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

Abstract

Here, we compare the regulation and localization of the Arabidopsis type III phosphatidylinositol (PtdIns) 4-kinases, AtPI4K␣1 and AtPI4K␤1, in Spodoptera frugiperda (Sf9) insect cells. We also explore the role of the pleckstrin homology (PH) domain in regulating AtPI4K␣1. Recombinant kinase activity was found to be differentially sensitive to PtdIns-4-phosphate (PtdIns4P), the product of the reaction. The specific activity of AtPI4K␣1 was inhibited 70% by 0.5 mm PtdIns4P. The effect of PtdIns4P was not simply due to charge because AtPI4K␣1 activity was stimulated approximately 50% by equal concentrations of the other negatively charged lipids, PtdIns3P, phosphatidic acid, and phosphatidyl-serine. Furthermore, inhibition of AtPI4K␣1 by PtdIns4P could be alleviated by adding recombinant AtPI4K␣1 PH domain, which selectively binds to PtdIns4P (Stevenson et al., 1998). In contrast, the specific activity of AtPI4K␤1, 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 AtPI4K␣1 was associated primarily with membranes in the perinuclear region, whereas AtPI4K␤1 was in the cytosol and associated with small vesicles throughout the cytoplasm. Expression of AtPI4K␣1 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 AtPI4K␣1 with fine actin filaments. These studies support the idea that the Arabidopsis type III PtdIns 4-kinases are responsible for distinct phosphoinositide pools.The negatively charged polyphosphorylated inositol lipids, phosphatidylinositol (PtdIns)-4-phosphate (PtdIns4P) and PtdIns(4,5)P 2 , are important biochemical cues for regulating vesicle formation and trafficking, cytoskeletal dynamics, membrane protein binding, and enzyme activity (Munnik et al., 1998;Stevenson et al., 2000;Wang, 2001;De Matteis et al., 2002) in addition to being precursors of the second messenger inositol(1,4,5)trisphosphate (Berridge and Irvine, 1989). Because of the multiple roles of the phosphoinositides (PIs) in cellular metabolism, regulation of PI biosynthesis is integral to both the response to and recovery from external stimuli. PtdIns 4-kinase, the enzyme that synthesizes PtdIns4P, is the first committed step in PtdIns(4,5)P 2 biosynthesis; therefore, regulation of PtdIns 4-kinase activity and/or its subcellular distribution could affect not only PtdIns4P biosynthesis but also the biosynthesis and distribution of PtdIns(4,5)P 2 .In plants, PtdIns 4-kinase activity has been reported to be associated with many cellular compartments such as the plasma membrane (Sommarin, 1988;Gross et al., 1992; Cho et al., 1993), cytosol (Okpodu et al., 1995), cytoskeleton (Tan and Boss, 1992;Xu et al., 1992), and the nucleus (Hendrix et al., 1989;...

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