ABSTRACT
Elucidating the molecular mechanisms regulating transduction of growth control signals and the discovery of the subversion of these pathways by oncogenes has proven critical in unraveling the biochemical factors leading to cellular transformation. One such line of investigation has been study of the effects of transforming p21Ras on platelet-derived growth factor type-beta receptor (PDGF-betaR) signaling. Platelet-derived growth factor is an important extracellular factor regulating the G0-S phase transition of mesenchymal cells. Expression of activated, oncogenic Kirsten- or Harvey-p21Ras in cells influences PDGF-betaR signaling at multiple levels. At least two separate mechanisms account for defective PDGF-betaR signaling in activated p21Ras-expressing cells: (i) transcriptional down-regulation of PDGF-betaR expression, and (ii) inhibition of ligand-induced PDGF-betaR phosphorylation by a factor which is present in the cellular membrane fraction of fibroblasts expressing activated p21Ras. The state of growth arrest in G0 is associated with increased expression of the PDGF-betaR, and oncogene-transformed cell lines, which fail to undergo growth-arrest following prolonged serum-deprivation, express constitutively low levels of the PDGF-betaR mRNA, and possess greatly reduced numbers of PDGF-BB-binding sites. This repression of PDGF-betaR expression by p21Ras is, at least in large part, transcriptional. The membrane-associated factor induced by oncogenic p21Ras provides a connection between cell morphology and cytoskeletal elements and control of ligand-dependent PDGF-betaR autophosphorylation. Reversion of the transformed phenotype results in the recovery of PDGF-betaR kinase activity. Conversely, disruption of the actin cytoskeleton of untransformed fibroblasts leads to the loss of PDGF-betaR function. These studies define two potential mechanisms for feedback control of PDGF-betaR function by downstream elements in the PDGF signaling pathway. In addition, the connection between cell morphology and the function of the PDGF-betaR established by these studies provides a new mechanistic link between the organization of the cytoskeleton, the Ras-related small G proteins, and the activity of membrane-bound receptor tyrosine kinases.
Subject(s)
Platelet-Derived Growth Factor/physiology , Proto-Oncogene Proteins p21(ras)/physiology , Receptors, Platelet-Derived Growth Factor/physiology , Signal Transduction , Animals , Cell Cycle , Cell Differentiation , Cell Division , Cell Line, Transformed , Cell Size , Cytoskeletal Proteins/metabolism , GTP-Binding Proteins/metabolism , Gene Expression Regulation , Humans , Integrins/physiology , Phosphoric Monoester Hydrolases/metabolism , Phosphorylation , Platelet-Derived Growth Factor/metabolism , Platelet-Derived Growth Factor/pharmacology , Proto-Oncogene Proteins p21(ras)/metabolism , Receptors, Platelet-Derived Growth Factor/antagonists & inhibitors , Receptors, Platelet-Derived Growth Factor/biosynthesis , Signal Transduction/drug effects , rac GTP-Binding Proteins , rho GTP-Binding ProteinsABSTRACT
When treated with millimolar concentrations of butyrate, many cell types undergo growth arrest in the G1 phase of the cell cycle. However, the molecular basis of butyrate-induced G1 arrest has not been elucidated. We have investigated the molecular mechanisms of butyrate-induced G1 arrest in synchronized cultures of untransformed 3T3 fibroblasts. We tested the hypothesis that butyrate-induced growth arrest might be mediated by the p21 cyclin-dependent kinase inhibitor. Sodium butyrate-treated 3T3 cells did, indeed, express elevated levels of p21 mRNA under conditions of G1 arrest. Surprisingly, however, primary cultures of fibroblasts from transgenic p21 "knockout" (p21-/-) mice and fibroblasts from wild-type p21-proficient (p21+/+) mice underwent butyrate-induced G1 arrest with similar dose dependencies. Therefore, p21 expression was not necessary for butyrate-induced G1 arrest. To identify other potential mechanisms of butyrate-induced growth arrest, we analyzed the butyrate sensitivity of key mitogenic signaling events during G1. We found that butyrate inhibited the mitogen-dependent transcriptional induction of cyclin D1 and phosphorylation of retinoblastoma (Rb), both in p21-proficient 3T3 cells and in p21+/+ and p21-/- mouse embryo fibroblasts. Butyrate treatment also prevented mitogen-dependent transcriptional induction of cyclin E and expression of cyclin A, cell cycle events that are temporally distal to expression of cyclin D and are necessary for entry into S phase. Abrogation of a requirement for cyclin D/cyclin-dependent kinase-dependent phosphorylation of Rb (by ectopic expression of the human papilloma virus E7 oncoprotein in 3T3 cells) resulted in decreased sensitivity to the antiproliferative actions of butyrate. Overall, these data show that butyrate-induced G1 arrest is, in large part, independent of p21 induction. Instead, butyrate-induced growth arrest appears to result from perturbation of the Rb signaling axis at the level of or at a stage prior to cyclin D1 expression.
Subject(s)
Butyrates/pharmacology , Cyclins/physiology , G1 Phase/drug effects , Retinoblastoma Protein/physiology , 3T3 Cells , Animals , Butyric Acid , Cyclin D1/metabolism , Cyclin-Dependent Kinase Inhibitor p21 , Cyclins/metabolism , Dose-Response Relationship, Drug , Gene Expression/drug effects , Mice , Mice, Knockout , Oncogene Proteins, Viral/metabolism , Phosphorylation/drug effects , Transfection , Tumor Suppressor Protein p53/physiologyABSTRACT
Ligand-stimulated Platelet-Derived Growth Factor (PDGF) type-beta receptor autophosphorylation, and tyrosine phosphorylation of receptor-associated signalling proteins, is blocked in cells expressing activated Ras genes. A factor present in membrane fractions of v-ras-expressing fibroblasts (Kbalb cells) dominantly inhibits the autophosphorylation of the PDGF type-beta receptor. Purification of this factor, via ion exchange, reveals that the inhibitor can be physically separated from the PDGF type-beta receptor, with reconstitution of PDGF type-beta receptor kinase activity in response to ligand binding. The inhibitor exhibited specificity for the PDGF type-beta receptor, and consistently co-purified with activated p21 ras, with Syp/PTP-2, and with Grb2. Neutralization of the p21 ras protein from the Kbalb cell membranes by p21 ras-specific monoclonal antibodies, however, completely removed the inhibition of PDGF type-beta receptor, rendering the PDGF type-beta receptor molecule capable of autophosphorylation in response to ligand. These results indicate that activated p21 ras either interacts directly with the PDGF type-beta receptor to inhibit autokinase activity, or complexes with different molecules such as Syp and/or Grb2 at the cell membrane to act on another effector which then inhibits PDGF type-beta receptor function.
Subject(s)
Proto-Oncogene Proteins p21(ras)/physiology , Receptors, Platelet-Derived Growth Factor/antagonists & inhibitors , Receptors, Platelet-Derived Growth Factor/physiology , 3T3 Cells/physiology , Amino Acid Sequence , Animals , Antibodies, Monoclonal/pharmacology , Humans , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Phosphorylation , Receptor, Platelet-Derived Growth Factor beta , Receptors, Platelet-Derived Growth Factor/metabolism , Sensitivity and SpecificityABSTRACT
Linkage data from 92 FAD kindreds were analyzed by lod score analysis under various assumptions of disease penetrance, marker allele frequencies, and heterogeneity. Multilocus linkage analysis supports the existence of a gene in 40%-65% of families with predominantly late-onset illness (after age 65) on chromosome 19 between D19S13 and ATP1A3. Evidence for a second FAD gene on chromosome 21 is weaker and stems primarily from a few families with early-onset disease. Our findings also indicate that choice of the genetic model for FAD and marker allele frequencies may be crucial to conclusions about linkage and heterogeneity.
