Biochemical requirements for PCBCK1 kinase activity, the Pneumocystis carinii MEKK involved in cell wall integrity

Fungal cell wall assembly is a complicated process involving Multiple enzymes and coordinated signaling pathways. The cell wall integrity MAPK pathway acts to stabilize the fungal cell wall during conditions of elevated temperature by regulation of glucan synthesis. The upstream kinase, BCK1, is a critical component of this pathway. Pneumonia is a significant cause of death from the fungal opportunistic pathog en Pneumocystis in immunocompromised states, especially with HIV infection. We have previously shown that PCBCK1 functions in the cell wall integrity pathway in yeast as a functional protein kinase. Kinases have specific requirements for enzymatic function which have not been investigated in fungi. Here we examine the biochemical requirements for PCBCK1 kinase activity expressed in Saccharomyces cerevisiae bck1Delta yeast. PCBCK1 requires 10 mM MgCl2, pH 6, temperature 30 degreesC, and 10 muM ATP for kinase activity. Interference of the Pneumocystis cell wall integrity pathway is an attractive target for drug development since glucan synthesis machinery is not present in humans. (C) 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.