Stem cell factor (SCF) is a growth factor that promotes viability and induces proliferation and differentiation of hematopoietic cells. In addition to its effects as a single factor, SCF acts synergistically with other growth factors to stimulate cellular proliferation. One research objective of this project is to delineate the biochemical mechanism of action of SCF. The receptor for SCF is the receptor tyrosine kinase (RTK) c-Kit. Similar to other RTKs, SCF activates the ras-raf-map kinase cascade and phosphatidylinositol-3-kinase (PI3K). In addition, our previous studies have shown that SCF activates Src family members, as well as components of the JAK/STAT pathway (JAK2 and Stat1). To define the role of JAK2 in SCF-mediated responses we have examined the capacity of SCF to induce proliferation of progenitor cells isolated from the fetal liver of JAK2-deficient mice. SCF-induced proliferation of JAK2-deficient fetal liver progenitor cells was significantly lower than wild-type progenitor cells. Studies examining the capacity of JAK2 deficient progenitor cells to survive and differentiate in response to SCF are presently in progress. We are also interested in the role of Src family members in SCF-mediated responses. Our previous studies have demonstrated that the Src family member Lyn is expressed at high levels in SCF responsive tissue, that SCF induces association of Lyn and c-Kit and that Lyn kinase activity increases during the G0/G1 transition, minutes after stimulation with SCF. We have subsequently found that treatment of cells with SCF activates a second wave of Lyn activation, during the G1/S transition of the cell cycle. These studies also showed that a portion of total cellular Lyn is constitutively associated with Cdk2, and that this interaction is required for SCF-mediated proliferation. These data demonstrate that Lyn is activated at multiple points during SCF-induced cell cycle progression. To address the role of Lyn in SCF-mediated responses, we examined the effect of a kinase inactive Lyn mutant on SCF-induced proliferation. Transfection of normal mast cells with this mutant dramatically impairs SCF-induced proliferation. We have also examined the capacity of cells from Lyn-deficient mice to respond to SCF. Both hematopoietic progenitor cells and mast cells were utilized since both these populations of cells express c-Kit and respond to SCF. SCF- mediated proliferation of Lyn-deficient mast cells and progenitor cells was significantly lower than wild-type cells. These data demonstrate that Lyn plays a role in c-Kit signaling pathways in both early progenitor cells as well as in terminally differentiated mast cells. In addition to studying the biochemical mechanism of action of wild- type c-Kit, a second objective of this project is to identify signal transduction pathways mediating cellular transformation by D816V c-Kit, an oncogenic mutant. This mutation is found in human patients with mastocytosis and likely plays a role in the development of this disease. We have found that the 85 kDa regulatory subunit of phosphatidylinositol-3-kinase (PI3K) is constitutively associated with D816V c-Kit and constitutively phosphorylated on tyrosine residues. Further, the amount of PI3K activity constitutively associated with D816V c-Kit was 75-fold greater than that associated with unstimulated wild type c-Kit. To examine the role of PI3K activity in factor- independent growth of cells expressing D816V c-Kit, we utilized the PI3K inhibitor wortmanin. Proliferation of cells expressing D816V c-Kit was more sensitive to the inhibitory effects of wortmanin than SCF- induced proliferation of cells expressing wild-type c-Kit. These findings demonstrate constitutive activation of PI3K by an oncogenic form of c-Kit and provides insight into mechanisms mediating transformation of hematopoietic cells by D816V c-Kit. - Cell signaling, Cytokines, growth factors, hematopoiesis, Signal Transduction,
| Shivakrupa, R; Linnekin, Diana (2005) Lyn contributes to regulation of multiple Kit-dependent signaling pathways in murine bone marrow mast cells. Cell Signal 17:103-9 |
| Lennartsson, Johan; Shivakrupa, R; Linnekin, Diana (2004) Synergistic growth of stem cell factor and granulocyte macrophage colony-stimulating factor involves kinase-dependent and -independent contributions from c-Kit. J Biol Chem 279:44544-53 |
| Shivakrupa, R; Bernstein, Alan; Watring, Nicole et al. (2003) Phosphatidylinositol 3'-kinase is required for growth of mast cells expressing the kit catalytic domain mutant. Cancer Res 63:4412-9 |
