Altered tyrosine phosphorylation of ERK1 MAP kinase and other macrophage molecules caused by Leishmania amastigotes

The involvement of tyrosine phosphorylation during macrophage infection wit h Leishmania amazonensis amastigotes was investigated. PTK antagonists such as genistein, herbimycin A, geldanamycin and tyrphostin 25 had no signific ant effect on adhesion to, or entry into, murine peritoneal macrophages, bu t increased parasite intracellular survival. LPS-induced tyrosine phosphory lation of target host proteins assessed by immunoprecipitation and Western blot was impaired or reversed by living amastigotes soon after 60 min-infec tion. Such reversion was not due to parasite-secreted molecules but was con tact-dependent, as assessed by cytochalasin D treatment of macrophage monol ayers prior to infection. Paraformaldehyde-fixed or sodium vanadate-treated amastigotes exerted no significant effect on overall macrophage tyrosine p hosphorylation. Immunoprecipitation of proteins employing 4G10 anti-phospho tyrosine antibody followed by Western blotting revealed that tyrosine phosp horylation of 120, 85, 60, 44 and 35 kDa proteins was selectively reversed by amastigote infection. Inhibition, measured by densitometry was from abou t 66-100% of uninfected cells. None of these proteins was immunoprecipitate d from amastigote-infected macrophage lysates but all of them except for p8 5 were recovered after treatment of parasites with 100 mu M sodium orthovan adate prior to infection, a treatment that inhibits Leishmania amastigote p rotein ecto-phosphatase. The 44 kDa protein was identified as ERK1 MAP kina se (MAPK) by Western blot. Amastigote infection also decreased tyrosine pho sphorylation induced by zymosan particles. Vanadate treatment of amastigote s prior to infection significantly decreased parasite intracellular surviva l. The action of a putative leishmanial ecto-protein phosphatase (PPase) is suggested. (C) 1999 Elsevier Science B.V. All rights reserved.