Inhibition of AP-1 binding and transcription by gold and selenium involving conserved cysteine residues in Jun and Fos.

Gold(I) salts and selenite, which have diverse therapeutic and biological effects, are noted for their reactivity with thiols. Since the binding of Jun-Jun and Jun-Fos dimers to the AP-1 DNA binding site is regulated in vitro by a redox process involving conserved cysteine residues, we hypothesized that some of the biological actions of gold and selenium are mediated via these residues. In electrophoretic mobility-shift analyses, AP-1 DNA binding was inhibited by gold(I) thiolates and selenite, with 50% inhibition occurring at approximately 5 microM and 1 microM, respectively. Thiomalic acid had no effect in the absence of gold(I), and other metal ions inhibited at higher concentrations, in a rank order correlating with their thiol binding affinities. Cysteine-to-serine mutants demonstrated that these effects of gold(I) and selenite require Cys272 and Cys154 in the DNA-binding domains of Jun and Fos, respectively. Gold(I) thiolates and selenite did not inhibit nonspecific protein binding to the AP-1 site and were at least an order of magnitude less potent as inhibitors of sequence-specific binding to the AP-2, TFIID, or NF1 sites compared with the AP-1 site. In addition, 10 microM gold(I) or 10 microM selenite inhibited expression of an AP-1-dependent reporter gene, but not an AP-2-dependent reporter gene. These data suggest a mechanism regulating transcription factor activity by inorganic ions which may contribute to the known antiarthritic action of gold and cancer chemoprevention by selenium.

Activation of the Lck tyrosine protein kinase by hydrogen peroxide requires the phosphorylation of Tyr-394.

Exposure of cells to H2O2 mimics many of the effects of treatment of cells with extracellular ligands. Among these is the stimulation of tyrosine phosphorylation. In this study, we show that exposure of cells to H2O2 increases the catalytic activity of the lymphocyte-specific tyrosine protein kinase p56lck (Lck) and induces tyrosine phosphorylation of Lck at Tyr-394, the autophosphorylation site. Using mutant forms of Lck, we found that Tyr-394 is required for H2O2-induced activation of Lck, suggesting that phosphorylation of this site may activate Lck. In addition, H2O2 treatment induced phosphorylation at Tyr-394 in a catalytically inactive mutant of Lck in cells that do not express endogenous Lck. This demonstrates that a kinase other than Lck itself is capable of phosphorylating Lck at the so-called autophosphorylation site and raises the possibility that this as yet unidentified tyrosine protein kinase functions as an activator of Lck. Such an activating enzyme could play an important role in signal transduction in T cells.

On the distribution of amino acid residues in transmembrane alpha-helix bundles.

The periodic distribution of residues in the sequence of 469 putative transmembrane alpha-helices from eukaryotic plasma membrane polytopic proteins has been analyzed with correlation matrices. The method does not involve any a priori assumption about the secondary structure of the segments or about the physicochemical properties of individual amino acid residues. Maximal correlation is observed at 3.6 residues per period, characteristic of alpha-helices. A scale extracted from the data describes the propensity of the various residues to lie on the same or on opposite helix faces. The most polar face of transmembrane helices, presumably that buried in the protein core, shows a strong enrichment in aromatic residues, while residues likely to face the fatty acyl chains of lipids are largely aliphatic.

Tyrosine-phosphorylated Stat1 and Stat2 plus a 48-kDa protein all contact DNA in forming interferon-stimulated-gene factor 3.

Interferon alpha induction of transcription operates through interferon-stimulated-gene factor 3 (ISGF), a transcription factor two components of which are members of the newly characterized Stat family of transcription factors. Interferon alpha induces tyrosine phosphorylation of Stat1 and Stat2 proteins that associate and, together with a 48-kDa protein, form ISGF3. Evidence is presented that a heterodimer of Stat1 and Stat2 is present in ISGF3 and that Stat1 and the 48-kDa protein make precise contact, while Stat2 makes general contact, with the interferon-stimulated response element, the binding site of the ISGF3.

The murine stk gene product, a transmembrane protein tyrosine kinase, is a receptor for macrophage-stimulating protein.

Macrophage-stimulating protein (MSP) was originally identified as an inducer of murine resident peritoneal macrophage responsiveness to chemoattractants. We recently showed that the product of RON, a protein tyrosine kinase cloned from a human keratinocyte library, is the receptor for MSP. Similarity of murine stk to RON led us to determine if the stk gene product is the murine receptor for MSP. Radiolabeled MSP could bind to NIH 3T3 cells transfected with murine stk cDNA (3T3/stk). Binding was saturable and was inhibited by unlabeled MSP but not by structurally related proteins, including hepatocyte growth factor and plasminogen. Specific binding to STK was demonstrated by cross-linking of 125I-labeled MSP to membrane proteins of 3T3/stk cells, which resulted in a protein complex with a molecular mass of 220 kDa. This radiolabeled complex comprised 125I-MSP and STK, since it could be immunoprecipitated by antibodies to the STK beta chain. Binding of MSP to stk cDNA-transfected cells induced tyrosine phosphorylation of the 150-kDa STK beta chain within 1 min and caused increased motile activity. These results establish the murine stk gene product as a specific transmembrane protein tyrosine kinase receptor for MSP. Inasmuch as the stk cDNA was cloned from a hematopoietic stem cell, our data suggest that in addition to macrophages and keratinocytes, a cell in the hematopoietic lineage may also be a target for MSP.

Differential activation of proliferation and cytotoxicity in human T-cell lymphotropic virus type I Tax-specific CD8 T cells by an altered peptide ligand.

Human T-cell leukemia virus type I (HTLV-I) gives rise to a neurologic disease known as HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Although the pathogenesis of the disease is unknown, the presence of a remarkably high frequency of Tax-specific, cytotoxic CD8 T cells may suggest a role of these cells in the development of HAM/TSP. Antigen-mediated signaling in a CD8 T-cell clone specific for the Tax(11-19) peptide of HTLV-I was studied using analog peptides substituted in their T-cell receptor contact residues defined by x-ray crystallographic data of the Tax(11-19) peptide in the groove of HLA-A2. CD8 T-cell stimulation with the wild-type peptide antigen led to activation of p56lck kinase activity, interleukin 2 secretion, cytotoxicity, and clonal expansion. A Tax analog peptide with an alanine substitution of the T-cell receptor contact residue tyrosine-15 induced T-cell-mediated cytolysis without activation of interleukin 2 secretion or proliferation. Induction of p56lck kinase activity correlated with T-cell-mediated cytotoxicity, whereas interleukin 2 secretion correlated with [3H]thymidine incorporation and proliferation. Moreover, Tax peptide analogs that activated the tyrosine kinase activity of p56lck could induce unresponsiveness to secondary stimulation with the wild-type peptide. These observations show that a single amino acid substitution in a T-cell receptor contact residue of Tax can differentially signal CD8 T cells and further demonstrate that primary activation has functional consequences for the secondary response of at least some Tax-specific CD8 T cells to HTLV-I-infected target cells.

Residue replacements of buried aspartyl and related residues in sensory rhodopsin I: D201N produces inverted phototaxis signals.

Residue replacements were made at five positions (Arg-73, Asp-76, Tyr-87, Asp-106, and Asp-201) in the Halobacterium salinarium phototaxis receptor sensory rhodopsin I (SR-I) by site-specific mutagenesis. The sites were chosen for their correspondence in position to residues of functional importance in the homologous light-driven proton pump bacteriorhodopsin found in the same organism. This work identifies a residue in SR-I shown to be of vital importance to its attractant signaling function: Asp-201. The effect of the substitution with the isosteric asparagine is to convert the normally attractant signal of orange light stimulation to a repellent signal. In contrast, similar neutral substitution of the four other ionizable residues near the photoactive site allows essentially normal attractant and repellent phototaxis signaling. Wild-type two-photon repellent signaling by the receptor is intact in the Asp-201 mutant, genetically separating the wild-type attractant and repellent signal generation processes. A possible explanation and implications of the inverted signaling are discussed. Results of neutral residue substitution for Asp-76 confirm our previous evidence that proton transfer reactions involving this residue are not important to phototaxis but that Asp-76 functions as the Schiff base proton acceptor in proton translocation by transducer-free SR-I.

Expression of the fms-like tyrosine kinase 4 gene becomes restricted to lymphatic endothelium during development.

We have recently cloned the human fms-like tyrosine kinase 4 gene FLT4, whose protein product is related to two vascular endothelial growth factor receptors FLT1 and KDR/FLK1. Here the expression of FLT4 has been analyzed by in situ hybridization during mouse embryogenesis and in adult human tissues. The FLT4 mRNA signals first became detectable in the angioblasts of head mesenchyme, the cardinal vein, and extraembryonally in the allantois of 8.5-day postcoitus (p.c.) embryos. In 12.5-day p.c. embryos, the FLT4 signal decorated developing venous and presumptive lymphatic endothelia, but arterial endothelia were negative. During later stages of development, FLT4 mRNA became restricted to vascular plexuses devoid of red cells, representing developing lymphatic vessels. Only the lymphatic endothelia and some high endothelial venules expressed FLT4 mRNA in adult human tissues. Increased expression occurred in lymphatic sinuses in metastatic lymph nodes and in lymphangioma. Our results suggest that FLT4 is a marker for lymphatic vessels and some high endothelial venules in human adult tissues. They also support the theory on the venous origin of lymphatic vessels.

Selective inhibition of the platelet-derived growth factor signal transduction pathway by a protein-tyrosine kinase inhibitor of the 2-phenylaminopyrimidine class.

The platelet-derived growth factor (PDGF) receptor is a member of the transmembrane growth factor receptor protein family with intrinsic protein-tyrosine kinase activity. We describe a potent protein-tyrosine kinase inhibitor (CGP 53716) that shows selectivity for the PDGF receptor in vitro and in the cell. The compound shows selectivity for inhibition of PDGF-mediated events such as PDGF receptor autophosphorylation, cellular tyrosine phosphorylation, and c-fos mRNA induction in response to PDGF stimulation of intact cells. In contrast, ligand-induced autophosphorylation of the epidermal growth factor (EGF) receptor, insulin receptor, and the insulin-like growth factor I receptor, as well as c-fos mRNA expression induced by EGF, fibroblast growth factor, and phorbol ester, was insensitive to inhibition by CGP 53716. In antiproliferative assays, the compound was approximately 30-fold more potent in inhibiting PDGF-mediated growth of v-sis-transformed BALB/c 3T3 cells relative to inhibition of EGF-dependent BALB/Mk cells, interleukin-3-dependent FDC-P1 cells, and the T24 bladder carcinoma line. When tested in vivo using highly tumorigenic v-sis- and human c-sis-transformed BALB/c 3T3 cells, CGP 53716 showed antitumor activity at well-tolerated doses. In contrast, CGP 53716 did not show antitumor activity against xenografts of the A431 tumor, which overexpresses the EGF receptor. These findings suggest that CGP 53716 may have therapeutic potential for the treatment of diseases involving abnormal cellular proliferation induced by PDGF receptor activation.