The product of rat gene 33 was identified as an ErbB-2-interacting protein in a two-hybrid screen employing the ErbB-2 juxtamembrane and kinase domains as bait. This interaction was reproduced in vitro with a glutathione S-transferase fusion protein spanning positions 282 to 395 of the 459-residue gene 33 protein.Activation of ErbB-2 catalytic function was required for ErbB-2-gene 33 physical interaction in living cells, whereas ErbB-2 autophosphorylation was dispensable. Expression of gene 33 protein was absent in growtharrested NIH 3T3 fibroblasts but was induced within 60 to 90 min of serum stimulation or activation of the ErbB-2 kinase and decreased sharply upon entry into S phase. New differentiation factor stimulation of mitogen-deprived mammary epithelial cells also caused accumulation of gene 33 protein, which could be found in a complex with ErbB-2. Overexpression of gene 33 protein in mouse fibroblasts inhibited (i) cell proliferation driven by ErbB-2 but not by serum, (ii) cell transformation induced by ErbB-2 but not by Ras or Src, and (iii) sustained activation of ERK 1 and 2 by ErbB-2 but not by serum. The gene 33 protein may convey inhibitory signals downstream to ErbB-2 by virtue of its association with SH3-containing proteins, including GRB-2, which was found to associate with gene 33 protein in living cells. These data indicate that the gene 33 protein is a feedback inhibitor of ErbB-2 mitogenic function and a suppressor of ErbB-2 oncogenic activity. We propose that the gene 33 protein be renamed with the acronym RALT (receptor-associated late transducer).Protein-protein interactions play a crucial role in the regulation of signal transduction pathways activated by receptor tyrosine kinases (RTKs) (58). SH2 (Src homology 2) and PTB (phosphotyrosine [PTyr] binding) domains recognize PTyr residues in the context of specific peptide sequences and can therefore bind to autophosphorylated receptors or to tyrosinephosphorylated RTK substrates (58,74). Modules based on PTyr-independent molecular recognition such as EH, PDZ, SH3, and WW domains (58,74) are also involved in signaling downstream to activated RTKs. In general, protein-protein interaction modules are found both in polypeptides possessing intrinsic catalytic properties and in adapter-scaffold proteins. In the former case protein-protein interactions may modulate the function of a given enzyme by simply regulating its subcellular distribution or by allosteric activation (58). Adapter-scaffold proteins, on the other hand, are essentially made up of protein-protein interaction domains that allow for the assembly of multiprotein complexes in which the functions of different enzymes are integrated both spatially and temporally (57).Upon ligand activation, RTKs target not only positive effectors but also enzymes involved in negative regulation of receptor signaling, such as tyrosine phosphatases (39), the Ras GTPase-activating protein (15), and c-Cbl (8,37,44). Adapter proteins such as Slap (67) and the SOCS gene family products (55) are also im...

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 DEBR, 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 DEBR 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 DEBR 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.

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 ubiquitinconjugated RALT species in living cells, suggesting that ubiquitinylation targets RALT for proteasome-dependent degradation. Such an integrated transcriptional and posttranslational control is likely to provide RALT with the ability to fluctuate timely in order to tune ErbB signals.

In Metazoans a number of cellular functions are controlled by receptor tyrosine kinases (RTKs) during development and in postnatal life. The execution of these programs requires that signals of adequate strength are delivered for the appropriate time within precise spatial boundaries. Several RTK inhibitors have been identified in invertebrate and mammalian organisms. Because they are involved in tuning and termination of receptor signals, negative regulators of RTK activity fulfill a fundamental function in the control of receptor signaling. ß