scholarly journals A new phosphospecific cell-based ELISA for p42/p44 mitogen-activated protein kinase (MAPK), p38 MAPK, protein kinase B and cAMP-response-element-binding protein

2000 ◽  
Vol 350 (3) ◽  
pp. 717-722 ◽  
Author(s):  
Henri H. VERSTEEG ◽  
Evert NIJHUIS ◽  
Gijs R. VAN DEN BRINK ◽  
Maaike EVERTZEN ◽  
Gwenda N. PYNAERT ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Protein Kinase ◽  
P38 Mapk ◽  
Protein Kinase B ◽  
Camp Response Element Binding ◽  
Mitogen Activated Protein Kinase ◽  
Camp Response Element ◽  
Response Element ◽  
Mitogen Activated Protein ◽  
Element Binding Protein

Assaying activation of signal transduction is laborious and does not allow the study of large numbers of samples, essential for high-throughput drug screens or for large groups of patients. Using phosphospecific antibodies, we have developed ELISA techniques enabling non-radioactive semi-quantitative assessment of the activation state of p42/p44 mitogen-activated protein kinase (MAPK), p38 MAPK, protein kinase B and the transcription factor cAMP-response-element-binding protein (CREB) in 96-well plates. This assay has been termed PACE (phosphospecific antibody cell-based ELISA) and was used successfully for both adherent and suspension cells. Various stimuli induced dose-dependent enzymic activity of which the kinetics closely correlated with those measured via classical methodology. Using PACE we have now characterized for the first time the concentration-dependent effects of various inflammatory prostaglandins on CREB phosphorylation in macrophages. PACE is a straightforward and novel technique enabling the large-scale analysis of signal transduction.

scholarly journals Estrogen Activation of Cyclic Adenosine 5′-Monophosphate Response Element-Mediated Transcription Requires the Extracellularly Regulated Kinase/Mitogen-Activated Protein Kinase Pathway

Endocrinology ◽  
2003 ◽  
Vol 144 (3) ◽  
pp. 832-838 ◽  
Author(s):  
Christian B. Wade ◽  
Daniel M. Dorsa
Keyword(s):  
Protein Kinase ◽  
Mapk Pathway ◽  
Camp Response Element Binding ◽  
Mitogen Activated Protein Kinase ◽  
Camp Response Element ◽  
Response Element ◽  
Creb Phosphorylation ◽  
Mitogen Activated Protein ◽  
Element Binding Protein

The ability of estrogen to rapidly initiate a variety of signal transduction cascades is increasingly recognized as playing an important role in a number of tissue-specific transcriptional actions of the hormone. In vivo, estrogen rapidly elicits phosphorylation of cAMP response element-binding protein (CREB). We have previously shown that both ERα and ERβ are capable of activating the MAPK pathway in response to a low dose of 17β-estradiol. In the present study, the ability of estrogen to act through both ERα and ERβ to increase CREB phosphorylation was evaluated in an immortalized hippocampal cell line stably expressing either receptor. Estrogen treatment promoted rapid CREB phosphorylation, reaching a maximum by 15 min. This activation is completely blocked by the antiestrogen ICI 182,780, suggesting an estrogen receptor-dependent mechanism. The addition of the mitogen/ERK kinase-1 inhibitor, PD98059, also blocked the ability of estrogen to signal to CREB phosphorylation. Estrogen also caused an increase in p90Rsk activity, a critical mediator of MAPK effects. Surprisingly, blockade of the protein kinase A pathway in cells treated with estrogen did not affect estrogen-mediated CREB phosphorylation. Thus, MAPK and p90Rsk appear to be the primary mediators of estrogen-induced gene transcription through ERα and ERβ.

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