Activation of tyrosine kinases is required for mitogenic signal transduction by many growth factor receptors. Stimulation of factor-dependent cells with cytokines such as IL-3 or GM-CSF results in the phosphorylation of multiple proteins including the receptors themselves. Both Janus and Src-family kinases are activated in a cytokine-dependent manner. Substrates for Janus kinases include STAT molecules which activate transcription of specific genes. It appears that the activation of JAK2 and STAT molecules may be necessary, but not sufficient, for GM-CSF-induced mitogenesis. Preliminary evidence by the applicant suggests that Src-like kinases are required for GM-CSF-induced mitogenesis, but it is not understood how their activation is regulated or what role they play in cytokine receptor signaling. It is hypothesized that Src-like kinases are required for mitogenic stimulation by cytokine receptors and, furthermore, that activation of the kinases is regulated by the binding of their SH2 domains to a specific phosphorylation site(s) in the cytoplasmic tail of the beta subunit of the IL-3/GM-CSF receptor. This proposal will identify Src-like kinases are able to transmit mitogenic signals by the IL-3/GM-CSF receptor, identify what signaling pathways are activated in a Src kinase dependent manner, and determine the mechanism by which src-like kinases are activated. In these studies they will utilize a chicken B-lymphocyte cell line that lacks expression of Src-like kinases, which has been transfected with the human GM-CSF receptor. In addition, dominant negative mutants of Src-like kinases will be expressed in the IL-3-dependent murine myeloid cell line 32Dc13 to assess the role of Src-like kinases in mitogenic signaling in these cells. Mutagenesis studies of a bacterial fusion protein containing a 236 amino acid region of the cytoplasmic tail of the beta subunit to which both Hck and Fyn bind, will be used to identify both phosphotyrosine-dependent and independent binding sites for the different domains of Hck and Fyn in the beta subunit. The importance of these binding sites in mitogenic signaling will be addressed by introducing mutations or deletions of these binding sites into receptors, and assessing the ability of the mutant receptors to induce proliferation. These approaches should also help to identify both substrates phosphorylated, as well as signaling pathways activated, in a Src kinase-dependent manner.
| Kassenbrock, C Kenneth; Anderson, Steven M (2004) Regulation of ubiquitin protein ligase activity in c-Cbl by phosphorylation-induced conformational change and constitutive activation by tyrosine to glutamate point mutations. J Biol Chem 279:28017-27 |
| Kassenbrock, C Kenneth; Hunter, Seija; Garl, Pamela et al. (2002) Inhibition of Src family kinases blocks epidermal growth factor (EGF)-induced activation of Akt, phosphorylation of c-Cbl, and ubiquitination of the EGF receptor. J Biol Chem 277:24967-75 |
| Hamilton, E; Miller, K M; Helm, K M et al. (2001) Suppression of apoptosis induced by growth factor withdrawal by an oncogenic form of c-Cbl. J Biol Chem 276:9028-37 |
