epidermal-growth-factor and lactacystin

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

Breast cancers often have increased MAPK activity; this pathway may drive breast cancer cell growth by targeting steroid hormone receptors. MAPK phosphorylates human progesterone receptors (PRs) on Ser294, thus regulating several aspects of PR activity. To study the role of PR Ser294 phosphorylation on subcellular distribution, we stably expressed wild-type (wt) or S294A (Ser294 to Ala) PR-B in several cell types. PRs phosphorylated on Ser294 were nuclear. Activation of MAPK induced Ser294 phosphorylation and rapid nuclear translocation of wt, but not S294A, PR-B; both receptors concentrated in the nucleus after progestin treatment. The MAPK kinase inhibitor, U0126, blocked epidermal growth factor but not progestin-induced Ser294 phosphorylation and translocation of wt PR, indicating a novel mechanism for nuclear localization. After progestin treatment, wt PR-B underwent ligand-dependent down-regulation, while S294A PR-B persisted in nuclei. Prolonged treatment with U0126 or the nuclear export inhibitor, leptomycin B, promoted nuclear accumulation of wt PR-B and blocked ligand-dependent PR down-regulation, suggesting that PR degradation occurs in the cytoplasm and requires MAPK-dependent nuclear export. Stabilization of PRs by leptomycin B also blocked PR transcriptional activity, indicating a link between nucleocytoplasmic shuttling, receptor stability, and function. These results support a regulatory role for MAPK in nuclear steroid hormone receptor subcellular localization and coupling to multiple PR functions.

Topics: Acetylcysteine; Active Transport, Cell Nucleus; Breast Neoplasms; Butadienes; Cell Nucleus; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Cytoplasm; Epidermal Growth Factor; Fatty Acids, Unsaturated; Humans; Ligands; Mitogen-Activated Protein Kinase Kinases; Multienzyme Complexes; Mutation; Nitriles; Phosphorylation; Promegestone; Proteasome Endopeptidase Complex; Receptors, Progesterone; Serine; Transcription, Genetic; Tumor Cells, Cultured

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

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

Studies in C. elegans and Drosophila melanogaster suggest that cbl proteins are inhibitors of epidermal growth factor receptor (EGFR) function. Here we describe that overexpression of cbl-b, a homologue of the c-cbl protooncogene, inhibits EGFR-induced apoptosis in MDA-MB-468 breast cancer cells. Overexpression of cbl-b results in a shortened duration of EGFR activation upon EGF stimulation. This is demonstrated by decreased amounts of phosphorylated EGFR as well as by inhibition of multiple downstream signaling pathways. The inhibition of signaling by cbl-b results from increased ubiquitination and degradation of the activated EGFR. The inhibitory effects of cbl-b overexpression on apoptosis and on EGFR signaling are reversed by blocking proteosomal degradation of the EGFR. These data demonstrate that the mechanism by which cbl-b inhibits EGFR-induced apoptosis is by activation-dependent degradation of the EGFR. They imply that this mechanism may be a general one whereby cbl proteins regulate intracellular signaling.

Topics: Acetylcysteine; Adaptor Proteins, Signal Transducing; Apoptosis; Breast Neoplasms; Carrier Proteins; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Epidermal Growth Factor; ErbB Receptors; Humans; Mitogen-Activated Protein Kinase Kinases; Multienzyme Complexes; Phosphoproteins; Proteasome Endopeptidase Complex; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-cbl; Signal Transduction; Tumor Cells, Cultured; Ubiquitin-Protein Ligases; Ubiquitins