Development of L-hydroxyamino acid dehydratase in rat liver

published_or_final_versionBiochemistryDoctoralDoctor of Philosoph

Induction of threonine dehydratase in developing rat liver

published_or_final_versionBiochemistryMasterMaster of Philosoph

Proteomic Approaches to the Analysis of Early Events in Colony-stimulating Factor-1 Signal Transduction

The exposure of cells to growth factors leads to the rapid tyrosine phosphorylation of proteins that play critical roles in initiating cellular responses. These proteins are associated with other nontyrosine-phosphorylated pro-teins. Together, they represent less than 0.02 % of the total cellular protein. To study their functions in growth factor signaling it is necessary to establish their identity, post-translational modifications, and interactions. We have focused on the characterization of this group of proteins during the early response of macrophages to the macrophage growth factor, colony-stimulating factor-1 (CSF-1). We review here the development of approaches to analysis of the rapid CSF-1-induced changes in the CSF-1 receptor tyrosine kinase and phosphotyrosyl sig-naling complexes. Recent advances in mass spectrome-try technology are greatly facilitating the characterization of such complexes. These methods strongly support and enhance genetic approaches that are being used to ana-lyze the function of individual signaling components an

Role and Regulation of CSF-1-Induced CSF-1 Receptor Interchain Disulfide Bonding in Receptor Activation in Macrophages

Abstract Colony stimulating factor-1 (CSF-1) is the major regulator of tissue macrophage development and function. The effects of CSF-1 are mediated by the CSF-1 receptor (CSF-1R), a class III receptor tyrosine kinase belonging to the PDGF receptor family. To study CSF-1R structure/function in macrophages using both genetic and proteomic approaches, we developed a novel, CSF-1R-deficient, mouse bone marrow macrophage (BMM) line (MacCsf1r−/−) (Yu et al., J. Leuk. Biol.84: in press, 2008). MacCsf1r−/− macrophages are maintained in GM-CSF. Retroviral expression of the wild type CSF-1R fully rescued the CSF-1-induced survival, proliferation, differentiation and morphological characteristics, which resemble those of primary BMM. We have studied CSF-1-induced covalent modifications of the receptor in MacCsf1r−/− cells. Activation of the CSF-1R involves ligand-induced receptor dimerization and trans-phosphorylation of the cytoplasmic domains. Besides tyrosine phosphorylation, CSF-1 stimulation results in disulfide bonding of the CSF-1R dimers and cysteine mutagenesis experiments revealed that specific residues in the kinase domain are involved. Inhibition of the disulfide bond formation significantly compromised CSF-1-induced CSF-1R tyrosine phosphorylation, indicating its requirement for full receptor activation. Analysis of the regulation of CSF-1-induced CSF-1R interchain disulfide bonding revealed the participation of third party molecules with additional CSF-1R post-translational modifications. The importance of ligand-induced disulfide bonding for class III receptor tyrosine kinase activation may not be restricted to the CSF-1R as interchain disulfide bonding of PDGF receptor dimers is also ligand-induced.</jats:p