The molecular mechanisms by which mammalian receptor tyrosine kinases are negatively regulated remain largely unexplored. Previous genetic and biochemical studies indicate that Kekkon-1, a transmembrane protein containing leucine-rich repeats and an immunoglobulin-like domain in its extracellular region, acts as a feedback negative regulator of epidermal growth factor (EGF) receptor signaling in Drosophila melanogaster development. Here we tested whether the related human LRIG1 (also called Lig-1) protein can act as a negative regulator of EGF receptor and its relatives, ErbB2, ErbB3, and ErbB4. We observed that in co-transfected 293T cells, LRIG1 forms a complex with each of the ErbB receptors independent of growth factor binding. We further observed that co-expression of LRIG1 with EGF receptor suppresses cellular receptor levels, shortens receptor half-life, and enhances ligand-stimulated receptor ubiquitination. Finally, we observed that co-expression of LRIG1 suppresses EGF-stimulated transformation of NIH3T3 fibroblasts and that the inducible expression of LRIG1 in PC3 prostate tumor cells suppresses EGF-and neuregulin-1-stimulated cell cycle progression. Our observations indicate that LRIG1 is a negative regulator of the ErbB family of receptor tyrosine kinases and suggest that LRIG1-mediated receptor ubiquitination and degradation may contribute to the suppression of ErbB receptor function.The four members of the ErbB family of receptor tyrosine kinases (epidermal growth factor (EGF) 1 receptor, ErbB2, ErbB3, and ErbB4) play key roles in mediating the development of a variety of tissues, and the aberrant activation of these receptors contributes to the growth and progression of numerous tumor types (1, 2). Binding of EGF-like family ligands to ErbB receptors stimulates receptor dimerization, kinase activation, autophosphorylation, and the engagement of multiple intracellular growth signaling pathways. Although considerable effort over the past two decades has gone into understanding mechanisms by which ErbB receptors are activated and signals are propagated, our understanding of the variety of molecular mechanisms underlying the suppression of growth factor receptor activity remains in its infancy.Growth factor-stimulated receptor down-regulation, involving receptor internalization and the cbl-mediated ubiquitination and trafficking of receptors to lysosomes (3, 4), represents one mechanism for preventing hypersignaling by the ErbB receptors. However, whereas EGF receptor (ErbB1 or EGFR) efficiently couples to cbl following stimulation with its ligand EGF, the ErbB2, ErbB3, and ErbB4 receptors do not efficiently couple to cbl following stimulation with neuregulin-1 (NRG1) (5) and do not undergo efficient NRG1-stimulated down-regulation (6, 7). Hence, other negative regulatory mechanisms may play major roles in suppressing ErbB receptor activity.Studies from the fruit fly Drosophila melanogaster point to the existence of several classes of proteins that negatively regulate EGF receptor activity in flies (...

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The activated B-cell factor (ABF)-1 cDNA was initially isolated from Epstein-Barr virus (EBV)-infected B cells and codes for a DNA-binding protein belonging to the basic helix-loop-helix (bHLH) family of transcription factors. In this study, we characterized the nuclear localization signal of ABF-1, mapped two distinct transcriptional repression domains, and identified one ABF-1-interacting protein, Id-2. By examining the subcellular location of deletion mutants of ABF-1 fused to green fluorescent protein (GFP), critical regions involved in nuclear localization were determined. Analysis of GFP-tagged ABF-1 deletion mutants revealed two separate regions capable of directing nuclear localization. One region mapped to the N-terminal amino acids 71 to 103, whereas the second region localized to the C-terminal bHLH domain. Transient transfection of ABF-1 deletion mutants demonstrated that the N-terminal amino acids 1 to 40 and the bHLH domain function together to achieve maximum repression of E2A activity. Taken together, these results indicate that ABF-1 is a nuclear transcriptional repressor with two distinct regions that function in a synergistic fashion to attenuate E2A-mediated gene activation.

Members of the basic helix-loop-helix (bHLH) family of transcription factors regulate a wide array of developmental processes in many cell types, including cell fate specification, differentiation and morphogenesis. Our studies describe the cloning of a gene from the nematode Caenorhabditis elegans that is closely related to the vertebrate-activated B-cell factor (ABF) gene. The nematode gene product CeABF-1 was detected by northern blot analysis from RNA isolated from pooled nematodes representing different developmental stages. The developmental expression profile of CeABF-1 was shown by RT-PCR analysis to be predominantly expressed in the larval stages L3 and L4, with lower levels observed in the L2 larval stage and adult. We also show that CeABF-1 is capable of forming heterodimers with E2A proteins and binding E-box target sites. Mammalian cells transfected with CeABF-1 expression plasmids were capable of blocking E2A-mediated gene transcription, but full repression activity required the presence of two conserved amino acid residues found within the first helix of the CeABF-1 bHLH domain. These results suggest a conserved mechanism of gene repression between certain class II bHLH and class I bHLH proteins found in vertebrates and invertebrates.

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