Melanie R. Mark

Protein tyrosine kinases

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Toll-like receptor-2 mediates lipopolysaccharide-induced cellular signalling

Vertebrates and invertebrates initiate a series of defence mechanisms following infection by Gram-negative bacteria by sensing the presence of lipopolysaccharide (LPS), a major component of the cell wall of the invading pathogen. In humans, monocytes and macrophages respond to LPS by inducing the expression of cytokines, cell-adhesion proteins, and enzymes involved in the production of small proinflammatory mediators. Under pathophysiological conditions, LPS exposure can lead to an often fatal syndrome known as septic shock. Sensitive responses of myeloid cells to LPS require a plasma protein called LPS-binding protein and the glycosylphosphatidylinositol-anchored membrane protein CD14. However, the mechanism by which the LPS signal is transduced across the plasma membrane remains unknown. Here we show that Toll-like receptor 2 (TLR2) is a signalling receptor that is activated by LPS in a response that depends on LPS-binding protein and is enhanced by CD14. A region in the intracellular domain of TLR2 with homology to a portion of the interleukin (IL)-1 receptor that is implicated in the activation of the IL-1–receptor-associated kinase is required for this response. Our results indicate that TLR2 is a direct mediator of signalling by LPS.

Reevaluation of the roles of protein S and gas6 as ligands for the receptor tyrosine kinase Rse/Tyro 3

Recently, Stitt et al. (1995) reported that protein S (PS), but not Gas6, is a potent ligand for the receptor tyrosine kinase known as Rse, Tyro 3, Brt, Sky, and Tif (hereafter referred to as Rse/Tyro 3). PS is an abundant serum protein previously characterized as an essential anticoagu-lant. Gas6, which was identified as a gene whose expression is increased by growth arrest, shares 43% amino acid identity and overall domain organization with PS (Manfio-letti et al., 1993). Stitt et al. (1995) based their conclusions on experiments describing interspecies interactions of bovine Gas6 with murine Rse/Tyro 3 (mRse/Tyro 3) and on interactions of bovine and human PS (hPS) with mRse/ Tyro 3. Consistent with the results of Stitt et al. (1995), we identified bovine PS as a ligand for human Rse/Tyro 3 (hRse/Tyro 3), and we also found that hPS can act as a ligand for mRse/Tyro 3. However, when we analyzed the more relevant intraspecies interactions, we obtained different results. We found that human Gas6 (hGas6), but not hPS, acted as a potent ligand for hRse/Tyro 3 (Figure 1). The hRse/Tyro 3 we have studied is very likely the true homolog of mRse/Tyro 3 since they share 90% amino acid identity and a similar expression pattern (Mark et al., 1994; Lai et al., 1994). To characterize the Rse/Tyro 3 ligand, we constructed soluble receptor proteins containing the extraceltular domain of either hRse/Tyro 3 or mRseFryro 3 fused to the Fc portion of human immunoglobulin G1 (hRse-lgG and mRse-lgG). A similar fusion protein (termed Tyro 3-Fc) was utilized by Stitt et al. (1995) to characterize the binding of bovine Gas6 and hPS to mRse/Tyro 3. We first determined whether hRse-lgG or mRse-lgG differed in its ability to bind to hPS or hGas6 containing an epitope tag that allows for side-by-side comparison of the binding properties of the two proteins. Either h Rse-lgG or mRse-lgG was incubated with conditioned medium containing putative ligands. Complexes were captured with protein A (specific for the IgG fusion protein) and visualized with an antibody specific for the epitope-tagged putative ligand. While hGas6 was bound by hRse-lgG, it was not efficiently bound by mRse-lgG (Figure 2). The reciprocal result was obtained in analysis of binding of hPS to hRse-lgG and mRse-lgG; hPS was bound by mRse-lgG but not by hRse-lgG. These results are consistent with an apparent difference in affinity of hPS for human as opposed …

Identification of a new member of the tumor necrosis factor family and its receptor, a human ortholog of mouse GITR

The tumor necrosis factor (TNF) and TNF receptor (TNFR) gene superfamilies regulate diverse biological functions, including cell proliferation, differentiation, and survival [1] [2] [3]. We have identified a new TNF-related ligand, designated human GITR ligand (hGITRL), and its human receptor (hGITR), an ortholog of the recently discovered murine glucocorticoid-induced TNFR-related (mGITR) protein [4]. The hGITRL gene mapped to chromosome 1q23, near the gene for the TNF homolog Fas/CD95 ligand [5]. The hGITR gene mapped to chromosome 1p36, near a cluster of five genes encoding TNFR homologs [1] [6]. We found hGITRL mRNA in several peripheral tissues, and detected hGITRL protein on cultured vascular endothelial cells. The levels of hGITR mRNA in tissues were generally low; in peripheral blood T cells, however, antigen-receptor stimulation led to a substantial induction of hGITR transcripts. Cotransfection of hGITRL and hGITR in embryonic kidney 293 cells activated the anti-apoptotic transcription factor NF-kappaB, via a pathway that appeared to involve TNFR-associated factor 2 (TRAF2) [7] and NF-kappaB-inducing kinase (NIK) [8]. Cotransfection of hGITRL and hGITR in Jurkat T leukemia cells inhibited antigen-receptor-induced cell death. Thus, hGITRL and hGITR may modulate T lymphocyte survival in peripheral tissues.

Structure-Function Analysis of Hepatocyte Growth Factor and its Tyrosine-Kinase Receptor c-Met

Hepatocyte growth factor (HGF) exhibits mitogenic and/or motogenic activities for a variety of cells (see for review Matsumoto and Nakamura, 1992). Structurally, it has similarities to kringle-containing serine-proteases, although it does not possess proteolytic activity. The HGF receptor (HGFr) is the product of the c-met proto-oncogene, a membrane-spanning tyrosine kinase receptor. The 190-kDa precursor is proteolytically processed within the extra-cellular domain (ECD) to a heterodimer consisting of a 50-kDa α subunit disufide-linked to a 145-kDa β subunit. A structure-activity relationship analysis of both HGF and its receptor was performed by functional analysis of ligand and receptor variants.