TNF-alpha inhibits flow and insulin signaling leading to NO production in aortic endothelial cells.

Endothelial cells release nitric oxide (NO) acutely in response to increased "flow" or fluid shear stress (FSS), and the increase in NO production is correlated with enhanced phosphorylation and activation of endothelial nitric oxide synthase (eNOS). Both vascular endothelial growth factor and FSS activate endothelial protein kinase B (PKB) by way of incompletely understood pathway(s), and, in turn, PKB phosphorylates eNOS at Ser-1179, causing its activation. In this study, we found that either FSS or insulin stimulated insulin receptor substrate-1 (IRS-1) tyrosine and serine phosphorylation and increased IRS-1-associated phosphatidylinositol 3-kinase activity, phosphorylation of PKB Ser-473, phosphorylation of eNOS Ser-1179, and NO production. Brief pretreatment of bovine aortic endothelial cells with tumor necrosis factor-alpha (TNF-alpha) inhibited the above described FSS- or insulin-stimulated protein phosphorylation events and almost totally inhibited FSS- or insulin-stimulated NO production. These data indicate that FSS and insulin regulate eNOS phosphorylation and NO production by overlapping mechanisms. This study suggests one potential mechanism for the development of endothelial dysfunction in disease states with alterations in insulin regulation and increased TNF-alpha levels.

A structure-function analysis of serine/threonine phosphorylation of the thrombopoietin receptor, c-Mpl.

Thrombopoietin (TPO), the critical regulator of platelet production, acts by binding to its cell surface receptor, c-Mpl. Numerous studies have shown that TPO binding leads to JAK2 kinase activation and Tyr phosphorylation of c-Mpl and several intracellular signaling intermediates, events vital for the biological activity of the hormone. In contrast, virtually nothing is known of the role of Ser or Thr phosphorylation of c-Mpl. By using phosphoamino acid analysis we found that Ser residues of c-Mpl were constitutively phosphorylated in receptor-bearing cells, levels that were increased following exposure of cells to TPO. To identify which residues were modified, and to determine the functional consequences of their phosphorylation, we generated a series of Ser to Ala mutations of a truncated c-Mpl receptor (T69) capable of supporting TPO-induced cell growth. Of the eight Ser within T69 we found that at least four are phosphorylated in TPO-stimulated cells. The mutation of each of these residues alone had minimal effects on TPO-induced proliferation, but substitution of all of the phosphoserine residues with Ala reduced the capacity of the receptor to support cell growth by over 50%. Additionally, the Ser at cytoplasmic position 18 is not detectably phosphorylated. However, the mutation of Ser-18 to Ala nearly abrogates TPO-induced proliferation and co-precipitation of JAK2 with Mpl. This study provides the first systematic analysis of the role of Ser residues in c-Mpl signaling.

Identification of flow-dependent endothelial nitric-oxide synthase phosphorylation sites by mass spectrometry and regulation of phosphorylation and nitric oxide production by the phosphatidylinositol 3-kinase inhibitor LY294002.

Endothelial cells release nitric oxide (NO) acutely in response to increased laminar fluid shear stress, and the increase is correlated with enhanced phosphorylation of endothelial nitric-oxide synthase (eNOS). Phosphoamino acid analysis of eNOS from bovine aortic endothelial cells labeled with [(32)P]orthophosphate demonstrated that only phosphoserine was present in eNOS under both static and flow conditions. Fluid shear stress induced phosphate incorporation into two specific eNOS tryptic peptides as early as 30 s after initiation of flow. The flow-induced tryptic phosphopeptides were enriched, separated by capillary electrophoresis with intermittent voltage drops, also known as "peak parking," and analyzed by collision-induced dissociation in a tandem mass spectrometer. Two phosphopeptide sequences determined by tandem mass spectrometry, TQpSFSLQER and KLQTRPpSPGPPPAEQLLSQAR, were confirmed as the two flow-dependent phosphopeptides by co-migration with synthetic phosphopeptides. Because the sequence (RIR)TQpSFSLQER contains a consensus substrate site for protein kinase B (PKB or Akt), we demonstrated that LY294002, an inhibitor of the upstream activator of PKB, phosphatidylinositol 3-kinase, inhibited flow-induced eNOS phosphorylation by 97% and NO production by 68%. Finally, PKB phosphorylated eNOS in vitro at the same site phosphorylated in the cell and increased eNOS enzymatic activity by 15-20-fold.

p90(RSK) is a serum-stimulated Na+/H+ exchanger isoform-1 kinase. Regulatory phosphorylation of serine 703 of Na+/H+ exchanger isoform-1.

The Na+/H+ exchanger isoform-1 (NHE-1) is the key member of a family of exchangers that regulates intracellular pH and cell volume. Activation of NHE-1 by growth factors is rapid, correlates with increased NHE-1 phosphorylation and cell alkalinization, and plays a role in cell cycle progression. By two-dimensional tryptic peptide mapping of immunoprecipitated NHE-1, we identify serine 703 as the major serum-stimulated amino acid. Mutation of serine 703 to alanine had no effect on acid-stimulated Na+/H+ exchange but completely prevented the growth factor-mediated increase in NHE-1 affinity for H+. In addition, we show that p90 ribosomal S6 kinase (p90(RSK)) is a key NHE-1 kinase since p90(RSK) phosphorylates NHE-1 serine 703 stoichiometrically in vitro, and transfection with kinase-inactive p90(RSK) inhibits serum-induced phosphorylation of NHE-1 serine 703 in transfected 293 cells. These findings establish p90(RSK) as a serum-stimulated NHE-1 kinase and a mediator of increased Na+/H+ exchange in vivo.

Data-dependent modulation of solid-phase extraction capillary electrophoresis for the analysis of complex peptide and phosphopeptide mixtures by tandem mass spectrometry: application to endothelial nitric oxide synthase.

Electrospray ionization (ESI) tandem mass spectrometry (MS/MS) of peptides in conjunction with automated sequence database searching of the resulting collision-induced dissociation (CID) spectra has become a powerful method for the identification of purified proteins or the components of protein mixtures. The success of the method is critically dependent on the manner by which the peptides are introduced into the mass spectrometer. In this report, we describe a capillary electrophoresis-based system for the automated, sensitive analysis of complex peptide mixtures. The system consists of an ESI-MS/MS instrument, a solid-phase extraction (SPE)-capillary zone electrophoresis (CZE) device for peptide concentration and separation, and an algorithm written in Instrument Control Language (ICL) which modulates the electrophoretic conditions in a data-dependent manner to optimize available time for the generation of high-quality CID spectra of peptides in complex samples. We demonstrate that the data-dependent modulation of the electric field significantly expands the analytical window for each peptide analyzed and that the sensitivity of the SPE-CZE technique is not noticeably altered by the procedure. By applying the technique to the analysis of in vivo phosphorylation sites of endothelial nitric oxide synthase (eNOS), we demonstrate the power of this system for the MS/MS analysis of minor peptide species in complex samples such as phosphopeptides generated by the proteolytic digestion of a large protein, eNOS, phosphorylated at low stoichiometry.

Metal-binding properties of a calcium-dependent monoclonal antibody.

The calcium-dependent mAb, M1 (also called anti-Flag or 4E11) was studied using a newly developed metal-sensitive enzyme-linked immunosorbent assay (ELISA). This antibody, specific for a calcium complex of the peptide antigen, Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys, has found widespread use as a mild purification reagent for Flag-epitope tagged recombinant proteins. Although M1 affinity columns release monovalent Flagged proteins in the absence of calcium, the antibody retains substantial affinity for the Flag sequence even in metal-free conditions, so that it has been impossible to use it to develop a metal-sensitive ELISA assay. This is due to the ability of the antibody to remain bound to polyvalent surface-coated antigen, for instance, when Flagged proteins are bound to ELISA plates or blotting filters. The resultant antigen polyvalence raises the avidity of the Flag antibody to a point where the reaction is essentially calcium-independent. However, when the antibody itself was made monovalent, by proteolytic cleavage to the Fab, this situation was reversed and the ELISA reaction became calcium-dependent. This new metal-dependent ELISA assay was used to explore the metal requirements of the antibody in detail. Among divalent metals, binding tapered off with increasing radius above that of calcium, or with decreasing radius below that of calcium. Several smaller metals, such as nickel, acted as inhibitors of the binding reaction. Substantial binding was demonstrated for heavy metals such as cadmium, lanthanum and samarium. Because it is of interest to use this antibody for the co-crystallization of recombinant Flag-fusion proteins, the ability to bind heavy metals was a significant finding.

Interleukin 2 stimulates serine phosphorylation of CD45 in CTLL-2.4 cells.

Ligation of interleukin 2 (IL2) is known to regulate both protein tyrosine and serine/threonine phosphorylation. A family of leukocyte transmembrane proteins whose cytoplasmic domain exhibits intrinsic protein tyrosine phosphatase activity is collectively called CD45 and is identified by a set of common cell surface epitopes. Although CD45 is known to be a phosphoprotein, it is not known how phosphorylation specifically regulates its function. We therefore identified a cell line, the IL4-dependent line CTLL-2.4, in which CD45 could be phosphorylated in response to addition of IL2. These cells are a variant of an IL2-dependent murine cell line which were selected for long-term growth on IL4 but which retain the ability to proliferate on exposure to IL2. Incubation of CTLL-2.4 in low serum concentrations followed by stimulation with IL2 caused a three- to fivefold increase in the phosphorylation of CD45 in a time- and concentration-dependent manner. CD45 in non-stimulated cells contained one major tryptic phosphopeptide, whereas, after exposure of the cells to IL2, two new phosphopeptides were present in CD45. The pattern of IL2-induced phosphorylation was different from that found following addition of phorbol 12-myristate 13-acetate (PMA) to the cells. Although IL2 induced rapid and potent tyrosine phosphorylation in CTLL-2.4 cells, all of the basal and cytokine-activated phosphorylation of CD45 occurred on serine residues. The IL2-stimulated phosphorylation caused no change in the amount of cell surface CD45 and no alteration of its catalytic activity using an artificial tyrosine phosphorylated substrate-RCM-lysozyme. We speculate that the increase in phosphorylation of CD45 may modify its association with potential substrates. The differences in the phosphorylation patterns induced by IL2 and PMA further suggest that more than one kinase can use CD45 as substrate and that IL2 activates a protein serine/threonine kinase different from protein kinase C.

Monoclonal antibodies block murine IL-4 receptor function.

IL-4 is a cytokine which can induce B-lymphocyte proliferation, increase cell-surface Ia expression, and induce some activated B cells to differentiate and begin to secrete IgE. IL-4 binds specifically to a cell-surface receptor (IL-4R) on cells from a variety of lineages including T and B cells. In general both primary cells and in vitro cell lines express less than 5000 receptors per cell. Utilizing a subclone of the cytotoxic T cell line CTLL-2 expressing a high level of IL-4R, mAb against the murine IL-4R were prepared. Two mAb have been identified which have different properties. These antibodies, designated M1 and M2, recognize sequences specific to the murine IL-4R. Immunoprecipitation studies with M1 and M2 on CTLL-2 cells have identified the receptor as a Mr = 145,000 cell-surface protein. Similar results have been obtained with the recently isolated full length murine IL-4R cDNA expressed in COS-7 cells. In addition the antibodies are capable of inhibiting IL-4 binding. One antibody, M1, is also a potent inhibitor of IL-4-induced proliferation. These antibodies will be useful in dissecting a wide array of activities attributed to IL-4.

IL-1 induces rapid phosphorylation of the IL-1 receptor.

The IL-1R on murine T cells is a Mr = 80,000 plasma membrane glycoprotein. cDNA cloning and transfection experiments have shown that this is an integral membrane protein, which binds both IL-1 alpha and IL-1 beta and transduces the IL-1 signal. A mAb, RM-5, which binds an epitope on the receptor which is distinct from the IL-1 binding site has been produced in rats. RM-5 has been used to immunoprecipitate the IL-1R from 32P-orthophosphate labeled CHO cells which express approximately 100,000 functional, murine rIL-1R/cell. Phosphorylation of the receptor was observed as early as 1 min after the addition of IL-1 and continued for periods of up to 30 min. Phosphorylation increases as the concentration of IL-1 increases from 10(-13) to 10(-8) M. Potassium hydroxide hydrolysis of the phosphorylated IL-1R shows that more than 90% of the phosphate is incorporated into serine or threonine. Thus, one of the earliest events after IL-1 binding to the IL-1R is activation of a serine/threonine protein kinase and phosphorylation of the IL-1R itself.

T-cell interleukin 1 receptor cDNA expressed in Chinese hamster ovary cells regulates functional responses to interleukin 1.

We have cloned a cDNA encoding a receptor identical to the native Mr 80,000 glycoprotein that binds interleukin (IL) 1 alpha and -beta in murine T cells. Chinese hamster ovary (CHO) cells transfected with this T-cell IL-1 receptor (IL-1R) [CHO(IL-1R)] cDNA express approximately 100,000 IL-1Rs per cell, compared to the less than 100 receptors present on control CHO cells. For two functional responses to IL-1, prostaglandin synthesis and cytokine secretion, CHO(IL-1R) cells were 1000 times more sensitive to IL-1 alpha than were control CHO cells. Northern blot analysis and antibody precipitation demonstrated that one of the cytokines induced was granulocyte colony-stimulating factor and that mRNA levels for this cytokine were increased in CHO(IL-1R) cells by IL-1 alpha concentrations that had no effect on control cells. To establish the role of the recombinant receptors in signal transduction, an IL-1R cDNA modified by deletion of the predicted cytoplasmic domain was expressed in the CHO cell line termed CHO(IL-1R delta CT). CHO(IL-1R delta CT) cells expressed approximately 100,000 high-affinity IL-1 binding sites per cell, but these cells were less sensitive than control lines to IL-1, as measured by prostaglandin and cytokine release. These results show that the IL-1R cDNA encodes the entire functional receptor and that the cytoplasmic domain is required for signal transduction but not ligand binding.

Antimicrobial activity of synthetic bactenecin.

Bactenecin is an antimicrobial peptide isolated from bovine neutrophils. Bactenecin was synthesized by solid-phase peptide synthesis and renatured to a fully disulfide bonded form. The peptide inhibits the growth of Escherichia coli and Staphylococcus aureus at the same concentration reported for the peptide purified from bovine neutrophils. Bactenecin inhibits the growth of other medically important bacteria and yeast, and it kills the fungus Trichophyton rubrum. Acetylation and amidation of the amino- and carboxy-termini of bactenecin do not change its potency, while replacement of its two cysteine residues with serine decreases the potency.

Human macrophage colony stimulating factor (M-CSF): alternate RNA splicing generates three different proteins that are expressed on the cell surface and secreted.

Human macrophage colony stimulating factor (M-CSF) cDNA clones were isolated from a pancreatic carcinoma cell line. Three different classes of M-CSF precursor protein (256, 554 and 438 amino acids in length) were predicted to be encoded by these cDNAs. Two of these, that we designate M-CSF alpha and M-CSF beta have already been described. The third, M-CSF gamma represents a novel class of M-CSF cDNA. All three precursors share a 32 amino acid signal sequence and the first 149 amino acids of the mature protein. At this position, M-CSF beta and gamma have insertions of 298 and 182 amino acids relative to M-CSF alpha. The first 182 amino acids of these insertions are shared between M-CSF beta and gamma. All three precursors share the C-terminal 75 amino acids that encode the transmembrane and cytoplasmic domains. Expression of all three cDNAs in COS-7 monkey kidney cells gave rise to soluble M-CSF activity, associated with proteins of subunit molecular weight 44 Kda (beta and gamma) or 28 Kda (alpha). In addition, M-CSF proteins could be detected on the surface of the transfected cells by indirect immunofluorescence. When the transmembrane and cytoplasmic domains of M-CSF alpha were removed by introducing a stop codon after amino acid 190, no membrane-bound M-CSF could be detected, but the truncated protein was secreted efficiently and was biologically active. This suggests that all three forms of M-CSF can exist as cell surface proteins, anchored by their hydrophobic transmembrane domains, and can be processed to soluble forms by proteolytic digestion. Although all soluble forms of M-CSF were biologically active in murine bone marrow colony and proliferation assays, they showed greatly reduced or no activity in similar assays using human bone marrow.

Human macrophage-colony stimulating factor: alternative RNA and protein processing from a single gene.

Macrophage-colony stimulating factor (M-CSF, CSF-1) has been reported to be required for the proliferation and differentiation of macrophages from hematopoietic progenitor cells. Recently, two human M-CSF cDNA clones were isolated encoding proteins of 256 and 554 amino acids. We report here the isolation of a third M-CSF cDNA that encodes a protein of 438 amino acids. The coding regions for the three cDNA clones share a common amino-terminus of 149 amino acids and a common carboxyl-terminus of 75 amino acids including a membrane spanning region. In addition, we isolated a genomic clone of human M-CSF. When each of the cDNA clones or the genomic clone were transfected into COS-7 monkey kidney cells, biologically active M-CSF was expressed as judged by the ability of transfected cell supernatants to stimulate proliferation and colony formation of murine bone marrow cells, as well as formation of monocytic colonies from human bone marrow cells. Surprisingly, proliferation of human bone marrow cells was not induced by recombinant human M-CSF. Analysis of the M-CSF proteins released by COS-7 cells revealed that monomer subunit proteins of 44 or 28 kDa were produced. In addition, we found that the membrane spanning region, present in all three forms of M-CSF cDNA, was not required for the synthesis of a biologically active protein. However, when the membrane spanning region was present in the three M-CSF cDNAs, cell surface associated forms of M-CSF could be readily detected.

Monoclonal antibody immunoprecipitation of cell membrane glycoproteins.

Procedural modifications facilitating the immunoprecipitation of cell surface-associated glycoproteins by monoclonal antibodies are presented. The use of complexes of antibodies coupled to protein A-Sepharose in place of antibodies directly coupled to Sepharose, and the inclusion of ATP and salt in the lysis buffer, is shown to markedly reduce the nonspecific binding of aggregated cytoskeletal proteins. These modifications result in low backgrounds while the specific membrane-associated proteins are still quantitatively immunoprecipitated.

Phosphorylation of the CD8 antigen on cytotoxic human T cells in response to phorbol myristate acetate or antigen-presenting B cells.

We have used the monoclonal antibody OKT8 to demonstrate that the cluster designation (CD)8 antigen on cytotoxic human T cells undergoes a previously unreported protein modification. Immunoprecipitation of CD8 with OKT8 from a CD8+ and CD4+ T cell line prelabeled with [32P]phosphate demonstrates that CD8 can be constitutively labeled with phosphate. CD8 undergoes rapid and intense phosphorylation in serine upon addition of phorbol myristate acetate to the cells. CD8 phosphorylation is induced upon addition of heterologous, Epstein-Barr virus-transformed B cells, which cause proliferation and are target cells for a cytotoxic CD8+CD4+ T cell line and a CD8+CD4- T cell clone. Phosphorylation induced by targets is dose-dependent, rapid, and followed by a fast dephosphorylation. Epstein-Barr virus-transformed B cells that do not induce proliferation of and are not targets for the CD8+CD4+ line and the CD8+CD4- clone do not induce CD8 phosphorylation. Cloned CD8+CD4- cells that proliferate in response to target cells, but lyse them only in the presence of lectin do not undergo target cell-induced CD8 phosphorylation. These data suggest that induction of CD8 phosphorylation is antigen-specific and is coincident with the cytotoxic response. Finally, preincubation of effector and target cells with an antibody to a monomorphic determinant of major histocompatibility complex class I antigens reduces target-induced CD8 phosphorylation to a greater extent than antibody to a major histocompatibility complex class II subregion (DR) monomorphic determinant, reinforcing the notion that major histocompatibility complex class I antigens interact with CD8+ cells.

Nature and specificity of lymphokine independence induced by a selectable retroviral vector expressing v-src.

A murine retroviral vector, LSNLsrc, has been constructed and examined for its ability to induce growth factor independence in cells normally dependent on interleukin 2 (IL-2) or interleukin 3 (IL-3) for growth. The LSNLsrc vector coexpressed the v-src gene of Rous sarcoma virus and the neo gene from transposon Tn5, allowing infected cells to be selected on the basis of G418 resistance. The murine cell lines CTLL-2 and FD.C/1, which are dependent for growth on IL-2 and IL-3, respectively, were both readily infected with the LSNLsrc virus. LSNLsrc-infected, G418-resistant cultures of FD.C/1 cells were able to give rise to IL-3-independent progeny, but all G418-resistant CTLL-2 cells retained normal IL-2 dependence. The induction of IL-3 independence by v-src was not a direct event, since limiting dilution analysis of the LSNLsrc-infected FD.C/1 cells showed that most of them were IL-3 dependent, despite expression of v-src mRNA and active pp60v-src kinase. However, clones selected from this population in the presence of IL-3 were able to undergo a subsequent progression event and generate IL-3-independent progeny. The generation of factor-independent variants in the clonal cultures was a rare event, as witnessed by the death of most of the cells in each clone when IL-3 was withdrawn. Together, these data indicate that a secondary event, in addition to v-src expression, was required to generate IL-3-independent growth. No evidence was found for an autocrine mechanism of transformation involving IL-2, IL-3, interleukin 4, or granulocyte-macrophage colony-stimulating factor.

Rapid phosphorylation and modulation of the T4 antigen on cloned helper T cells induced by phorbol myristate acetate or antigen.

A cell surface protein known as T4 (CD4, Leu3), Mr = 55,000, is expressed on the subset of human T lymphocytes which provides helper function for B cell and cytotoxic T cell activities. We show that T4 is constitutively phosphorylated and that phorbol myristate acetate (PMA) induces a rapid serine phosphorylation which is followed by a fast dephosphorylation. Within 5 min after PMA treatment, there is a 24% reduction of T4 on the cell surface, by 4 h the loss is nearly complete, and by 20 h T4 is re-expressed. Addition of antigen to a T4+ antigen-reactive T cell clone induces both phosphorylation and dephosphorylation with kinetics similar to that described for PMA. Antigen also causes reduction of cell surface T4, although to a lesser degree than stimulation with PMA. The rapid phosphorylation/dephosphorylation of T4 as well as its movement from the cell surface suggest that T4 functions as a receptor for an unknown ligand.

Characterization of the phosphotyrosyl protein phosphatase activity of calmodulin-dependent protein phosphatase.

Calmodulin-dependent protein phosphatase from bovine brain and heart was assayed for phosphotyrosine and phosphoserine phosphatase activity using several substrates: 1) smooth muscle myosin light chain (LC20) phosphorylated on tyrosine or serine residues, 2) angiotensin I phosphorylated on tyrosine, and 3) synthetic phosphotyrosine- or phosphoserine-containing peptides with amino acid sequences patterned after the autophosphorylation site in Type II regulatory subunit of the cAMP-dependent protein kinase. The phosphatase was activated by Ni2+ and Mn2+, and stimulated further by calmodulin. In the presence of Ni2+ and calmodulin, it exhibited similar kinetic constants for the dephosphorylation of phosphotyrosyl LC20 (Km = 0.9 microM, and Vmax = 350 nmol/min/mg) and phosphoseryl LC20 (Km = 2.6 microM, Vmax = 690 nmol/min/mg). Dephosphorylation of phosphotyrosyl LC20 was inhibited by phosphoseryl LC20 with an apparent Ki of 2 microM. Compared to the reactions with phosphotyrosyl LC20 as the substrate, reactions with phosphotyrosine-containing oligopeptides exhibited slightly higher Km and lower Vmax values. The reaction with the phosphoseryl peptide based on the Type II regulatory subunit sequence exhibited a slightly higher Km (23 microM), but a much higher Vmax (4400 nmol/min/mg) than that with its phosphotyrosine-containing counterpart. Micromolar concentrations of Zn2+ inhibited the phosphatase activity; vanadate was less potent, and 25 mM NaF was ineffective. The study provides quantitative data to serve as a basis for comparing the ability of the calmodulin-dependent protein phosphatase to act on phosphotyrosine- and phosphoserine-containing substrates.