Plant and Cell Physiology Advance Access originally published online on February 2, 2009
Plant and Cell Physiology 2009 50(3):595-609; doi:10.1093/pcp/pcp018
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Characterization of Phosphatidylinositol Phosphate Kinases from the Moss Physcomitrella patens: PpPIPK1 and PpPIPK2
1Department of Biochemistry, Center for Chemistry and Chemical Engineering, Lund University, PO Box 124, SE-22100 Lund, Sweden
2School of Biosciences, University of Birmingham, Birmingham B15 2TT, UK
3National Institute for Basic Biology, Okazaki, 444-8585 Japan
4School of Life Science, The Graduate University for Advanced Studies (SOKENDAI), Okazaki, 444-8585 Japan
5Faculty of Fisheries Sciences, Hokkaido University, Hakodate, 041-8611 Japan
6Department of Plant Physiology, Umeå Plant Science Centre, Umeå University, SE-90187, Umeå, Sweden
*Corresponding authors: Marianne Sommarin, E-mail, marianne.sommarin{at}plantphys.umu.se; Fax, +46-907866676; Koji Mikami, E-mail, komikami{at}fish.hokudai.ac.jp; Fax, +81-138408899.
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Abstract |
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Phosphoinositides (PIs) play a major role in eukaryotic cells, despite being a minor component of most membranes. This is the first report on PI metabolism in a bryophyte, the moss Physcomitrella patens. Moss PI composition is similar to that of other land plants growing under normal conditions. In contrast to the large number of PIPK genes present in flowering plants, the P. patens genome encodes only two type I/II PIPK genes, PpPIPK1 and PpPIPK2, which are very similar at both the nucleotide and protein product levels. However, the expression of the two genes is differentially regulated, and in vitro biochemical characterization shows that the resulting enzymes have different substrate specificities. PpPIPK1 uses PtdIns4P and PtdIns3P with similar preference and also metabolizes PtdIns(3,4)P2 to produce PtdIns(3,4,5)P3, a PI not yet detected in intact plant cells. PpPIPK2 prefers PtdIns as substrate and is much less active towards PtdIns4P and PtdIns3P. Thus, PpPIPK2 shows properties reminiscent of both PtdInsP-kinase and PtdIns-kinases. Moreover, a substitution of glutamic acid by alanine in the activation loop drastically reduced PpPIPK1 activity and altered the substrate specificity to PtdIns5P being the preferred substrate compared with PtdIns4P and PtdIns3P. These findings demonstrate that the substrate specificity of plant PIPKs is determined in a plant-specific manner, which provides new insights into the regulatory modes of PIPK activity in plants.
Keywords: Membrane occupation and recognition nexus (MORN) - Phosphatidylinositol phosphate kinase (PIPK) - Phosphoinositide metabolism - Phosphoinositides - Physcomitrella patens.
Abbreviations: BSA, bovine serum albumin; DAG, diacylglycerol; EST, expressed sequence tag; GFP, green fluorescent protein; [3H]GroPInsP, [3H]glycero-phosphoinositol phosphates; GST, glutathione S-transferase; Ins(1,4,5)P3, inositol-1,4,5-trisphosphate; MORN, membrane occupation and recognition nexus; ORF, open reading frame; PI, phosphoinositide; PIPK, PtdInsP kinase; PtdIns, phosphatidylinositol; PtdInsP2, PtdIns bisphosphate; PtdInsP, PtdIns phosphate; PtdIns(4,5)P2, PtdIns-4,5-bisphosphate; PtdIns(3,4)P2, PtdIns-3,4-bisphosphate; PtdIns(3,5)P2, PtdIns-3,5-bisphosphate; PtdIns(3,4,5)P3, PtdIns-3,4,5-trisphosphate; PtdOH, phosphatidic acid; RACE, rapid amplification of cDNA ends; RT–PCR, reverse transcription–PCR; TLC, thin-layer chromatography
(Received November 4, 2008; Accepted January 27, 2009)
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