ABSTRACT
The metalloprotease-dependent extracellular domain cleavage of the adhesion molecule CD44 is frequently observed in human tumors and is thought to promote metastasis. This cleavage is followed by gamma-secretase-dependent release of CD44 intracellular domain (CD44-ICD), which exhibits nuclear signaling activity. Using a reversible Ret-dependent oncogenic conversion model and a restricted proteomic approach, we identified a positive correlation between the neoplastic transformation of Rat-1 cells and the expression of standard CD44. In these transformed cells, CD44 was found to undergo a sequential metalloprotease and gamma-secretase cleavage, resulting in an increase in expression of CD44-ICD. We showed that this proteolytic fragment possesses a transforming activity. In support of this role, a significant and specific reduction in Ret-induced transformation of Rat-1 cells was observed following drug-mediated inhibition of gamma-secretase. Taken together, these findings suggest that the shedding of CD44 may not only modulate metastasis but also affects earlier events in tumorigenesis through the release of CD44-ICD.
Subject(s)
Cell Transformation, Neoplastic/metabolism , Endopeptidases/metabolism , Hyaluronan Receptors/metabolism , Amino Acid Sequence , Amyloid Precursor Protein Secretases , Animals , Aspartic Acid Endopeptidases , Cell Membrane/drug effects , Cell Membrane/metabolism , Cell Transformation, Neoplastic/genetics , Detergents/pharmacology , Fibroblasts/enzymology , Fibroblasts/metabolism , Fibroblasts/pathology , Hyaluronan Receptors/biosynthesis , Hyaluronan Receptors/genetics , Molecular Sequence Data , Mutagenesis, Site-Directed , Protein Isoforms , Protein Structure, Tertiary , Rats , Up-RegulationSubject(s)
Lysophospholipids/physiology , Multienzyme Complexes , Animals , Catalytic Domain , Cell Movement/physiology , Glucose-6-Phosphate Isomerase/metabolism , Glycoproteins/metabolism , Humans , Lysophospholipids/chemistry , Lysophospholipids/pharmacology , Phosphodiesterase I , Phosphoric Diester Hydrolases/genetics , Phosphoric Diester Hydrolases/metabolism , Pyrophosphatases , Receptors, Cell Surface/physiology , Signal Transduction/physiologyABSTRACT
BACKGROUND: Cellular cholesterol is a vital component of the cell membrane. Its concentration is tightly controlled by mechanisms that remain only partially characterized. In this study, we describe a late endosome/lysosomes-associated protein whose expression level affects cellular free cholesterol content. METHODOLOGY/PRINCIPAL FINDINGS: Using a restricted proteomic analysis of detergent-resistant membranes (DRMs), we have identified a protein encoded by gene C11orf59. It is mainly localized to late endosome/lysosome (LE/LY) compartment through N-terminal myristoylation and palmitoylation. We named it Pdro for protein associated with DRMs and endosomes. Very recently, three studies have reported on the same protein under two other names: the human p27RF-Rho that regulates RhoA activation and actin dynamics, and its rodent orthologue p18 that controls both LE/LY dynamics through the MERK-ERK pathway and the lysosomal activation of mammalian target of rapamycin complex 1 by amino acids. We found that, consistent with the presence of sterol-responsive element consensus sequences in the promoter region of C11orf59, Pdro mRNA and protein expression levels are regulated positively by cellular cholesterol depletion and negatively by cellular cholesterol loading. Conversely, Pdro is involved in the regulation of cholesterol homeostasis, since its depletion by siRNA increases cellular free cholesterol content that is accompanied by an increased cholesterol efflux from cells. On the other hand, cells stably overexpressing Pdro display reduced cellular free cholesterol content. Pdro depletion-mediated excess cholesterol results, at least in part, from a stimulated low-density lipoprotein (LDL) uptake and an increased cholesterol egress from LE/LY. CONCLUSIONS/SIGNIFICANCE: LDL-derived cholesterol release involves LE/LY motility that is linked to actin dynamics. Because Pdro regulates these two processes, we propose that modulation of Pdro expression in response to sterol levels regulates LDL-derived cholesterol through both LDL uptake and LE/LY dynamics, to ultimately control free cholesterol homeostasis.
Subject(s)
Carrier Proteins/metabolism , Cholesterol/metabolism , Endosomes/metabolism , Homeostasis , Lysosomes/metabolism , Amino Acid Sequence , Base Sequence , Biological Transport , Carrier Proteins/chemistry , Carrier Proteins/genetics , DNA Primers , Flow Cytometry , Fluorescent Antibody Technique , Gene Knockdown Techniques , Humans , Intracellular Signaling Peptides and Proteins , Lipoproteins, LDL/metabolism , Molecular Sequence Data , Reverse Transcriptase Polymerase Chain Reaction , Tandem Mass SpectrometryABSTRACT
Germ line alterations in BRCA1 (breast cancer susceptibility gene 1) are associated with an increased susceptibility to breast and ovarian cancer. BRCA1 acts as a scaffold protein implicated in multiple cellular functions, such as transcription, DNA repair, and ubiquitination. However, the molecular mechanisms responsible for tumorigenesis are not yet fully understood. We have recently demonstrated that BRCA1 interacts in vivo with acetyl coenzyme A carboxylase alpha (ACCA) through its tandem of BRCA1 C terminus (BRCT) domains. To understand the biological function of the BRCA1.ACCA complex, we sought to determine whether BRCA1 is a regulator of lipogenesis through its interaction with ACCA. We showed here that RNA inhibition-mediated down-regulation of BRCA1 expression induced a marked increase in the fatty acid synthesis. We then delineated the biochemical characteristics of the complex and found that BRCA1 interacts solely with the phosphorylated and inactive form of ACCA (P-ACCA). Finally, we demonstrated that BRCA1 affects lipid synthesis by preventing P-ACCA dephosphorylation. These results suggest that BRCA1 affects lipogenesis through binding to P-ACCA, providing a new mechanism by which BRCA1 may exert a tumor suppressor function.
Subject(s)
Acetyl-CoA Carboxylase/chemistry , BRCA1 Protein/physiology , Gene Expression Regulation, Neoplastic , Lipids/chemistry , Acetyl-CoA Carboxylase/metabolism , BRCA1 Protein/metabolism , Cell Line, Tumor , DNA Repair , Down-Regulation , Fatty Acids/metabolism , Gene Silencing , Genes, Tumor Suppressor , Genetic Vectors , Glutathione Transferase/metabolism , Humans , Immunoblotting , Immunoprecipitation , Phosphorylation , Plasmids/metabolism , Protein Binding , Protein Structure, Tertiary , RNA/chemistry , RNA Interference , Recombinant Fusion Proteins/chemistry , Reverse Transcriptase Polymerase Chain Reaction , Time Factors , Transfection , Ubiquitin/chemistry , Up-RegulationABSTRACT
Dominant-activating mutations in the RET (rearranged during transfection) proto-oncogene, a receptor tyrosine kinase, are causally associated with the development of multiple endocrine neoplasia type 2A (MEN2A) syndrome. Such oncogenic RET mutations induce its ligand-independent constitutive activation, but whether it spreads identical signaling to ligand-induced signaling is uncertain. To address this question, we designed a cellular model in which RET can be activated either by its natural ligand, or alternatively, by controlled dimerization of the protein that mimics MEN2A dimerization. We have shown that controlled dimerization leaves proximal RET signaling intact but impacts substantially on the tuning of the distal AKT kinase activation (delayed and sustained). In marked contrast, distal activation of ERK remained unaffected. We further demonstrated that specific temporal adjustment of ligand-induced AKT activation is dependent upon a lipid-based cholesterol-sensitive environment, and this control step is bypassed by MEN2A RET mutants. Therefore, these studies revealed that MEN2A mutations propagate previously unappreciated subtle differences in signaling pathways and unravel a role for lipid rafts in the temporal regulation of AKT activation.
Subject(s)
Glial Cell Line-Derived Neurotrophic Factor Receptors/metabolism , Multiple Endocrine Neoplasia Type 2a/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Proto-Oncogene Proteins c-ret/metabolism , Signal Transduction , Dimerization , Glial Cell Line-Derived Neurotrophic Factor/metabolism , Glial Cell Line-Derived Neurotrophic Factor Receptors/genetics , Humans , Mitogen-Activated Protein Kinase 3/metabolism , Mutation , Proto-Oncogene Mas , Proto-Oncogene Proteins c-ret/geneticsABSTRACT
Lysophosphatidic acid (LPA) is a receptor-active lipid mediator with a broad range of biological effects. Ovarian cancer cells synthesize LPA, which promotes their motility, growth, and survival. We show that a murine homolog of a human protein previously reported to hydrolyze LPA is a highly selective detergent-stimulated LPA phosphatase that can be used to detect and quantitate LPA. Use of this protein in novel enzymatic assay demonstrates that SK-OV-3 ovarian cancer cells release physiologically relevant levels of biologically active LPA into the extracellular space. LPA release is markedly increased by nucleotide agonists acting through a P2Y4 purinergic receptor. Promotion of LPA formation by nucleotides is accompanied by stimulation of phospholipase D (PLD) activity. Overexpression of both PLD1 and PLD2 in SK-OV-3 cells produces active enzymes, but only overexpression of PLD2 results in significant amplification of both nucleotide-stimulated PLD activity and LPA production. SK-OV-3 cells express and secrete a phospholipase A2 activity that can generate LPA from the lipid product of PLD, phosphatidic acid. Our results identify a novel role for nucleotides in the regulation of ovarian cancer cells and suggest an indirect but critical function for PLD2 in agonist-stimulated LPA production.