ABSTRACT
Stem cell factor (SCF) binds to c-Kit and is an important mediator of survival, growth, and function of hematopoietic progenitor cells and mast cells. Lyn and other Src family members are activated by SCF and associate with phosphorylated tyrosine residues in the c-Kit juxtamembrane region. However, studies using c-Kit mutants incapable of directly recruiting Src family members suggest this kinase family plays a minimal role in c-Kit stimulus-response coupling mechanisms. The objective of this study was to specifically target Lyn and subsequently address its role in SCF-mediated responses of primary hematopoietic progenitor cells and mast cells. To this end, a dominant-inhibitory Lyn mutant and Lyn-deficient mice were used. Transfection of normal murine mast cells with kinase-inactive Lyn impaired SCF-induced growth. Further, SCF-induced proliferation and chemotaxis of Lyn-deficient mast cells were less than for wild-type mast cells. SCF-induced growth of progenitor cells lacking Lyn was also reduced compared with that of wild-type progenitor cells. Impairment of SCF-mediated responses of Lyn-deficient mast cells and progenitor cells did not result from reductions in surface expression of c-Kit. These studies demonstrate that Lyn is required for normal SCF-mediated responses of primary progenitors and for a differentiated lineage.
Subject(s)
Cell Division/drug effects , Chemotaxis/drug effects , Hematopoietic Stem Cells/cytology , Stem Cell Factor/pharmacology , src-Family Kinases/physiology , Animals , Bone Marrow Cells/cytology , Bone Marrow Cells/metabolism , Cells, Cultured , Gene Expression , Humans , Mast Cells/cytology , Mast Cells/metabolism , Mice , Mice, Knockout , Mutation , Tetradecanoylphorbol Acetate/pharmacology , Transfection , src-Family Kinases/deficiency , src-Family Kinases/geneticsABSTRACT
At least 70% of small cell lung cancers (SCLCs) express the Kit receptor tyrosine kinase and its ligand, stem cell factor (SCF). In an effort to define the signal transduction pathways activated by Kit in SCLC, we focused on Src family kinases and, in particular, Lck, a Src-related tyrosine kinase that is expressed in hemopoietic cells and certain tumors, including SCLC. SCF treatment of the H526 cell line induced a physical association between Kit and Lck that, in vitro, was dependent on phosphorylation of the juxtamembrane domain of Kit. Stimulation of Kit with recombinant SCF resulted in a rapid 3-6-fold increase in the specific activity of Lck, which was similar in magnitude to the activation of Lck resulting from the cross-linking of the T-cell receptor complex of Jurkat cells. Lck activity peaked by 5 min after SCF addition, and the elevated activity persisted for at least 30 min in the presence of SCF, with kinetics similar to the activation of mitogen-activated protein kinase. PP1, an inhibitor of Src family kinases with selectivity for Lck, completely inhibited SCF-mediated growth but had little effect on insulin-like growth factor-I-mediated growth. PP1 antagonized both SCF-mediated proliferation and inhibition of apoptosis. PP1 had no effect on Kit kinase activity but was shown to block total Lck activity by at least 90% by immune complex kinase assay. Low levels of Src, Hck, and Yes were also expressed in the H526 cell line; only Yes showed a consistent increase in specific activity, which was also inhibited by PP1 following SCF treatment. These data demonstrate that, in the H526 SCLC cell line, Lck and, possibly, Yes are downstream of Kit in a signal transduction pathway; the inhibition by PP1 of SCF-mediated proliferation and inhibition of apoptosis suggests that Src family kinases are intermediates in the signaling pathways that regulate these processes.
Subject(s)
Carcinoma, Small Cell/drug therapy , Enzyme Inhibitors/pharmacology , Lung Neoplasms/drug therapy , Lymphocyte Specific Protein Tyrosine Kinase p56(lck)/physiology , Proto-Oncogene Proteins c-kit/physiology , Stem Cell Factor/antagonists & inhibitors , src-Family Kinases/antagonists & inhibitors , Carcinoma, Small Cell/metabolism , Humans , Jurkat Cells , Lung Neoplasms/metabolism , Pyrazoles/pharmacology , Pyrimidines/pharmacology , Signal Transduction , Stem Cell Factor/pharmacologyABSTRACT
Interaction of stem cell factor (SCF), a haematopoietic growth factor, with the receptor tyrosine kinase c-kit leads to autophosphorylation of c-kit as well as tyrosine phosphorylation of various substrates. Little is known about the role of the JAK/STAT pathway in signal transduction via receptor tyrosine kinases, although this pathway has been well characterized in cytokine receptor signal transduction. We recently found that the Janus kinase Jak2 associates with c-kit and that SCF induces rapid and transient phosphorylation of Jak2. Here we present evidence that SCF activates the transcription factor Stat1. Phosphorylated c-kit co-immunoprecipitates with Stat1 within 1 min of SCF stimulation of the human cell line MO7e. Co-precipitation experiments using glutathione S-transferase fusion proteins indicate that association with c-kit is mediated by the Stat1 SH2 domain. Stat1 is rapidly tyrosine-phosphorylated in response to SCF in MO7e cells, the murine cell line FDCP-1 and normal progenitor cells. SCF-induced phosphorylation of Jak2 and Stat1 was also observed in murine 3T3 fibroblasts stably transfected with full-length human c-kit receptor. Furthermore c-kit directly phosphorylates Stat1 fusion proteins in in vitro kinase assays. Electrophoretic mobility-shift assays with nuclear extracts from SCF-stimulated cell lines and normal progenitor cells indicate that activated Stat1 binds the m67 oligonucleotide, a high-affinity SIE promoter sequence. These results demonstrate that Stat1 is activated in response to SCF, and suggest that Stat1 is a component of the SCF signal-transduction pathway.
Subject(s)
DNA-Binding Proteins/metabolism , Proto-Oncogene Proteins c-kit/metabolism , Proto-Oncogene Proteins , Stem Cell Factor/pharmacology , Trans-Activators/metabolism , Animals , DNA/metabolism , DNA-Binding Proteins/chemistry , Electrophoresis, Polyacrylamide Gel , Glutathione Transferase/genetics , Humans , Janus Kinase 2 , Mice , Nuclear Proteins/metabolism , Phosphorylation , Precipitin Tests , Protein-Tyrosine Kinases/metabolism , Recombinant Fusion Proteins/metabolism , STAT1 Transcription Factor , Signal Transduction/physiology , Trans-Activators/chemistry , Transcriptional Activation/physiology , Transfection , Tumor Cells, Cultured , src Homology DomainsABSTRACT
Stem cell factor (SCF) is a cytokine critical for normal hematopoiesis. The receptor for SCF is c-Kit, a receptor tyrosine kinase. Our laboratory is interested in delineating critical components of the SCF signal transduction pathway in hematopoietic tissue. The present study examines activation of Src family members in response to SCF. Stimulation of cell lines as well as normal progenitor cells with SCF rapidly increased tyrosine phosphorylation of the Src family member Lyn. Peak responses were noted 10-20 min after SCF treatment, and phosphorylation of Lyn returned to basal levels 60-90 min after stimulation. SCF also induced increases in Lyn kinase activity in vitro. Lyn coimmunoprecipitated with c-Kit, and studies with GST fusion proteins demonstrated that Lyn readily associated with the juxtamembrane region of c-Kit. Treatment of cells with either Lyn antisense oligonucleotides or PP1, a Src family inhibitor, resulted in dramatic inhibition of SCF-induced proliferation. These data demonstrate that SCF rapidly activates Lyn and suggest that Lyn is critical in SCF-induced proliferation in hematopoietic cells.
Subject(s)
Hematopoietic Stem Cells/metabolism , Liver/cytology , Proto-Oncogene Proteins c-kit/chemistry , Proto-Oncogene Proteins c-kit/metabolism , Stem Cell Factor/pharmacology , src-Family Kinases/metabolism , Binding Sites , Cell Division/drug effects , Cell Line , Cell Membrane/metabolism , Cells, Cultured , Enzyme Activation , Granulocyte-Macrophage Colony-Stimulating Factor/pharmacology , Hematopoietic Stem Cells/cytology , Humans , Kinetics , Liver/embryology , Phosphorylation , Proto-Oncogene Proteins c-kit/isolation & purification , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/metabolism , Recombinant Proteins/pharmacology , Tetradecanoylphorbol Acetate/pharmacology , src-Family Kinases/biosynthesis , src-Family Kinases/isolation & purificationABSTRACT
Recent work has demonstrated the importance of Janus family kinases (JAKs) and signal transducers and activators of transcription (STATs) in the stimulus-response coupling of receptors lacking intrinsic tyrosine kinase activity. In particular, the JAK-STAT pathway appears critical in signal transduction by interferon as well as numerous hematopoietic growth factors interacting with members of the hemapoietin receptor superfamily. Although ligands that interact with receptor tyrosine kinases (RTK), such as epidermal growth factor (EGF), platelet-derived growth factor (PDGF) and colony stimulating factor-1 (CSF-1), have been shown to induce increases in phosphorylation of both JAKs and STATs, little is known about activation of this pathway by stem cell factor (SCF). This review will summarize what is known about the JAK/STAT pathway in relation to SCF signal transduction.
Subject(s)
DNA-Binding Proteins/physiology , Protein-Tyrosine Kinases/metabolism , Proto-Oncogene Proteins , Signal Transduction , Stem Cell Factor/physiology , Transcriptional Activation , Animals , Cerebrospinal Fluid/metabolism , Epidermal Growth Factor/metabolism , Hematopoiesis , Humans , Interferons/physiology , Janus Kinase 2 , Mice , Phosphorylation , Platelet-Derived Growth Factor/metabolism , Protein-Tyrosine Kinases/physiology , Receptor Protein-Tyrosine Kinases/metabolismABSTRACT
Stem cell factor (SCF) is a hematopoietic growth factor that interacts with the receptor tyrosine kinase, c-kit. We have found that SCF-stimulates rapid and transient tyrosine phosphorylation of JAK2 in human and murine cell lines, as well as in normal human progenitor cells. JAK2 and c-kit were associated in unstimulated cells with further recruitment of JAK2 to the c-kit receptor complex after SCF stimulation. Treatment of cells with JAK2 antisense oligonucleotides resulted in a 46% decrease in SCF-induced proliferation. These data demonstrate that SCF induces tyrosine phosphorylation of JAK2 and suggest that JAK2 is a component of the SCF signal transduction pathway.
Subject(s)
Protein-Tyrosine Kinases/metabolism , Proto-Oncogene Proteins c-kit/metabolism , Proto-Oncogene Proteins , Signal Transduction/drug effects , Stem Cell Factor/pharmacology , Animals , Base Sequence , Cell Line , Humans , Janus Kinase 2 , Mice , Molecular Sequence Data , Phosphorylation/drug effects , Proto-Oncogene MasABSTRACT
Stem cell factor (SCF) interacts with the receptor tyrosine kinase c-Kit and has potent effects on hematopoiesis. We have examined the role of JAK2 in the SCF signal transduction pathway. JAK2 and c-Kit were constitutively associated, and treatment with SCF resulted in rapid and transient tyrosine phosphorylation of JAK2. Incubation of cells with JAK2 antisense oligonucleotides resulted in significant decreases in SCF-induced proliferation. These data suggest that JAK2 plays a role in SCF-induced proliferation.
Subject(s)
Protein-Tyrosine Kinases/metabolism , Proto-Oncogene Proteins c-kit/metabolism , Proto-Oncogene Proteins , Signal Transduction , Stem Cell Factor/pharmacology , Animals , Base Sequence , Blotting, Western , Cell Division/drug effects , Cell Line , DNA, Antisense , Electrophoresis, Polyacrylamide Gel , Humans , Janus Kinase 2 , Mice , Molecular Sequence Data , Phosphoproteins/analysis , Phosphorylation/drug effects , Phosphotyrosine/metabolism , Tumor Cells, CulturedABSTRACT
Although isokinetic strength testing has been in use for more than two decades, and numerous studies have addressed isokinetic performance of the lumbar spine, the effect of instructions on isokinetic trunk strength has not been studied. In a sample of 30 healthy women, this study examined the effect of "high-demand" instructions on lumbar strength performance. High-demand instructions were found to have a substantial positive effect on performance variability, reliability, absolute magnitude, and validity. Under these conditions, isokinetic trunk strength was found to be predictive of performance in a frequent lifting-lowering task.