F., and Lopez, J. A. (2002) J. Exp. Med. 196, 1057-1066). Lipid rafts are membrane microdomains enriched in cholesterol and sphingolipids implicated in localizing membrane ligands and in cellular signaling. We now show that factor XI was localized to lipid rafts in activated platelets ( approximately 8% of total bound) but not in resting platelets. Optimal binding of factor XI to membrane rafts required prothrombin (and Ca2+) or high molecular weight kininogen (and Zn2+), which are required for factor XI binding to platelets. An antibody to GPIb (SZ-2) that disrupts factor XI binding to the GPIb-IX-V complex also disrupted factor XI-raft association. The isolated recombinant Apple 3 domain of factor XI, which mediates factor XI binding to platelets, also completely displaces factor XI from membrane rafts. To investigate the physiological relevance of the factor XI-raft association, the structural integrity of lipid rafts was disrupted by cholesterol depletion utilizing methyl-beta-cyclodextrin. Cholesterol depletion completely prevented FXI binding to lipid rafts, and initial rates of factor XI activation by thrombin on activated platelets were inhibited >85%. We conclude that factor XI is localized to GPIb in membrane rafts and that this association is important for promoting the activation of factor XI by thrombin on the platelet surface.

Blood Platelets↗
PubMed2002

Toll-like receptor 4-MD-2 complex mediates the signal transduction induced by flavolipin, an amino acid-containing lipid unique to Flavobacterium meningosepticum.

Flavolipin, an amino acid-containing lipid isolated from Flavobacterium meningosepticum, induces many immune responses. It has been shown that flavolipin does not induce an immune response of macrophages derived from C3H/HeJ mice, which possess a point mutation in Toll-like receptor 4 (TLR4). To determine whether TLR4 or the molecular complex of TLR4 and TLR4 association molecule MD-2 mediates the flavolipin signal, flavolipin responsiveness was examined by measuring NF-kappaB activation in Ba/F3 cells and Ba/F3 transfectants expressing TLR4 or both TLR4 and MD-2. Flavolipin-induced NF-kappaB activation was detected in the cells expressing both TLR4 and MD-2, but not in the other cells. Expression of CD14 in the transfectant expressing both TLR4 and MD-2 increased the sensitivity to flavolipin. Furthermore, flavolipin stereoisomers were chemically synthesized, and their abilities to induce NF-kappaB activation were examined. (R)-Flavolipin, in which the configuration of the lipid moiety is R, induced NF-kappaB activation via the TLR4-MD-2 complex, but (S)-flavolipin did not. In this study, we demonstrated the involvement of TLR4-MD-2 and CD14 in flavolipin signaling and the importance of the (R)-configuration of the flavolipin lipid moiety for the induction of an immune response via TLR4-MD-2.

Animals↗
PubMed1998

The yeast Ada complex mediates the ligand-dependent activation function AF-2 of retinoid X and estrogen receptors.

Nuclear receptors can function as ligand-inducible transregulators in both mammalian and yeast cells, indicating that important features of control of transcription have been conserved throughout evolution. Here, we report the isolation and characterization of a yeast protein that exhibits properties expected for a coactivator/mediator of the ligand-dependent activation function AF-2 present in the ligand-binding domain (LBD, region E) of the retinoid X (RXRalpha) and estrogen (ERalpha) receptors. This protein is identical to Ada3, a component of the yeast Ada coactivator complex. We demonstrate that: (1) the region encompassing residues 347-702 of Ada3 interacts with the LBD of RXRalpha and ERalpha in a ligand-dependent manner in yeast; (2) this interaction corresponds to a direct binding and requires the integrity of the core of the AF-2 activating domain (AF-2 AD) of both RXRalpha and ERalpha; (3) Ada3 as well as Ada2 and Gcn5, two other components of the Ada complex, are required for maximal AF-2 activity in yeast; and (4) Ada3 is able to enhance the AF-2 activity of RXRalpha and ERalpha when overexpressed in yeast and mammalian cells. Taken together, these data indicate that ligand-dependent transactivation by RXRalpha and ERalpha in yeast is mediated at least in part by the Ada complex, in which the Ada3 subunit directly binds to the holoreceptor LBD.

Animals↗
PubMedDate not supplied

Influence of membrane-active peptides on lipospermine/DNA complex mediated gene transfer.

To explore whether endosomal release presents a major barrier to lipospermine-mediated gene delivery, acidic membrane-active peptides derived from influenza virus or artificial sequences were incorporated into DNA/dioctadecylamidoglycylspermine (= Transfectam) complexes. Depending on the cell line used, gene expression levels are approximately 3-30-fold higher than those obtained by applying DNA complexed to optimal amounts of Transfectam alone. In addition, gene transfer efficiency of DNA complexes with lower amounts of Transfectam (1.5-2 charge equiv) is increased by a factor of up to 1000 by peptides INF6 (influenza virus derived sequence) and INF10 (artificial sequence). The helper lipids 1,2-dioleoylphosphatidylethanolamine, egg phosphatidylethanolamine, and 1,2-dioleoyl-racglycerol also can enhance the gene transfer. Thus, endosomal escape seems to be only a moderate barrier for optimized, positively charged DNA/Transfectam complexes, but a substantial bottleneck for less positively charged complexes.

Amino Acid Sequence↗
PubMed2000

Selective inhibition of adaptor complex-mediated vesiculation.

A short while ago, we could only inhibit post-Golgi membrane traffic with crude, unselective tools, such as low temperature or high extracellular sucrose. Molecular dissection of vesiculation steps has revealed unexpected complexity in the coating machinery that has initiated a search for more specific inhibitors. We have learned that membrane vesiculation is driven by a tightly regulated multicomponent, membrane-associated protein machine held together by carefully specified interaction domains. An experimental advantage of such complex interacting machinery is that it is very susceptible to disruption by dominant negative inhibitors or by overexpression. As a result, we now have much more specific inhibitors of post-Golgi membrane traffic. Some, such as dynamin K44A, may be general inhibitors, whereas others can distinguish classes of endocytotic events (10), binding events that require clathrin from those that do not (42), or specific steps of endocytosis (62). Ligand-mediated uptake of EGF and numerous, but not all, GPCRs can be inhibited by overexpression of an ARF GTPase-activating protein that has no effect on transferrin uptake (67). We can look forward to increasingly powerful and selective inhibitors that should help us to navigate successfully the complex routes of post-Golgi membrane traffic.

Adaptor Protein Complex alpha Subunits↗
PubMed2006

BKCa-Cav channel complexes mediate rapid and localized Ca2+-activated K+ signaling.

Large-conductance calcium- and voltage-activated potassium channels (BKCa) are dually activated by membrane depolarization and elevation of cytosolic calcium ions (Ca2+). Under normal cellular conditions, BKCa channel activation requires Ca2+ concentrations that typically occur in close proximity to Ca2+ sources. We show that BKCa channels affinity-purified from rat brain are assembled into macromolecular complexes with the voltage-gated calcium channels Cav1.2 (L-type), Cav2.1 (P/Q-type), and Cav2.2 (N-type). Heterologously expressed BKCa-Cav complexes reconstitute a functional "Ca2+ nanodomain" where Ca2+ influx through the Cav channel activates BKCa in the physiological voltage range with submillisecond kinetics. Complex formation with distinct Cav channels enables BKCa-mediated membrane hyperpolarization that controls neuronal firing pattern and release of hormones and transmitters in the central nervous system.

Amino Acid Sequence↗
PubMed1984

Inhibitory effect of honey bee venom on immune complex mediated leukocyte migration into rabbit knee-joints.

The anti-inflammatory effect of purified honey bee venom (HBV) was studied using a recently described animal model in which preformed immune complexes were injected into rabbit knee-joints. As little as a single injection of 1.2 micrograms HBV/kg body weight subcutaneously significantly reduced the immune complex induced joint inflammation as measured by reduction in leukocyte counts in the joint fluid. This decrease was obvious 3 and 6 but not 9 hours after induction of the inflammation. There was no significant effect on leukocyte random migration, chemotactic responsiveness or phagocytosis, indicating that HBV did not interfere with normal phagocyte motility and ingestion. The modifying effects by HBV on the inflammatory response to immune complexes in vivo is most likely due to interference with other components of the inflammatory response.

Animals↗
PubMed2005

A WAVE2-Abi1 complex mediates CSF-1-induced F-actin-rich membrane protrusions and migration in macrophages.

Colony-stimulating factor 1 (CSF-1) is an important physiological chemoattractant for macrophages. The mechanisms by which CSF-1 elicits the formation of filamentous actin (F-actin)-rich membrane protrusions and induces macrophage migration are not fully understood. In particular, very little is known regarding the contribution of the different members of the Wiskott-Aldrich Syndrome protein (WASP) family of actin regulators in response to CSF-1. Although a role for WASP itself in macrophage chemotaxis has been previously identified, no data was available regarding the function of WASP family verprolin-homologous (WAVE) proteins in this cell type. We found that WAVE2 was the predominant isoform to be expressed in primary macrophages and in cells derived from the murine monocyte/macrophage RAW264.7 cell line (RAW/LR5). CSF-1 treatment of macrophages resulted in WAVE2 accumulation in F-actin-rich protrusions induced by CSF-1. Inhibition of WAVE2 function by expressing a dominant-negative mutant or introducing anti-WAVE2 antibodies in RAW/LR5 cells, as well as reduction of endogenous WAVE2 expression by RNA-mediated interference (RNAi), resulted in a significant reduction of CSF-1-elicited F-actin protrusions. WAVE2 was found in a protein complex together with Abelson kinase interactor 1 (Abi1) in resting or stimulated cells. Both WAVE2 and Abi1 were recruited to and necessary for the formation of F-actin protrusions in response to CSF-1. Reducing the levels of WAVE2, directly or by targeting Abi1, resulted in an impaired cell migration to CSF-1. Altogether these data identify a WAVE2-Abi1 complex crucial for the normal actin cytoskeleton reorganization and migration of macrophages in response to CSF-1.

Actins↗
PubMed2005

Dynein-dynactin complex mediates protein kinase A-dependent clustering of Weibel-Palade bodies in endothelial cells.

OBJECTIVE: Perinuclear clustering is observed for several different organelles and illustrates dynamic regulation of the secretory pathway and organelle distribution. Previously, we observed that a subset of Weibel-Palade bodies (WPBs), endothelial cell-specific storage organelles, undergo centralization when endothelial cells are stimulated with cAMP-raising agonists of von Willebrand factor (vWF) secretion. In this study, we investigated this phenomenon of WPB clustering in more detail. METHODS AND RESULTS: Our results demonstrate that the clustered WPBs are localized at the microtubule organizing center and that cluster formation depends on an intact microtubule network. Disruption of the microtubules by nocodazole completely abolished clustering, whereas treatment with the actin depolymerizing compound cytochalasin B had no effect on WPB clustering. Interfering with the dynein-dynactin interaction by overexpression of the p50 dynamitin subunit or the CC1 domain of the p150glued subunit of the dynactin complex completely inhibited perinuclear clustering of WPBs, suggesting that dynein activity mediates this process. Furthermore, we found that inhibition of dephosphorylation resulted in an increase in clustering, whereas inhibition of protein kinase A (PKA) markedly reduced WPB clustering. CONCLUSIONS: These results suggest that perinuclear clustering of WPBs involves PKA-dependent regulation of the dynein-dynactin complex. Endothelial cell stimulation with epinephrine results in retrograde movement of a subset of WPBs to the microtubule organizing center. This minus-end directed transport requires an intact microtubular network and is mediated by the motor protein dynein. Together, our results suggest that epinephrine-induced clustering of WPBs involves PKA-dependent regulation of the dynein-dynactin complex.

1-Methyl-3-isobutylxanthine↗
PubMed1999

Active transcriptional repression by the Rb-E2F complex mediates G1 arrest triggered by p16INK4a, TGFbeta, and contact inhibition.

Rb inhibits progression from G1 to S phase of the cell cycle. It associates with a number of cellular proteins; however, the nature of these interactions and their relative significance in cell cycle regulation are still unclear. We present evidence that Rb must normally interact with the E2F family of transcription factors to arrest cells in G1, and that this arrest results from active transcriptional repression by the Rb-E2F complex, not from inactivation of E2F. Thus, a major role of E2F in cell cycle regulation is assembly of this repressor complex. We demonstrate that active repression by Rb-E2F mediates the G1 arrest triggered by TGFbeta, p16INK4a, and contact inhibition.

Animals↗