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Co-purification of mitogen-activated protein kinases with phorbol ester-induced c-Jun kinase activity in U937
B J Pulverer1, K Hughes, C C Franklin
1Ontario Cancer Institute, Princess Margaret Hospital, Toronto, Canada.
Abstract:
Phorbol esters, such as phorbol myristate acetate (PMA), cause differentiation of U937 human monomyelocytic cells along the macrophage pathway. Within 15 min of PMA treatment DNA binding of the c-jun transcription factor is increased and is accompanied by rapid changes in the phosphate content of the c-jun protein. Phorbol esters stimulate phosphorylation of serines 63 and 73 located within the A1 transactivation domain of c-Jun that have previously been shown to positively regulate activity. A protein kinase activity is detectable in extracts of phorbol ester-treated U937 cells that specifically targets these two serines. Using novel assays, the protein kinase activity has been purified over 1000-fold. The major portion of protein kinase activity co-chromatographs over three columns with pp42/44 mitogen-activated protein kinases as judged by immunological methods. The significance of these results with respect to mitogen-induced transcription of AP-1-responsive genes is discussed.
Insights
Phorbol myristate acetate (PMA) triggers macrophage differentiation in U937 cells by rapidly increasing c-Jun transcription factor DNA binding. This involves phosphorylation of key c-Jun serines by a novel protein kinase activity identified as mitogen-activated protein kinase.
Area of Science:
- Cellular Biology
- Molecular Biology
- Biochemistry
Background:
- Phorbol esters, like PMA, induce differentiation of U937 cells into macrophages.
- Transcription factor c-Jun plays a crucial role in cellular responses to stimuli.
Purpose of the Study:
- To investigate the molecular mechanisms underlying PMA-induced U937 cell differentiation.
- To identify the protein kinase responsible for c-Jun phosphorylation in response to PMA.
Main Methods:
- Treatment of U937 cells with phorbol myristate acetate (PMA).
- Assessing DNA binding of the c-Jun transcription factor.
- Analyzing changes in c-Jun protein phosphorylation.
- Purifying and characterizing the responsible protein kinase activity.
- Immunological identification of the kinase using mitogen-activated protein kinase (MAPK) assays.
Main Results:
- PMA treatment rapidly increased c-Jun DNA binding and phosphorylation within 15 minutes.
- Specific phosphorylation occurred at serines 63 and 73 in the c-Jun A1 transactivation domain.
- A novel protein kinase activity targeting these serines was detected and purified over 1000-fold.
- The purified kinase activity predominantly co-chromatographed with pp42/44 mitogen-activated protein kinases.
Conclusions:
- PMA-induced U937 cell differentiation involves rapid modulation of c-Jun activity.
- Mitogen-activated protein kinases are likely involved in the phosphorylation of c-Jun at regulatory sites.
- This phosphorylation event may contribute to the transcription of AP-1-responsive genes during differentiation.