ABSTRACT
The short isoform of ErbB3-binding protein 1 (Ebp1), p42, is considered to be a potent tumor suppressor in a number of human cancers, although the mechanism by which it exerts this tumor-suppressive activity is unclear. Here, we report that p42 interacts with the cSH2 domain of the p85 subunit of phosphathidyl inositol 3-kinase (PI3K), leading to inhibition of its lipid kinase activity. Importantly, we found that p42 induces protein degradation of the p85 subunit and further identified HSP70/CHIP complex as a novel E3 ligase for p85 that is responsible for p85 ubiquitination and degradation. In this process, p42 couples p85 to the HSP70/CHIP-mediated ubiquitin-proteasomal system (UPS), thereby promoting a reduction of p85 levels both in vitro and in vivo. Thus, the tumor-suppressing effects of p42 in cancer cells are driven by negative regulation of the p85 subunit of PI3K.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Brain Neoplasms/enzymology , Class Ia Phosphatidylinositol 3-Kinase/metabolism , Glioma/enzymology , HSP70 Heat-Shock Proteins/metabolism , Nuclear Proteins/metabolism , Proteasome Endopeptidase Complex/metabolism , RNA-Binding Proteins/metabolism , Ubiquitin-Protein Ligases/metabolism , Adaptor Proteins, Signal Transducing/genetics , Animals , Binding Sites , Brain Neoplasms/genetics , Brain Neoplasms/pathology , Class Ia Phosphatidylinositol 3-Kinase/genetics , DNA-Binding Proteins , Glioma/genetics , Glioma/pathology , HEK293 Cells , HSP70 Heat-Shock Proteins/genetics , Humans , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Nude , Nuclear Proteins/genetics , PC12 Cells , Protein Isoforms , Protein Stability , Proteolysis , RNA Interference , RNA-Binding Proteins/genetics , Rats , Time Factors , Transfection , Tumor Burden , Ubiquitin-Protein Ligases/deficiency , Ubiquitin-Protein Ligases/genetics , src Homology DomainsABSTRACT
We performed a screening program to identify telomerase inhibitors from our drug source obtained from fungus fermentations, and found that two compounds, CRM646-A and thielavin B, inhibited telomerase activity at doses of 3.2 and 32 microM, respectively. These compounds also inhibited the activity of viral reverse transcriptase at almost the same dose levels which inhibited telomerase activity.
Subject(s)
Enzyme Inhibitors/pharmacology , Hydroxybenzoates/pharmacology , Telomerase/antagonists & inhibitors , Enzyme Inhibitors/chemistry , Humans , Hydroxybenzoates/chemistry , Molecular Structure , Telomerase/metabolism , U937 CellsSubject(s)
Acremonium/chemistry , Acremonium/metabolism , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Heparin Lyase/antagonists & inhibitors , Hydroxybenzoates/chemistry , Hydroxybenzoates/pharmacology , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/isolation & purification , Antineoplastic Agents/pharmacology , Dose-Response Relationship, Drug , Drug Evaluation, Preclinical , Drug Screening Assays, Antitumor , Enzyme Inhibitors/isolation & purification , Fermentation , Heparin/metabolism , Hydroxybenzoates/isolation & purification , Inhibitory Concentration 50 , Magnetic Resonance Spectroscopy , Melanoma/drug therapy , Melanoma/pathology , Mice , Molecular Structure , Neoplasm Invasiveness , Tumor Cells, CulturedABSTRACT
In tsRSV-infected NRK (tsNRK) cells, pp60(v-src) reactivation by temperature-shift from a nonpermissive temperature, 39 C, to a permissive one, 32 degrees C, induced the production of inositol phosphates (IPt) and phosphatidylethanol (PEt). This was accompanied by an increase in membrane-associated protein kinase C (PKC) activity in the absence of exogenous growth factors. However, with serum-stimulation, the amounts of IPt and PEt at 32 degrees C were less than those at 39 degrees C. Pretreatment with PKC inhibitors, Ro-31-8220 and staurosporine, enhanced the accumulation of IPt but not of PEt at 32 degrees C. The tyrosine phosphorylation of phospholipase Cgamma1 (PLCgamma1) was increased either by serum or by pp60(v-src) reactivation. These results suggest that serum transduces its signal through PLCgamma1 mediation, and that pp60(v-src), possibly through the PKC mediation, negatively affects serum-induced PLCgamma1 activation.
Subject(s)
Glycerophospholipids/metabolism , Inositol Phosphates/metabolism , Oncogene Protein pp60(v-src)/metabolism , Animals , Benzoquinones , Cell Line, Transformed , Culture Media , Enzyme Inhibitors/pharmacology , Genes, src , Indoles/pharmacology , Isoenzymes/metabolism , Kidney , Kinetics , Lactams, Macrocyclic , Phospholipase C gamma , Phosphotyrosine/metabolism , Protein Kinase C/antagonists & inhibitors , Protein Kinase C/metabolism , Quinones/pharmacology , Rats , Rifabutin/analogs & derivatives , Staurosporine/pharmacology , Temperature , Type C Phospholipases/metabolismABSTRACT
The effect on the phospholipase C gamma 1 activity of eleven prenylated flavonoids from Sophora flavescens was investigated. These flavonoids exhibited relatively strong inhibitory activity with IC50 values ranged from 7.5 x 10(-6) M to 35 x 10(-5) M with the exception of kushenol H (4) (IC50 value; > 5.3 x 10(-4) M). The presence of C3-OH resulted in a significant diminution of activity and the configuration of C3-OH is likely to be another factor influencing the activity. In addition, hydration of the C-4"'-C-5"' double bond of the lavandulyl side chain caused complete loss of activity. These data suggest that the presence and configuration of C3-OH are related to the inhibitory activity and the lavandulyl side chain is also important for high inhibitory activity against PLC gamma 1.
Subject(s)
Enzyme Inhibitors/pharmacology , Flavonoids/pharmacology , Isoenzymes/antagonists & inhibitors , Type C Phospholipases/antagonists & inhibitors , Animals , Cattle , Cerebellum/enzymology , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/isolation & purification , Flavonoids/chemistry , Flavonoids/isolation & purification , Molecular Structure , Phospholipase C gamma , Structure-Activity RelationshipABSTRACT
Herbimycin A, an inhibitor of protein tyrosine kinases, dose-dependently reduced PDGF-induced inositol phosphates (IPt) accumulation without effect on phosphatidylethanol (PEt) formation in PLC-gamma 1-overexpressing NIH 3T3 (NIH 3T3 gamma 1) cells. The compound also reduced tyrosine phosphorylations of some proteins including PLC-gamma 1 in response to PDGF. On the other hand, phorbol 12-myristate 13-acetate (PMA)-induced phospholipase D (PLD) activation was reduced by herbimycin A in the cells, indicating that the pathways for PLD activation by PDGF and PMA are different from each other. Also, these results suggest that PLC-gamma 1 activation is not always an upstream event for PLD activation and that tyrosine phosphorylation of one or more proteins not affected by herbimycin A should be indispensable for PLD activation in PDGF-stimulated NIH 3T3 gamma 1 cells.
