Moloecular cloning of a novel human cDNA encoding a zinc finger protein that binds to the IL-3 promoter

Abstracts I 563

PS October 2: Signal Transduction

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Association 2 Receptor

of JAK Subunits

Tyrosine

Kinases

with

N. Tanaka, H. Asao, K. Ohbo, T. Takeshita, M. Nakamura Sugamura Department of Microbiology, Tohoku University of Medicine, Sendai 980-77, Japan

the

and K. School

The functional imerleukin 2 (lL-2) receptors contain the p and y chains which are necessary for the transduction of cell growth signals. We here demonstrate that the p and y chains associate with JAKl and JAK2-related kinases, respectively. Monoclonal antibodies specific for the p chain and y chain coimmunoprecipitated JAKl and JAKZ-related tyrosine kinases, respectively, from the lysate of MOLTP, a subline of MOLT4 stably uansfected with the p chain. Tyrosine phosphorylation of JAKl and JAKZrelated kinase was induced upon IL-2 stimulation, and IL-2 activated the JAKZrelated kinase. These results demonstrate that the JAKl and JAKZrelated tyrosine kinases are physically associated with the p chain and y chain respectively, and suggest that these kinases may be implicated in IL-2.induced signal transduction.

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DIFFERENTIAL REGULATION OF REL/NFxB ACTIVATION AND FUNCTION IN MACROPHAGES AND ENDOTHELIAL CELLS C. Wu, Y. Ohmori and T. A. Hamilton Cleveland Clinic Foundation, Cleveland, Ohio, 44195 In the present studies, LPS-stimulated activation of KB binding proteins was examined in a murine macrophage-like cell line (RAW 264.7) and a uolvoma-transformed endothelioma cell line (H5Vj. LPS was a’potent’st&ulus of xB binding activity in both cell t&s.‘IJsing EMSA, three complexes were observed in LPS-treated RAW 264.7 cells while only two were observed in the H5V cells. Use of Rel specific antibodies (recogninzing NFrBl, RelA or c-Rel) either in combination with EMSA or in western blots demonstrated that the two complexes formed in both cell types contained predominantly NFKB 1 homodimers aad NFxBl/RelA heterodimers. The complex found only in LPS-stimulated RAW 264.7 cells contained c-Rel. The differential effect of LPS stimulation was also seen in cell lines transfected with RelA or c-Rel responsive rB motifs linked to CAT. Co-transfection of macrophages with either RelA or c-Rel expression vectors effectively promoted rB-dependent transcription. In contrast, co-transfection of H5V endothelial cells with RelA efficiently promoted a-dependent uanscription while c-Rel was only marginally active. Collectively these findings indicate that the LPS-dependent stimulation of c-Rel is differentially regulated in endothelial cells and macrophages. Furthermore, c-Rel functions differentially in the two cell types as well. Supported by USPHS HL29589, CA39621.

MURINE I&a NEGATIVELY REGULATES uB-DEPENDENT TRANSCRIPTION IN LPS-STIMULATED RAW 264.7 MACROPHAGES. J.M. Tebo, C. Wu, Y. Ohmori, and T.A. Hamilton Department of Immunology, Cleveland Clinic Foundation, Cleveland, Ohio, 44195 LPS-induced transcription in macrophages is believed to involve activation of members of the Rel homology family of aanscription factors and may be negatively regulated by cytoplasmic inhibitor proteins collectively termed IKBs. To evaluate the role of IKBS in LPS-stimulated macrophages, murine IrBa (mIrBa) has been expressed as a GST fusion protein and examined for its ability to control KB binding activities in nuclear extracts from LPS-treated RAW 264.7 macrophages. mIwBu-GST inhibits KB binding activity from RAW 264.7 cells in a phosphorylation-dependent manner. Transfection of macrophages with an expression vector encoding mIltBa inhibits LPS-stimulated transcription driven by a 243 base minimal promoter obtained from the 5’ flanking region of the murine IP-IO gene. The minimal promoter contains two rB motifs which have been shown to be critical fo LPS-dependent reporter gene transcription. The uB sites appear to be the specific target of mIvBa function as reporter gene aanscription driven by these motifs in the context of a heterologous TK promoter is also inhibited by cotransfection with mIrBa. These observations indicate that mIwBr plays an important role in conaolling xB-dependent transcription in stimulated murine macmphages. Supported by USPHS CA39621.

Tyrosine phoephorylation and activation of JAK family tyrosine kinases by interleukin-9. T. yin and Y.-C. yang Indiana Univ. Med. c!tr., Indianapolis, IN 46202 Interleukin (IL)-9 is a multifunctional lymphokine produced by activated CLJ4+ T cells. The targets of IL-P include T cells, B cells, mast cells and hematopoietic progenitors. Barever, the signaling pathway6 mediated by IL-9 are still elusive. We report here that IL-9 not only stimulatestyroeinephosphorylationof J&K familykinasee but also enhances JAK in vitro kinase activity in a human megakaryocytic leukemia cell line, HO7B. The kinetic studies indicated that tyrosine phosphorylation and activation of JAR kinases occurred within 1 min. peaked by 5 to 10 min. and persisted at least for 45 min. We have also examined the involvement of Src family tyrosine kinases including Src, Lyn and Fyn in IL-9 signaling. there is no evidence that SrC family kinases aretyrosine phosphorylated and activated by IL-9. Furthermore, we demonstrated that Stat 91 and related transcriptional factors are activated following IL-9 stimulation. These results suggest that JAK familytyroeine kinases and stat 91 family of transcriptional factors are most likely candidates for the signaling molecules or transducers activated by IL-g.

Effect of the Protein Tyrosine Kinase Inhibitor, Geldanamycin and the Cytokine Synthesis Inhibitor SKF 86002 on Inflammatory Mediator Expression in Human Umbilical Vein Endothelial cells.

MOLOECULAR CLONING OF A NOVEL HUMAN cDNA ENCODING A ZINC FINGER PROTEIN THAT BINDS TO THE IL-3 PROMOTER. T. Yokota, N. Koyano-Nakagawa, J. Nishida and K. Arai Inst. Med. Science, Univ. of Tokyo, Tokyo, Japan 108, DNAX Res. Inst., Palo Alto, CA 94304 The CT/GC-rich sequence (at positions -76 to -47) of the human IL-3 gene is one of the upstream elements responding to HTLV-I Tax. We isolated cDNAs encoding DBI, a novel zinc-finger DNA binding protein, as well as previously described EGRI and EGR2, all of which bind to this CT/GC-rich sequence. DBl has six Cys2/His2-type zinc finger motifs and glutamine-streth which is often found in other transcription factors. Electrophoretic mobility shift assay, using specific antibodies, showed that DBl constitutively binds to this region whereas EGRI binds in a T cell activation-dependent manner. Overexpression of EGRl, EGR2, or DBI protein in Jurkat cells increased the transcription activity of the IL-3 promoter in the presence of upstream elements and T cell activation signals. These results suggest that these zinc finger proteins support the constitutive and inducible promoter activities of the IL-3 gene through CT/GC-rich sequence.

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John R. White, Judy M. Lee, Christina M. Sciallo and John C. Lee. Dept. Cellular Biochemisuy. SmithKline Beecham, King of Prussia, PA 19406 USA. Tumor Necrosis Factor a (TNF). Lipopolysachaide (LPS) and Interleukin-I n and f3 (IL-I) iare potent inducers of Interleukin-X (IL-X) and adhesion molecule expnxsion in hum.an umbilical vein endothelial cells (HUVEC). IL-X and adhesion molecules are intimnely involved in neutrophil dispedesis ‘and activation. However, the role of protein kinases and the action of specific cytokme synthesis inhibitors on signal transduction pathways initiated by IL-I. LPS or TNF have not been well defined. The protein tyrosine kinnse tiibiturs geldanamycin and herbimycin A inhibited IL-l. TNFa and LPS induced II-X, E-selectin and ICAM-I production in a reversible non-tuxic nmnner (ICsO = 12.5