Abstract
The Src family of protein-tyrosine kinases (SFKs) participates in a variety of signal transduction pathways, including promotion of cell growth, prevention of apoptosis, and regulation of cell interactions and motility. In particular, SFKs are required for the mitogenic response to platelet-derived growth factor (PDGF). However, it is not clear whether there is a discrete SFK-specific pathway leading to enhanced gene expression or whether SFKs act to generally enhance PDGF-stimulated gene expression. To examine this, we treated quiescent NIH3T3 cells with PDGF in the presence or absence of small molecule inhibitors of SFKs, phosphatidylinositol 3-kinase (PI3K), and MEK1/2. Global patterns of gene expression were analyzed by using Affymetrix Gene-Chip arrays, and data were validated by using reverse transcription-PCR and ribonuclease protection assay. We identified a discrete set of immediate early genes induced by PDGF and inhibited in the presence of the SFK-selective inhibitor SU6656. A subset of these SFK-dependent genes was induced by PDGF even in the presence of the MEK1/2 inhibitor U0126 or the PI3K inhibitor LY294002. By using ribonuclease protection assays and nuclear run-off assays, we further determined that PDGF did not stimulate the rate of transcription of these SFK-dependent immediate early genes but rather promoted mRNA stabilization. Our data suggest that PDGF regulates gene expression through an SFK-specific pathway that is distinct from the Ras-MAPK and PI3K pathways, and that SFKs signal gene expression by enhancing mRNA stability.
Highlights
The protein-tyrosine kinase Src was first described more than 20 years ago
Our laboratory has recently focused on the growth factor-stimulated production of Myc, a transcription factor required for efficient cell cycle transit
Previous work demonstrated that the block to platelet-derived growth factor (PDGF)-stimulated DNA synthesis caused by dominant negative SFKs could be overcome by expression of Myc [10]
Summary
Cell Line and Reagents—NIH3T3 fibroblast cells were maintained in Dulbecco’s modified Eagle’s medium with 2 mM L-glutamine, 1.5 mg/ml glucose, 100 units/ml penicillin, 100 g/ml streptomycin, and 10% heatinactivated fetal bovine serum. The primer and RNA were denatured at 70 °C for 10 min and placed on ice, and cDNA was synthesized by using SuperScript II RT (Invitrogen) according to the manufacturer’s instructions. Five g of RNeasy mini kit-purified total RNA was reverse-transcribed with SuperScript II RT using oligo(dT) primers (Invitrogen) according to the manufacturer’s instructions. The nuclei were resuspended in 2 ml of ice-cold reaction buffer (20 mM Tris, pH 7.5, 10 mM MgCl2, 140 mM KCl, 1 mM dithiothreitol, and 20% glycerol) and pelleted by centrifugation for 5 min at 2000 rpm at 4 °C. Following 10 min of incubation at 37 °C, the nascent RNA was extracted using Trizol and hybridized to specific DNA probes blotted on Hybond-N membrane (Amersham Biosciences). Bound antibodies were detected using horseradish peroxidase-linked secondary antibodies (Sigma) and detected using SuperSignal West Pico chemiluminescent substrate (Pierce)
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