Atsushi Iwama

STK/RON receptor tyrosine kinase mediates both apoptotic and growth signals via the multifunctional docking site conserved among the HGF receptor family.

STK/RON tyrosine kinase, a member of the hepatocyte growth factor (HGF) receptor family, is a receptor for macrophage‐stimulating protein (MSP). To examine the STK/RON signalling pathway, we generated STK/ RON transfectants showing opposite features in growth. STK/RON‐expressing Ba/F3 pro‐B cells (BaF/STK) exhibited MSP‐dependent growth, whereas STK/ RON‐expressing mouse erythroleukaemia cells (MEL/ STK) displayed MSP‐induced apoptosis. This apoptosis was accompanied by the prolonged activation of c‐Jun N‐terminal kinase (JNK), which has recently been implicated in the initiation of apoptosis. Co‐immunoprecipitation analyses showed that autophosphorylated STK/RON associated with PLC‐gamma, P13‐kinase, Shc and Grb2 in both transfectants. However, major tyrosine‐phosphorylated proteins, p61 and p65, specifically associated with STK/RON in MEL/STK cells. Mutations at two C‐terminal tyrosine residues, Y1330 and Y1337, in the counterpart of the multifunctional docking site of the HGF receptor abolished both MSP‐induced growth and apoptosis. Analyses of these mutants and in vitro association revealed that signalling proteins including p61 and p65 directly bound to the phosphotyrosines in the multifunctional docking site. These results demonstrate that positive or negative signals toward cell growth are generated through the multifunctional docking site and suggest the involvement of p61 and p65 as well as JNK in apoptosis. Our findings provide the first evidence for apoptosis via a receptor tyrosine kinase.

The receptor tyrosine kinase, Cek8, is transiently expressed on subtypes of motoneurons in the spinal cord during development

Receptor tyrosine kinases (RTKs) play important roles in cellular proliferation, differentiation, and survival. We performed reverse transcriptase-polymerase chain reactions (RT-PCR) from enriched embryonic day 5 (E5) chick motoneurons by panning to identify RTKs involved in the early development of motoneuron. In situ hybridization revealed that Cek8, a member of the eph family, was specifically expressed on motoneurons at the brachial and lumbar segments of the spinal cord which innervate limb muscles, and disappeared after the naturally occurring cell death period (E6-E11). Immunohistochemistry using an anti-Cek8 monoclonal antibody showed the localization of Cek8 protein at the cell bodies and axonal fibers of motoneurons and muscles. The unique expression of Cek8 suggests its involvement in cellular survival or cell-cell interactions for specific subpopulations of developing motoneurons.

Role of macrophage-stimulating protein and its receptor, RON tyrosine kinase, in ciliary motility.

Macrophage-stimulating protein (MSP) is an 80-kD serum protein with homology to hepatocyte growth factor (HGF). Its receptor, RON tyrosine kinase, is a new member of the HGF receptor family. The MSP-RON signaling pathway has been implicated in the functional regulation of mononuclear phagocytes. However, the function of this pathway in other types of cells has not been elucidated. Here we show that in contrast to the HGF receptor, which was expressed at the basolateral surface, RON was localized at the apical surface of ciliated epithelia in the airways and oviduct. In addition, MSP was found in the bronchoalveolar space at biologically significant concentrations. MSP bound to RON on normal human bronchial epithelial cells with a high affinity (Kd = 0.5 nM) and induced autophosphorylation of RON. Activation of RON by MSP led to a significant increase in ciliary beat frequency of human nasal cilia. These findings indicate that the ciliated epithelium of the mucociliary transport apparatus is a novel target of MSP. Ciliary motility is critical for mucociliary transport. Our findings suggest that the MSP-RON signaling pathway is a novel regulatory system of mucociliary function and might be involved in the host defense and fertilization.

Identification and Characterization of a Ligand for Receptor Protein-Tyrosine Kinase HTK Expressed in Hematopoietic Cells

HTK is a receptor tyrosine kinase (RTK) which belongs to the Eph subfamily of RTK. Using a BlAcore, a surface plasmon resonance detection system, it was determined that a colon cancer cell line C-1 expressed HTK ligand (HTKL). From the conditioned medium of C-1 cells, a soluble form of HTKL was purified by receptor affinity chromatography. The N-terminal amino acid sequence of purified HTKL was determined and the full-length cDNA of HTKL was isolated from a C-1 cell cDNA library. HTKL is a type I transmembrane protein which consists of 333 amino acids. HTK receptor tyrosine phosphorylation was induced by membrane-bound or clustered soluble ligands but not by unclustered soluble ligands, indicating that HTKL requires cell-to-cell interaction for receptor phosphorylation. FACS analysis of human bone marrow mononuclear cells showed that HTK receptor was expressed in CD34lowc-KIT+ hematopoietic progenitor cell fraction. These findings suggest the interaction of hematopoietic progenitor cells with HTKL-expressing cells in bone marrow and the involvement of HTKL in the differentiation and/or proliferation of these progenitor cells.