Isolation and functional characterization of the human 90K promoter.

90K is a secreted protein thought to be involved in the body's defense against pathogens and cancer. To elucidate its transcriptional regulation, the promoter of human 90K (HGMW-approved symbol LGAL S3BP) was isolated and characterized. Analysis of the 3. 3-kb 5'-flanking region revealed that it is a TATA-less promoter, but neither GC-rich nor dependent on SP1 sites. RNase protection assays detected one major transcription start site (+1) and several minor transcription start sites upstream and downstream. Deletion studies defined a minimal promoter (-103 --> -49) and indirectly suggested positive synergism between different elements within it. Consistent with the proposed function of 90K, its promoter activity could be stimulated by poly(I). poly(C), mimicking viral infection. Two regions mediating induction by poly(I). poly(C) (-171 --> -112, -32 --> 46) were identified by deletion mutants. A small region around the minimal promoter (-99 --> -12) was highly homologous between human and mouse. While both human and mouse minimal promoters contained an interferon-responsive element (IRF-E), the human minimal promoter was not inducible by poly(I). poly(C) in contrast to that of the mouse. Point mutations 30 bp upstream of the IRF-E, however, conferred inducibility to the human minimal promoter, suggesting interaction between different promoter elements.

Differential effect on TCR:CD3 stimulation of a 90-kD glycoprotein (gp90/Mac-2BP), a member of the scavenger receptor cysteine-rich domain protein family.

We studied the effects of a 90-kD glycoprotein (gp90/Mac-2BP) belonging to the scavenger receptor family, present in normal serum and at increased levels in inflammatory disease and cancer patients, on some T cell function parameters. Whereas the lymphocyte proliferative response to non-specific mitogens such as phytohaemagglutinin (PHA) and concanavalin A (Con A), but not pokeweed mitogen (PWM), was strongly reduced, probably due to the lectin-binding properties of gp90/Mac-2BP, the response to T cell receptor (TCR) agonists such as superantigens and allogeneic cells was potentiated. When lymphocytes were stimulated with different anti-TCR:CD3 MoAbs, both in soluble and solid-phase form, gp90/Mac-2BP was able to down-regulate the proliferative response to anti-CD3 MoAb, whereas the response to anti-TCR alphabeta MoAb was enhanced. A similar differential effect was observed when a MoAb against CD5 (another member of the scavenger receptor superfamily) was added to anti-CD3 or anti-TCR-stimulated cells; anti-CD5 MoAb strongly down-modulated the CD3-mediated response, whereas its presence in culture was associated with potentiation of the response to TCR alphabeta agonists. gp90/Mac-2BP was able per se to up-regulate Ca2+ levels in freshly isolated lymphocytes; moreover, its presence in culture was associated with increased Ca2+ mobilization following stimulation with anti-TCR alphabeta, but not anti-CD3 MoAb. These data indicate that gp90/Mac-2BP could be able to influence some immune responses, possibly through multiple homologous interactions with other members of the scavenger receptor family; moreover, our findings suggest that signalling through the different components of the TCR:CD3 complex may follow distinct activation pathways into the cells.

A family of proteins that inhibit signalling through tyrosine kinase receptors.

Phosphotyrosine phosphatases are critical negative or positive regulators in the intracellular signalling pathways that result in growth-factor-specific cell responses such as mitosis, differentiation, migration, survival, transformation or death. The SH2-domain-containing phosphotyrosine phosphatase SHP-2 is a positive signal transducer for several receptor tyrosine kinases (RTKs) and cytokine receptors. To investigate its mechanism of action we purified a tyrosine-phosphorylated glycoprotein which in different cell types associates tightly with SHP-2 and appears to serve as its substrate. Peptide sequencing in conjunction with complementary DNA cloning revealed a new gene family of at least fifteen members designated signal-regulatory proteins (SIRPs). They consist of two subtypes distinguished by the presence or absence of a cytoplasmic SHP-2-binding domain. The transmembrane polypeptide SIRP alpha1 is a substrate of activated RTKs and in its tyrosine-phosphorylated form binds SHP-2 through SH2 interactions and acts as its substrate. It also binds SHP-1 and Grb2 in vitro and has negative regulatory effects on cellular responses induced by growth factors, oncogenes or insulin. Our findings indicate that proteins belonging to the SIRP family generally regulate signals defining different physiological and pathological processes.

Characterization of the PEST family protein tyrosine phosphatase BDP1.

Using a polymerase chain reaction (PCR) amplification strategy, we identified a novel protein tyrosine phosphatase (PTPase) designated Brain Derived Phosphatase (BDP1). The full length sequence encoded an open reading frame of 459 amino acids with no transmembrane domain and had a calculated molecular weight of 50 kDa. The predicted amino acid sequence contained a PEST motif and accordingly, BDP1 shared the greatest homology with members of the PTP-PEST family. When transiently expressed in 293 cells BDP1 hydrolyzed p-Nitrophenylphosphate, confirming it as a functional protein tyrosine phosphatase. Northern blot analysis indicated that BDP1 was expressed not only in brain, but also in colon and several different tumor-derived cell lines. Furthermore, BDP1 was found to differentially dephosphorylate autophosphorylated tyrosine kinases which are known to be overexpressed in tumor tissues.

Isolation and developmental expression analysis of Enx-1, a novel mouse Polycomb group gene.

Members of the Polycomb group (Pc-G) of genes encode transcriptional regulators that control the expression of key developmental effector genes in Drosophila melanogaster. Although multiple Pc-G genes have been identified and characterized in Drosophila, information about these important regulatory proteins in vertebrates, including their precise expression patterns, has remained scarce. We report here the cloning of Enx-1, a novel vertebrate Pc-G gene, which encodes the murine homolog of the Drosophila Enhancer of zeste (E(z)) gene. Drosophila E(z) controls the expression of several homeobox genes as well as some segmentation genes and its disruption causes multiple phenotypes in Drosophila development. Analysis of the primary structure of murine Enx-1 reveals the conservation of several regions, including the previously described SET domain and a newly defined CXC domain. In addition, we find the SET domain to be conserved in evolutionarily distant species ranging from vertebrates to plants and fungi. The expression pattern analysis of Enx-1 reveals ubiquitous expression throughout early embryogenesis, while in later embryonic development Enx-1 expression becomes restricted to specific sites within the central and peripheral nervous system and to the major sites of fetal hematopoiesis. In adult stages we also find Enx-1 expression to be restricted to specific tissues, including spleen, testis and placenta.

Colon carcinoma kinase-4 defines a new subclass of the receptor tyrosine kinase family.

Complementary DNA (cDNA) encoding a novel member of the receptor tyrosine kinase (RTK) family has been isolated from colon carcinoma tissue. Colon carcinoma kinase 4 (CCK-4) mRNA is highly expressed in human lung tissue and at lower levels in the thyroid gland and ovary. While no mRNA was found in human adult colon tissues, expression varied remarkably in colon carcinoma-derived cell lines. CCK-4 cDNA encodes a chicken KLG-related, 1071 amino acid-long transmembrane glycoprotein containing several genetic alterations within the RTK consensus sequences. These define CCK-4 as a catalytically inactive member of the RTK family of proteins and, in analogy to HER3, suggest a potentially tumor-characteristic role as a signal amplifier or modulator for an as yet unidentified kinase-competent partner.

Selective down-regulation of the insulin receptor signal by protein-tyrosine phosphatases alpha and epsilon.

Binding of insulin to its receptor (IR) causes rapid autophosphorylation with concomitant activation of its tyrosine kinase which transmits the signal by phosphorylating cellular substrates. The IR activity is controlled by protein-tyrosine phosphatases, but those directly involved in regulating the insulin receptor and its signaling pathways have not yet been identified. Using baby hamster kidney cells overexpressing the IR and a novel insulin-based selection principle, we established stable cell lines with functionally coupled expression of the IR and protein-tyrosine phosphatases. The two closely related protein-tyrosine phosphatases alpha and epsilon were identified as negative regulators of IR tyrosine kinase.

The receptor tyrosine kinase p185HER2 is expressed on a subset of B-lymphoid blasts from patients with acute lymphoblastic leukemia and chronic myelogenous leukemia.

The class I receptor tyrosine kinase (RTK) HER2 is an oncoprotein that is frequently involved in the pathogenesis of tumors of epithelial origin. Here we report mRNA expression in peripheral blood and bone marrow cells from healthy donors in hematopoietic cell lines and leukemic blasts from patients with acute lymphoblastic leukemia (ALL), acute myeloblastic leukemia (AML), chronic lymphoblastic leukemia (CLL), and chronic myeloid leukemia (CML). However, cell surface expression of HER2 protein (p185HER2) was found exclusively on a subset of leukemic cells of the B-lymphoblastic lineage. p185HER2 expression was found on blasts in 2 of 15 samples from infants, 9 of 19 samples from adult patients with C-ALL (CD19+CD10+), and 1 of 2 samples from patients with pro-B ALL (CD19+CD10-), whereas none of the leukemic cells from patients with AML (0/30), T-ALL (0/7), CLL (0/5) (CD19+CD5+), or CML in chronic and accelerated phase (0/5) or in blast crisis with myeloid differentiation (0/14) were positive for p185HER2. However, cells from 3 of 4 patients with CML in B-lymphoid blast crisis (CD19+CD10+) expressed high levels of p185HER2, which was also found on the surface of the CML-derived B-cell lines BV-173 and Nalm-1. Our study shows p185HER2 expression on malignant cells of hematopoietic origin for the first time. Aberrant expression of this oncogenic receptor tyrosine kinase in hematopoietic cell types may be an oncogenic event contributing to the development of a subset of B-lymphoblastic leukemias.

The gene (LGALS3BP) encoding the serum protein 90K, associated with cancer and infection by the human immunodeficiency virus, maps at 17q25.

Levels of a 90-kDa tumor associated protein, designated 90K (gene symbol LGALS3BP), have been found elevated in the serum of patients with cancer and in those infected by the human immunodeficiency virus (HIV). 90K appears to be implicated in immune response associated with natural killer (NK) and lymphokine-activated killer (LAK) cell cytotoxicity. Using fluorescence in situ hybridization the full length 90K cDNA has been localized to chromosome 17q25.

Src kinase associates with a member of a distinct subfamily of protein-tyrosine phosphatases containing an ezrin-like domain.

A 6.2-kb full-length clone encoding a distinct protein-tyrosine phosphatase (PTP; EC 3.1.3.48), PTPD1, was isolated from a human skeletal muscle cDNA library. The cDNA encodes a protein of 1174 amino acids with N-terminal sequence homology to the ezrin-band 4.1-merlin-radixin protein family, which also includes the two PTPs H1 and MEG1. The PTP domain is positioned in the extreme C-terminal part of PTPD1, and there is an intervening sequence of about 580 residues without any apparent homology to known proteins separating the ezrin-like and the PTP domains. Thus, PTPD1 and the closely related, partially characterized, PTPD2 belong to the same family as PTPH1 and PTPMEG1, but because of distinct features constitute a different PTP subfamily. Northern blot analyses indicate that PTPD1 and PTPD2 are expressed in a variety of tissues. In transient coexpression experiments PTPD1 was found to be efficiently phosphorylated by and associated with the src kinase pp60src.

The secreted tumor-associated antigen 90K is a potent immune stimulator.

Immunization of mice with conditioned media from human breast cancer cells yielded the monoclonal antibody SP-2, which recognized an antigen of approximately 90-95 kDa. This protein, designated 90K, was found to be present in the serum of healthy individuals and at elevated levels in the serum of subpopulations of patients with various types of cancer and AIDS. Here we report the primary structure of the SP-2 antigen and demonstrate its relationship to a family of proteins which carry a scavenger receptor cysteine-rich domain. Northern blot analysis of normal tissues, primary tumors, and tumor-derived cell lines indicates a broad expression spectrum of the 90K gene at widely varying levels. Functional characterization reveals stimulatory effects of 90K on host defense systems, such as natural killer cell and lymphokine-activated killer cell activity, and indicates that its immunostimulatory effects may be mediated through the induction of interleukin-2 and possibly other cytokines.

Breast cancer is associated with loss of the c-kit oncogene product.

The proto-oncogene c-kit encodes a tyrosine kinase receptor related to the PDGF/CSF-1 receptors. Mutations of this gene result in impairment of hematopoiesis, melanogenesis and gametogenesis. Using monoclonal antibodies to the c-kit gene product, we have analyzed its expression in normal and transformed human tissues. Unexpectedly, the receptor was found to be expressed in normal mammary epithelium. While in benign breast lesions, the c-kit gene product was detected at variable levels in 82% of the instances, in primary tumors, no product could be identified in 87% of the cases. This phenotype is maintained in metastatic foci. These findings were confirmed by paired Northern blot analysis of RNA preparations from normal and tumor tissues. These results demonstrate that the c-kit receptor may also be involved in the growth control of mammary epithelium and that this function may be impaired following malignant transformation and de-differentiation.

Expression of c-kit receptor in normal and transformed human nonlymphoid tissues.

The protooncogene c-kit encodes a tyrosine kinase with a molecular weight of 145,000, highly related to the platelet derived growth factor/colony stimulating factor receptors. Mutations of the murine gene result in impairment of hematopoiesis, gametogenesis, and of the melanocyte cell lineage. In order to elucidate c-kit functions in development and oncogenesis we have analyzed immunohistochemically its expression in human normal and transformed nonlymphoid tissues. The receptor has been detected in spermatogonia, melanocytes, and unexpectedly, in astrocytes, renal tubules, parotid cells, thyrocytes, and breast epithelium. While the gene product is expressed in seminoma, lung tumors, and melanoma of low invasiveness, no detectable levels have been detected in thyroid and breast carcinomas, astrocytomas, and invasive melanomas. In breast tumors these findings were confirmed by paired, Northern blot analysis of RNA preparations from normal and transformed tissue. The present results demonstrate that the c-kit receptor plays a role in the development of a larger spectrum of cell lineages. Furthermore, on the basis of the transformation associated changes, we speculate that, while in some cell types, c-kit expression positively regulates mitogenesis and is selected for in neoplastic transformation, in other tissues the c-kit pathway is involved in morphogenesis and differentiation and is, therefore, negatively selected in the course of tumor progression.

The genes for the frog skin peptides GLa, xenopsin, levitide and caerulein contain a homologous export exon encoding a signal sequence and part of an amphiphilic peptide.

From genomic libraries of Xenopus laevis, parts of the genes coding for the precursors of the skin peptides GLa (peptide with amino-terminal glycine and carboxy-terminal leucinamide), xenopsin and levitide have been isolated and sequenced. The gene for prepropeptide GLa comprises four exons, separated by relatively small introns. The gene for preproxenopsin is composed of five exons, of which all but the last one have been analyzed. This is a large gene encompassing at least 25,000 base pairs. In addition, two exons of the gene for preprolevitide have been isolated. A comparison of these genes reveals the presence of a homologous exon. This exon contains 161 bp, starts one base pair prior to the initiation codon and encodes a signal peptide and part of a pro region with processing sites. In addition, the two genes for preprocaerulein analyzed previously [Vlasak et al. (1987) Eur. J. Biochem. 169, 53-58] also contain a similar exon. This demonstrates the existence of a homologous export exon in genes encoding the precursors of different skin peptides.

Xenopsin: the neurotensin-like octapeptide from Xenopus skin at the carboxyl terminus of its precursor.

We have synthesized two oligodeoxyribonucleotide mixtures that contain sequences complementary to different parts of the hypothetical mRNA sequence of xenopsin, a biologically active octapeptide found in skin extracts from Xenopus laevis. The two primer pools were independently used to initiate reverse transcription on skin poly(A)+ RNA and the resulting cDNAs were then used to screen in parallel a cDNA library prepared from skin poly(A)+ RNA. One of the clones that hybridized with both probes was subjected to sequence analysis. It contains a nearly full-length DNA copy of a mRNA of approximately equal to 490 nucleotides that encodes a xenopsin precursor protein. The deduced precursor is 80 amino acids long, exhibits a putative signal sequence at the NH2 terminus, and contains the biologically active peptide at the COOH terminus. The region corresponding to the NH2-terminal portion of the xenopsin precursor shows a striking nucleotide and amino acid sequence homology with the precursor of PYLa, another recently described peptide from Xenopus skin.

Structural analysis of repetitive sequence elements transcribed in early development of Xenopus laevis.

A cDNA library prepared from mRNA of Xenopus laevis embryos was screened with a genomic DNA fragment containing various transcribed repetitive sequence elements. Comparison of the nucleotide sequence of two isolated cDNAs and their genomic relatives allows one to define two transcribed repetitive sequence elements. One of them belongs to a highly reiterated family and consists of a tandem array of homologous subunits of 77-80 bp. The other is reiterated approximately 2200 times, has a size of 260 bp and displays a conserved region of 135 bp. The data are consistent with the presence of repetitive sequence transcripts in the 3' part of mRNA molecules.

The nucleotide and amino acid coding sequence of a gene for H1 histone that interacts with euchromatin. The early embryonic H1 gene of the sea urchin Strongylocentrotus purpuratus.

We have determined the nucleotide sequence of the gene that codes for an H1 histone associated with euchromatin in the sea urchin Strongylocentrotus purpuratus. The gene codes for a protein of 205 amino acids. The nucleotide sequence of this gene is homologous to the sequence of H1 mRNA that is expressed during early embryonic development. We have compared the nucleotide and protein coding sequences of this H1 gene to those of the early H1 histone gene of the sea urchin Psammechinus miliaris. There is considerable drift by nucleotide substitution between the two genes randomly distributed across the mRNA coding region. Despite this divergence of nucleotide sequence, there are local constraints on amino acid substitutions throughout the molecule and especially in its central region. We have also compared the amino acid sequence in this central hydrophobic region of the euchromatic S. purpuratus H1 histone to the same region in H1 and H5 histones associated with heterochromatin. We show that certain amino acids are conserved and aligned in frameworks in all the sequenced H1 proteins.

Genetic variation in the human insulin gene.

Four recombinant lambda phages containing nucleotide sequences complementary to a cloned human preproinsulin DNA probe have been isolated from human DNA. Restriction analyses in conjunction with Southern hybridizations reveal two types of gene sequences. One isolate of each type was subjected to complete nucleotide sequence determination. The sequences contain the entire preproinsulin messenger RNA region, two intervening sequence. 260 nucleotides upstream from the messenger RNA capping site, and 35 nucleotides beyond the polyadenylate attachment site. Our results strongly suggest that these two gene types are allelic variants of a single insulin gene.