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Steel factor-induced tyrosine phosphorylation in murine mast cells. Common elements with IL-3-induced signal
1Biomedical Research Centre, University of British Columbia, Vancouver, Canada.
Abstract:
The c-kit/W gene encodes a transmembrane protein tyrosine kinase, which is the receptor for Steel factor (SLF). SLF shares many general characteristics of hemopoietic growth factors, stimulating the survival, proliferation, and differentiation of stem and progenitor cells. We have investigated the tyrosine phosphorylation events that ensue after SLF binding to the c-kit protein using primary cultures of murine mast cells as a model system and have compared the effects of SLF and IL-3. Proteins that became phosphorylated on tyrosine after treatment of cells with SLF included c-kit itself, and major protein substrates designated p130, p122, p118, p115, p112, p100, p77, p55, p44, and p42. The majority of these proteins were cytosolic and maximally phosphorylated within 2 min of growth factor treatment. Combinations of immunoprecipitation and immunoblotting with antibodies specific for proteins known to be associated with signaling pathways demonstrated that none of the major tyrosine-phosphorylated species correlated with phospholipase C-gamma 1, GTPase activating protein, or phosphatidylinositol 3' kinase. However, stimulation with SLF led to a modest increase in tyrosine phosphorylation of the 85-kDa subunit of the phosphatidylinositol 3' kinase and increased association with a 150-kDa phosphotyrosyl protein, likely to be c-kit. Two species that did correlate with known elements were the 44- and 42-kDa polypeptides, shown to be members of the mitogen-activated protein kinase family. A subset of these proteins (p130, p115/112, p100, p55, p44, p42) were also tyrosine-phosphorylated when cells were stimulated by IL-3. MonoQ ion-exchange chromatography and two dimensional gel analyses were used to demonstrate that at least the p55, p44, and p42 substrates were identical, as well as some more minor species of molecular weights 50, 38, and 36 kDa, thus indicating common pathways of signaling in hemopoietic cells. Whereas in the case of SLF the dose-response characteristics of the proliferative response and the induction of tyrosine phosphorylation were similar, in the case of IL-3, much lower concentrations were required for maximal proliferation than maximal tyrosine phosphorylation. These studies form the basis for further molecular characterization of common components of signal transduction pathways in hemopoietic cells.
Insights
Steel factor (SLF) binding to c-kit triggers tyrosine phosphorylation in mast cells, identifying key signaling proteins like mitogen-activated protein kinases. Some phosphorylation pathways are shared with IL-3, indicating common signaling mechanisms in hematopoietic cells.
Area of Science:
- Cell biology
- Molecular biology
- Hematopoiesis
Background:
- The c-kit receptor tyrosine kinase, activated by Steel factor (SLF), is crucial for hematopoietic stem and progenitor cell survival, proliferation, and differentiation.
- Understanding the downstream signaling events triggered by SLF binding to c-kit is essential for elucidating hematopoietic cell regulation.
Purpose of the Study:
- To investigate the tyrosine phosphorylation events following SLF binding to c-kit in murine mast cells.
- To compare the signaling effects of SLF with those of IL-3 (interleukin-3).
Main Methods:
- Primary cultures of murine mast cells were treated with SLF and IL-3.
- Tyrosine phosphorylation was analyzed using immunoprecipitation and immunoblotting.
- Protein identification involved antibodies against known signaling proteins and techniques like MonoQ ion-exchange chromatography and 2D gel electrophoresis.
Main Results:
- SLF treatment induced tyrosine phosphorylation of c-kit itself and several other proteins, including p44 and p42, identified as mitogen-activated protein kinases.
- While SLF and IL-3 share some common tyrosine-phosphorylated substrates (e.g., p55, p44, p42), their dose-response relationships for proliferation and phosphorylation differ.
- SLF binding led to increased tyrosine phosphorylation of the phosphatidylinositol 3'-kinase 85-kDa subunit and association with a 150-kDa phosphotyrosyl protein, likely c-kit.
Conclusions:
- SLF signaling involves tyrosine phosphorylation of multiple cytosolic proteins, including MAP kinases.
- Common signaling pathways exist between SLF and IL-3 in hematopoietic cells.
- The studies provide a foundation for further molecular characterization of signal transduction in hematopoietic cells.