Protein Kinase Activation and Protein Phosphorylation in Naegleria fowleri Amebae in Response to Normal Human Serum

Authors

  • DAN-MY T. CHU,

    1. Department of Microbiology and Immunology, Medical College of Virginia Campus Virginia Commonwealth University, Richmond, Virginia 23298–0678, USA
    Search for more papers by this author
  • TAMMY J. FERGUSON,

    1. Department of Microbiology and Immunology, Medical College of Virginia Campus Virginia Commonwealth University, Richmond, Virginia 23298–0678, USA
    Search for more papers by this author
  • FRANCINE MARCIANO-CABRAL

    Corresponding author
    1. Department of Microbiology and Immunology, Medical College of Virginia Campus Virginia Commonwealth University, Richmond, Virginia 23298–0678, USA
      Corresponding Author: F. Marciano-Cabral—Telephone number: 804–828–9742; FAX number: 804–828–8220; Email: fmcabral@hsc.vcu.edu
    Search for more papers by this author

Corresponding Author: F. Marciano-Cabral—Telephone number: 804–828–9742; FAX number: 804–828–8220; Email: fmcabral@hsc.vcu.edu

Abstract

ABSTRACT. 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 phosphory lation 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.

Ancillary