Scatter Factor (SF) is a fibroblast‐secreted protein which promotes motility and matrix invasion of epithelial cells. Hepatocyte Growth Factor (HGF) is a powerful mitogen for hepatocytes and other epithelial tissues. SF and HGF, purified according to their respective biological activities, were interchangeable and equally effective in assays for cell growth, motility and invasion. Both bound with identical affinities to the same sites in target cells. The receptor for SF and HGF was identified as the product of the MET oncogene by: (i) ligand binding and coprecipitation in immunocomplexes; (ii) chemical crosslinking to the Met beta subunit; (iii) transfer of binding activity in insect cells by a baculovirus carrying the MET cDNA; (iv) ligand‐induced tyrosine phosphorylation of the Met beta subunit. SF and HGF cDNA clones from human fibroblasts, placenta and liver had virtually identical sequences. We conclude that the same molecule (SF/HGF) acts as a growth or motility factor through a single receptor in different target cells.

The Tpl‐2 protein serine/threonine kinase was originally identified, in a C‐terminally deleted form, as the product of an oncogene associated with the progression of Moloney murine leukemia virus‐induced T cell lymphomas in rats. The kinase domain of Tpl‐2 is homologous to the Saccharomyces cerevisiae gene product, STE11, which encodes a MAP kinase kinase kinase. This suggested that Tpl‐2 might have a similar activity. Consistent with this hypothesis, immunoprecipitated Tpl‐2 and Tpl‐2deltaC (a C‐terminally truncated mutant) phosphorylated and activated recombinant fusion proteins of the mammalian MAP kinase kinases, MEK‐1 and SEK‐1, in vitro. Furthermore, transfection of Tpl‐2 into COS‐1 cells or Jurkat T cells. markedly activated the MAP kinases, ERK‐1 and SAP kinase (JNK), which are substrates for MEK‐1 and SEK‐1, respectively. Tpl‐2, therefore, is a MAP kinase kinase kinase which can activate two MAP kinase pathways. After Raf and Mos, Tpl‐2 is the third serine/threonine oncoprotein kinase that has been shown to function as a direct activator of MEK‐1.

The human c-MET oncogene encodes a transmembrane tyrosine kinase (p190c-met) with structural and functional features of a growth-factor receptor. Monoclonal antibodies (MAbs) have been used to investigate the distribution of the c-Met protein in human normal and neoplastic tissues. By immunofluorescence microscopy homogeneous expression was detected in normal hepatocytes as well as in epithelial cells lining the stomach, the small and the large intestine. Positive staining was also found in epithelial cells of the endometrium and ovary, and in basal keratinocytes of esophagus and skin. By Northern blot analysis, high levels of c-met messenger RNA were detected in specimens of liver, gastro-intestinal tract and kidney. c-met-specific mRNA was also found in thyroid, pancreas and placenta, in which organs c-Met protein was barely detectable by immunofluorescence. The antibodies revealed expression of c-MET protein in hepatomas (11/14), carcinomas of colon and rectum (19/21), stomach (11/22), kidney (16/19), ovary (9/17) and skin (7/17). Carcinomas of the lung (13/20), thyroid (11/13) and pancreas (5/7) were also positive. In these last cases (lung, thyroid and pancreas) tumor cells were homogeneously stained by the antibodies, whereas in their normal counterparts staining was barely detectable. These data suggest that the receptor encoded by c-MET plays a physiological role in epithelial cell growth and that its expression is altered in human carcinomas.

Members of a family of highly conserved proteins, termed 14-3-3 proteins, were found by several experimental approaches to associate with Raf-1, a central component of a key signal transduction pathway. Optimal complex formation required the amino-terminal regulatory domain of Raf-1. The association of 14-3-3 proteins and Raf-1 was not substantially affected by the activation state of Raf.

In multiple myeloma (MM), migration is necessary for the homing of tumor cells to bone marrow (BM), for expansion within the BM microenvironment, and for egress into the peripheral blood. In the present study we characterize the role of vascular endothelial growth factor (VEGF) and ␤ 1 integrin (CD29) in MM cell migration. We show that protein kinase C (PKC) ␣ is translocated to the plasma membrane and activated by adhesion of MM cells to fibronectin and VEGF. We identify ␤ 1 integrin modulating VEGF-triggered MM cell migration on fibronectin. We show that transient enhancement of MM cell adhesion to fibronectin triggered by VEGF is dependent on the activity of both PKC and ␤ 1 integrin. Moreover, we demonstrate that PKC␣ is constitutively associated with ␤ 1 integrin. These data are consistent with PKC␣-dependent exocytosis of activated ␤ 1 integrin to the plasma membrane, where its increased surface expression mediates binding to fibronectin; conversely, catalytically active PKC␣-driven internalization of ␤ 1 integrin results in MM cell de-adhesion. We show that the regulatory subunit of phosphatidylinositol (PI) 3-kinase (p85) is constitutively associated with FMS-like tyrosine kinase-1 (Flt-1). VEGF stimulates activation of PI 3-kinase, and both MM cell adhesion and migration are PI 3-kinase-dependent. Moreover, both VEGF-induced PI 3-kinase activation and ␤ 1 integrin-mediated binding to fibronectin are required for the recruitment and activation of PKC␣. Time-lapse phase contrast video microscopy (TLVM) studies confirm the importance of these signaling components in VEGFtriggered MM cell migration on fibronectin.

The anti-angiogenic effect of thrombospondin-1 has been shown to be mediated through binding of the type-1 repeat (TSR) domain to the CD36 transmembrane receptor. We now report that the TSR domain can inhibit VEGF-induced migration in human umbilical vein endothelial cells (HUVEC), cells that lack CD36. Moreover, we identified β1 integrins as a critical receptor in TSR-mediated inhibition of migration in HUVEC. Using pharmacological inhibitors of downstream VEGF receptor effectors, we found that phosphoinositide 3-kinase (PI3k) was essential for TSR-mediated inhibition of HUVEC migration, but that neither PLCγ nor Akt was necessary for this response. Furthermore, β1 integrins were critical for TSR-mediated inhibition of microvascular endothelial cells, cells that express CD36. Together, our results indicate that β1 integrins mediate the anti-migratory effects of TSR through a PI3k-dependent mechanism.

The biological effects of hepatocyte growth factor/scatter factor are mediated by autophosphorylation of its receptor, the Met tyrosine kinase, on two carboxyl-terminal tyrosines. These phosphotyrosines (Y1349VHVNATY1356VNV) are multifunctional docking sites for several effectors. Grb2, the adaptor for the Ras guanyl-nucleotide exchanger SOS, binds to Tyr1356 in the YVNV motif. By site-directed mutagenesis we either abrogated or duplicated the Grb2 consensus, without interfering with the other effectors. Loss of the link with Grb2 severely impaired transformation. The same mutation, however, had no effect on the "scattering" response, indicating that the level of signal which can be reached by Grb2-independent routes is permissive for motility. Duplication of the Grb2 binding site enhanced transformation and left motility unchanged. Thus, two Met-mediated biological responses, motility and growth, can be dissociated on the basis of their differential requirement for a direct link with Ras.

JAK2, a member of the Janus kinase superfamily was found to interact functionally with Raf-1, a central component of the ras/mitogen-activated protein kinase signal transduction pathway. Interferon-y and several other cytokines that are known to activate JAK2 kinase were also found to stimulate Raf-I kinase activity toward MEK-1 in mammalian cells. In the baculovirus coexpression system, Raf-1 was activated by JAK2 in the presence of p2lras. Under these conditions, a ternary complex of p2lras, JAK2, and Raf-1 was observed. In contrast, in the absence of p2lras, coexpression of JAK2 and Raf-1 resulted in an overall decrease in the Raf-1 kinase activity. In addition, JAK2 phosphorylated Raf-1 at sites different from those phosphorylated by pp6Ov-src. In mammalian cells treated with either erythropoietin or interferon-'y, a small fraction of Raf-1 coimmunoprecipitated with JAK2 in lysates of cells in which JAK2 was activated as judged by its state of tyrosine phosphorylation. Taken together, these data suggest that JAK2 and p21ras cooperate to activate Raf-1.The serine/threonine kinase Raf-1 is activated by numerous growth factors (1, 2) and is believed to play a central role in cell growth and differentiation. Binding of a growth factor to its tyrosine kinase receptor at the cell surface leads to p21ras activation that in turn recruits Raf-1 to the plasma membrane (3, 4) through a direct interaction with the N-terminal region of Raf-1. At the cell membrane, Raf-1 is activated in a p21ras-independent manner. Activated Raf-1 phosphorylates and activates MEK-1, leading to activation of the mitogenactivated protein (MAP) kinase cascade (5, 6). Unlike members of the tyrosine kinase receptor superfamily, the cytokine receptors have no intrinsic tyrosine kinase activity, but are dependent mainly upon the JAK family of tyrosine kinases with which they form a stable complex (7-10). Binding of a cytokine to its receptor causes the receptor to dimerize, thereby activating the associated JAK kinases through tyrosine phosphorylation. Activated JAK kinases in turn phosphorylate a number of important cellular substrates, chief among which are members of the STAT family of transcription factors (11)(12)(13)(14). Previous studies have documented that tyrosine phosphorylation of STATs leads to their oligomerization and is essential for activation of their DNA binding activity (15, 16).However, recently it has become apparent that subsequent phosphorylation of STATs on serine/threonine residues is required for their full activation as transcription factors (17,18). It has also been reported that JAK2, p2lras, and Raf-1 are required for the activation of MAP kinases by growth hormone stimulation (19), and in the interferon (IFN) system, STAT activation requires functionally activated MAP kinases (20). This focuses particular interest on the molecular mechanisms by which cytokine receptor family members couple to Raf-1 and to the MAP kinase cascade. Before the discovery of either MEK-1 as a physiological substrate of Raf-1 or ...