Signal transduction from the membrane to the nucleus: variations on common themes

Platelet aggregation at sites of vascular injury releases both peptide growth factors and vasoactive compounds. Although significant attention has been focused on peptide growth factors, very little is known about the mitogenic effect of vasoactive compounds. We evaluated the effect of serotonin (5-HT) and thromboxane A2 (TXA2) mimetic U46619 alone and in combination on aortic endothelial cells. Stimulation of endothelial cells by 5-HT resulted in an increase in tritiated thymidine uptake and an increase in cell number, whereas U46619 did not have any significant effect. However, when endothelial cells were exposed to both compounds, U46619 potentiated the mitogenic effect of 5-HT on endothelial cells. When endothelial cells were preincubated with LY281067 (a 5-HT2 receptor antagonist) or ridogrel (a combined TXA2 synthase inhibitor and receptor antagonist), LY281067 blocked the mitogenic effect of 5-HT and ridogrel blocked the potentiating effect of U46619 on 5-HT2-induced tritiated thymidine incorporation. When endothelial cells were preincubated with both antagonists, the effects of both 5-HT and U46619 were blocked. Recent studies have indicated that regenerating endothelial cells at sites of vascular injury may release growth factors for vascular smooth muscle cells, leading to smooth muscle cell proliferation and development of neointima. This study suggests that the combined use of 5-HT and TXA2 receptor antagonists may inhibit the growth of endothelial cells at sites of vascular injury and attenuate the formation of neointima.     

A truncated HIV-1 Tat protein basic domain rapidly translocates through the plasma membrane and accumulates in the cell nucleus

Tat is an 86-amino acid protein involved in the replication of human immunodeficiency virus type 1 (HIV-1). Several studies have shown that exogenous Tat protein was able to translocate through the plasma membrane and to reach the nucleus to transactivate the viral genome. A region of the Tat protein centered on a cluster of basic amino acids has been assigned to this translocation activity. Recent data have demonstrated that chemical coupling of a Tat-derived peptide (extending from residues 37 to 72) to several proteins allowed their functional internalization into several cell lines or tissues. A part of this same domain can be folded in an alpha-helix structure with amphipathic characteristics. Such helical structures have been considered as key determinants for the uptake of several enveloped viruses by fusion or endocytosis. In the present study, we have delineated the main determinants required for Tat translocation within this sequence by synthesizing several peptides covering the Tat domain from residues 37 to 60. Unexpectedly, the domain extending from amino acid 37 to 47, which corresponds to the alpha-helix structure, is not required for cellular uptake and for nuclear translocation. Peptide internalization was assessed by direct labeling with fluorescein or by indirect immunofluorescence using a monoclonal antibody directed against the Tat basic cluster. Both approaches established that all peptides containing the basic domain are taken up by cells within less than 5 min at concentrations as low as 100 nM. In contrast, a peptide with a full alpha-helix but with a truncated basic amino acid cluster is not taken up by cells. The internalization process does not involve an endocytic pathway, as no inhibition of the uptake was observed at 4 degrees C. Similar observations have been reported for a basic amino acid-rich peptide derived from the Antennapedia homeodomain (1). Short peptides allowing efficient translocation through the plasma membrane could be useful vectors for the intracellular delivery of various non-permeant drugs including antisense oligonucleotides and peptides of pharmacological interest.     

Blocked signal transduction to the ERK and JNK protein kinases in anergic CD4+ T cells

T cells activated by antigen receptor stimulation in the absence of accessory cell-derived costimulatory signals lose the capacity to synthesize the growth factor interleukin-2 (IL-2), a state called clonal anergy. An analysis of CD3- and CD28-induced signal transduction revealed reduced ERK and JNK enzyme activities in murine anergic T cells. The amounts of ERK and JNK proteins were unchanged, and the kinases could be fully activated in the presence of phorbol 12-myristate 13-acetate. Dephosphorylation of the calcineurin substrate NFATp (preexisting nuclear factor of activated T cells) also remained inducible. These results suggest that a specific block in the activation of ERK and JNK contributes to defective IL-2 production in clonal anergy.     

Signal transduction by the IL-1 type I receptor: evidence for the involvement of a receptor-coupled protein kinase

A novel serine/threonine specific protein kinase was found to be associated with the type I IL-1 receptor in the murine T cell lines D10N and EL-4. This kinase was identified in immunoprecipitates from IL-1 stimulated T-cells by its ability to phosphorylate exogenous substrates in the presence of radiolabeled ATP. An endogenous protein, most likely a member of the IL-1 R1 complex, was also phosphorylated. The activation of the kinase is specific for IL-1, neither TNF nor phorbol esters were able to activate the IL-1 RI associated kinase activity. The IL-1 receptor antagonist had no intrinsic activity and inhibited the activation of the kinase. The activation of the kinase was rapid and detectable after 30 seconds of IL-1 stimulation. A minimal model of the IL-RI signal transduction complex is discussed, presenting this novel serine/threonine kinase as a constituent of the complex.     

Epstein-Barr virus protein LMP2A regulates reactivation from latency by negatively regulating tyrosine kinases involved in sIg-mediated signal transduction

Like other herpesviruses, Epstein-Barr Virus (EBV) persists in its host through an ability to establish latent infection with episodic reactivations. In latent infection EBV expresses an integral membrane protein LMP2A that regulates reactivation from latency. LMP2A is constitutively tyrosine phosphorylated and is associated with lyn and syk tyrosine kinases. The activity of lyn is substantially reduced. In EBV-infected cells in which LMP2A is expressed, crosslinking of sIg fails to trigger the protein tyrosine kinase signal cascade, tyrosine phosphorylation of cell proteins does not change, second messengers are not generated, and lytic EBV infection is not induced. In contrast, crosslinking of sIg on cells infected with EBV recombinants with null mutations in LMP2A results in transient tyrosine phosphorylation of lyn, syk, phospholipase C gamma 2 and phosphatidylinositol-3' kinase, transiently increased intracellular free calcium, and reactivation of lytic EBV infection. These studies describe a novel molecular regulator of herpesvirus latency and focus attention on the importance of transmembrane signal transduction in herpes virus reactivation from latency. They support the working hypothesis that the identification of ligand-receptor interactions that can result in the induction of reactivation will provide an important inroad toward the delineation of the molecular mechanism, which govern herpesvirus reactivation from latency.     

Uptake of long chain fatty acids by human placental choriocarcinoma (BeWo) cells: role of plasma membrane fatty acid-binding protein

In order to understand the mechanisms by which fatty acids are taken up by the placenta, the uptake of oleic, linoleic, arachidonic, and docosahexaenoic acids by cultured human placental choriocarcinoma (BeWo) cells was examined. Fatty acid uptake by BeWo cells was temperature-dependent and exhibited saturable kinetics. Oleic acid was taken up least and docosahexaenoic acid most by these cells. Moreover, competitive studies of fatty acid uptake by BeWo cells also indicated preferential uptake compared with oleic acid in the order of docosahexaenoic acid, arachidonic acid, and linoleic acid. Western blot analysis demonstrated that BeWo cells express a protein immunoreactive with antibodies to the human placental plasma membrane fatty acid-binding protein (p-FABPpm). Furthermore, pre-treatment of BeWo cells with these antibodies inhibited most of the uptake of docosahexaenoic (64%) and arachidonic acids (68%) whereas oleic acid uptake was inhibited only 32% compared with the controls treated with preimmune serum. These results clearly demonstrate that the pFABPpm may be involved in the preferential uptake of essential fatty acids (EFA) and their long chain polyunsaturated fatty acids (LCPUFA) by these cells. Studies on the distribution of radiolabeled fatty acids in the cellular lipids of BeWo cells showed that docosahexaenoic acid was incorporated mainly in the triacylglycerol fraction, followed by the phospholipid fraction, whereas for arachidonic acid the reverse was true. The preferential incorporation of docosahexaenoic acid into triacylglycerol suggests that triacylglycerol may play an important role in the placental transport of docosahexaenoic acid to the fetal circulation. Together these results demonstrate the preferential uptake of EFA/LCPUFA by BeWo cells that is most probably mediated via the pFABPpm. We thus propose that the p-FABPpm may be involved in the sequestration of maternal plasma LCPUFA by the placenta.     

Signal sequence recognition in posttranslational protein transport across the yeast ER membrane

We have analyzed how the signal sequence of prepro-alpha-factor is recognized during the first step of posttranslational protein transport into the yeast endoplasmic reticulum. Cross-linking studies indicate that the signal sequence interacts in a Kar2p- and ATP-independent reaction with Sec61p, the multispanning membrane component of the protein-conducting channel, by intercalation into transmembrane domains 2 and 7. While bound to Sec61p, the signal sequence forms a helix that is contacted on one side by Sec62p and Sec71p. The binding site is located at the interface of the protein channel and the lipid bilayer. Signal sequence recognition in cotranslational translocation in mammals appears to occur similarly. These results suggest a general mechanism by which the signal sequence could open the channel for polypeptide transport.     

Signal transduction from the extracellular matrix--a role for the focal adhesion protein-tyrosine kinase FAK

The therapy concept of topical factor XIII application was developed on the basis of clinical and experimental investigations on improvement of wound healing, as well as on the morphological and pathophysiological topical site of venous ulcers. Topical treatment with factor XIII is special with regard to mode of application as well as efficacy, since a lot of other medications used for local wound treatment contain a fibrinolytic component. In the last 32 months, 23 inpatients (17 female, 6 male) averaging 62.9 +/- 14.1 years and suffering ulcerative leg disease were treated with topically applied factor XIII. The average period of distal venous ulcer in these patients was 3.3 +/- 2.04 years. The extent of the ulcer surface ranged between 2.5 x 3.0 cm (minimum) and 18.5 x 8.0 cm (maximum). All patients had been in medical consultation for several years. The venous ulcers were based upon a "postthrombotic syndrome" in 15 patients. Six patients were suffering from ulcerations due to arterial and venous mixed disease, and there were 2 patients with ulcerations of unknown etiology. In 5 patients, 2 with unknown etiology and 3 with arterial and venous mixed disease, local treatment with factor XIII was discontinued after 4 weeks because there was definitely no improvement in wound healing. Additionally, 3 patients with ulcerations due to arterial and venous mixed disease were treated for a maximum of 6 weeks with only moderate improvement in healing. Twelve of the 15 patients (79.3%) with extended, chronic ulceration on the basis of a postthrombotic syndrome showed such a distinct improvement of topical site after an average of only 3.15 +/- 1.14 weeks that they were released for further ambulatory treatment. Three patients had to be treated for a maximum period of 6 weeks, also with distinct improvement in wound healing. Apart from a wound surface reduction and a clinically documentable improvement of granulation tendency, there was a marked reduction of secretion and bleeding tendency within the ulcer area observed in all patients. None of the patients showed any systemic or local allergic reactions.     

Intracellular protein transport to the thyrocyte plasma membrane: potential implications for thyroid physiology

Calculations of stopping power ratios, water to air, for the determination of absorbed dose to water in clinical proton beams using ionization chamber measurements have been undertaken using the Monte Carlo method. A computer code to simulate the transport of protons in water (PETRA) has been used to calculate sw.air-data under different degrees of complexity, ranging from values based on primary protons only to data including secondary electrons and high-energy secondary protons produced in nonelastic nuclear collisions. All numerical data are based on ICRU 49 proton stopping powers. Calculations using primary protons have been compared to the simple continuous slowing-down approximation (c.s.d.a.) analytical technique used in proton dosimetry protocols, not finding significant differences that justify elaborate Monte Carlo simulations except beyond the mean range of the protons (the far side of the Bragg peak). The influence of nuclear nonelastic processes, through the detailed generation and transport of secondary protons, on the calculated stopping-power ratios has been found to be negligible. The effect of alpha particles has also been analysed, finding differences smaller than 0.1% from the results excluding them. Discrepancies of up to 0.6% in the plateau region have been found, however, when the production and transport of secondary electrons are taken into account. The large influence of nonelastic nuclear interactions on proton depth-dose distributions shows that the removal of primary protons from the incident beam decreases the peak-to-plateau ratio by a large factor, up to 40% at 250 MeV. It is therefore emphasized that nonelastic nuclear reactions should be included in Monte Carlo simulations of proton beam depth-dose distributions.     

An essential role for autophosphorylation in the dissociation of activated protein kinase C from the plasma membrane

The cellular localization of protein kinase C (PKC) is intimately associated with the regulation of its biological activity. Previously we have demonstrated that the redistribution of PKC to the plasma membrane in response to physiological stimuli is followed by a rapid returning of PKC back to the cytoplasm (Feng, X., Zhang, J., Barak, L. S., Meyer, T., Caron, M. G., and Hannun, Y. A. (1998) J. Biol. Chem. 273, 10755-10762). Although the process of PKC membrane targeting has been extensively studied, the molecular mechanism underlying the dissociation of membrane-bound PKC remains unclear. In the present study, by examining the dynamic distribution of wild-type PKC betaII and its kinase-deficient mutant (K371R), we demonstrate that kinase activity is required for PKC membrane dissociation. Moreover, the inability of PKC betaII(K371R) to dissociate from the plasma membrane in cells overexpressing wild-type PKC betaII suggests that autophosphorylation activity of the kinase might be essential for its membrane dissociation. This was further supported by mutational analysis of two in vivo autophosphorylation sites on PKC betaII. The replacement of Ser660 or Thr641 by alanine (S660A or T641A) was found to synergistically reduce the reversal of PKC betaII membrane translocation, whereas the replacement of the same amino acids by glutamic acid (S660E or T641E), an amino acid commonly used to mimic phosphate, results in mutants behaving similar to wild-type PKC betaII. These findings point to an essential role for autophosphorylation in the dissociation of activated PKC from the plasma membrane and suggest that, like PKC membrane translocation, the returning of PKC to the cytoplasm after its activation is also delicately regulated.     

Signal transduction by T- and B-cell antigen receptors: converging structures and concepts

Recent evidence demonstrates that the antigen receptor complexes of T and B lymphocytes are very similar in general architecture and primary and secondary structure of component polypeptides, and that they use common mechanisms for transmembrane signal transduction. Most importantly, multiple subunits of each receptor (Ig-alpha, Ig-beta and Ig-gamma, CD3 gamma, CD3 delta and CD3 epsilon, and T-cell receptor zeta and eta) possess a motif of approximately 26 amino-acids, denoted ARH1, which appears to carry sufficient structural information for receptor-mediated lymphocyte activation.     

Janus kinases and focal adhesion kinases play in the 4.1 band: a superfamily of band 4.1 domains important for cell structure and signal transduction

The band 4.1 domain was first identified in the red blood cell protein band 4.1, and subsequently in ezrin, radixin, and moesin (ERM proteins) and other proteins, including tumor suppressor merlin/schwannomin, talin, unconventional myosins VIIa and X, and protein tyrosine phosphatases. Recently, the presence of a structurally related domain has been demonstrated in the N-terminal region of two groups of tyrosine kinases: the focal adhesion kinases (FAK) and the Janus kinases (JAK). Additional proteins containing the 4.1/JEF (JAK, ERM, FAK) domain include plant kinesin-like calmodulin-binding proteins (KCBP) and a number of uncharacterized open reading frames identified by systematic DNA sequencing. Phylogenetic analysis of amino acid sequences suggests that band 4.1/JEF domains can be grouped in several families that have probably diverged early during evolution. Hydrophobic cluster analysis indicates that the band 4.1/JEF domains might consist of a duplicated module of approximately 140 residues and a central hinge region. A conserved property of the domain is its capacity to bind to the membrane-proximal region of the C-terminal cytoplasmic tail of proteins with a single transmembrane segment. Many proteins with band 4.1/JEF domains undergo regulated intra- or intermolecular homotypic interactions. Additional properties common to band 4.1/JEF domains of several proteins are binding of phosphoinositides and regulation by GTPases of the Rho family. Many proteins with band 4. 1/JEF domains are associated with the actin-based cytoskeleton and are enriched at points of contact with other cells or the extracellular matrix, from which they can exert control over cell growth. Thus, proteins with band 4.1/JEF domain are at the crossroads between cytoskeletal organization and signal transduction in multicellular organisms. Their importance is underlined by the variety of diseases that can result from their mutations.     

Multiple pathways for protein transport into or across the thylakoid membrane

Many thylakoid proteins are cytosolically synthesized and have to cross the two chloroplast envelope membranes as well as the thylakoid membrane en route to their functional locations. In order to investigate the localization pathways of these proteins, we over-expressed precursor proteins in Escherichia coli and used them in competition studies. Competition was conducted for import into the chloroplast and for transport into or across isolated thylakoids. We also developed a novel in organello method whereby competition for thylakoid transport occurred within intact chloroplasts. Import of all precursors into chloroplasts was similarly inhibited by saturating concentrations of the precursor to the OE23 protein. In contrast, competition for thylakoid transport revealed three distinct precursor specificity groups. Lumen-resident proteins OE23 and OE17 constitute one group, lumenal proteins plastocyanin and OE33 a second, and the membrane protein LHCP a third. The specificity determined by competition correlates with previously determined protein-specific energy requirements for thylakoid transport. Taken together, these results suggest that thylakoid precursor proteins are imported into chloroplasts on a common import apparatus, whereupon they enter one of several precursor-specific thylakoid transport pathways.     

Distinct biochemical and topological properties of the 31- and 27-kilodalton plasma membrane intrinsic protein subgroups from red beet

Plasma membrane vesicles from red beet (Beta vulgaris L.) storage tissue contain two prominent major intrinsic protein species of 31 and 27 kD (X. Qi, C.Y Tai, B.P. Wasserman [1995] Plant Physiol 108: 387-392). In this study affinity-purified antibodies were used to investigate their localization and biochemical properties. Both plasma membrane intrinsic protein (PMIP) subgroups partitioned identically in sucrose gradients; however, each exhibited distinct properties when probed for multimer formation, and by limited proteolysis. The tendency of each PMIP species to form disulfide-linked aggregates was studied by inclusion of various sulfhydryl agents during tissue homogenization and vesicle isolation. In the absence of dithiothreitol and sulfhydryl reagents, PMIP27 yielded a mixture of monomeric and aggregated species. In contrast, generation of a monomeric species of PMIP31 required the addition of dithiothreitol, iodoacetic acid, or N-ethylmaleimide. Mixed disulfide-linked heterodimers between the PMIP31 and PMIP27 subgroups were not detected. Based on vectorial proteolysis of right-side-out vesicles with trypsin and hydropathy analysis of the predicted amino acid sequence derived from the gene encoding PMIP27, a topological model for a PMIP27 was established. Two exposed tryptic cleavage sites were identified from proteolysis of PMIP27, and each was distinct from the single exposed site previously identified in surface loop C of a PMIP31. Although the PMIP31 and PMIP27 species both contain integral proteins that appear to occur within a single vesicle population, these results demonstrate that each PMIP subgroup responds differently to perturbations of the membrane.     

Targeting of the Yersinia pestis YopM protein into HeLa cells and intracellular trafficking to the nucleus

The YopM virulence protein of Yersinia pestis has been described as binding human alpha-thrombin and inhibiting thrombin-induced platelet aggregation in vitro. However, recent studies have shown that a YopM-CyaA fusion protein could be targeted vectorially into eukaryotic cells through the Yersinia type III secretion system. In this study, our objective was to characterize YopM's fate in more detail. We followed YopM in the culture medium and inside infected HeLa cells. We confirmed that the native YopM is targeted into HeLa cells, where it is insensitive to exogenous trypsin. The bacteria must be surface located to target YopM, and YopB and YopD are necessary, whereas the LcrE protein (called also YopN) makes this process more efficient. Immunofluorescence localization revealed that YopM, in contrast to YopE, is not only targeted to the cytoplasm but also trafficks to the cell's nucleus by means of a vesicle-associated pathway that is strongly inhibited by brefeldin A, perturbed by monensin or bafilomycin A1 and dependent upon microtubules (decreased by colchicine and nocodazole). These findings revealed a novel interaction of Yersinia pestis with its eukaryotic host.     

The central structural feature of the membrane fusion protein subunit from the Ebola virus glycoprotein is a long triple-stranded coiled coil

The ectodomain of the Ebola virus Gp2 glycoprotein was solubilized with a trimeric, isoleucine zipper derived from GCN4 (pIIGCN4) in place of the hydrophobic fusion peptide at the N terminus. This chimeric molecule forms a trimeric, highly alpha-helical, and very thermostable molecule, as determined by chemical crosslinking and circular dichroism. Electron microscopy indicates that Gp2 folds into a rod-like structure like influenza HA2 and HIV-1 gp41, providing further evidence that viral fusion proteins from diverse families such as Orthomyxoviridae (Influenza), Retroviridae (HIV-1), and Filoviridae (Ebola) share common structural features, and suggesting a common membrane fusion mechanism.     

Human regulatory subunit RI beta of cAMP-dependent protein kinases: expression, holoenzyme formation and microinjection into living cells

The human regulatory subunit RI beta of cAMP-dependent protein kinases was expressed in Escherichia coli as a fusion protein with glutathione S-transferase. Purification was performed by affinity chromatography on glutathione-agarose beads after cleavage with thrombin. The human recombinant RI beta protein migrated at 55 kDa on SDS-PAGE and displayed immunoreactivity with an anti-human RI beta antiserum. Furthermore, the purified recombinant RI beta protein was shown to exist as a dimer that was able to form holoenzyme with the catalytic subunit C alpha. The rate of RI beta 2C alpha 2 holoenzyme formation was faster in the presence than in the absence of MgATP. The kinase activity measured before and after adding cAMP to the holoenzyme showed that the presence of cAMP resulted in holoenzyme dissociation and release of active C alpha-subunit, due to cAMP binding to RI beta. Compared to a RI alpha 2C alpha 2 holoenzyme, the RI beta 2C alpha 2 holoenzyme exhibited a more than twofold higher sensitivity to cAMP. The subcellular localization of RI beta was analyzed in quiescent REF-52 fibroblasts and Wistar rat thyroid (WRT) cells after microinjection of fluorescently labeled proteins into the cytoplasm. A cytoplasmic distribution was observed when free RI beta was injected, whereas free C alpha injected into the cytoplasm appeared in the nucleus. When holoenzymes with labeled RI beta and unlabeled C alpha, or unlabeled RI beta and labeled C alpha, were injected, unstimulated cells showed fluorescence in the cytoplasm of both cell types. REF-52 cells stimulated with 8-bromo-cAMP (8-Br-cAMP) and WRT cells treated with thyrotropin (TSH) showed fluorescence mainly in the cytoplasm when RI beta was the labeled subunit of the in vivo dissociated holoenzyme. In contrast, nuclear fluorescence was evident from the release and translocation of labeled C alpha from the holoenzyme complex after stimulation with 8-Br-cAMP or TSH.