Localization and signaling of the receptor protein tyrosine kinase Tyro3 in cortical and hippocampal neurons.

Protein phosphorylation serves as a critical biochemical regulator of short-term and long-term synaptic plasticity. Receptor protein tyrosine kinases (RPTKs) including members of the trk, eph and erbB subfamilies have been shown to modulate signaling cascades that influence synaptic function in the central nervous system (CNS). Tyro3 is one of three RPTKs belonging to the "TAM" receptor family, which also includes Axl and Mer. Tyro3 is the most widely expressed of these receptors in the CNS. Despite recent advances suggesting roles for members of this receptor family in the reproductive and immune systems, their functions in the CNS remain largely unexplored. In an effort to elucidate the roles of Tyro3 and its ligand, the protein growth arrest-specific gene6 (Gas6) in the hippocampus and cortex, we performed a detailed study of the localization and signaling of Tyro3 polypeptides in rat hippocampal and cortical neurons. Tyro3 was readily detected in dendrites and in the soma where it was distributed in a punctate pattern. Tyro3 exhibited only a limited level of co-localization with postsynaptic density protein-95 (PSD-95), suggesting that while located within dendrites, it was not confined to the postsynaptic compartment. In addition, Tyro3 was also identified in the axons and growth cones of immature neurons. The prominent expression of Tyro3 in dendrites suggested that it may be capable of modulating signaling pathways triggered by synaptic transmission. We have provided evidence in support of this role by demonstrating that Gas6 induced the phosphorylation of Tyro3 in cortical neurons in vitro, resulting in the recruitment of the mitogen-activated protein kinase (MAPK) and the phosphoinositide-3 kinase (PI(3)K) signaling pathways. As these pathways play critical roles in the induction of hippocampal long-term potentiation (LTP), these findings suggest that Tyro3 signaling may influence synaptic plasticity in the dendritic compartment of hippocampal and cortical neurons.

Gas6 anti-apoptotic signaling requires NF-kappa B activation.

The growth arrest-specific 6 gene product Gas6 is a growth and survival factor related to protein S. Gas6 is the ligand of Axl receptor tyrosine kinase; upon binding to its receptor Gas6 activates the phosphatidylinositol 3-OH kinase (PI3K) and its downstream targets S6K and Akt. Gas6 anti-apoptotic signaling was previously shown to require functional PI3K and Akt and to involve Bad phosphorylation in serum-starved NIH 3T3 cells. Here we demonstrate that Gas6 induces a rapid and transient increase in nuclear NF-kappa B binding activity coupled to transcription activation from NF-kappa B-responsive promoters and increase in Bcl-x(L) protein level. Gas6 survival function is impaired in cells lacking p65/RelA and in NIH 3T3 cells transfected with a dominant negative I kappa B, indicating that NF-kappa B activation plays a central role in promoting survival in this system. Moreover, NF-kappa B activation can be blocked by a dominant negative Akt and by wortmannin, an inhibitor of PI3K, thus suggesting that NF-kappa B activation is a downstream event with respect to PI3K and Akt, as already described for other growth factors. In addition, we show that glycogen synthase kinase 3, which is phosphorylated in response to Gas6, can physically associate with NFKB1/p105 in living cells and can phosphorylate it in vitro. Furthermore, Gas6 treatment is coupled to a decrease in p105 protein level. Altogether these data suggest the involvement of NF-kappa B and glycogen synthase kinase 3 in Gas6 anti-apoptotic signaling and unveil a possible link between these survival pathways.

Gas 6 promotes Axl-mediated survival in pulmonary endothelial cells.

We examined Gas 6-Axl interactions in human pulmonary artery endothelial cells (HPAEC) and in Axl-transduced HPAEC to test Gas 6 function during endothelial cell survival. We identified the 5.0-kb Axl, 4.2-kb Rse, and 2.6-kb Gas 6 mRNAs in HPAEC. Immunoprecipitation and Western blotting confirmed the presence of these proteins. Gas 6 is present in cell-associated and secreted fractions of growth-arrested HPAEC, independent of cell density. In addition, the Axl receptor is constitutively phosphorylated in growth-arrested cultures, and exogenous Gas 6 enhanced Axl phosphorylation threefold. Gas 6 added to growth-arrested HPAEC resulted in a significant increase in cell number (1.5 nM Gas 6 increased cell number 35%). Flow cytometry revealed that Gas 6 treatment resulted in 28% fewer apoptosing cells. Transduction of a full-length Axl cDNA into HPAEC resulted in 54% fewer apoptosing cells after Gas 6 treatment. Collectively, the data demonstrate antiapoptotic activities for Gas 6 in HPAEC and suggest that Gas 6 signaling may be relevant to endothelial cell survival in the quiescent environment of the vessel wall.

Gas6 regulates mesangial cell proliferation through Axl in experimental glomerulonephritis.

Proliferation of mesangial cells is a hallmark of glomerular disease, and understanding its regulatory mechanism is clinically important. Previously, we demonstrated that the product of growth arrest-specific gene 6 (Gas6) stimulates mesangial cell proliferation through binding to its cell-surface receptor Axl in vitro. We also showed that warfarin and the extracellular domain of Axl conjugated with Fc portion of human IgG1 (Axl-Fc) inhibit mesangial cell proliferation by interfering the Gas6/Axl pathway in vitro. In the present study, therefore, we examined in vivo roles of Gas6 and Axl in an experimental model of mesangial proliferative glomerulonephritis induced by the injection of anti-Thy1.1 antibody (Thy1 GN). In Thy1 GN, expression of Gas6 and Axl was markedly increased in glomeruli, and paralleled the progression of mesangial cell proliferation. Administration of warfarin or daily injection of Axl-Fc inhibited mesangial cell proliferation, and abolished the induction of platelet-derived growth factor-B mRNA and protein in Thy1 GN. Moreover, the anti-proliferative effect of warfarin was achieved at lower concentrations than those in routine clinical use. These findings indicate that the Gas6/Axl pathway plays a key role in mesangial cell proliferation in vivo, and could be a potentially important therapeutic target for the treatment of renal disease.

Gas6 induces growth, beta-catenin stabilization, and T-cell factor transcriptional activation in contact-inhibited C57 mammary cells.

Gas6 is a growth factor related to protein S that was identified as the ligand for the Axl receptor tyrosine kinase (RTK) family. In this study, we show that Gas6 induces a growth response in a cultured mammalian mammary cell line, C57MG. The presence of Gas6 in the medium induces growth after confluence and similarly causes cell cycle reentry of density-inhibited C57MG cells. We show that Axl RTK but not Rse is efficiently activated by Gas6 in density-inhibited C57MG cells. We have analyzed the signaling required for the Gas6 proliferative effect and found a requirement for PI3K-, S6K-, and Ras-activated pathways. We also demonstrate that Gas6 activates Akt and concomitantly inhibits GSK3 activity in a wortmannin-dependent manner. Interestingly, Gas6 induces up-regulation of cytosolic beta-catenin, while membrane-associated beta-catenin remains unaffected. Stabilization of beta-catenin in C57MG cells is correlated with activation of a T-cell factor (TCF)-responsive transcriptional element. We thus provide evidence that Gas6 is mitogenic and induces beta-catenin proto-oncogene stabilization and subsequent TCF/Lef transcriptional activation in a mammary system. These results suggest that Gas6-Axl interaction, through stabilization of beta-catenin, may have a role in mammary development and/or be involved in the progression of mammary tumors.

Noninsulin-dependent diabetes mellitus occurs in mice ectopically expressing the human Axl tyrosine kinase receptor.

The axl tyrosine kinase receptor is aberrantly expressed on myeloid cells of many individuals afflicted with chronic myelogenous leukemia (CML) and other myeloid leukemias. Although previous studies demonstrated this kinase to have oncogenic potential, it is not known whether axl actively participates in the onset and/or progression of CML. We addressed this question by generating transgenic mice possessing constitutive ectopic expression of human axl throughout cells of the myeloid hematopoietic lineage through the use of the granulocyte colony-stimulating factor (GCSF) receptor promoter. The transgenics did not exhibit hematopoietic malignancies, but did exhibit phenotypic characteristics associated with noninsulin-dependent diabetes mellitus (NIDDM) including hyperglycemia and hyperinsulinemia, severe insulin resistance, progressive obesity, hepatic lipidosis, and pancreatic islet dysplasia. The obese-diabetes phenotype was similar to that observed in the agouti and melanocortin-4(-/-) mutants, however the axl transgenics were not hyperphagic. Axl transgenic animals expressed elevated serum tumor necrosis factor (TNF)-alpha levels that were further enhanced upon in vitro lipopolysaccharide (LPS) stimulation of peripheral blood. Administration of the axl ligand, gas6, to peripheral transgenic blood samples eliminated excessive TNF-alpha production in response to LPS stimulation. As a means to better understand axl-gas6 biology, transgenic animals were produced which systemically expressed the gas6-binding axl proteolytic cleavage product. A more severe NIDDM phenotype occurred in these mice. The observed phenotypes may be related to the axl receptor or proteolytic cleavage product competing with related axl family receptors for binding of the gas6 ligand. We conclude that axl expression in myeloid cells in itself does not lead to the onset or progression of leukemia and suggest that ectopic axl expression affects endogenous modulation of TNF-alpha production indirectly resulting in the NIDDM phenotype.

Novel neurotrophin-1/B cell-stimulating factor-3: a cytokine of the IL-6 family.

We have identified a cytokine of the IL-6 family and named it novel neurotrophin-1/B cell-stimulating factor-3 (NNT-1/BSF-3). NNT-1/BSF-3 cDNA was cloned from activated Jurkat human T cell lymphoma cells. Its sequence predicts a 225-aa protein with a 27-aa signal peptide, a molecular mass of 22 kDa in mature form, and the highest homology to cardiotrophin-1 and ciliary neurotrophic factor. The gene for NNT-1/BSF-3 is on chromosome 11q13. A murine equivalent to NNT-1/BSF-3 also was identified, which shows 96% homology to human NNT-1/BSF-3. NNT-1/BSF-3 mRNA is found mainly in lymph nodes and spleen. NNT-1/BSF-3 induces tyrosine phosphorylation of glycoprotein 130 (gp130), leukemia inhibitory factor receptor beta, and signal transducer and activator of transcription 3 in the SK-N-MC human neuroblastoma cells. NNT-1/BSF-3 shows activities typical of IL-6 family members. In vitro, it supports the survival of chicken embryo motor and sympathetic neurons. In mice, it induces serum amyloid A, potentiates the induction by IL-1 of corticosterone and IL-6, and causes body weight loss and B cell hyperplasia with serum IgG and IgM increase. NNT-1/BSF-3 is a gp130 activator with B-cell stimulating capability.

Gas6-mediated survival in NIH3T3 cells activates stress signalling cascade and is independent of Ras.

Gas6 is a growth factor membrane of the vitamin K-dependent family of proteins which is preferentially expressed in quiescent cells. Gas6 was identified as the ligand for Axl tyrosine kinase receptor family. Consistent with this, Gas6 was previously reported to induce cell cycle re-entry of serum-starved NIH3T3 cells and to prevent cell death after complete growth factor withdrawal, the survival effect being uncoupled from Gas6-induced mitogenesis. We have previously demonstrated that both Gas6 mitogenic and survival effects are mediated by Src and the phosphatidylinositol3-OH kinase (PI3K). Here we report that Ras is required for Gas6 mitogenesis but is dispensable for its survival effect. Gas6-induced survival requires the activity of the small GTPases of the Rho family, Rac and Rho, together with the downstream kinase Pak. Overexpression of the respective dominant negative constructs abrogates Gas6-mediated survival functions. Addition of Gas6 to serum starved cells results in the activation of AKT/PKB and in the phosphorylation of the Bcl-2 family member, Bad. By ectopic expression of a catalytically inactive form of AKT/PKB, we demonstrate that AKT/PKB is necessary for Gas6-mediated survival functions. We further show evidence that Gas6 stimulation of serum starved NIH3T3 cells results in a transient ERK, JNK/SAPK and p38 MAPK activation. Blocking ERK activation did not influence Gas6-induced survival, suggesting that such pathway is not involved in Gas6 protection from cell death. On the contrary we found that the late constitutive increase of p38 MAPK activity associated with cell death was downregulated in Gas6-treated NIH3T3 cells thus suggesting that Gas6 might promote survival by interfering with this pathway. Taken together the evidence here provided identity elements involved in Gas6 signalling more specifically elucidating the pathway responsible for Gas6-induced cell survival under conditions that do not allow cell proliferation.

Growth arrest-specific gene 6 (Gas6)/adhesion related kinase (Ark) signaling promotes gonadotropin-releasing hormone neuronal survival via extracellular signal-regulated kinase (ERK) and Akt.

We identified Ark, the mouse homolog of the receptor tyrosine kinase Axl (Ufo, Tyro7), in a screen for novel factors involved in GnRH neuronal migration by using differential-display PCR on cell lines derived at two windows during GnRH neuronal development. Ark is expressed in Gn10 GnRH cells, developed from a tumor in the olfactory area when GnRH neurons are migrating, but not in GT1-7 cells, derived from a tumor in the forebrain when GnRH neurons are postmigratory. Since Ark (Ax1) signaling protects from programmed cell death in fibroblasts, we hypothesized that it may play an antiapoptotic role in GnRH neurons. Gn10 (Ark positive) GnRH cells were more resistant to serum withdrawal-induced apoptosis than GT1-7 (Ark negative) cells, and this effect was augmented with the addition of Gas6, the Ark (Ax1) ligand. Gas6/Ark stimulated the extracellular signal-regulated kinase, ERK, and the serine-threonine kinase, Akt, a downstream component of the phosphoinositide 3-kinase (PI3-K) pathway. To determine whether ERK or Akt activation is required for the antiapoptotic effects of Gas6/Ark in GnRH neurons, cells were serum starved in the absence or presence of Gas6, with or without inhibitors of ERK and PI3-K signaling cascades. Gas6 rescued Gn10 cells from apoptosis, and this effect was blocked by coincubation of the cells with the mitogen-activated protein/ERK kinase (MEK) inhibitor, PD98059, or wortmannin (but not rapamycin). These data support an important role for Gas6/Ark signaling via the ERK and PI3-K (via Akt) pathways in the protection of GnRH neurons from programmed cell death across neuronal migration.

GAS6 inhibits granulocyte adhesion to endothelial cells.

GAS6 is a ligand for the tyrosine kinase receptors Rse, Axl, and Mer, but its function is poorly understood. Previous studies reported that both GAS6 and Axl are expressed by vascular endothelial cells (EC), which play a key role in leukocyte extravasation into tissues during inflammation through adhesive interactions with these cells. The aim of this work was to evaluate the GAS6 effect on the adhesive function of EC. Treatment of EC with GAS6 significantly inhibited adhesion of polymorphonuclear cells (PMN) induced by phorbol 12-myristate 13-acetate (PMA), platelet-activating factor (PAF), thrombin, interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha), but not that induced by FMLP and IL-8. GAS6 did not affect adhesion to resting EC. Titration experiments showed that high concentrations of GAS6 were needed to inhibit PMN adhesion and that inhibition was dose-dependent at the concentration range of 0.1 to 1 microg/mL. One possibility was that high concentrations were needed to overwhelm the effect of endogenous GAS6 produced by EC. In line with this possibility, treatment of resting EC with soluble Axl significantly potentiated PMN adhesion. Analysis of localization of GAS6 by confocal microscopy and cytofluorimetric analysis showed that it is concentrated along the plasma membrane in resting EC and treatment with PAF induces depletion and/or redistribution of the molecule. These data suggest that GAS6 functions as a physiologic antiinflammatory agent produced by resting EC and depleted when proinflammatory stimuli turn on the proadhesive machinery of EC.

Identification and tissue expression of a splice variant for the growth arrest-specific gene gas6.

The growth arrest-specific gene gas6 encodes a secreted protein (Gas6) which is a member of the vitamin K-dependent protein family and was identified as a ligand for the Ax1 tyrosine kinase receptor family. Gas6 shares significant similarity with protein S and a similar domain organisation: an extensively gamma-carboxylated amino-terminal, four epidermal growth factor-like motifs and a large carboxy-terminal region, known as the D domain. Here we report on the isolation of a splice variant (gas6SV) characterised by an in-frame 129 bp insertion between the fourth EGF domain and the D domain. The gene gas6 was previously mapped on chromosome 13. The genomic organisation of gas6 has been investigated demonstrating the presence of alternative splicing consensus sites. Expression of gas6SV has been investigated in various human tissues and found to have a similar distribution pattern as gas6, with the exception of the spleen where gas6SV seems to be the predominant form.

The product of a gas6 splice variant allows the release of the domain responsible for Axl tyrosine kinase receptor activation.

The product of gas6 (Gas6) is a growth factor with high level of similarity to protein S and was identified as the ligand for Axl family of tyrosine kinase receptors. Gas6 contains an N-terminal gamma-carboxylated domain (Gla), four epidermal growth factor like domains and a large C-terminal D region. An alternative Gas6 spliced form (Gas6SV) having an additional 43 amino acids between fourth EFG like and D domain was characterised. Here we show data indicating that Gas6SV is specifically cleaved within the inserted sequence, thereby splitting the D domain from the remaining part of the protein. The resulting two proteolytic products of 36 kDa and 50 kDa were separated and the 50 kDa fragment corresponding to region D was shown to be responsible for Axl receptor activation. Furthermore a deletion mutant of Gas6 containing only the D domain was shown to similarly activate Axl receptor phosphorylation unequivocally demonstrating that D domain can act as a signalling molecule. The possible roles of the proteolytic processing of Gas6SV in the regulation of growth factor availability are discussed.

GAS6, the ligand of Axl and Rse receptors, is expressed in hematopoietic tissue but lacks mitogenic activity.

GAS6, a gene previously identified as growth arrest specific, has been demonstrated to be the ligand of Axl, a novel tyrosine kinase receptor widely expressed in both normal and neoplastic hematopoietic tissue. We have observed previously that GAS6 mRNA was present in whole bone marrow. This preliminary finding prompted us to investigate the presence of GAS6 in hematopoietic tissue and the possible role of this molecule in controlling the proliferation of hematopoietic precursors. We report here that the protein GAS6 is diffusely present in hematopoietic tissue, both in stromal and in hematopoietic cells, and that, among these cells, positivity is observed in megakaryocytes and myelomonocytic precursors. Furthermore, our data suggest that GAS6 is not a growth factor for hematopoietic progenitors or stromal fibroblasts. Despite the fact that both the Axl receptor and its ligand, GAS6, are expressed in hematopoietic tissue, the biological role of their interactions remains to be determined.

GAS6 mediates adhesion of cells expressing the receptor tyrosine kinase Axl.

Axl is a receptor tyrosine kinase that contains both immunoglobulin and fibronectin III repeats in its extracellular domain reminiscent of cell adhesion molecules. Expression of the receptor tyrosine kinase Axl in the 32D myeloid cell line permits aggregation of cells in response to treatment with the native ligand GAS6; this aggregation was not observed in untreated 32D-Axl cells nor in treated parental cells. This aggregation can be blocked by the addition of excess Axl extracellular domain peptide and does not require intracellular Axl kinase activity. Cell surface binding activity of GAS6 was mapped to distinct plasma membrane interacting domains that are separate from the GAS6 motifs that engage the Axl receptor. This suggests that aggregation is mediated by a heterotypic intercellular mechanism whereby cell-bound GAS6 interacts with Axl receptor on an adjacent cell. This mechanism is supported by our observation that GAS6 binds to 32D parental cells which then permits their aggregation with untreated 32D-Axl cells. We have recently demonstrated that the GAS6-Axl interaction does not initiate mitogenesis in 32D cells. When considered with the adhesion results, these data suggest that an important biological function of the Axl-GAS6 interaction is to mediate cell-cell binding.

Requirement of phosphatidylinositol 3-kinase-dependent pathway and Src for Gas6-Axl mitogenic and survival activities in NIH 3T3 fibroblasts.

Gas6 is a secreted protein previously identified as the ligand of the Axl receptor tyrosine kinase. We have shown that Gas6 is able to induce cell cycle reentry of serum-starved NIH 3T3 cells and to efficiently prevent apoptosis after complete growth factor removal, a survival effect uncoupled from Gas6-induced mitogenesis. Here we report that the mitogenic effect of Gas6 requires phosphatidylinositol 3-kinase (PI3K) activity since it is abrogated both by the specific inhibitor wortmannin and by overexpression of the dominant negative P13K p85 subunit. Consistently, Gas6 activates the P13K downstream targets S6K and Akt, whose activation is abrogated by addition of wortmannin. Moreover, rapamycin treatment blocks Gas6-induced entry into the S phase of serum-starved NIH 3T3 cells. We also demonstrate the requirement of Src tyrosine kinase for Gas6 signalling since stable or transient expression of a catalytically inactive form of Src significantly inhibited Gas6-stimulated entry into the S phase. Accordingly, Gas6 addition to serum-starved NIH 3T3 cells causes activation of the intrinsic Src kinase activity. When specifically analyzed in a survival assay, these elements were found to be required for the survival effect of Gas6. Taken together, the evidence presented here identifies elements involved in the Gas6 transduction pathway that are responsible for its antiapoptotic effect and suggests that Src is involved in the events regulating cell survival.

Human chondrocyte expression of growth-arrest-specific gene 6 and the tyrosine kinase receptor axl: potential role in autocrine signaling in cartilage.

OBJECTIVE: To determine if human articular chondrocytes express the axl tyrosine kinase receptor and its ligand Gas-6, a protein product of growth-arrest-specific gene 6, and to determine if Gas-6 and axl function in the regulation of chondrocyte growth and survival. METHODS: The presence of Gas-6 and axl was examined in situ in human articular cartilage by immunohistochemistry and in vitro in cell culture studies using primary human chondrocytes and immortalized human chondrocytes. The ability of recombinant Gas-6 to mediate adhesion of chondrocytes and to stimulate chondrocyte axl phosphorylation was determined. Studies of the role of Gas-6 and axl in cell proliferation and survival were also performed. RESULTS: Both Gas-6 and axl were detected in cartilage by immunohistochemical staining. Gas-6 and axl messenger RNA (mRNA) and protein were also detected in cultures of primary and immortalized human chondrocytes. Compared with cells cultured in medium containing 10% serum, Gas-6 mRNA levels were increased in immortalized chondrocytes cultured in serum-free medium, while axl expression decreased. Chondrocytes attached to Gas-6-coated plastic, and the attachment was blocked by a soluble Ig fusion protein containing the axl extracellular domain. Recombinant human Gas-6 and serum-free conditioned medium from primary and immortalized human chondrocyte cultures stimulated chondrocyte axl tyrosine phosphorylation. A mitogenic effect was noted both when immortalized chondrocytes were stimulated with recombinant Gas-6 or when they were made to overexpress axl by transfection. Addition of recombinant Gas-6 to serum-free medium resulted in increased survival of primary chondrocytes cultured at low density in agarose. CONCLUSION: These findings present evidence for an autocrine signaling pathway in cartilage involving Gas-6 and the axl tyrosine kinase adhesion receptor. Stimulation of axl by Gas-6 may play an important role in the control of chondrocyte growth and survival.

Differential activation of the Ras/extracellular-signal-regulated protein kinase pathway is responsible for the biological consequences induced by the Axl receptor tyrosine kinase.

To understand the mechanism of Axl signaling, we have initiated studies to delineate downstream components in interleukin-3-dependent 32D cells by using a chimeric receptor containing the recombinant epidermal growth factor (EGF) receptor extracellular and transmembrane domains and the Axl kinase domain (EAK [for EGF receptor-Axl kinase]). We have previously shown that upon exogenous EGF stimulation, 32D-EAK cells are capable of proliferation in the absence of interleukin-3. With this system, we determined that EAK-induced cell survival and mitogenesis are dependent upon the Ras/extracellular-signal-regulated protein kinase (ERK) cascade. Although the phosphatidylinositol-3 kinase pathway is activated upon EAK signaling, it appears to be dispensable for the biological actions of the Axl kinase. Furthermore, we demonstrated that different threshold levels of Ras/ERK activation are needed to induce a block to apoptosis or proliferation in 32D cells. Recently, we have identified an Axl ligand, GAS6. Surprisingly, GAS6-stimulated 32D-Axl cells exhibited no blockage to apoptosis or mitogenic response which is correlated with the absence of Ras/ERK activation. Taken together, these data suggest that different extracellular domains dramatically alter the intracellular response of the Axl kinase. Furthermore, our data suggest that the GAS6-Axl interaction does not induce mitogenesis and that its exact role remains to be determined.

Megakaryocyte growth and development factor (MGDF) moderately enhances in-vitro platelet aggregation.

Megakaryocyte growth and development factor (MGDF) is a novel cytokine which promotes the development of immature megakaryocytes into platelets. We tested the hypothesis that MGDF would alter the sensitivity of platelets to aggregating agents as assessed by in-vitro platelet aggregometry. Platelet aggregation in the presence or absence of MGDF was tested with single doses of clinically relevant aggregating agents. A dose-dependent enhancement of the aggregation response to epinephrine was noted in MGDF treated platelets. When a range of concentrations of ADP were used to generate an aggregation dose-response curve, the addition of MGDF to platelet rich plasma shifted the dose response curve to the left. The effect of MGDF on platelet aggregation was partially prevented by the coincubation of platelets with a soluble form of the receptor for MGDF, the extracellular domain of c-mpl. In addition, we demonstrate that exogenous MGDF is able to induce tyrosine phosphorylation of platelet proteins with apparent molecular weights of 85 kDa and 130 kDa. From these data we conclude that exogenously added MGDF moderately increases the sensitivity of platelets to aggregating agents through a mechanism which appears to involve tyrosine phosphorylation of platelet proteins.

Receptor tyrosine kinases expressed in metastatic colon cancer.

Using a PCR-based cloning technique, we have isolated a series of DNA fragments coding for tyrosine kinases that are expressed in a metastatic human colon tumor, and have subsequently analyzed their expression pattern at the protein level in human tumors. We identified both the alpha and the beta forms of the platelet-derived growth factor receptor (PDGFR), axl and 8 other genes, including 3 cytoplasmic tyrosine kinases. To study their expression in human colon cancer, we performed Western blots of matched sets of normal tissues and of carcinomas from the same patient. These revealed that the alpha-PDGFR migrates predominantly as a 200-kDa band in 8/8 normal tissues, and as a 170-kDa band in 17/17 malignant tissues, as well as in colonic polyps, suggesting that expression of an isoform of this receptor may be a marker for the progression of colon cancer. Additional studies showed that the Axl receptor tyrosine kinase was expressed at 10-fold higher levels in a peritoneal metastatic nodule than in other normal and malignant tissues. Immunohistochemistry revealed Axl over-expression specifically in the malignant cells of the tumor. This indicates that over-expression and possibly a differential processing event of tyrosine kinase receptors may be involved in colon cancer, and that they are potential markers for the progression of this disease.

Axl receptor tyrosine kinase stimulated by the vitamin K-dependent protein encoded by growth-arrest-specific gene 6.

The Axl receptor tyrosine kinase was identified as a protein encoded by a transforming gene from primary human myeloid leukaemia cells by DNA-mediated transformation of NIH 3T3 cells. Axl is the founding member of a family of related receptors that includes Eyk, encoded by a chicken proto-oncogene originally described as a retroviral transforming gene, and c-Mer, encoded by a human proto-oncogene expressed in neoplastic B- and T-cell lines. The transforming activity of Axl demonstrates that the receptor can drive cellular proliferation. The function of Axl in non-transformed cells and tissues is unknown, but may involve the stimulation of cell proliferation in response to an appropriate signal, namely a ligand that activates the receptor. We report here the purification of an Axl stimulatory factor, and its identification as the product of growth-arrest-specific gene 6 (ref. 6). This is, to our knowledge, the first description of a ligand for the Axl family of receptors.