Altered tyrosine phosphorylation of ERK1 MAP kinase and other macrophage molecules caused by Leishmania amastigotes.
The involvement of tyrosine phosphorylation during macrophage infection with Leishmania amazonensis amastigotes was investigated. PTK antagonists such as genistein, herbimycin A, geldanamycin and tyrphostin 25 had no significant effect on adhesion to, or entry into, murine peritoneal macrophages, but increased parasite intracellular survival. LPS-induced tyrosine phosphorylation of target host proteins assessed by immunoprecipitation and Western blot was impaired or reversed by living amastigotes soon after 60 min-infection. Such reversion was not due to parasite-secreted molecules but was contact-dependent, as assessed by cytochalasin D treatment of macrophage monolayers prior to infection. Paraformaldehyde-fixed or sodium vanadate-treated amastigotes exerted no significant effect on overall macrophage tyrosine phosphorylation. Immunoprecipitation of proteins employing 4G10 anti-phosphotyrosine antibody followed by Western blotting revealed that tyrosine phosphorylation of 120, 85, 60, 44 and 35 kDa proteins was selectively reversed by amastigote infection. Inhibition, measured by densitometry was from about 66-100% of uninfected cells. None of these proteins was immunoprecipitated from amastigote-infected macrophage lysates but all of them except for p85 were recovered after treatment of parasites with 100 microM sodium orthovanadate prior to infection, a treatment that inhibits Leishmania amastigote protein ecto-phosphatase. The 44 kDa protein was identified as ERK1 MAP kinase (MAPK) by Western blot. Amastigote infection also decreased tyrosine phosphorylation induced by zymosan particles. Vanadate treatment of amastigotes prior to infection significantly decreased parasite intracellular survival. The action of a putative leishmanial ecto-protein phosphatase (PPase) is suggested.
Martiny
A
A
Instituto de Biofisica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Centro de Ciênias da Saúde, Ilha do Fundão, RJ, Brazil.
Meyer-Fernandes
J R
JR
de Souza
W
W
Vannier-Santos
M A
MA
D000818:Animals; D015153:Blotting, Western; D006801:Humans; D007891:Leishmania / Q000201:enzymology / Q000502:physiology*; D017737:Macrophages, Peritoneal / Q000378:metabolism / Q000469:parasitology*; D008297:Male; D051379:Mice; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism*; D011233:Precipitin Tests; D014774:Virulence
D010750:Phosphoproteins; D019000:Phosphotyrosine; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D010749:Phosphoprotein Phosphatases