Biochemical characterization of mitogen-activated protein (MAP) kinase activity in Toxoplasma gondii.
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.
Roisin
M P
MP
UFR Cochin Université René Descartes, Paris, France. roisin@cochin.univ-paris 5.fr
Robert-Gangneux
F
F
Creuzet
C
C
Dupouy-Camet
J
J
D000818:Animals; D002118:Calcium / Q000378:metabolism; D015151:Immunoblotting / Q000379:methods; D051379:Mice; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D014122:Toxoplasma / Q000201:enzymology*; D014443:Tyrosine / Q000378:metabolism
D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D020928:Mitogen-Activated Protein Kinases; D020929:Mitogen-Activated Protein Kinase Kinases; D002118:Calcium