Ken-ichi Arai

Cytokine receptors and signal transduction.

: Cytokines play a vital role in coordinating immune and inflammatory responses. Unlike growth factor receptors with a tyrosine kinase, cytokine receptors have no intrinsic tyrosine kinase activity. Based on their structure, cytokine receptors are classified into several groups. High affinity receptors for IL-2, IL-3, IL-5, IL-6, and GM-CSF are composed of at least two distinct subunits, alpha and beta. The alpha subunits are primary cytokine binding proteins, and the beta subunits are required for formation of high affinity binding sites as well as for signal transduction. The GM-CSF, IL-3, and IL-5 receptors appear to share the same beta subunit in human, and therefore cross-talk among these cytokines may occur at the receptor level. High affinity receptors presumably are linked to various signal transduction pathways that lead to different cytokine functions. Differential expression of the cytokine receptors as well as reorganization of intracellular signalling pathways are critical for development of hemopoietic cells.

Expression cloning of the human IL-3 receptor cDNA reveals a shared β subunit for the human IL-3 and GM-CSF receptors

A cDNA for a human interleukin-3 (hIL-3) binding protein has been isolated by a novel expression cloning strategy: a cDNA library was coexpressed with the cDNA for the beta subunit of human granulocyte/macrophage colony-stimulating factor (GM-CSF) receptor (hGMR beta) in COS7 cells and screened by binding of 125I-labeled IL-3. The cloned cDNA (DUK-1) encodes a mature protein of 70 kd, which belongs to the cytokine receptor family and which alone binds hIL-3 with extremely low affinity (Kd = 120 +/- 60 nM). A high affinity IL-3-binding site (Kd = 140 +/- 30 pM) was reconstituted by coexpressing the DUK-1 protein and hGMR beta, indicating that hIL-3R and hGMR share the beta subunit. Therefore, we designated DUK-1 as the alpha subunit of the hIL-3R. As in human hematopoietic cells, hIL-3 and hGM-CSF complete for binding in fibroblasts expressing the cDNAs for hIL-3R alpha, GMR alpha, and the common beta subunit, indicating that different alpha subunits compete for a common beta subunit.

Signal transduction by the high-affinity GM-CSF receptor: two distinct cytoplasmic regions of the common beta subunit responsible for different signaling.

The high‐affinity receptors for granulocyte‐macrophage colony stimulating factor (GM‐CSF), interleukin 3 (IL‐3) and IL‐5 consist of two subunits, alpha and beta. The alpha subunits are specific to each cytokine and the same beta subunit (beta c) is shared by these three receptors. Although none of these receptor subunits has intrinsic kinase activity, these cytokines induce protein tyrosine phosphorylation, activation of Ras, Raf‐1 and MAP kinase, and transcriptional activation of nuclear proto‐oncogenes such as c‐myc, c‐fos and c‐jun. In this paper, we describe a detailed analysis of the signaling potential of the beta c subunit by using a series of cytoplasmic deletion mutants. The human beta c consists of 881 amino acid residues. A C‐terminal deletion mutant of beta c at amino acid 763 (beta 763) induced phosphorylation of Shc and activation of Ras, Raf‐1, MAP kinase and p70 S6 kinase, whereas a deletion at amino acid 626 (beta 626) induced none of these effects. The beta 763 mutant, as well as the full‐length beta c, induced transcription of c‐myc, c‐fos and c‐jun. Deletions at amino acid 517 (beta 517) and 626 (beta 626) induced c‐myc and pim‐1, but no induction of c‐fos and c‐jun was observed. GM‐CSF increased phosphatidylinositol 3 kinase (PI3‐K) activity in anti‐phosphotyrosine immunoprecipitates from cells expressing beta 763 as well as beta c, whereas it was only marginally increased from cells expressing beta 517 or beta 626. Thus, there are at least two distinct regions within the cytoplasmic domain of beta c that are responsible for different signals, i.e. a membrane proximal region of approximately 60 amino acid residues upstream of Glu517 is essential for induction of c‐myc and pim‐1, and a distal region of approximately 140 amino acid residues (between Leu626 and Ser763) is required for activation of Ras, Raf‐1, MAP kinase and p70 S6 kinase, as well as induction of c‐fos and c‐jun.

Suppression of apoptotic death in hematopoietic cells by signalling through the IL-3/GM-CSF receptors.

Interleukin 3 (IL‐3) and granulocyte‐macrophage colony stimulating factor (GM‐CSF) exert their biological functions through acting on a specific receptor which consists of a ligand‐specific alpha subunit and the shared common beta subunit. Inhibition by genistein of a subset of IL‐3/GM‐CSF‐mediated signals, including c‐myc induction, resulted in the abrogation of DNA synthesis, however, IL‐3 still protected cells from apoptotic cell death. Conversely, a C‐terminal truncated form of the GM‐CSF receptor, which is missing a critical cytoplasmic region required for activation of the Ras/Raf‐1/MAP kinase pathway, induced DNA synthesis, but failed to prevent cell death in response to GM‐CSF. Consequently, cells died by apoptosis in the presence of GM‐CSF, despite displaying a transient mitogenic response. However, expression of activated Ras protein complemented defective signalling through the mutant receptor and supported long‐term proliferation in concert with GM‐CSF. These results indicate that IL‐3 and GM‐CSF prevent apoptosis of hematopoietic cells by activating a signalling pathway distinct from the induction of DNA synthesis and that long‐term cell proliferation requires the activation of both pathways.

GPA1, a haploid-specific essential gene, encodes a yeast homolog of mammalian G protein which may be involved in mating factor signal transduction

GPA1 protein of Saccharomyces cerevisiae is homologous to the alpha subunit of mammalian G protein. GPA1 transcript was found in haploid cells but was not detected in diploid cells. Disruption of GPA1 resulted in a haploid-specific lethal phenotype, indicating that GPA1 is a haploid-specific essential gene for cell growth. Upon regulation of expression of GPA1 by the galactose-inducible GAL1 promoter, the loss of GPA1 function was found to lead to cell-cycle arrest at the late G1 phase. Mutants that suppress the lethality of the gpa1::HIS3 mutation showed a sterile phenotype that was not cell-type-specific. These results suggest that GPA1 protein may control the signal for mating-factor-mediated cell-cycle arrest.

Nucleotide sequences of STE2 and STE3, cell type-specific sterile genes from Saccharomyces cerevisiae

The nucleotide sequences of STE2 and STE3, cell type‐specific sterile genes of Saccharomyces cerevisiae, were determined; major open reading frames encode 431 and 470 amino acids, respectively. STE2 and STE3 proteins seem to be folded in a similar fashion and are likely to be membrane‐bound. Both consist of seven hydrophobic segments in each NH2‐terminal region with a long hydrophilic domain in each COOH‐terminal region. However, the two putative gene products do not exhibit extensive sequence homology. The STE2 protein has no obvious hydrophobic signal peptide; the NH2 terminus of the STE3 protein might serve as a signal peptide. The STE2 transcript, 1.7 kb, was detected in MATa strains but not in MATα strains, while the STE3 transcript, also 1.7 kb, was detected only in MATα cells. In STE2, two canonical TATA sequences are located 18 and 27 bp upstream of the mRNA start site, which has been mapped 32 bp before the initiator ATG codon, while STE3 contains a similar sequence (TATAGA), which is preceded by a long AT sequence, 140 bp upstream of the initiator ATG codon. Transcription of STE2 in a cells seems to be enhanced by exogenous α‐factor.

NFATx, a Novel Member of the Nuclear Factor of Activated T Cells Family That Is Expressed Predominantly in the Thymus

The nuclear factor of activated T cells (NFAT) regulates cytokine gene expression in T cells through cis-acting elements located in the promoters of cytokine genes. Here, we report the cDNA cloning, chromosomal localization, and initial characterization of a transcription factor related to NFATp and NFATc. The novel molecule, designated NFATx, exhibits in its middle a region very similar to the Rel homology domain in NFATc and NFATp. The amino-terminal region of NFATx also shows significant similarities to corresponding sequences in NFATc and NFATp and contains three copies of a conspicuous 17-residue motif of unknown function. We provide evidence showing that NFATx can reconstitute binding to the NFAT-binding site from the interleukin 2 promoter when combined with AP1 (c-Fos/c-Jun) polypeptides and that NFATx is capable of activating transcription of the interleukin 2 promoter in COS-7 cells when stimulated with phorbol ester and calcium ionophore. NFATx mRNA is preferentially and remarkably found in the thymus and at lower levels in peripheral blood leukocytes. The expression pattern of NFATx, together with its functional activity, strongly suggests that NFATx plays a role in the regulation of gene expression in T cells and immature thymocytes.

Structure of the chromosomal gene for granulocyte-macrophage colony stimulating factor: comparison of the mouse and human genes.

A cDNA clone that expresses granulocyte‐macrophage colony stimulating factor (GM‐CSF) activity in COS‐7 cells has been isolated from a pcD library prepared from mRNA derived from concanavalin A‐activated mouse helper T cell clones. Based on homology with the mouse GM‐CSF cDNA sequence, the mouse GM‐CSF gene was isolated. The human GM‐CSF gene was also isolated based on homology with the human GM‐CSF cDNA sequence. The nucleotide sequences determined for the genes and their flanking regions revealed that both the mouse and human GM‐CSF genes are composed of three introns and four exons. The organization of the mouse and human GM‐CSF genes are highly homologous and strong sequence homology between the two genes is found both in the coding and non‐coding regions. A ‘TATA’‐like sequence was found 20‐25 bp upstream from the transcription initiation site. In the 5′‐flanking region, there is a highly homologous region extending 330 bp upstream of the putative TATA box. This sequence may play a role in regulation of expression of the GM‐CSF gene. These structures are compared with those of different lymphokine genes and their regulatory regions.

Coordinate regulation of immune and inflammatory responses by T cell-derived lymphokines.

In response to antigenic stimulation, helper T cells secrete a set of protein mediators called lymphokines that regulate proliferation, differentiation, and maturation of lymphocytes and hemopoietic cells. Because all known lymphokines are composed of a single polypeptide chain, their coding sequences can be isolated by functional expression in appropriate host cells. Based on this expression cloning protocol, a number of T cell lymphokine genes have been isolated, their primary structure has been determined, and biological properties of their recombinant products have been examined. These studies revealed the existence of a regulatory network between lymphoid cells and hemopoietic cells mediated by the actions of multiple pleiotropic lymphokines produced by activated T cells. Because all or a part of this network can be activated in different ways by unique combinations of lymphokines, it is clear that T cells can play a vital role in coordinating the function of different body compartments in the immune and inflammatory responses. The activation of lymphokine genes in T cells by antigen is rapid and temporal. Therefore, an inflammatory response that involves proliferation and maturation of target cells may be restricted to the site of lymphokine production. This inducible hemopoiesis appears to be differentially regulated from the steady state or constitutive hemopoiesis that occurs in the bone marrow micro‐environment in the absence of immunological stimuli.— MIYAJIMA, A.; MIYATAKE, S.; SCHREURS, J.; de Vries, J.; Arai, N.; Yokota, T.; Arai, K. Coordinate regulation of immune and inflammatory responses by T cell‐derived lymphokines. FASEB J. 2: 2462‐2473; 1988.

Molecular Biology of Interleukin 4 and Interleukin 5 Genes and Biology of their Products that Stimulate B Cells, T Cells and Hemopoietic Cells

Lymphokines produced by helper T cells activated by antigen mediate numerous efTector functions of T cells. Unlike immunoglobulin or the T cell receptor, lymphokines do not bear antigen specificity and stimulate proliferation and differentiation of lymphocytes and hemopoietic cells (Arai et al. 1986). Since many lymphokines are composed of single polypeptide chains, their coding sequences can be isolated by functional expression in appropriate host cells. Using a pcD cDNA expression vector (Okayama & Berg 1983), we have developed a screening procedure employing transfection of plasmid DNAs into mammalian cells followed by assay of transfected cell supernatants for lymphokine activities of interest (Yokota et al. 1984. 1985, 1987c). Based on this expression cloning protocol, many T cell lymphokine genes have been isolated and their primary structures determined. These studies have revealed a regulatory network formed between lymphoid cells and hemopoietic cells through the action of multiple lymphokines produced by activated T cells. For example, interleukin 2 (IL-2) stimulates predominantly T cells whereas interleukin 3 (lL-3) and granulocytemacrophage colony stimulating factor (GM-CSF) stimulate hemopoietic cells. A number of molecules stimulate B cells. Among them, interleukin 4 (IL-4), interleukin 5 (IL-5) and B cell stimulatory factor 2 (BSF-2) have been chemically defined by molecular cloning (Lee et al. 1986, Noma et al. 1986, Yokota et al. !986,