epidermal-growth-factor and benzyloxycarbonylleucyl-leucyl-leucine-aldehyde

Protein ubiquitylation regulates diverse cellular processes via distinct ubiquitin chains that differ by linkage type and length. However, a comprehensive method for measuring these properties has not been developed. Here we describe a method for assessing the length of substrate-attached polyubiquitin chains, "ubiquitin chain protection from trypsinization (Ub-ProT)." Using Ub-ProT, we found that most ubiquitylated substrates in yeast-soluble lysate are attached to chains of up to seven ubiquitin molecules. Inactivation of the ubiquitin-selective chaperone Cdc48 caused a dramatic increase in chain lengths on substrate proteins, suggesting that Cdc48 complex terminates chain elongation by substrate extraction. In mammalian cells, we found that ligand-activated epidermal growth factor receptor (EGFR) is rapidly modified with K63-linked tetra- to hexa-ubiquitin chains following EGF treatment in human cells. Thus, the Ub-ProT method can contribute to our understanding of mechanisms regulating physiological ubiquitin chain lengths and composition.

Topics: Adaptor Proteins, Signal Transducing; Autophagy-Related Proteins; Carrier Proteins; Cell Cycle Proteins; Epidermal Growth Factor; ErbB Receptors; HeLa Cells; Humans; Leupeptins; Polyubiquitin; Proteins; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Ubiquitin; Ubiquitinated Proteins; Ubiquitination; Valosin Containing Protein

Protein responses to extracellular cues are governed by gene transcription, mRNA degradation and translation, and protein degradation. In order to understand how these time-dependent processes cooperate to generate dynamic responses, we analyzed the response of human mammary cells to the epidermal growth factor (EGF). Integrating time-dependent transcript and protein data into a mathematical model, we inferred for several proteins their pre-and post-stimulus translation and degradation coefficients and found that they exhibit complex, time-dependent variation. Specifically, we identified strategies of protein production and degradation acting in concert to generate rapid, transient protein bursts in response to EGF. Remarkably, for some proteins, for which the response necessitates rapidly decreased abundance, cells exhibit a transient increase in the corresponding degradation coefficient. Our model and analysis allow inference of the kinetics of mRNA translation and protein degradation, without perturbing cells, and open a way to understanding the fundamental processes governing time-dependent protein abundance profiles.

Topics: Computer Simulation; Early Growth Response Protein 1; Epidermal Growth Factor; Genes, Immediate-Early; Humans; Leupeptins; Phenotype; Proteasome Inhibitors; Protein Biosynthesis; Proteolysis; Proto-Oncogene Proteins c-myc; RNA Precursors; RNA, Messenger; Time Factors

ACK1 (activated Cdc42-associated kinase 1) is a cytoplasmic tyrosine kinase implicated in trafficking through binding to epidermal growth factor (EGF) receptor and clathrin. Here, we have identified a new ACK1-binding partner, the E3 ubiquitin ligase Nedd4-2, which binds ACK1 via a conserved PPXY-containing region. We show that this motif also binds Nedd4-related proteins and several other WW domain-containing proteins, including the tumor suppressor oxidoreductase Wwox. In HeLa cells ACK1 colocalizes with Nedd4-2 in clathrin-rich vesicles, requiring this PPXY motif. Nedd4-2 strongly down-regulates ACK1 levels when coexpressed, and this process can be blocked by proteasome inhibitor MG132. ACK1 degradation via Nedd4 requires their mutual interaction and a functional E3 ligase; it is also driven by ACK1 activity. ACK1 is polyubiquitinated in vivo, and dominant inhibitory Nedd4 blocks endogenous ACK1 turnover in response to acute EGF treatment. Because EGF stimulation activates ACK1 ( Galisteo, M., Y., Y., Urena, J., and Schlessinger, J. (2006) Proc. Natl. Acad. Sci. U. S. A. 103, 9796-9801 ), our result suggest that EGF receptor-mediated ACK1 activation allows Nedd4-2 to drive kinase degradation. Thus the interplay between Nedd4-2-related E3 ligases that regulate ACK1 levels and Cbl that modifies EGF receptor impinges on cell receptor dynamics. These processes are particularly pertinent given the report of genomic amplification of the ACK1 locus in metastatic tumors.

Topics: Amino Acid Motifs; Animals; Chlorocebus aethiops; Clathrin; Clathrin-Coated Vesicles; COS Cells; Cysteine Proteinase Inhibitors; Down-Regulation; Endosomal Sorting Complexes Required for Transport; Enzyme Activation; Epidermal Growth Factor; ErbB Receptors; Gene Expression Regulation, Enzymologic; HeLa Cells; Humans; Leupeptins; Nedd4 Ubiquitin Protein Ligases; Neoplasm Metastasis; Neoplasms; Oxidoreductases; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein Binding; Protein Structure, Tertiary; Protein Transport; Protein-Tyrosine Kinases; Proto-Oncogene Proteins c-cbl; Tumor Suppressor Proteins; Ubiquitin-Protein Ligases; Ubiquitination; WW Domain-Containing Oxidoreductase

We describe here an important function of the novel calmodulin kinase I isoform, pregnancy-upregulated nonubiquitous calmodulin kinase (Pnck). Pnck (also known as CaM kinase Ibeta(2)) was previously shown to be differentially overexpressed in a subset of human primary breast cancers, compared with benign mammary epithelial tissue. In addition, during late pregnancy, Pnck mRNA was shown to be strongly upregulated in epithelial cells of the mouse mammary gland exhibiting decreased proliferation and terminal differentiation. Pnck mRNA is also significantly upregulated in confluent and serum-starved cells, compared with actively growing proliferating cells (Gardner HP, Seung HI, Reynolds C, Chodosh LA. Cancer Res 60: 5571-5577, 2000). Despite these suggestive data, the true physiological role(s) of, or the signaling mechanism(s) regulated by Pnck, remain unknown. We now report that epidermal growth factor receptor (EGFR) levels are significantly downregulated in a ligand-independent manner in human embryonic kidney-293 (HEK-293) cells overexpressing Pnck. MAP kinase activation was strongly inhibited by EGFR downregulation in the Pnck-overexpressing cells. The EGFR downregulation was not the result of reduced transcription of the EGFR gene but from protea-lysosomal degradation of EGFR protein. Knockdown of endogenous Pnck mRNA levels by small interfering RNA transfection in human breast cancer cells resulted in upregulation of unliganded EGFR, consistent with the effects observed in the overexpression model of Pnck-mediated ligand-independent EGFR downregulation. Pnck thus emerges as a new component of the poorly understood mechanism of ligand-independent EGFR degradation, and it may represent an attractive therapeutic target in EGFR-regulated oncogenesis.

Topics: Adaptor Proteins, Signal Transducing; Calcium-Calmodulin-Dependent Protein Kinase Type 1; Cell Line; Down-Regulation; Epidermal Growth Factor; Epithelial Cells; ErbB Receptors; Extracellular Signal-Regulated MAP Kinases; Gene Expression; Humans; Intracellular Signaling Peptides and Proteins; Leupeptins; Ligands; Lysosomes; Macrolides; Phosphoproteins; Phosphorylation; Protease Inhibitors; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein Kinase Inhibitors; Quinazolines; Recombinant Fusion Proteins; RNA, Small Interfering; Shc Signaling Adaptor Proteins; Src Homology 2 Domain-Containing, Transforming Protein 1; Transfection; Tyrphostins

Numerous studies implicate proteasomes in the regulation of EGF receptor (EGFR) endocytosis on the basis of the ability of inhibitors to decrease EGFR degradation, but the exact mechanisms remain obscure. We demonstrated that EGFR itself is not a direct target for proteasome, since it is delivered to lysosomes intact. Evidence is presented that the inhibitory effect of MG132 on EGF degradation is due mostly to free ubiquitin depletion resultant from the suppression of proteasomal functioning by MG132. By subcellular fractionation, we show two MG132-sensitive steps in the EGFR degradation pathway: sorting from early (EE) to late (LE) endosomes, and late stage of LE maturation. MG132 treatment resulted in stabilization of EGFR tyrosine phosphorylation and its association with c-Cbl. Nevertheless, ubiquitination of EGFR at late stages of endocytosis was significantly lower than that in control cells. Highly ubiquitinated forms of EGFR demonstrated more sensitivity to MG132 treatment.

Topics: Cell Line, Tumor; Endocytosis; Endosomes; Epidermal Growth Factor; ErbB Receptors; Humans; Leupeptins; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein Processing, Post-Translational; Proto-Oncogene Proteins c-cbl; Signal Transduction; Time Factors; Ubiquitin

EGF suppresses proteolysis via class 1 phosphatidylinositol 3-kinase (PI3K) in renal tubular cells. EGF also increases the abundance of glycolytic enzymes (e.g., glyceraldehyde-3-phosphate dehydrogenase [GAPDH]) and transcription factors (e.g., pax2) that are degraded by the lysosomal pathway of chaperone-mediated autophagy. To determine if EGF regulates chaperone-mediated autophagy through PI3K signaling, this study examined the effect of inhibiting PI3K and its downstream mediators Akt and the mammalian target of rapamycin (mTOR). Inhibition of PI3K with LY294002 prevented EGF-induced increases in GAPDH and pax2 abundance in NRK-52E renal tubular cells. Similar results were seen with an adenovirus encoding a dominant negative Akt (DN Akt). Expression of a constitutively active Akt increased GAPDH and pax2 abundance. An mTOR inhibitor, rapamycin, did not prevent EGF-induced increases in these proteins. Neither DN Akt nor rapamycin alone had an effect on total cell protein degradation, but both partially reversed EGF-induced suppression of proteolysis. DN Akt no longer affected proteolysis after treatment with a lysosomal inhibitor, methylamine. In contrast, methylamine or the inhibitor of macroautophagy, 3-methyladenine, did not prevent rapamycin from partially reversing the effect of EGF on proteolysis. Notably, rapamycin did not increase autophagasomes detected by monodansylcadaverine staining. Blocking the proteasomal pathway with either MG132 or lactacystin prevented rapamycin from partially reversing the effect of EGF on proteolysis. It is concluded that EGF regulates pax2 and GAPDH abundance and proteolysis through a PI3K/Akt-sensitive pathway that does not involve mTOR. Rapamycin has a novel effect of regulating proteasomal proteolysis in cells that are stimulated with EGF.

Topics: Acetylcysteine; Adenine; Animals; Autophagy; Cell Line; Chromones; Epidermal Growth Factor; Glyceraldehyde-3-Phosphate Dehydrogenases; Kidney Tubules; Leupeptins; Lysosomes; Methylamines; Morpholines; PAX2 Transcription Factor; Peptide Hydrolases; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Proteasome Endopeptidase Complex; Protein Kinases; Proto-Oncogene Proteins c-akt; Rats; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

In human parturition, uterotonic prostaglandins (PGs) arise predominantly via increased expression of cyclooxygenase-2 (COX-2 [also known as prostaglandin synthase 2]) within intrauterine tissues. Interleukin-1 (IL-1) and epidermal growth factor (EGF), both inducers of COX-2 transcription, are among numerous factors that accumulate within amniotic fluid with advancing gestation. It was previously demonstrated that EGF could potentiate IL-1beta-driven PGE(2) production in amnion and amnion-derived (WISH) cells. To define the mechanism for this observation, we hypothesized that EGF and IL-1beta might exhibit synergism in regulating COX-2 gene expression. In WISH cells, combined treatment with EGF and IL-1beta resulted in a greater-than-additive increase in COX-2 mRNA relative to challenge with either agent independently. Augmentation of IL-1beta-induced transactivation by EGF was not observed in cells harboring reporter plasmids bearing nuclear factor-kappa B (NFkappaB) regulatory elements alone, but was evident when a fragment (-891/ +9) of the COX-2 gene 5'-promoter was present. Both agents transiently activated intermediates of multiple signaling pathways potentially involved in the regulation of COX-2 gene expression. The 26 S proteasome inhibitor, MG-132, selectively abrogated IL-1beta-driven NFkappaB activation and COX-2 mRNA expression. Only pharmacologic blockade of the p38 mitogen-activated protein kinase eliminated COX-2 expression following EGF stimulation. We conclude that EGF and IL-1beta appear to signal through different signaling cascades leading to COX-2 gene expression. IL-1beta employs the NFkappaB pathway predominantly, while the spectrum of EGF signaling is broader and includes p38 kinase. The synergism observed between IL-1beta and EGF does not rely on augmented NFkappaB function, but rather, occurs through differential use of independent response elements within the COX-2 promoter.

Topics: Amnion; Cell Line; Cyclooxygenase 2; Drug Synergism; Enzyme Inhibitors; Epidermal Growth Factor; Female; Gene Expression; Humans; Interleukin-1; Leupeptins; Membrane Proteins; NF-kappa B; p38 Mitogen-Activated Protein Kinases; Promoter Regions, Genetic; Prostaglandin-Endoperoxide Synthases; Recombinant Proteins; RNA, Messenger; Signal Transduction; Time Factors; Up-Regulation

The effect of proteasomal activity suppression induced by MG132, a synthetic proteasomal inhibitor of EGF-receptor complexes endocytosis in human epidermoid carcinoma A431 cell line, was studied. Using subcellular fractionation in 17% Percoll gradient, it was demonstrated that the addition of MG132 to the cells 15 min following stimulation of EGF endocytosis resulted in a slight accumulation of 125I-EGF in early endosomes, and in much more significant accumulation of the labeled growth factor in late endosomes/lysosomes, as compared to untreated cells. The release of 125I-EGF degradation products into the incubation medium was significantly (3-12-fold) inhibited in the presence of MG132. At the same time biochemical analysis has demonstrated that the EGF receptor itself is not a direct target of proteasomes, since it is revealed as a full-length protein with native mol. mass (170 kDa) in fractions of early and late endosomes and lysosomes. Possible mechanisms of the MG132 effect on intracellular processing of EGF-receptor complexes are discussed.

Topics: Cell Fractionation; Cell Line, Tumor; Endocytosis; Epidermal Growth Factor; ErbB Receptors; Humans; Iodine Radioisotopes; Leupeptins; Proteasome Endopeptidase Complex; Proteasome Inhibitors

Phospholipase C gamma 1 (PLC gamma 1), an enzyme participating in phosphoinositide turnover, is one of the key elements in cell signaling. Here it is shown that treatment of A431 carcinoma cells with proteasome inhibitors Mg132 and lactacystin results in increasing the PLC gamma 1 intracellular level. Simultaneously, several additional bands with lower electrophoretic mobilities were detected on immunoblots, using anti-PLC gamma 1 antibodies. PLC gamma 1 ubiquitinilation was shown using immunoprecepitation. In control A 431 cells, PLC gamma 1 is ubiquitinilated, but the addition of EGF greatly induces the ubiquitinilation of the protein. Association of PLC gamma 1 with ubiquitin-ligase c-Cb1 was shown. Dynamics of ubiquitinilation under EGF treatment is in a close agreement with that of association of PLC gamma 1 and c-Cb1. It is concluded that PLC gamma 1 is ubiquitinilated and degraded by proteasomes. PLC gamma 1 ubiquitinilation is an EGF-dependent process.

Topics: Acetylcysteine; Carcinoma, Squamous Cell; Cell Line, Tumor; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Epidermal Growth Factor; Epithelial Cells; Humans; Leupeptins; Multienzyme Complexes; Phospholipase C gamma; Proteasome Endopeptidase Complex; Type C Phospholipases; Ubiquitins

Unstimulated PC12 pheochromocytoma cells contain many proteins that bound to 14-3-3s in competition with a 14-3-3-binding peptide. Additional proteins, including one of 39 kDa (p39), became capable of binding to 14-3-3s in phosphatidylinositol 3-kinase-dependent responses to epidermal growth factor or nerve growth factor in vivo. The growth factor regulation was unaffected by inhibitors of the mitogen- or stress-activated protein kinase pathways, or by glucose starvation, but was blocked by amino acid starvation and only partially blocked by rapamycin. p39 in extracts of unstimulated, nutrient-fed cells, but not nutrient-starved cells, was able to bind to 14-3-3s after phosphorylation by protein kinase B (PKB) in vitro. Nutrient starvation did not affect the growth factor-stimulated activation of PKB in vivo. Either cycloheximide (CHX) or the cysteine protease inhibitor, MG132, restored the responsiveness of p39 to growth factors in nutrient-starved cells. In contrast, MG132 could not replace amino acids in supporting the growth factor-stimulated phosphorylation of two downstream targets of mTOR (mammalian target of rapamycin), namely eukaryotic initiation factor 4E binding protein 1 (4E-BP1) and p70 S6 kinase. CHX permitted complete growth factor-stimulated phosphorylation of both 4E-BP1 and p70 S6 kinase in nutrient- starved cells; however, unlike p39, phosphorylation of these proteins was blocked by rapamycin. These findings implicate PKB (or an enzyme with similar specificity) in the growth factor-triggered phosphorylation of p39. In addition, amino acid starvation induces a CHX- and MG132-sensitive pathway that targets p39 and appears to be distinct from the mechanism of regulation of 4E-BP1 and p70 S6 kinase.

Topics: 14-3-3 Proteins; Amino Acids; Animals; Carrier Proteins; Cell Extracts; Chromones; Cycloheximide; Enzyme Inhibitors; Epidermal Growth Factor; Glucose; Growth Substances; Intracellular Signaling Peptides and Proteins; Leupeptins; Morpholines; Nerve Growth Factor; PC12 Cells; Phosphoproteins; Phosphorylation; Protein Serine-Threonine Kinases; Proteins; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Rats; Ribosomal Protein S6 Kinases, 70-kDa; Tyrosine 3-Monooxygenase

EGF, but not TGF alpha, efficiently induces degradation of the EGF receptor (EGFR). We show that EGFR was initially polyubiquitinated to the same extent upon incubation with EGF and TGF alpha, whereas the ubiquitination was more sustained by incubation with EGF than with TGF alpha. Consistently, the ubiquitin ligase c-Cbl was recruited to the plasma membrane upon activation of the EGFR with EGF and TGF alpha, but localized to endosomes only upon activation with EGF. EGF remains bound to the EGFR upon endocytosis, whereas TGF alpha dissociates from the EGFR. Therefore, the sustained polyubiquitination is explained by EGF securing the kinase activity of endocytosed EGFR. Overexpression of the dominant negative N-Cbl inhibited ubiquitination of the EGFR and degradation of EGF and EGFR. This demonstrates that EGF-induced ubiquitination of the EGFR as such is important for lysosomal sorting. Both lysosomal and proteasomal inhibitors blocked degradation of EGF and EGFR, and proteasomal inhibitors inhibited translocation of activated EGFR from the outer limiting membrane to inner membranes of multivesicular bodies (MVBs). Therefore, lysosomal sorting of kinase active EGFR is regulated by proteasomal activity. Immuno-EM showed the localization of intact EGFR on internal membranes of MVBs. This demonstrates that the EGFR as such is not the proteasomal target.

Topics: Acetylcysteine; Ammonium Chloride; Animals; Cell Membrane; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Cytoplasmic Vesicles; Endocytosis; Endopeptidases; Epidermal Growth Factor; ErbB Receptors; Humans; Immunohistochemistry; Intracellular Membranes; Leupeptins; Microscopy, Confocal; Microscopy, Electron; Multienzyme Complexes; Protease Inhibitors; Proteasome Endopeptidase Complex; Protein Transport; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-cbl; Transforming Growth Factor alpha; Ubiquitin-Protein Ligases; Ubiquitins

Kinin B(1) receptor (B(1)R) expression and the importance of the transcription factor nuclear factor (NF)-kappa B in this process were evaluated in models based on the rabbit aorta: freshly isolated tissue (postisolation induction) and cultured smooth muscle cells (SMCs). A 3-h incubation of freshly isolated tissues determined a sharp B(1)R mRNA increase (RT-PCR). Coincubation of tissues with a stimulus (interleukin-1 beta, fetal bovine serum, epidermal growth factor, or cycloheximide) further increased mRNA levels. Cultured SMCs possessed a basal population of surface B(1)Rs ([(3)H]Lys-des-Arg(9)-bradykinin binding) that was upregulated by treatments with the same set of stimuli (binding, mRNA, nuclear runon). Pharmacological inhibitors of NF-kappa B (MG-132, BAY 11-7082, dexamethasone) or actinomycin D reduced the postisolation induction of B(1)Rs in fresh aortic tissue (contractility or mRNA) and the cytokine effect on cells (mRNA, binding). NF-kappa B may be a common mediator of various stimuli that increase B(1)R gene transcription in the rabbit aorta, including tissue isolation, but cycloheximide also stabilizes B(1)R mRNA. The SMC models faithfully mimic the in vivo situation with regard to B(1)R regulation.

Topics: Animals; Antineoplastic Agents; Aorta; Blood Proteins; Cells, Cultured; Dexamethasone; Dose-Response Relationship, Drug; Epidermal Growth Factor; Interleukin-1; Leupeptins; Male; Muscle, Smooth, Vascular; NF-kappa B; Nitriles; Nucleic Acid Synthesis Inhibitors; Organic Chemicals; Protein Synthesis Inhibitors; Rabbits; Receptor, Bradykinin B1; Receptors, Bradykinin; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sulfones; Up-Regulation; Vasoconstrictor Agents; Vasodilator Agents

Epidermal growth factor (EGF) receptor-overexpressing p53-deficient A431 cells response to toxic dose of EGF by G1 arrest and apoptosis was studied. We previously reported an increased expression of growth arrest and DNA-damage-inducible gene, Gadd45, in EGF-overexposed A431 cells. The mechanism for this induction was increased half-lives of mRNA and protein. In this study, using phorbol ester (a PKC activator) and specific inhibitors of PKC isoforms, we showed that protein kinase C-delta (PKCdelta) was involved in the increase of Gadd45 protein stability. We further demonstrated that Gadd45 is ubiquitinated and is regulated by proteolysis. While EGF induced ubiquitination of total cellular proteins, there was a decrease in Gadd45 ubiquitination, which could be inhibited by Rottlerin, a PKCdelta-specific inhibitor. These results suggest that an increase in Gadd45 stability may involve PKCdelta-dependent ubiquitin-proteasome pathway.

Topics: Acetophenones; Benzopyrans; Cell Division; Cycloheximide; DNA Damage; Enzyme Activation; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Flavonoids; GADD45 Proteins; Humans; Imidazoles; Intracellular Signaling Peptides and Proteins; Isoenzymes; Kinetics; Leupeptins; Mitogen-Activated Protein Kinases; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Protein Kinase C; Protein Kinase C-delta; Proteins; Pyridines; Recombinant Proteins; Staurosporine; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured; Ubiquitins

Tumor necrosis factor alpha (TNF-alpha) has been suggested to induce chondrocytic chondrolysis in both inflammatory and degenerative joint diseases. However, its intracellular signaling pathway leading to the chondrolysis has not been studied in detail. Thus, we investigated whether TNF-alpha activates a transcription factor nuclear factor kappaB (NF-kappaB) in human chondrocyte-like cells (HCS-2/8) and induces the expression of genes involved in the degradation of cartilage matrix. Treatment of the cells with TNF-alpha markedly increased the levels of matrix metalloproteinase 1 (MMP-1), MMP-3, intercellular adhesion molecule 1 (ICAM-1), and cyclo-oxygenase 2 (COX-2) messenger RNAs (mRNAs). The increase in the mRNAs was associated with the activation of p65/p50 heterodimer NF-kappaB. IkappaB-alpha and IkappaB-beta, cytoplasmic molecules preventing the nuclear translocation of NF-kappaB, were degraded rapidly by TNF-alpha followed by their synthesis to the basal level. Treatment with proteasome inhibitors inhibited the degradation of both IkappaB-alpha and IkappaB-beta and prevented the TNF-alpha-dependent nuclear translocation of p65. Furthermore, the inhibitors completely prevented the TNF-alpha-dependent induction of MMP-1, MMP-3, ICAM-1, and COX-2 mRNAs. Thus, it is shown that the activation of p65/p50 NF-kappaB by TNF-alpha plays a cardinal role in inducing the expression of MMP-1, MMP-3, ICAM-1, and COX-2 genes, which are involved in matrix degradation and inflammatory reaction in chondrocytes, leading to chondrocytic chondrolysis.

Topics: Blotting, Western; Chondrocytes; Cyclooxygenase 2; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; DNA-Binding Proteins; Electrophoretic Mobility Shift Assay; Enzyme Induction; Epidermal Growth Factor; Humans; I-kappa B Proteins; Intercellular Adhesion Molecule-1; Isoenzymes; Leupeptins; Matrix Metalloproteinase 1; Matrix Metalloproteinase 3; Membrane Proteins; Multienzyme Complexes; NF-kappa B; NF-KappaB Inhibitor alpha; Prostaglandin-Endoperoxide Synthases; Proteasome Endopeptidase Complex; RNA, Messenger; Time Factors; Transcriptional Activation; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

c-Cbl plays a negative regulatory role in tyrosine kinase signaling by an as yet undefined mechanism. We demonstrate here, using the yeast two-hybrid system and an in vitro binding assay, that the c-Cbl RING finger domain interacts with UbcH7, a ubiquitin-conjugating enzyme (E2). UbcH7 interacted with the wild-type c-Cbl RING finger domain but not with a RING finger domain that lacks the amino acids that are deleted in 70Z-Cbl, an oncogenic mutant of c-Cbl. The in vitro interaction was enhanced by sequences on both the N- and C-terminal sides of the RING finger. In vivo and in vitro experiments revealed that c-Cbl and UbcH7 synergistically promote the ligand-induced ubiquitination of the epidermal growth factor receptor (EGFR). In contrast, 70Z-Cbl markedly reduced the ligand-induced, UbcH7-mediated ubiquitination of the EGFR. MG132, a proteasome inhibitor, significantly prolonged the ligand-induced phosphorylation of both the EGFR and c-Cbl. Thus, c-Cbl plays an essential role in the ligand-induced ubiquitination of the EGFR by a mechanism that involves an interaction of the RING finger domain with UbcH7. This mechanism participates in the down-regulation of tyrosine kinase receptors and loss of this function, as occurs in the naturally occurring 70Z-Cbl isoform, probably contributes to oncogenic transformation.

Topics: Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Epidermal Growth Factor; ErbB Receptors; Leupeptins; Ligands; Ligases; Models, Biological; Multienzyme Complexes; Phosphorylation; Proteasome Endopeptidase Complex; Protein Binding; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-cbl; Recombinant Fusion Proteins; Two-Hybrid System Techniques; Tyrosine; Ubiquitin-Conjugating Enzymes; Ubiquitin-Protein Ligases; Ubiquitins; Zinc Fingers

The combination of epidermal growth factor (EGF) plus transforming growth factor-beta 1 (TGF-beta 1) causes hypertrophy in renal epithelial cells. One mechanism contributing to hypertrophy is that EGF induces activation of the cell cycle and increases protein synthesis, whereas TGF-beta 1 prevents cell division, thereby converting hyperplasia to hypertrophy. To assess whether suppression of proteolysis is another mechanism causing hypertrophy induced by these growth factors, we measured protein degradation in primary cultures of proximal tubule cells and in cultured NRK-52E kidney cells. A concentration of 10(-8) M EGF alone or EGF plus 10(-10) M TGF-beta 1 decreased proteolysis by approximately 30%. TGF-beta 1 alone did not change protein degradation. Using inhibitors, we examined which proteolytic pathway is suppressed. Neither proteasome nor calpain inhibitors prevented the antiproteolytic response to EGF + TGF-beta 1. Inhibitors of lysosomal proteases eliminated the antiproteolytic response to EGF + TGF-beta 1, suggesting that these growth factors act to suppress lysosomal proteolysis. This antiproteolytic response was not caused by impaired EGF receptor signaling, since lysosomal inhibitors did not block EGF-induced protein synthesis. We conclude that suppression of lysosomal proteolysis contributes to growth factor-mediated hypertrophy of cultured kidney cells.

Topics: Animals; Calpain; Cell Cycle; Cell Line; Cells, Cultured; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Epidermal Growth Factor; Humans; Hypertrophy; Kidney Tubules, Proximal; Kinetics; Leucine; Leupeptins; Mitogens; Multienzyme Complexes; Proteasome Endopeptidase Complex; Proteins; Rabbits; Rats; Recombinant Proteins; Transforming Growth Factor beta