Keting Chu

Discovery of a cytokine and its receptor by functional screening of the extracellular proteome.

To understand the system of secreted proteins and receptors involved in cell-cell signaling, we produced a comprehensive set of recombinant secreted proteins and the extracellular domains of transmembrane proteins, which constitute most of the protein components of the extracellular space. Each protein was tested in a suite of assays that measured metabolic, growth, or transcriptional responses in diverse cell types. The pattern of responses across assays was analyzed for the degree of functional selectivity of each protein. One of the highly selective proteins was a previously undescribed ligand, designated interleukin-34 (IL-34), which stimulates monocyte viability but does not affect responses in a wide spectrum of other assays. In a separate functional screen, we used a collection of extracellular domains of transmembrane proteins to discover the receptor for IL-34, which was a known cytokine receptor, colony-stimulating factor 1 (also called macrophage colony-stimulating factor) receptor. This systematic approach is thus useful for discovering new ligands and receptors and assessing the functional selectivity of extracellular regulatory proteins.

Modulation of human cytomegalovirus immediate‐early gene enhancer by mitogen‐activated protein kinase kinase kinase‐1

The immediate‐early (IE) promoter of human cytomegalovirus (HCMV) constitutes a primary genetic switch, which determines the progression of viral infection. Earlier reports by others have shown mitogen‐activated protein kinase kinase kinase‐1 (MEKK1) to be able to up‐regulate HCMV‐IE promoter through downstream mitogen‐activated protein kinase (MAPK) pathways. However, we noticed that the activation of the HCMV‐IE promoter by constitutively active MEKK1 (MEKK1‐TRU) might not be through the MAPK pathways. Using a HCMV‐IE enhancer/promoter (− 522 to + 72) driving a luciferase reporter, we demonstrated that the downstream MAPK activation actually repressed the up‐regulation of the promoter by MEKK1 in CHO‐K1 and human 293 cells. We further found that the up‐regulation of HCMV‐IE promoter by MEKK1 could be in great extent suppressed by over‐expression of IκBα. Deletion of the NFκB/rel sites in the HCMV‐IE enhancer region by mutagenesis proportionally reduced the transcriptional activation by MEKK1‐TRU, whereas deletion of the ATF/CREB binding sites or cyclic AMP response elements (CRE) had no effects. Furthermore, the NFκB/rel deletion mutant also showed repression on the basic transcription activity of the HCMV‐IE promoter. Our results indicate that the NFκB/rel sites are not only responsible for the modulation of HCMV‐IE enhancer activity by MEKK1 but also control the basic transcription activity of the HCMV‐IE promoter. On the other hand, the four consensus CRE sites were found to have no function in the activation of the promoter by MEKK1. J. Cell. Biochem. 83: 563–573, 2001.