CAPP Ceramide Activated Protein Phosphatase in yeast. (360G-Wellcome-088104_Z_08_Z)

In eukaryotes, excess levels of the sphingolipid ceramide can block cell proliferation. This proposal aims to provide further insights into ceramide toxicity by studying a ceramide-activated protein phosphatase (CAPP) complex that is hardly understood. Using the yeast Saccharomyces cerevisiae as a model system, we will identify the sphingoid signal required for CAPP activity and elucidate the subunit composition of the CAPP complex. The latter will involve immuneprecipitation and multi-copy comp etition studies to identify the regulatory subunits that in concert with the Sit4 phosphatase catalytic subunit assemble into the CAPP complex. Based on our previously published observations that a subunit of the Elongator complex is a Sit4-dependent phosphoprotein and required for ceramide toxicity, we will study whether CAPP targets Elongator as a ceramide-dependent substrate. A more comprehensive substrate spectrum of CAPP will be obtained by identifying a kinase suppressor of ceramide which will be screened for ceramide resistance in a kinome-wide fashion overexpressing yeast protein kinases. Eventually, we anticipate that our proposal will provide a much clearer picture of how dephosphorylation by CAPP is involved in ceramide signalling and cell death, how CAPP activity is regulated by sphingolipids and what protein phosphorylation events CAPP may potentially cross-talk with.