Algal Toxins as Guidance to Identify Phosphoproteins with Key Roles inApoptotic Cell Death

T.      Solstad; K.   E.   Fladmark

Abstract

The protein phosphatase inhibiting toxins microcystin and nodularin act rapidly to induce apoptotic cell death.Their inhibitory effect on protein phosphatases 1 and 2A can be utilized as tools to understand the phosphorylation-dependent regulatory mechanism underlying the early stage of apoptosis. The incubation of freshly isolated hepatocyteswith these toxins results in a rapid hyperphosphorylation of cellular proteins before any morphological signs of apoptosisappears [Fladmark, K. E., Brustugun, O. T., Hovland, R., Boe, R., Gjertsen, B. T., Zhivotovsky, B. and Doskeland, S. O.(1999) Cell Death Differ. 6, 1099-108]. Proteins subjected to phosphorylation in this early phase of apoptosis may playkey roles in this cellular process and become valuable targets for drug development. The ultra-rapid apoptosis-inductionby microcystin and nodularin provides a unique amount of synchronized apoptotic cells with "large" amounts of mainlyserine/threonine phosphorylated proteins. This ultra-rapid toxin-induced u p-concentration of phosphorylated proteins re-duces the material needed as well as simplifies our effort in order to obtain enough phosphoproteins for mass spectromet-ric identification and characterization. We will here give an overview of our strategy for identification of low-abundancephosphoproteins involved in algal toxin-induced apoptosis and most likely also in a general apoptotic pathway.

Keywords: Phosphatase inhibitor, microcystin, nodularin, apoptosis, phosphorylation, phosphopeptide enrichment, massspectrometry

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