Atsushi Miyajima

Interleukin-3, granulocyte-macrophage colony stimulating factor and interleukin-5 transduce signals through two STAT5 homologs.

Interleukin‐3 (IL‐3) is an important regulator of hemopoiesis and considerable effort has been directed towards the study of its mechanism of signal transduction. In this paper, we describe the first molecular identification of a STAT transcription factor that is activated by IL‐3. STATs exist in a cytoplasmic, transcriptionally inactive form which, in response to extracellular signals, become tyrosine phosphorylated and translocate to the nucleus where they bind to specific DNA elements. Several of these DNA elements were found which bind proteins in an IL‐3‐responsive manner. Analysis of these bandshift complexes with available antibodies to the known STATs suggests that IL‐3 activates the DNA‐binding ability of STAT5, a protein which was originally characterized as a prolactin‐responsive transcription factor in sheep. IL‐5 and granulocyte‐macrophage colony stimulating factor (GM‐CSF), which share a common signaling receptor subunit with IL‐3, also activate STAT5. Unexpectedly, two murine STAT5 homologs, 96% identical to each other at the amino acid level, were isolated and IL‐3‐dependent GAS binding could be reconstituted in COS cells transfected with IL‐3 receptor and either STAT5 cDNA. In IL‐3‐dependent hemopoietic cells, both forms of STAT5 are expressed and activated in response to IL‐3.

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.

Critical cytoplasmic domains of the common beta subunit of the human GM-CSF, IL-3 and IL-5 receptors for growth signal transduction and tyrosine phosphorylation.

The high‐affinity receptors for human granulocyte‐macrophage colony‐stimulating factor (GM‐CSF), interleukin 3 (IL‐3) and interleukin 5 (IL‐5) are composed of two distinct subunits, alpha and beta c. The alpha subunits are specific for each cytokine, whereas the beta subunit (beta c) is shared by the three receptors and is an essential component of signal transduction. We have made a series of mutant beta c cDNAs that delete various regions of the cytoplasmic domain and examined the function of these mutants by coexpressing them with the alpha subunit of the human GM‐CSF receptor (hGMR) in an IL‐3‐dependent mouse pro‐B cell line BaF3. Two domains in the membrane‐proximal portion of beta c were found to be important for transducing the hGM‐CSF‐mediated growth signals: one domain between Arg456 and Phe487 appears to be essential for proliferation, and the second domain between Val518 and Asp544 enhances the response to GM‐CSF, but is not absolutely required for proliferation. The region between Val518 and Leu626 was responsible for major tyrosine phosphorylation of 95 and 60 kDa proteins. Thus, beta c‐mediated major tyrosine phosphorylation of these proteins was apparently separated from proliferation. However, the beta 517 mutant lacking residues downstream of Val518 transmitted a herbimycin‐sensitive proliferation signal, suggesting that beta 517 still activates a tyrosine kinase(s). We also evaluated the role of the cytoplasmic domain of the GMR alpha subunit and the results suggest that it is involved in the hGM‐CSF‐mediated signal transduction, but is not essential.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

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.

Interleukin 2 and erythropoietin activate STAT5/MGF via distinct pathways.

Signal transducers and activators of transcription (STAT) proteins play an important role in cytokine signal transduction in conjunction with Janus kinases (JAKs). MGF/STAT5 is known as prolactin regulated STAT. Here we demonstrate that interleukin 2 (IL‐2) as well as erythropoietin (EPO) stimulate STAT5 and induce tyrosine phosphorylation of STAT5. These IL‐2‐ and EPO‐induced STATs have an identical DNA binding specificity and immunoreactivity. We also show that IL‐4 induces a DNA binding factor which possesses similar, but distinct, DNA binding specificity from that of STAT5 and is immunologically different from STAT5. Analysis of two EPO receptor (EPOR) transfected CTLL‐2 cell lines discloses that IL‐2 activates JAK1 and JAK3 as well as STAT5, while EPO stimulates STAT5 and JAK2 in EPO‐responsive CTLL‐2 cells (ERT/E2). On the contrary, EPO activates neither JAK2 nor STAT5 in other cell lines that failed to respond to EPO (ERT cells). EPOR and JAK2 associate with each other regardless of EPO presence in ERT/E2 cells, however, such an interaction is not present in ERT cells. Thus, EPOR and JAK2 association seems to be important for EPO responsiveness in CTLL‐2 cells.

Identification of the second subunit of the murine interleukin-5 receptor: interleukin-3 receptor-like protein, AIC2B is a component of the high affinity interleukin-5 receptor

Murine interleukin‐5 (IL‐5) binds to its receptor with high and low affinity. It has been shown that the high affinity IL‐5 receptor (IL‐5‐R) is composed of at least two membrane protein subunits and is responsible for IL‐5‐mediated signal transduction. One subunit of the high affinity IL‐5‐R is a 60 kDa membrane protein (p60 IL‐5‐R) whose cDNA was isolated using the anti‐IL‐5‐R monoclonal antibody (mAb), H7. This subunit alone binds IL‐5 with low affinity. The second subunit does not bind IL‐5 by itself, and is expressed not only on IL‐5‐dependent cell lines but also on an IL‐3‐dependent cell line, FDC‐P1. Expression of the p60 IL‐5‐R cDNA in FDC‐P1 cells, which do not bind IL‐5, reconstituted the high affinity IL‐5‐R. We have characterized the second subunit of the IL‐5‐R by using another anti‐IL‐5‐R mAb, R52.120, and the anti‐IL‐3‐R mAb, anti‐Aic‐2. The anti‐Aic‐2 mAb down‐regulated binding of IL‐5 to an IL‐5‐dependent cell line, Y16. Both R52.120 and anti‐Aic‐2 mAbs recognized membrane proteins of 130–140 kDa expressed on FDC‐P1 and Y16 cells. The R52.120 mAb recognized both murine IL‐3‐R (AIC2A) and its homologue (AIC2B) expressed on L cells transfected with suitable cDNAs. The high affinity IL‐5‐R was reconstituted on an L cell transfectant co‐expressing AIC2B and p60 IL‐5‐R, whereas only the low affinity IL‐5‐R was detected on a transfectant co‐expressing AIC2A and p60 IL‐5‐R.(ABSTRACT TRUNCATED AT 250 WORDS)

The amino-terminal helix of GM-CSF and IL-5 governs high affinity binding to their receptors.

Transduction of the biological effects of granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) and interleukin‐5 (IL‐5) requires the interaction of each cytokine with at least two cell surface receptor components, one of which is shared between these two cytokines. A strategy is presented that allowed us to identify receptor binding determinants in GM‐CSF and IL‐5. Mixed species (human and mouse) receptors were used to locate unique receptor binding domains on a series of human‐mouse hybrid GM‐CSF and IL‐5 cytokines. Results show that the interaction of these two cytokines with the shared subunit of their high affinity receptor complexes is governed by a very small part of their peptide chains. The presence of a few key residues in the amino‐terminal alpha‐helix of each ligand is sufficient to confer specificity to the interaction. Comparison with other cytokines suggests that the amino‐terminal helix of many of these proteins may contain the recognition element for the formation of high affinity binding sites with the alpha subunit of their multi‐component receptors.

Common signal transduction system shared by STE2 and STE3 in haploid cells of Saccharomyces cerevisiae: autocrine cell-cycle arrest results from forced expression of STE2

Induction of STE2 expression using the GAL1 promoter both in a wild‐type MATα strain and in a MATα ste3 strain caused transient cell‐cycle arrest and changes in morphology (‘shmoo’‐like phenotype) in a manner similar to a cells responding to α‐factor. In addition, STE2 expressed in a MATα ste3 mutant allowed the cell to conjugate with a cells but at an efficiency lower than that of wild‐type α cells. This result indicates that signal(s) generated by a‐factor in α cells can be substituted by signal(s) generated by the interaction of α‐factor with the expressed STE2 product. When STE2 or STE3 was expressed in a matα1 strain (insensitive to both α‐ and a‐factors), the cell became sensitive to α‐ or a‐factor, respectively, and resulted in morphological changes. These results suggest that STE2 and STE3 are the sole determinants for α‐factor and a‐factor sensitivity, respectively, in this strain. On the other hand, expression of STE2 in an a/α diploid cell did not affect the α‐factor insensitive phenotype. Haploid‐specific components may be necessary to transduce the α‐factor signal. These results are consistent with the idea that STE2 encodes an α‐factor receptor and STE3 encodes an a‐factor receptor, and suggest that both α‐ and a‐factors may generate an exchangeable signal(s) within haploid cells.

The Hematopoietic Cytokine Receptors

The growth and differentiation of hematopoietic stem cells to form the vast repertoire of mature blood cells that exists in vivo is orchestrated by an array of intercellular signals, mediated by cytokines in association with a complex stromal microenvironment. Cytokines are a diverse group of glycoproteins, expressed constitutively or inducibly by a wide variety of cell types, in membrane-bound or secreted forms (reviewed in Nicola, 1989; Arai et al., 1990; Howard et al., 1993). In addition to controlling hematopoietic development, cytokines mediate many physiological responses, such as immunity, inflammation, and antiviral activity. A single cytokine can exhibit multiple functions depending on its target cell type, and different cytokines often show similar biological functions on the same target cell population (Metcalf, 1986). Combinations of cytokines can interact synergistically (Metcalf and Nicola, 1991; Heyworth et al., 1988, 1992) or antagonistically (reviewed in Graham and Pragnell,1990; Ruscetti et al.,1991) to give novel responses. In addition, many cytokines trigger the release of other cytokines (Dinarello et al., 1986; Fibbe et al., 1986; Yang et al.,1988). Thus, a complex network is formed among various types of cells through cytokines.