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Biochemical characterization of mitogen-activated protein (MAP) kinase activity in Toxoplasma gondii
M P Roisin1, F Robert-Gangneux, C Creuzet
1UFR Cochin Université René Descartes, Paris, France. roisin@cochin.univ-paris 5.fr
Abstract:
Mitogen-activated protein (MAP) kinase or extracellular signal-regulated kinase (ERK) are activated by many extracellular stimuli. In this study, we investigated whether MAP kinase and tyrosine kinases were involved in transducing signals in Toxoplasma gondii. Using anti-phosphotyrosine and anti-active ERK antibodies, we identified several phosphorylated proteins in Toxoplasma. In particular, phosphorylation of a 47 kDa and a 43 kDa protein increased strongly after calcium influx. MAP kinase activity, caused by calcium influx, was determined using either a specific synthetic peptide, or an in gel kinase assay. Conversely, calcium chelators (BAPTA and EGTA) and a calcium channel blocker (nifedipine) inhibited this activation. Also, a specific inhibitor of MAP kinase kinase (PD 098059) blocked MAP kinase activity. Three specific anti-MAP kinase antibodies recognized the 47 kDa and 43 kDa proteins, which were putatively identified as ERK1- and ERK2-homologs, respectively. These findings provide early evidence of signal transduction involving members of the MAP kinase family in T. gondii.
Insights
Toxoplasma gondii utilizes mitogen-activated protein (MAP) kinase signaling pathways for calcium-dependent signal transduction. Researchers identified specific MAP kinase homologs involved in this process.
Area of Science:
- Parasitology
- Cellular Biology
- Molecular Biology
Background:
- Mitogen-activated protein (MAP) kinases, also known as extracellular signal-regulated kinases (ERKs), are crucial in cellular signaling pathways responding to various extracellular stimuli.
- Understanding signal transduction mechanisms in parasites like Toxoplasma gondii is vital for developing targeted interventions.
Purpose of the Study:
- To investigate the involvement of MAP kinase and tyrosine kinase signaling in Toxoplasma gondii.
- To identify specific proteins and pathways activated by extracellular stimuli in T. gondii.
Main Methods:
- Utilized anti-phosphotyrosine and anti-active ERK antibodies to detect phosphorylated proteins.
- Employed calcium influx, calcium chelators (BAPTA, EGTA), a calcium channel blocker (nifedipine), and a MAP kinase kinase inhibitor (PD 098059) to study pathway activation.
- Performed in-gel kinase assays and used specific synthetic peptides to measure MAP kinase activity.
- Used three specific anti-MAP kinase antibodies for protein identification.
Main Results:
- Identified several phosphorylated proteins in Toxoplasma, with a notable increase in 47 kDa and 43 kDa proteins following calcium influx.
- Demonstrated that calcium influx activates MAP kinase activity, while calcium chelators and a channel blocker inhibit it.
- Confirmed that PD 098059 effectively blocks MAP kinase activity.
- Putatively identified the 47 kDa and 43 kDa proteins as ERK1 and ERK2 homologs, respectively, based on antibody recognition.
Conclusions:
- Provided early evidence for signal transduction involving MAP kinase family members in Toxoplasma gondii.
- Established a link between calcium influx and MAP kinase activation in this parasite.
- Highlighted the potential role of ERK homologs in T. gondii signaling pathways.