ABSTRACT
The ErbB-2 interacting protein receptor-associated late transducer (RALT) was previously identified as a feedback inhibitor of ErbB-2 mitogenic signals. We now report that RALT binds to ligand-activated epidermal growth factor receptor (EGFR), ErbB-4 and ErbB-2.ErbB-3 dimers. When ectopically expressed in 32D cells reconstituted with the above ErbB receptor tyrosine kinases (RTKs) RALT behaved as a pan-ErbB inhibitor. Importantly, when tested in either cell proliferation assays or biochemical experiments measuring activation of ERK and AKT, RALT affected the signalling activity of distinct ErbB dimers with different relative potencies. RALT deltaEBR, a mutant unable to bind to ErbB RTKs, did not inhibit ErbB-dependent activation of ERK and AKT, consistent with RALT exerting its suppressive activity towards these pathways at a receptor-proximal level. Remarkably, RALT deltaEBR retained the ability to suppress largely the proliferative activity of ErbB-2.ErbB-3 dimers over a wide range of ligand concentrations, indicating that RALT can intercept ErbB-2.ErbB-3 mitogenic signals also at a receptor-distal level. A suppressive function of RALT deltaEBR towards the mitogenic activity of EGFR and ErbB-4 was detected at low levels of receptor occupancy, but was completely overcome by saturating concentrations of ligand. We propose that quantitative and qualitative aspects of RALT signalling concur in defining identity, strength and duration of signals generated by the ErbB network.
Subject(s)
Carrier Proteins/chemistry , Carrier Proteins/metabolism , ErbB Receptors/metabolism , Intracellular Signaling Peptides and Proteins , Protein Serine-Threonine Kinases , Receptor, ErbB-2/metabolism , Signal Transduction , 3T3 Cells , Animals , Cell Division , Cell Line , DNA/metabolism , Dimerization , Dose-Response Relationship, Drug , Enzyme Activation , Enzyme Inhibitors/pharmacology , Glutathione Transferase/metabolism , Immunochemistry , Immunohistochemistry , Ligands , Mice , Microscopy, Confocal , Microscopy, Fluorescence , Mitogen-Activated Protein Kinases/metabolism , Models, Genetic , Mutation , Platelet-Derived Growth Factor/metabolism , Protein Binding , Protein Structure, Tertiary , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-akt , Receptor Protein-Tyrosine Kinases/antagonists & inhibitors , Receptor, ErbB-4 , Recombinant Fusion Proteins/metabolism , TransfectionABSTRACT
Over-expression studies have demonstrated that RALT (receptor associated late transducer) is a feedback inhibitor of ErbB-2 mitogenic and transforming signals. In growth-arrested cells, expression of endogenous RALT is induced by mitogenic stimuli, is high throughout mid to late G1 and returns to baseline as cells move into S phase. Here, we show that physiological levels of RALT effectively suppress ErbB-2 mitogenic signals. We also investigate the regulatory mechanisms that preside to the control of RALT expression. We demonstrate that pharmacological ablation of extracellular signal-regulated kinase (ERK) activation leads to blockade of RALT expression, unlike genetic and/or pharmacological interference with the activities of PKC, Src family kinases, p38 SAPK and PI-3K. Tamoxifen-dependent activation of an inducible Raf : ER chimera was sufficient to induce RALT expression. Thus, activation of the Ras-Raf-ERK pathway is necessary and sufficient to drive RALT expression. The RALT protein is labile and was found to accumulate robustly upon pharmacological inhibition of the proteasome. We were able to detect ubiquitin-conjugated RALT species in living cells, suggesting that ubiquitinylation targets RALT for proteasome-dependent degradation. Such an integrated transcriptional and post-translational control is likely to provide RALT with the ability to fluctuate timely in order to tune ErbB signals.
