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BIOCHEMICAL PROCESSES AND MACROMOLECULAR STRUCTURES

Differential Regulation of Two Arabidopsis Type III Phosphatidylinositol 4-Kinase Isoforms. A Regulatory Role for the Pleckstrin Homology Domain¹

Department of Botany, North Carolina State University, Raleigh, North Carolina 27695

Here, we compare the regulation and localization of the Arabidopsis type III phosphatidylinositol (PtdIns) 4-kinases, AtPI4K1 and AtPI4K1, in Spodoptera frugiperda (Sf9) insect cells. We also explore the role of the pleckstrin homology (PH) domain in regulating AtPI4K1. Recombinant kinase activity was found to be differentially sensitive to PtdIns-4-phosphate (PtdIns4P), the product of the reaction. The specific activity of AtPI4K1 was inhibited 70% by 0.5 mM PtdIns4P. The effect of PtdIns4P was not simply due to charge because AtPI4K1 activity was stimulated approximately 50% by equal concentrations of the other negatively charged lipids, PtdIns3P, phosphatidic acid, and phosphatidyl-serine. Furthermore, inhibition of AtPI4K1 by PtdIns4P could be alleviated by adding recombinant AtPI4K1 PH domain, which selectively binds to PtdIns4P (Stevenson et al., 1998). In contrast, the specific activity of AtPI4K1, which does not have a PH domain, was stimulated 2-fold by PtdIns4P but not other negatively charged lipids. Visualization of green fluorescent protein fusion proteins in insect cells revealed that AtPI4K1 was associated primarily with membranes in the perinuclear region, whereas AtPI4K1 was in the cytosol and associated with small vesicles throughout the cytoplasm. Expression of AtPI4K1 without the PH domain in the insect cells compromised PtdIns 4-kinase activity and caused mislocalization of the kinase. The green fluorescent protein-PH domain alone was associated with intracellular membranes and the plasma membrane. In vitro, the PH domain appeared to be necessary for association of AtPI4K1 with fine actin filaments. These studies support the idea that the Arabidopsis type III PtdIns 4-kinases are responsible for distinct phosphoinositide pools.

¹ This work was supported in part by the National Science Foundation (grant no. MCD 0091090) and in part by the North Carolina Agricultural Research Service.

² Present address: Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC 27710.

³ Present address: Department of Plant Sciences, University of Cambridge, Downing Site, Cambridge CB2 3EA, UK.

^* Corresponding author; e-mail wendy_boss{at}ncsu.edu; fax 919–515–3436.

Received February 5, 2003; returned for revision February 19, 2003; accepted February 19, 2003.

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