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Series GSE4043 Query DataSets for GSE4043
Status Public on Mar 07, 2006
Title Gene profiling analysis of Src chemical rescue
Organism Mus musculus
Experiment type Expression profiling by array
Summary The restoration of catalytic activity to mutant enzymes by small molecules is well-established for in vitro systems. Here we show that the protein tyrosine kinase Src R388A mutant can be rescued in live cells using the small molecule imidazole. Cellular rescue of a v-Src homolog was rapid and reversible and conferred predicted oncogenic properties. Using chemical rescue in combination with mass spectrometry, six known Src kinase substrates were confirmed, and several new protein targets identified. Chemical rescue data suggests that c-Src is active under basal conditions. Rescue of R388A c-Src also allowed contributions of Src to the MAP kinase pathway to be clarified. This chemical rescue approach is likely to be of broad utility in cell signaling.
We were also interested in examining the impact of Src rescue on the kinetics of gene expression. Chronic gene expression changes in v-Src transformed colon cancer and NIH3T3 cells have been reported, but the chemical rescue method permits insights into rapid kinetic changes. We used gene microarray analysis of imidazole activated Src, 1 h after imidazole treatment of 8A7F cells as well as 6N7F control cells, a set of several genes show increases (>1.7-fold) at 1 h in the 8A7F cells and another set show decreases (>1.7-fold) at 1 h with minimal changes in 6N7F control cells. Thirteen of these genes were further analyzed using real-time RT-PCR and most of the genes tested showed similar changes using both techniques. These gene changes were not reported in cells chronically transformed with v-Src or rapidly stimulated with growth factors suggesting that rapid initiation of Src-mediated tyrosine phosphorylation may induce a specialized pattern of gene expression changes. However, these earlier experiments were done under different conditions which may also contribute to gene effects.
Keywords: time course, cell type comparison
Overall design SrcR388A/Y527F or SrcD386N/Y527F stably transfected SYF cells were serum-starved overnight and then treated with imidazole 5 mM for 1 h or 12 h. RNeasy Miniprep column (Qiagen) was used to prepare total RNA from each treatment group. All samples were run in commercial arrays from Affymetrix,
using Murine Genome MOE430 2.0 GeneChips as described in the Affymetrix website.
Contributor(s) Qiao Y, Murillo FM, Jie C, Cole PA
Citation(s) 16513984
Submission date Jan 13, 2006
Last update date Feb 11, 2019
Contact name Philip A Cole
Phone 410-614-0322
Fax 410-614-7717
Organization name Johns Hopkins Medical School
Department Pharmacology and Molecular Sciences
Lab Dr. Philip A. Cole
Street address 725 N Wolfe St
City Baltimore
State/province MD
ZIP/Postal code 21205
Country USA
Platforms (1)
GPL1261 [Mouse430_2] Affymetrix Mouse Genome 430 2.0 Array
Samples (6)
GSM92297 Src R388A/Y527F-0 hr
GSM92459 Src R388A/Y527F-1 hr
GSM92524 Src R388A/Y527F-12 hr
BioProject PRJNA95177

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SOFT formatted family file(s) SOFTHelp
MINiML formatted family file(s) MINiMLHelp
Series Matrix File(s) TXTHelp

Supplementary file Size Download File type/resource
GSE4043_Analysis_ProbeInfo1.xls 13.4 Mb (ftp)(http) XLS
GSE4043_Analysis_ProbeInfo2.xls 6.4 Mb (ftp)(http) XLS
GSE4043_Analysis_ProbeInfo3.xls 7.5 Mb (ftp)(http) XLS
GSE4043_Analysis_ProbeInfo4.xls 16.4 Mb (ftp)(http) XLS
GSE4043_Analysis_Signals.xls 8.8 Mb (ftp)(http) XLS
GSE4043_RAW.tar 36.0 Mb (http)(custom) TAR (of CEL)

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