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.     

Ras2 and Ras1 protein phosphorylation in Saccharomyces cerevisiae

This work describes the phosphorylation of Saccharomyces cerevisiae Ras proteins and explores the physiological role of the phosphorylation of Ras2 protein. Proteins expressed from activated alleles of RAS were less stable and less phosphorylated than proteins from cells expressing wild-type alleles of RAS. This difference in phosphorylation level did not result from increased signaling through the Ras-cAMP pathway or reflect the primarily GTP-bound nature of activated forms of Ras protein per se. In addition, phosphorylation of Ras protein was not dependent on proper localization of the Ras2 protein to the plasma membrane nor on the interaction of Ras2p with its exchange factor, Cdc25p. The preferred phosphorylation site on Ras2 protein was identified as serine 214. This site, when mutated to alanine, led to promiscuous phosphorylation of Ras2 protein on nearby serine residues. A decrease in phosphorylation may lead to a decrease in signaling through the Ras-cAMP pathway.     

Insulin-induced translocation of protein kinase B to the plasma membrane in rat adipocytes

Translation termination in vivo was studied in the yeast Saccharomyces cerevisiae using a translation-assay system. Codon changes that were made at position -2 relative to the stop codon, gave a 3.5-fold effect on termination in a release-factor-defective (sup45) mutant strain, in line with the effect observed in a wild-type strain. The influence of the -2 codon could be correlated to the charge of the corresponding amino acid residue in the nascent peptide; an acidic residue favoring efficient termination. Thus, the C-terminal end of the nascent peptide influences translation termination both in the bacterium Escherichia coli and to a lesser extent in the yeast S. cerevisiae. However, the sensitivity to the charge of the penultimate amino acid is reversed when the E. coli and S. cerevisiae are compared. Changing - 1 (P-site) codons in yeast gave a 10-fold difference in effect on the efficiency of termination. This effect could not be related to any property of the encoded last amino acid in the nascent peptide. Iso-codons read by the same tRNA (AAA/G, GAA/G) gave similar readthrough values. Codons for glutamine (CAA/G), glutamic acid (GAA/G) and isoleucine (AUA/C) that are read by different isoaccepting tRNAs are associated with an approximately twofold difference in each case in termination efficiency. This suggests that the P-site tRNA is able to influence termination at UGAC in yeast.     

The C2 domain of the Ca(2+)-independent protein kinase C Apl II inhibits phorbol ester binding to the C1 domain in a phosphatidic acid-sensitive manner

There are two protein kinase Cs (PKCs) in the Aplysia nervous system, PKC Apl I, which is homologous to the Ca(2+)-activated PKC family, and PKC Apl II, which is homologous to the Ca(2+)-independent PKCs epsilon and eta. Purified PKC Apl I requires much less phosphatidylserine for activation than does purified PKC Apl II, and this may explain why the neurotransmitter serotonin activates PKC Apl I but not PKC Apl II in the intact nervous system [Sossin, W. S., Fan, X., and Baseri, F. (1996) J. Neurosci. 16, 10-18]. PKC Apl II's requirement for high levels of phosphatidylserine may be mediated by its C2 domain, since removal of this domain allows PKC Apl II to be activated at lower concentrations of phosphatidylserine. To begin to understand how this inhibition is mediated, we generated fusion proteins containing the C1 and C2 domains from PKC Apl II and determined their lipid dependence for phorbol ester binding. Our results indicate that the presence of the C2 domain lowers the affinity of protein kinase C activators for the C1 domains and this inhibition can be removed by phosphatidylserine. Phosphatidic acid, however, is much more potent than phosphatidylserine in reducing C2 domain-mediated inhibition, suggesting that phosphatidic acid may be a required cofactor for the activation of PKC Apl II.     

Characterization of the in vitro reconstituted cyclin A or B1-dependent cdk2 and cdc2 kinase activities

Human cyclins A and B1 were assembled with the cdk2 or cdc2 protein to reconstitute their respective kinase activities in vitro. Both cyclins complemented either cdk2 or cdc2, yielding kinase activities that supported the phosphorylation of histone H1. Activation of cdk2-catalyzed H1 kinase activity by cyclin A required a 10-min preincubation of the two components, whereas cdc2 kinase supported phosphate incorporation without a detectable time lag upon the addition of cyclin B1, suggesting a slower association rate of cdk2 with cyclin A compared with cdc2 and cyclin B1. Both cdk2 and cyclin A, as well as cdc2 and cyclin B1, formed stable complexes in the absence of ATP and substrate that could be isolated after glycerol gradient centrifugation. Incubation of the isolated complexes with ATP and histone H1 supported the phosphorylation of the substrate. Cyclin A-activated cdk2 or cdc2 phosphorylated p107, a pRB-related cellular protein, 10 times more effectively than the cyclin B1-complexed kinases. This was most likely due to a direct association of cyclin A with p107 (Ewen, M. E., Faha, B., Harlow, E., and Livingston, D. (1992) Science 255, 85-87; Faha, B., Ewen, M. E., Tsai, L.-H., Livingston, D., and Harlow, E. (1992) Science 255, 87-90). The reconstituted cdc2-cyclin B1 complex incorporated 4-5-fold more phosphate into the p34 subunit of the three-subunit (p70, p34, and p14) human single-stranded DNA-binding protein (also called RP-A), a DNA replication and DNA repair factor, than cdc2-cyclin A. No detectable phosphorylation of the p34 protein was observed with cdk2 complexed with either cyclin B1 or A. These data indicate that both cyclins as well as the catalytic subunits are important factors in controlling the rate of phosphorylation of a given substrate. The cyclin-activated cdc2 family kinases may target their cellular substrates through cyclin-mediated protein-protein interactions.     

N-WASP, a novel actin-depolymerizing protein, regulates the cortical cytoskeletal rearrangement in a PIP2-dependent manner downstream of tyrosine kinases

Here we identify a 65 kDa protein (N-WASP) from brain that binds the SH3 domains of Ash/Grb2. The sequence is homologous to Wiskott-Aldrich syndrome protein (WASP). N-WASP has several functional motifs, such as a pleckstrin homology (PH) domain and cofilin-homologous region, through which N-WASP depolymerizes actin filaments. When overexpressed in COS 7 cells, the wild-type N-WASP causes several surface protrusions where N-WASP co-localizes with actin filaments. Epidermal growth factor (EGF) treatment induces the complex formation of EGF receptors and N-WASP, and produces microspikes. On the other hand, two mutants, C38W (a point mutation in the PH domain) and deltaVCA (deletion of the actin binding domain), localize predominantly in the nucleus and do not cause a change in the cytoskeleton, irrespective of EGF treatment. Interestingly, the C38W PH domain binds less effectively to phosphatidylinositol 4,5-bisphosphate (PIP2) than the wild-type PH domain. These results suggest the importance of the PIP2 binding ability of the PH domain and the actin binding for retention in membranes. Collectively, we conclude that N-WASP transmits signals from tyrosine kinases to cause a polarized rearrangement of cortical actin filaments dependent on PIP2.     

Dopamine-induced recruitment of dopamine D1 receptors to the plasma membrane

The recruitment of G protein-coupled receptors from the cytoplasm to the plasma membrane generally is believed to be a constitutive process. We show here by the use of both confocal microscopy and subcellular fractionation that, for at least one such receptor, this recruitment is regulated and not constitutive. Cells from a proximal tubular-like cell line, LLCPK1 cells, were incubated with either a D1 agonist, a dopamine precursor, or an inhibitor of dopamine metabolism to increase dopamine availability in the cell. Each of the three procedures led to a rapid translocation of dopamine D1 receptors from the cytosol to the plasma membrane.     

Leukotriene D4 activates mitogen-activated protein kinase through a protein kinase Calpha-Raf-1-dependent pathway in human monocytic leukemia THP-1 cells

Leukotriene D4 (LTD4) is a major lipid mediator involved in inflammatory and allergic disorders including bronchial asthma. Despite its potent biological activity, little is known about the receptor and intracellular signaling pathways. Here we analyzed the signal transduction mechanisms through LTD4 receptors using human monocytic leukemia THP-1 cells. When these cells were stimulated with LTD4, intracellular calcium concentration was increased and mitogen-activated protein kinase (MAP kinase) was activated severalfold. This activation was inhibited by staurosporine or GF109203X treatment or abolished by protein kinase C depletion. Cytosolic protein kinase Calpha was translocated to the membrane, and Raf-1 was activated by LTD4 treatment in a similar time course. LTD4-induced Raf-1 activation was diminished by protein kinase C depletion in the cells. A chemotactic response of THP-1 cells toward LTD4 was observed which was inhibited by pertussis toxin (PTX) pretreatment. Thus, LTD4 has at least two distinct signaling pathways in THP-1 cells, a PTX-insensitive mitogen-activated protein kinase activation through protein kinase Calpha and Raf-1 and a PTX-sensitive chemotactic response. This cellular signaling can explain in part the versatile activities of LTD4 in macrophages under inflammatory and allergic conditions.     

Possible role of alteration in pH, but not in ionic strength, in the modulation of Ca(2+)-dependent and diacylglycerol-induced association of protein kinase C-alpha with plasma membrane

In an attempt to elucidate the mechanism of the membrane binding of protein kinase C-alpha (PKC-alpha), effects of pH and ionic strength were examined on Ca(2+)-dependent and diacylglycerol-induced association of this PKC with rat liver plasma membrane. In the absence of diacylglycerol, the membrane-bound PKC-alpha increased according to the changes in pH from 6 to 8. However, these reactions were not seriously influenced by alterations in ionic strength (0-240 mM NaCl). In the presence of diacylglycerol, this pH-modulated binding of PKC-alpha to plasma membrane was more effective under lower Ca2+ concentration at pH 8. These results suggest that alkaline pH conditions seem to be important for preferable association of PKC-alpha with plasma membrane.     

GCKR links the Bcr-Abl oncogene and Ras to the stress-activated protein kinase pathway

The Bcr-Abl oncogene, found in Philadelphia chromosome-positive myelogenous leukemia (CML), activates Ras and triggers the stress-activated protein kinase (SAPK or Jun NH2-terminal kinase [JNK]) pathway. Interruption of Ras or SAPK activation dramatically reduces Bcr-Abl-mediated transformation. Here, we report that Bcr-Abl through a Ras-dependent pathway signals the serine/threonine protein kinase GCKR (Germinal Center Kinase Related) leading to SAPK activation. Either an oncogenic form of Ras or Bcr-Abl enhances GCKR catalytic activity and its activation of SAPK, whereas inhibition of GCKR impairs Bcr-Abl-induced SAPK activation. Bcr-Abl mutants that are impaired for GCKR activation are also unable to activate SAPK. Consistent with GCKR being a functional target in CML, GCKR is constitutively active in CML cell lines and found in association with Bcr-Abl. Our results indicate that GCKR is a downstream target of Bcr-Abl and strongly implicate GCKR as a mediator of Bcr-Abl in its transformation of cells.     

Inhibition of HIV-1 gp160-dependent membrane fusion by a furin-directed alpha 1-antitrypsin variant

Furin is a membrane-associated calcium-dependent serine endoprotease that cleaves proproteins on the carboxyl side of the consensus sequence -Arg-X-Lys/Arg-Arg-. Using site-directed mutagenesis, a variant alpha 1-antitrypsin (alpha 1-AT) was constructed which contains in its reactive site -Arg-X-X-Arg-, the minimal sequence required for efficient processing by furin (Molloy, S. S., Bresnahan, P. A., Leppla, S. H., Klimpel, K. R., and Thomas, G. (1992) J. Biol. Chem. 267, 16396-16402). This alpha 1-AT variant, [Arg355 Arg358]alpha 1-AT (alpha 1-PDX), is greater than 3,000-fold more effective than [Arg358]alpha 1-AT (alpha 1-AT Pittsburgh, alpha 1-PIT) at inhibiting furin in vitro (K0.5 = 0.03 microgram/ml). Furthermore, the P4 Arg in alpha 1-PDX greatly attenuates the thrombin inhibitory properties of this serpin (> 300-fold) compared with alpha 1-PIT (which contains a P4 Ala), thus increasing the selectivity of alpha 1-PDX for furin. Expression studies show that alpha 1-PDX, and not alpha 1-PIT, blocks the processing of two furin substrates, pro-beta-nerve growth factor and human immunodeficiency virus (HIV)-1 gp160 in transfected cells. In addition, a syncytium assay shows that alpha 1-PDX blocks the membrane fusogenic properties of HIV-1 gp160. The potential use of alpha 1-PDX in manipulating the activation of proproteins in a tissue- and time-specific manner is discussed.     

Human Sos1: a guanine nucleotide exchange factor for Ras that binds to GRB2

A human complementary DNA was isolated that encodes a widely expressed protein, hSos1, that is closely related to Sos, the product of the Drosophila son of sevenless gene. The hSos1 protein contains a region of significant sequence similarity to CDC25, a guanine nucleotide exchange factor for Ras from yeast. A fragment of hSos1 encoding the CDC25-related domain complemented loss of CDC25 function in yeast. This hSos1 domain specifically stimulated guanine nucleotide exchange on mammalian Ras proteins in vitro. Mammalian cells overexpressing full-length hSos1 had increased guanine nucleotide exchange activity. Thus hSos1 is a guanine nucleotide exchange factor for Ras. The hSos1 interacted with growth factor receptor-bound protein 2 (GRB2) in vivo and in vitro. This interaction was mediated by the carboxyl-terminal domain of hSos1 and the Src homology 3 (SH3) domains of GRB2. These results suggest that the coupling of receptor tyrosine kinases to Ras signaling is mediated by a molecular complex consisting of GRB2 and hSos1.     

Protein kinase C phosphorylates the "a" forms of plasma membrane Ca2+ pump isoforms 2 and 3 and prevents binding of calmodulin

Phosphorylation by protein kinase C of the "a" and "b" variants of plasma membrane Ca2+ pump isoforms 2 and 3 was studied. Full-length versions of these isoforms were assembled and expressed in COS cells. Whereas the "a" forms were phosphorylated easily with PKC, isoform 2b was phosphorylated only a little, and isoform 3b was not phosphorylated at all. Phosphorylation of isoforms 2a and 3a did not affect their basal activity, but prevented the stimulation of their activity by calmodulin and their binding to calmodulin-Sepharose. This indicated that phosphorylation prevented activation of these isoforms by preventing calmodulin binding. Based on these results, phosphorylation of the pump with PKC would be expected to increase free intracellular Ca2+ levels in those cells where isoforms 2a and 3a are expressed.     

Protein kinase C and calmodulin effects on the plasma membrane Ca2+-ATPase from excitable and nonexcitable cells

We have purified Ca2+-ATPase from synaptosomal membranes (SM)1 from rat cerebellum by calmodulin affinity chromatography. The enzyme was identified as plasma membrane Ca2+-ATPase by its interaction with calmodulin and monoclonal antibodies produced against red blood cell (RBC) Ca2+-ATPase, and by thapsigargin insensitivity. The purpose of the study was to establish whether two regulators of the RBC Ca2+-ATPase, calmodulin and protein kinase C (PKC), affect the Ca2+-ATPase isolated from excitable cells and whether their effects are comparable to those on the RBC Ca2+-ATPase. We found that calmodulin and PKC activated both enzymes. There were significant quantitative differences in the phosphorylation and activation of the SM versus RBC Ca2+-ATPase. The steady-state Ca2+-ATPase activity of SM Ca2+-ATPase was approximately 3 fold lower and significantly less stimulated by calmodulin. The initial rate of PKC catalyzed phosphorylation (in the presence of 12-myristate 13-acetate phorbol) was approximately two times slower for SM enzyme. While phosphorylation of RBC Ca2+-ATPase approached maximum level at around 5 min, comparable level of phosphorylation of SM Ca2+-ATPase was observed only after 30 min. The PKC-catalyzed phosphorylation resulted in a statistically significant increase in Ca2+-ATPase activity of up to 20-40%, higher in the SM Ca2+-ATPase. The differences may be associated with diversities in Ca2+-ATPase function in erythrocytes and neuronal cells and different isoforms composition.     

KSR stimulates Raf-1 activity in a kinase-independent manner

Kinase suppressor of Ras (KSR) is an evolutionarily conserved component of Ras-dependent signaling pathways. Here, we find that murine KSR (mKSR1) translocates from the cytoplasm to the plasma membrane in the presence of activated Ras. At the membrane, mKSR1 modulates Ras signaling by enhancing Raf-1 activity in a kinase-independent manner. The activation of Raf-1 is mediated by the mKSR1 cysteine-rich CA3 domain and involves a detergent labile cofactor that is not ceramide. These findings reveal another point of regulation for Ras-mediated signal transduction and further define a noncatalytic role for mKSR1 in the multistep process of Raf-1 activation.     

The oestrogen receptor regulates NFkappaB and AP-1 activity in a cell-specific manner

It has recently been identified the PEPT2 cDNA encodes the high affinity proton-coupled peptide transporter in rabbit kidney cortex. PEPT2 represents a 729 amino acid protein with 12 putative transmembrane domains that mediates H+/H3O+ dependent electrogenic transmembrane transport of di- and tripeptides and of selected peptidomimetics. Here the functional expression of PEPT2 in the methylotropic yeast Pichia pastoris is described under the control of a methanol inducible promoter. Western blot analysis of Pichia cell membranes prepared from a recombinant clone identified a protein with an apparent molecular mass of about 85-87 kDa. Peptide uptake into cells expressing PEPT2 was up to 80 times higher than in control cells. Cells of recombinant clones showed a saturable peptide transport activity for the hydrolysis resistant dipeptide 3H-D-Phe-Ala with an app. K0.5 of 0.143 +/- 0.016 mM. Inhibition of 3H-D-Phe-Ala uptake by selected di- and tripeptides and beta-lactam antibiotics revealed the same substrate specificity as obtained in renal membrane vesicles or for PEPT2 when expressed in Xenopus laevis oocytes. A novel fluorescence based assay for assessing transport function based on a coumarin-labeled fluorescent peptide analogue has also been developed. Moreover, using a histidyl auxotrophe strain a PEPT2 expressing cell clone in which transport function can be monitored by a simple yeast growth test was established. In conclusion, this is one of only a few reports on successful functional expression of mammalian membrane transport proteins in yeast. The high expression level will provide a simple means for future studies either on the structure-affinity relationship for substrate interaction with PEPT2 or for selection of mutants generated by random mutagenesis.     

Cloned Ca(2+)-dependent K+ channel modulated by a functionally associated protein kinase

Calcium-dependent potassium (KCa) channels carry ionic currents that regulate important cellular functions. Like some other ion channels, KCa channels are modulated by protein phosphorylation. The recent cloning of complementary DNAs encoding Slo KCa channels has enabled KCa channel modulation to be investigated. We report here that protein phosphorylation modulates the activity of Drosophila Slo KCa channels expressed in Xenopus oocytes. Application of ATP-gamma S to detached membrane patches increases Slo channel activity by shifting channel voltage sensitivity. This modulation is blocked by a specific inhibitor of cyclic AMP-dependent protein kinase (PKA). Mutation of a single serine residue in the channel protein also blocks modulation by ATP-gamma S, demonstrating that phosphorylation of the Slo channel protein itself modulates channel activity. The results also indicate that KCa channels in oocyte membrane patches can be modulated by an endogenous PKA-like protein kinase which remains functionally associated with the channels in the detached patch.