Novel mechanism of C/EBP beta (NF-M) transcriptional control: activation through derepression.

Phosphorylation of transcription factors is regarded as a major mechanism to control their activity in regulation of gene expression. C/EBP beta is a transcription factor that becomes activated after phosphorylation to induce genes involved in inflammation, acute-phase response, cytokine expression, cell growth, and differentiation. The chicken homolog NF-M collaborates with Myb and various kinase oncogenes in normal myeloid differentiation as well as in the leukemic transformation of myelomonocytic cells. Here, we examined the structure of NF-M and its mechanism of activation. We show that NF-M is a repressed transcription factor with concealed activation potential. Derepressed NF-M exhibits enhanced transcriptional efficacy in reporter assays. More importantly, NF-M activates resident chromatin-embedded, myelomonocyte-specific target genes, even in heterologous cell types such as fibroblasts or erythroblasts. We identified two regions within NF-M that act to repress trans-activation. Repression is abolished by deletion of these regions, activation of signal transduction kinases including v-erbB, polyoma middle T, ras and mil/raf, or point mutation of a critical phosphorylation site for MAP kinases. We provide evidence that phosphorylation plays a unique role to derepress rather than to enhance the trans-activation domain as a novel mechanism to regulate gene expression by NF-M/C/EBP beta.

Activation of Src family kinases by colony stimulating factor-1, and their association with its receptor.

The receptor for the macrophage colony stimulating factor-1 (CSF-1R) is a transmembrane glycoprotein with intrinsic tyrosine kinase activity. CSF-1 stimulation promotes the growth of cells of the macrophage lineage and of fibroblasts engineered to express CSF-1R. We show that CSF-1 stimulation resulted in activation of three Src family kinases, Src, Fyn and Yes. Concomitant with their activation, all three Src family kinases were found to associate with the ligand-activated CSF-1 receptor. These interactions were also demonstrated in SF9 insect cells co-infected with viruses encoding the CSF-1 receptor and Fyn, and the isolated SH2 domain of Fyn was capable of binding the CSF-1R in vitro. Analysis of mutant CSF-1Rs revealed that the 'kinase insert' (KI) domain of CSF-1R was not required for interactions with Src family kinases, but that mutation of one of the receptor autophosphorylation sites, Tyr809, reduced both their binding and enzymatic activation. Because fibroblasts expressing this receptor mutant are unable to form colonies in semi-solid medium or to grow in chemically defined medium in the presence of CSF-1, the Src family kinases may play a physiological role in the mitogenic response to CSF-1.

Association of Fyn with the activated platelet-derived growth factor receptor: requirements for binding and phosphorylation.

Three members of the Src family of tyrosine kinases [pp60c-src (Src), p59fyn (Fyn) and pp62c-yes (Yes)] are ubiquitously expressed, and are thus likely to have general roles in growth control. We have previously shown that, after addition of platelet-derived growth factor (PDGF) to quiescent cells, all three kinases become activated and associated with the PDGF receptor. We have now addressed the requirements for this association. First, we have used a baculovirus expression system to show that Fyn associates with the activated PDGF receptor in vitro in the absence of other proteins, demonstrating that the association between the two molecules is direct. Second, by generating cell lines expressing chimeric molecules consisting of Fyn sequences fused to a portion of beta-galactosidase, we found that the SH2 domain of Fyn is necessary for ligand-stimulated association with the PDGF receptor in vivo. Third, those fusion proteins that associated with the PDGF receptor also became phosphorylated in vivo following PDGF treatment, and in in vitro kinase assays, suggesting that the amino-terminal half of Fyn contains the sites of PDGF-stimulated phosphorylation. Partially purified, kinase-negative Fyn also became phosphorylated in the activated PDGF receptor complex in vitro, demonstrating that the PDGF receptor phosphorylates Fyn, rather than the novel phosphorylations occurring by autophosphorylation.