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BBB seminar: Clive S. D'Santos

Identification of phosphorylation sites within the lipid kinase phosphatidylinositol 5-phosphate 4-kinase in response to UV stimulation

Clive S. D'Santos
The Norwegian Proteomics Centre (PROBE), University of Bergen

Mass spectrometry based proteomic approach to analyse protein samples is a rapid and sensitive technique aimed at and providing detailed information on protein function. We have used mass spectrometry to investigate the role of lipid kinases as regulated signalling enzymes in response to extracellular stress.

Little is known about the function of the lipid phosphatidylinositol 5-phosphate and its metabolising enzyme phosphatidylinositol 5-phosphate 4-kinase (PIPkinase) type IIß. Recently, however, the ING family of tumour suppressor transcription factors has been demonstrated to interact with phosphatidylinositol 5-phosphate in vitro via PHD domains. These proteins bind phosphatidylinositol 5-phosphate and subsequently translocate to the nucleus in response to UV stimulation (Gozani O et al., 2003, Cell, 114:99-111). Using mass spectrometry we identified two sites of phosphorylation on immunoprecipitated samples of PIPkinase type IIß. Thr322 and Ser326 were both found to be phosphorylated while in semi-quantitative analyses Ser326 phosphorylation was found to be UV sensitive. Mass spectrometry has contributed to provide the first definitive sites of phosphorylation of PIPkinase type IIß.

The potential of this and other mass spectrometry based approaches directed at determining protein structure and function will be highlighted.