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 human congenital syndromes ectrodactyly ectodermal dysplasia-cleft lip/palate syndrome, ankyloblepharon ectodermal dysplasia clefting, and split-hand/foot malformation are all characterized by ectodermal dysplasia, limb malformations, and cleft lip/palate. These phenotypic features are a result of an imbalance between the proliferation and differentiation of precursor cells during development of ectoderm-derived structures. Mutations in the p63 and interferon regulatory factor 6 (IRF6) genes have been found in human patients with these syndromes, consistent with phenotypes. Here, we used human and mouse primary keratinocytes and mouse models to investigate the role of p63 and IRF6 in proliferation and differentiation. We report that the ΔNp63 isoform of p63 activated transcription of IRF6, and this, in turn, induced proteasomemediated ΔNp63 degradation. This feedback regulatory loop allowed keratinocytes to exit the cell cycle, thereby limiting their ability to proliferate. Importantly, mutations in either p63 or IRF6 resulted in disruption of this regulatory loop: p63 mutations causing ectodermal dysplasias were unable to activate IRF6 transcription, and mice with mutated or null p63 showed reduced Irf6 expression in their palate and ectoderm. These results identify what we believe to be a novel mechanism that regulates the proliferation-differentiation balance of keratinocytes essential for palate fusion and skin differentiation and links the pathogenesis of 2 genetically different groups of ectodermal dysplasia syndromes into a common molecular pathway.

BackgroundMicroRNAs (miRNAs) are a class of small non-coding RNAs that have recently emerged as important regulators of gene expression. They negatively regulate gene expression post-transcriptionally by translational repression and target mRNA degradation. miRNAs have been shown to play crucial roles in muscle development and in regulation of muscle cell proliferation and differentiation.Methodology/Principal FindingsBy comparing miRNA expression profiling of proliferating myoblasts versus differentiated myotubes, a number of modulated miRNAs, not previously implicated in regulation of myogenic differentiation, were identified. Among these, miR-221 and miR-222 were strongly down-regulated upon differentiation of both primary and established myogenic cells. Conversely, miR-221 and miR-222 expression was restored in post-mitotic, terminally differentiated myotubes subjected to Src tyrosine kinase activation. By the use of specific inhibitors we provide evidence that expression of miR-221 and miR-222 is under the control of the Ras-MAPK pathway. Both in myoblasts and in myotubes, levels of the cell cycle inhibitor p27 inversely correlated with miR-221 and miR-222 expression, and indeed we show that p27 mRNA is a direct target of these miRNAs in myogenic cells. Ectopic expression of miR-221 and miR-222 in myoblasts undergoing differentiation induced a delay in withdrawal from the cell cycle and in myogenin expression, followed by inhibition of sarcomeric protein accumulation. When miR-221 and miR-222 were expressed in myotubes undergoing maturation, a profound alteration of myofibrillar organization was observed.Conclusions/SignificancemiR-221 and miR-222 have been found to be modulated during myogenesis and to play a role both in the progression from myoblasts to myocytes and in the achievement of the fully differentiated phenotype. Identification of miRNAs modulating muscle gene expression is crucial for the understanding of the circuits controlling skeletal muscle differentiation and maintenance.

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.

1. Five presynaptic toxins have been isolated in pure from from the venom of Bungarus multicinctus and Bungarus caeruleus and named β1, β2, β3, β4, and β‐ceruleotoxin. 2. They differ in electrophoretic mobility and amino acid composition, while all have the same molecular weight (22000) and are composed of two subunits of molecular weight 9000 and 12000. 3. The toxins have phospholipase A activity when assayed with both natural and synthetic phospholipids, and this activity requires the presence of Ca2+ ions. 4. β‐Bungarotoxin (β3) binds 1 mol of Ca2+ per mol of protein and this binding induces a conformational change as detected by fluorescence measurements in the presence of the dye 8‐anilino‐1‐naphthalene sulfonic acid. 5. The phospholipase activity of all the toxins is lost when a critical histidine residue is modified with p‐bromophenancyl bromide. 6. As a result of the modification the lethality of the toxins is greatly reduced. 7. Native toxin causes a rapid decrease in amplitude of end‐plate potentials, followed by a transient increase and subsequent decrease, until transmitter release is completely abolished. The modified toxin still causes the early decrease in release but toxin action does not progress to complete block. 8. The rate of blockage of transmitter release by native toxin is reduced in the presence of modified toxin. 9. It is concluded that phospholipase activity plays an important role in the action of this class of toxins at the neuromuscular junction.