Algal toxins as guidance to identify phosphoproteins with key roles in apoptotic cell death.
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 hepatocytes with these toxins results in a rapid hyperphosphorylation of cellular proteins before any morphological signs of apoptosis appears [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 play key roles in this cellular process and become valuable targets for drug development. The ultra-rapid apoptosis-induction by microcystin and nodularin provides a unique amount of synchronized apoptotic cells with "large" amounts of mainly serine/threonine phosphorylated proteins. This ultra-rapid toxin-induced up-concentration of phosphorylated proteins reduces the material needed as well as simplifies our effort in order to obtain enough phosphoproteins for mass spectrometric identification and characterization. We will here give an overview of our strategy for identification of low-abundance phosphoproteins involved in algal toxin-induced apoptosis and most likely also in a general apoptotic pathway.
Solstad
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Proteomic Unit, University of Bergen (PROBE), Jonas Lies vei 91, N-5009, Norway. therese.solstad@biomed.uib.no
Fladmark
K E
KE
D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D000458:Cyanobacteria / Q000737:chemistry*; D015180:Electrophoresis, Gel, Two-Dimensional; D022781:Hepatocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D013058:Mass Spectrometry; D052998:Microcystins; D010456:Peptides, Cyclic / Q000302:isolation & purification / Q000494:pharmacology*; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors; D010750:Phosphoproteins / Q000032:analysis* / Q000378:metabolism / Q000502:physiology
D052998:Microcystins; D010456:Peptides, Cyclic; D010750:Phosphoproteins; C063998:nodularin; C078588:microcystin; D010749:Phosphoprotein Phosphatases