The Pneumocystis carinii cdc2 cell division cycle homologue exhibits characteristic protein kinase acttvity

Purpose: Pneumocystis carinii (PC) causes severe pneumonia in patients with impaired immunity. Little is known about life cycle control of PC. The cell cycle of eukaryotes is regulated by expression and activation of molecules known as cell division cycle (cdc) proteins. Of these, the cdc2 gene encodes an essential serine-threonine kinase containing a conserved PSTAIR amino-acid motif. We hypothesized that PC must Methods: First, immunoblotting was performed with a potyclonal rabbit antibody generated against the conserved PSTAIR sequence. PC were purified by differential centrifugation from corticosteroid immunosuppressed rats, reserved by SDS-PAGE and transferred to nitrocellulose. To demonstrate protein kinase activity, PC were dissolved in Triton X-100 and immunoprecipited with the anti-PSTAIR antibody. Additionally, separated populations of cysts and trophozoites was studied by filtering trophozoites through a 3μ filter (cysts are retained), normalizing each population for protein concentration, and immunoprecipiting with the anti-PSTAIR antibody. Results: The anti-PSTAIR antibody bound to a PC molecule with apparent molecular mass of 38kDa. Non-immune rabbit IgG did not react. Furthermore, pre-treatment of anti-PSTAIR with the cognate peptide blocked immunoreactivity. The P. carinii cdc2 homologue possesses charactersitic protein kinase activity as demonstrated by the immunoprecipitate's ability to phosphorylate H1 histone when incubated with a mixture of ³²P-γATP and H1 histone. Again, non-immune rabbit IgG or blocked anti-PSTAIR run as concurrent controls did not demonstrate kinase activity. In addition, rat lung proteins prepared from uninfected rats using identical methods and immunoprecipited with anti-PSTAIR failed to show any significant kinase activity, verifying that the protein kinase activity was derived from the PC cdcZ homologue and was not a host cell contaminant. Protein kinase activity appeared greater in the trophozoite population than in cysts when normalized for protein concentration. Non-immune controls run concurrently failed to show any significant kinase activity in either population. Conclusions: These studies document a functional cdc2 homologue in P. carinii which appears to exhibit greater kinase activity in PC trophozoites than cysts. Clinical Implications: A better understanding of PC life cycle may yield important new insights for treatment of PC pneumonia.