Association of hematopoietic cell phosphatase with c-Kit after stimulation with c-Kit ligand.

Protein tyrosine phosphorylation and dephosphorylation have been implicated in the growth and functional responses of hematopoietic cells. Recent studies have identified a novel protein tyrosine phosphatase, termed hematopoietic cell phosphatase (HCP) or PTP1C, that is predominantly expressed in hematopoietic cells. HCP encodes a cytoplasmic phosphatase that contains two src homology 2 (SH2) domains. Since SH2 domains have been shown to target the association of signal-transducing molecules with activated growth factor receptors containing intrinsic protein kinase activity, we assessed the association of HCP with two hematopoietic growth factor receptors, c-Kit and c-Fms. The results demonstrate that HCP transiently associates with ligand-activated c-Kit but not c-Fms and that this association occurs through the SH2 domains. In both colony-stimulating factor 1- and stem cell factor-stimulated cells, there is a marginal increase in tyrosine phosphorylation of HCP. Lastly, HCP can dephosphorylate autophosphorylated c-Kit and c-Fms in in vitro reactions. The potential role of HCP in stem cell factor signal transduction is discussed.

Complementary DNA cloning and characterization of truncated form of c-kit in human colon carcinoma cells.

We have obtained a novel c-kit complementary DNA (cDNA) from a colon carcinoma cell line, Colo201, and characterized its structure. The size of the transcript in Colo201 was approximately 3.5 kilobases and it hybridized to the c-kit cDNA fragments encompassing the kinase domain, but not to the cDNA fragments encoding extracellular and transmembrane domains. The predicted protein encoded by those cDNAs was composed of 257 amino acids containing the NH2-terminal 25 unique amino acids in frame by the COOH terminal of the KIT protein. Of interest, these 25 amino acids were encoded by intron 15 of the c-kit gene. The aberrant mRNA was also detected in another colon carcinoma cell line, BM314. The translation of this message in Colo201 was confirmed by flow cytometry and immunoblot analysis. This is the first report describing the aberrant transcript of c-kit in human tumor cells, and it is suggested that truncated form of c-kit might play a role in the onset and development of human colon carcinoma.

Characterization of a fusion protein composed of the extracellular domain of c-kit and the Fc region of human IgG expressed in a baculovirus system.

The extracellular domain of c-kit, which is the receptor for stem cell factor (SCF), was fused genetically to the Fc portion of human immunoglobulin G1. This chimeric protein, c-kitFc, was then expressed in the baculovirus system. The fusion product was secreted into the serum-free culture medium as a soluble dimeric form of approximately 210 Kda. The recombinant protein was easily purified by protein A affinity chromatography at approximately 25 micrograms protein per ml of medium. Binding assay and cross-linking assay showed that the fusion protein retained high affinity for binding SCF (Kd = 0.3 nM). Addition of the chimeric protein into the culture medium of SCF-dependent cells inhibited cell proliferation in a dose-dependent manner. These results suggest that the dimeric c-kitFc protein can be used as an antagonist of SCF for the study of hematopoietic progenitor cells.

Identification and characterization of a soluble c-kit receptor produced by human hematopoietic cell lines.

Stem cell factor (SCF) triggers cell growth by binding to cell surface c-kit receptors. Soluble forms of several cytokine receptors have been described and may play a role in the modulation of cytokine activity in vivo. For these reasons, we investigated whether human hematopoietic cells produce soluble c-kit receptors. The human leukemia cell lines OCIM1 and MO7e display approximately 80,000 and approximately 35,000 high-affinity cell surface c-kit receptors, respectively. Soluble c-kit receptors were detected by enzyme immunoassay in OCIM1 and MO7e culture supernatants. We determined the molecular weight and binding affinity of soluble c-kit receptor produced by OCIM1 cells, soluble c-kit receptor purified from human serum, and recombinant soluble c-kit receptor expressed in CHO cells. The three soluble c-kit receptors each have a molecular weight of 98 kD. Quantitative binding experiments with 125I-SCF indicate that the soluble c-kit receptors obtained from human serum or OCIM1 cells have binding affinities for SCF of approximately 200 to 300 pmol/L, in contrast to the recombinant form, which has a binding affinity of approximately 1.5 nmol/L. All three forms of the soluble c-kit receptor were able to compete with c-kit receptors on OCIM1 cells for 125I-SCF binding. Thus human hematopoietic cells can produce a soluble form of the c-kit receptor that retains high-affinity SCF binding activity. We speculate that the soluble c-kit receptor may bind SCF and function as a receptor antagonist in vivo.

Human kit ligand (stem cell factor) modulates platelet activation in vitro.

The human stem cell factor (SCF), also termed c-Kit ligand (KL), is a hematopoietic growth factor produced by mesenchymal cells that induces proliferation of bone marrow progenitor cells, megakaryocytes, and mast cells via interaction with c-Kit, its cognate receptor. Expression of the c-kit gene was identified in human platelets by the polymerase chain reaction technique. The presence of the c-Kit receptor was demonstrated by the specific binding of 125I-KL/SCF to ADP-stimulated platelets. The identity of the c-Kit protein was confirmed by immunoreactivity with an anti-c-Kit-specific antibody and by its characterization as a phosphotyrosine-containing protein. Under constitutive conditions, c-Kit was found to be tyrosine-phosphorylated and was associated with a 85-kDa phosphoprotein that could be a fragment of phosphatidylinositol 3-kinase. These data indicate the presence of a new platelet surface molecule that could function in platelet activation. We demonstrate that the secondary wave of platelet aggregation and serotonin secretion induced by epinephrine and ADP, but not by the thromboxane analog U46619, was augmented by KL/SCF. The effect of KL/SCF on epinephrine/ADP-induced platelet activation appeared to be mediated in part through the thromboxane pathway. These data suggest that KL/SCF could modulate hemostasis via interaction with platelets, particularly in conditions where mesenchymal cells are exposed to circulating blood elements, such as in wound healing or atherosclerosis.