Protein microarrays for multiplex analysis of signal transduction pathways.
We have developed a multiplexed reverse phase protein (RPP) microarray platform for simultaneous monitoring of site-specific phosphorylation of numerous signaling proteins using nanogram amounts of lysates derived from stimulated living cells. We first show the application of RPP microarrays to the study of signaling kinetics and pathway delineation in Jurkat T lymphocytes. RPP microarrays were used to profile the phosphorylation state of 62 signaling components in Jurkat T cells stimulated through their membrane CD3 and CD28 receptors, identifying a previously unrecognized link between CD3 crosslinking and dephosphorylation of Raf-1 at Ser259. Finally, the potential of this technology to analyze rare primary cell populations is shown in a study of differential STAT protein phosphorylation in interleukin (IL)-2-stimulated CD4(+)CD25(+) regulatory T cells. RPP microarrays, prepared using simple procedures and standard microarray equipment, represent a powerful new tool for the study of signal transduction in both health and disease.
Chan
Steven M
SM
Division of Immunology and Rheumatology, Department of Medicine, Stanford University School of Medicine, Stanford, California 94305, USA.
Ermann
Joerg
J
Su
Leon
L
Fathman
C Garrison
CG
Utz
Paul J
PJ
D017252:CD3 Complex / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D006801:Humans; D019169:Jurkat Cells / Q000378:metabolism* / Q000502:physiology; D007700:Kinetics; D008894:Milk Proteins / Q000378:metabolism; D010766:Phosphorylation; D040081:Protein Array Analysis / Q000379:methods*; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism; D050794:STAT1 Transcription Factor; D050796:STAT3 Transcription Factor; D050799:STAT5 Transcription Factor; D015398:Signal Transduction / Q000502:physiology*; D013601:T-Lymphocytes / Q000378:metabolism; D015534:Trans-Activators / Q000378:metabolism
D017252:CD3 Complex; D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; D008894:Milk Proteins; D050794:STAT1 Transcription Factor; C494079:STAT1 protein, human; D050796:STAT3 Transcription Factor; C494086:STAT3 protein, human; D050799:STAT5 Transcription Factor; D015534:Trans-Activators; D019908:Proto-Oncogene Proteins c-raf