Entamoeba histolytica: tyrosine kinase activity induced by fibronectin through the beta1-integrin-like molecule.
Previously, we characterized a 140-kDa protein from Entamoeba histolytica as a beta1-integrin-like molecule that binds fibronectin. In this work we present data showing that the amoebic receptor is associated with another surface molecule, the 220-kDa lectin, and with protein tyrosine kinase activity. By immunoprecipitation with the alphabeta1Eh antibody, we demonstrated by immune complex assays for tyrosine protein kinases that the amoebic fibronectin receptor was associated with two phosphorylated proteins of 50 and 70 kDa when internal membranes were used as the source of antigen. When cells were stimulated with fibronectin, two proteins of 55 and 90 kDa were tyrosine phosphorylated, as shown by Western blot with alphaPY20, its phosphorylation being time dependent after fibronectin stimulation. However, when the actin cytoskeleton of fibronectin-stimulated trophozoites was stabilized with phalloidin, the level and the pattern of phosphorylated proteins were different. In this case, a high-molecular-weight component, heavily phosphorylated, was present, which may include the 220-kDa lectin. We also present data confirming that the signaling pathway that is activated by fibronectin is specific. This was demonstrated by comparing the pattern of phosphoproteins obtained in immune complexes prepared with alphabeta1Eh, alphaL220, and alphaPY20 from total extracts obtained in the presence of phalloidin, from cells that had been exposed to fibronectin, soluble concanavalin A, or concanavalin-A-coated substrate. The presence of tyrosine kinases associated with the beta1-integrin-like amoebic molecule was confirmed by immunoprecipitation assays along with the combined use of a tyrosine kinase-specific substrate, the peptide RR-SRC, and a tyrosine kinase inhibitor, genistein.
Hernández-Ramírez
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VI
Experimental Pathology Department, CINVESTAV-IPN, Avenida I.P.N. No. 2508, Col. San Pedro Zacatenco, México, DF 07360, México.
Anaya-Ruiz
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M
Rios
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A
Talamás-Rohana
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P
D000818:Animals; D015153:Blotting, Western; D003599:Cytoskeleton / Q000502:physiology; D004591:Electrophoresis, Polyacrylamide Gel; D004748:Entamoeba histolytica / Q000201:enzymology* / Q000378:metabolism; D004790:Enzyme Induction / Q000187:drug effects; D004791:Enzyme Inhibitors; D005353:Fibronectins / Q000378:metabolism / Q000494:pharmacology* / Q000502:physiology; D019833:Genistein; D019012:Integrin beta1 / Q000276:immunology / Q000378:metabolism*; D010455:Peptides; D010590:Phalloidine; D010766:Phosphorylation; D011233:Precipitin Tests; D011505:Protein-Tyrosine Kinases / Q000096:biosynthesis* / Q000502:physiology; D017469:Receptors, Fibronectin / Q000378:metabolism; D015398:Signal Transduction
D004791:Enzyme Inhibitors; D005353:Fibronectins; D019012:Integrin beta1; D010455:Peptides; D017469:Receptors, Fibronectin; D010590:Phalloidine; C047660:arginyl-arginyl-leucyl-isoleucyl-glutamyl-aspartyl-alanyl-glutamyl-tyrosyl-alanyl-alanyl-arginyl-glycine; D019833:Genistein; D011505:Protein-Tyrosine Kinases