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 (...
The Met receptor tyrosine kinase regulates a complex array of cellular behaviors collectively known as "invasive growth." While essential for normal development and wound repair, this program is frequently co-opted by tumors to promote their own growth, motility, and invasion. Met is overexpressed in a variety of human tumors, and this aberrant expression correlates with poor patient prognosis. Previous studies indicate that Met receptor levels are governed in part by cbl-mediated ubiquitination and degradation, and uncoupling of Met from cbl-mediated ubiquitination promotes its transforming activity. Here we describe a novel mechanism for Met degradation. We find that the Met receptor interacts with the transmembrane protein LRIG1 independent of hepatocyte growth factor (HGF) stimulation and that LRIG1 destabilizes the Met receptor in a cbl-independent manner. Overexpression of LRIG1 destabilizes endogenous Met receptor in breast cancer cells and impairs their ability to respond to HGF. LRIG1 knockdown increases Met receptor half-life, indicating that it plays an essential role in Met degradation. Finally, LRIG1 opposes Met synergy with the ErbB2/Her2 receptor tyrosine kinase in driving cellular invasion. We conclude that LRIG1 is a novel suppressor of Met function, serving to regulate cellular receptor levels by promoting Met degradation in a ligand-and cbl-independent manner.
Protein inhibitors of activated STATs (PIAS) family members are ubiquitin-protein isopeptide ligase-small ubiquitin-like modifier ligases for diverse transcription factors. However, the regulation of PIAS protein activity in cells is poorly understood. Previously, we reported that expression of Trim32, a RING domain ubiquitin-protein isopeptide ligase-ubiquitin ligase mutated in human limb-girdle muscular dystrophy type 2H (LGMD2H) and Bardet-Biedl syndrome, is elevated during mouse skin carcinogenesis, protecting keratinocytes from apoptosis induced by UVB and tumor necrosis factor-␣ (TNF␣). Here we report that Trim32 interacts with Piasy and promotes Piasy ubiquitination and degradation. Ubiquitination of Piasy by Trim32 could be reproduced in vitro using purified components. Their interaction was induced by treatment with UVB/ TNF␣ and involved redistribution of Piasy from the nucleus to the cytoplasm, where it accumulated in cytoplasmic granules that colocalized with Trim32. Piasy destabilization and ubiquitination required an intact RING domain in Trim32. The LGMD2H-associated missense point mutation prevented Trim32 binding to Piasy, and human Piasy failed to colocalize with human Trim32 in fibroblasts isolated from an LGMD2H patient. Trim32 expression increased the transcriptional activity of NFB in epidermal keratinocytes, both under basal treatment and after UVB/TNF␣ treatment. Conversely, Piasy inhibited NFB activity under the same conditions and sensitized keratinocytes to apoptosis induced by TNF␣ and UVB. Our results indicate that, by controlling Piasy stability, Trim32 regulates UVB-induced keratinocyte apoptosis through induction of NFB and suggests loss of function of Trim32 in LGMD2H.Protein ubiquitination is a fundamental process in eukaryotic cells, controlling the degradation of proteins through the 26 S proteasome. E3 4 -ubiquitin ligases catalyze the last step of the process and provide substrate specificity. The activity of many cellular factors is controlled by their abundance and stability in the cell, properties that depend on the rate of ubiquitination. By providing substrate specificity, E3 ligases become a point of control for ubiquitination and stability of many cellular factors. A variety of otherwise structurally unrelated E3 ligases contain a common RING domain that provides interaction with the E2 ubiquitin-conjugating enzyme (1). In Trim32, the RING domain is present in the amino terminus of the protein, as part of the RBCC (Ring/B-Box/Coiled-coil) or tripartite motif that defines the TRIM family of proteins (2). Members of the TRIM family may be located in the cytoplasm or nucleus and are thought to form large multimeric complexes, forming subcellular structures resembling spheres, speckles, or ribbons. Different TRIM family members are characterized by a specific carboxyl-terminal domain. In Trim32, the carboxyl terminus contains six repeats of the NHL (NCL-1, HT2A and LIN-41) motif, thought to mediate protein/protein interactions (3). TRIM family members have been implicated...
Environmental cues are transmitted to the interior of the cell via a complex network of signaling hubs. Receptor tyrosine kinases (RTKs) and trimeric G proteins are two such major signaling hubs in eukaryotes. Conventionally, canonical signal transduction via trimeric G proteins is thought to be triggered exclusively by G protein-coupled receptors. Here we used molecular engineering to develop modular fluorescent biosensors that exploit the remarkable specificity of bimolecular recognition, i.e., of both G proteins and RTKs, and reveal the workings of a novel platform for activation of G proteins by RTKs in single living cells. Comprised of the unique modular makeup of guanidine exchange factor Gα-interacting vesicle-associated protein (GIV)/girdin, a guanidine exchange factor that links G proteins to a variety of RTKs, these biosensors provide direct evidence that RTK-GIV-Gαi ternary complexes are formed in living cells and that Gαi is transactivated within minutes after growth factor stimulation at the plasma membrane. Thus, GIV-derived biosensors provide a versatile strategy for visualizing, monitoring, and manipulating the dynamic association of Gαi with RTKs for noncanonical transactivation of G proteins in cells and illuminate a fundamental signaling event regulated by GIV during diverse cellular processes and pathophysiologic states.
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