Protein kinase activation and protein phosphorylation in Naegleria fowleri amebae in response to normal human serum.
Activation of signal transduction pathways in response to serum complement in Naegleria fowleri amebae was investigated. We examined the activation of protein kinases and changes in the phosphorylation state of proteins in N. fowleri stimulated by normal human serum (NHS). To determine differences in phosphorylation of proteins when amebae were exposed to NHS or heat inactivated serum (HIS) lacking complement, amebae were labeled with [32P] orthophosphate. An increase in phosphorylation of relatively low molecular weight proteins was noted in N. fowleri incubated in NHS with a concomitant decrease in phosphorylation of high molecular mass polypeptides. To investigate whether serine/threonine or tyrosine kinases were stimulated by NHS, amebae were treated with protein kinase inhibitors H7, staurosporine or genistein, prior to serum exposure and examined for susceptibility to complement. Treatment with each of these inhibitors resulted in increased complement lysis. Incubation of N. fowleri with genistein specifically inhibited tyrosine phosphorylation of proteins stimulated by NHS. A tyrosine kinase activity assay using exogenous polyGlu-Tyr substrate demonstrated differential activation of tyrosine kinases in amebae treated with NHS when compared to treatment with HIS. The results suggest that activation of protein kinases and subsequent protein phosphorylation are important in mediating complement resistance in N. fowleri.
Chu
D M
DM
Department of Microbiology and Immunology, Medical College of Virginia Campus Virginia Commonwealth University, Richmond 23298-0678, USA.
Ferguson
T J
TJ
Marciano-Cabral
F
F
D000562:Amebiasis / Q000469:parasitology; D000818:Animals; D003165:Complement System Proteins / Q000276:immunology* / Q000494:pharmacology; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019833:Genistein / Q000494:pharmacology; D006801:Humans; D008590:Meningoencephalitis / Q000469:parasitology; D016848:Naegleria fowleri / Q000254:growth & development / Q000276:immunology / Q000378:metabolism*; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D015800:Protozoan Proteins / Q000378:metabolism*; D015398:Signal Transduction; D019311:Staurosporine / Q000494:pharmacology
D004791:Enzyme Inhibitors; D015800:Protozoan Proteins; D019000:Phosphotyrosine; D003165:Complement System Proteins; D019833:Genistein; D011505:Protein-Tyrosine Kinases; D019311:Staurosporine