Thrombin activates transcription factors sp1, NF-kappaB, and CREB: importance of the use of phosphatase inhibitors during nuclear protein extraction for the assessment of transcription factor DNA-binding activities.
Thrombin, a serine protease, is an important effector of many cellular processes and has been shown to up-regulate the expression of several genes. The mechanisms underlying thrombin-mediated regulation of gene transcription remain poorly understood. The original aim of this work was to study the effects of thrombin on the activation of transcription factors, Sp1, NF-kappaB, and CREB by means of electrophoretic mobility-shift assays (EMSA). However, an inconsistent pattern of results was observed. We raised the possibility that some EMSA results may have been erroneous by the fact that during the nuclear protein extraction and EMSA procedure, transcription factors are dephosphorylated by cellular phosphatases and hence their DNA-binding capacities are modified. Therefore, we have altered the original nuclear extraction protocol by including a mixture of phosphatase inhibitors during protein extraction and subsequent EMSA steps. We show here that this simple measure led to significant changes in both basal and thrombin-induced levels of activation of Sp1 and CREB, but not of NF-kappaB. In light of the data presented here, it would be important to reexamine the conclusions of many reports in which EMSA was used to assess the basal and agonist-induced levels of transcription factor DNA-binding activities.
Edmead
C
C
Molecular Cell Biology Laboratory, Thrombosis Research Institute, Manresa Road, London, SW3 6LR, United Kingdom.
Kanthou
C
C
Benzakour
O
O
D002478:Cells, Cultured; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism*; D004247:DNA / Q000378:metabolism; D017931:DNA Primers; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006801:Humans; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism*; D009687:Nuclear Proteins / Q000302:isolation & purification* / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000037:antagonists & inhibitors; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D013917:Thrombin / Q000494:pharmacology*
D017362:Cyclic AMP Response Element-Binding Protein; D017931:DNA Primers; D004791:Enzyme Inhibitors; D016328:NF-kappa B; D009687:Nuclear Proteins; D016329:Sp1 Transcription Factor; D004247:DNA; D010744:Phosphoric Monoester Hydrolases; D013917:Thrombin