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Originally published In Press as doi:10.1074/jbc.M611728200 on March 21, 2007

J. Biol. Chem., Vol. 282, Issue 19, 14121-14131, May 11, 2007
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Arabidopsis Phosphatidylinositol Phosphate Kinase 1 Binds F-actin and Recruits Phosphatidylinositol 4-Kinase beta1 to the Actin Cytoskeleton*Formula

Amanda J. Davis{ddagger}, Yang Ju Im{ddagger}, Joshua S. Dubin§, Kenneth B. Tomer§, and Wendy F. Boss{ddagger}1

From the {ddagger}Plant Biology, North Carolina State University, Raleigh, North Carolina 27695 and §Laboratory of Structural Biology, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709

Received for publication, December 21, 2006 , and in revised form, March 14, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The actin cytoskeleton can be influenced by phospholipids and lipid-modifying enzymes. In animals the phosphatidylinositol phosphate kinases (PIPKs) are associated with the cytoskeleton through a scaffold of proteins; however, in plants such an interaction was not clear. Our approach was to determine which of the plant PIPKs interact with actin and determine whether the PIPK-actin interaction is direct. Our results indicate that AtPIPK1 interacts directly with actin and that the binding is mediated through a predicted linker region in the lipid kinase. AtPIPK1 also recruits AtPI4Kbeta1 to the cytoskeleton. Recruitment of AtPI4Kbeta1 to F-actin was dependent on the C-terminal catalytic domain of phosphatidylinositol-4-phosphate 5-kinase but did not require the presence of the N-terminal 251 amino acids, which includes 7 putative membrane occupation and recognition nexus motifs. In vivo studies confirm the interaction of plant lipid kinases with the cytoskeleton and suggest a role for actin in targeting PIPKs to the membrane.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Phosphatidylinositol phosphate kinases (PIPKs)2 are a family of enzymes that phosphorylate phosphatidylinositol phosphates (PtdInsP) to phosphatidylinositol bisphosphates (PtdInsP2). Arabidopsis has 11 predicted isoforms of PtdInsP kinases (1). AtPIPK1 and AtPIPK10 have been characterized biochemically, and PtdIns4P is the primary substrate making the predominant product phosphatidylinositol (4, 5) bisphosphate (PtdIns(4,5)P2), suggesting that they are similar to the type I PIPKs found in humans (2–5). However, unlike the mammalian, yeast or Caenorhabditis elegans PIPKs, AtPIPK1–9 contain N-terminal putative membrane occupation and recognition nexus (MORN) motifs similar to those first reported in junctophilins (1, 6). MORN motifs are unique to this family of enzymes and have not been reported in any other eukaryotic lipid kinases.

In eukaryotic models PtdInsP kinases supply PtdIns(4,5)P2 for many cellular functions, and it is well known that phospholipids play an integral role in regulating the structure and dynamics of the cytoskeleton through the many actin-binding proteins that interact with PtdIns(4,5)P2 (7–10). For example, the absence of PtdInsP kinases in yeast has an adverse affect on the yeast cell morphology (11) and causes them to be unable to properly form actin cables (12). In some animal cell lines overexpression of PtdInsP kinases results in the formation of actin comet tails and stress fibers (13, 14) and disruption of membrane trafficking (14). In these animal cell lines, a functional PtdInsP kinase producing PtdIns(4,5)P2 was necessary to cause the changes in cytoskeletal structure (13, 15). In mammalian systems, PIPK activity has been recovered with an F-actin fraction, and the {alpha}, beta, and {gamma} isoforms have been identified as co-purifying with F-actin. The data support a model where these PIPKs do not directly interact with F-actin but that this interaction is mediated by the presence of Racs, small GTP-binding proteins (16). In plants, PtdIns kinase activity (17, 18) and PtdInsP kinase activity co-purify with an F-actin-enriched fraction (18), but it is not clear from these studies whether plant PtdInsP kinases directly bind F-actin or, like animal PIPKs, associate with a scaffold of actin-binding proteins.

Actin remodeling in root hairs and pollen tubes, sites of rapid growth in plants, is sensitive to alterations in PtdIns(4,5)P2 biosynthesis. It was first noted that expression of mutant Arabidopsis Rac2 in tobacco pollen tubes decreased plasma membrane PtdIns(4,5)P2 and disrupted the normal actin filament orientation (19). The GFP-AtRac2 was localized to the apical tip of the pollen tube, and PtdInsP kinase activity was co-immunoprecipitated with Rac2 antibodies. More recent work in pollen tubes suggested that phospholipase C-mediated PtdIns(4,5)P2 turnover also affected actin structure and suggested that PtdIns(4,5)P2 metabolism is necessary in normal pollen growth (20, 21). Expression of inactive mutants of PLC isoforms resulted in increased levels PtdIns(4,5)P2 throughout the entire pollen tube and enriched at the apical tip (20, 21). In both tobacco (21) and petunia (20), disruption of PLC hydrolysis of PtdIns(4,5)P2 using inactive mutants or chemical inhibitors of PLC resulted in tip swelling, suggesting that PLC-mediated PtdIns(4,5)P2 turnover was essential for normal tip-directed growth. In root hairs, decreased tip-localized PtdIns(4,5)P2 resulting from a loss of the inositol phospholipid transfer protein, Atsfh1p, also resulted in unfocused root hair growth and disruption of the tip-directed actin filament orientation (22). Both in pollen and root hairs the data suggested that tip-localized PtdIns(4,5)P2 affected tip growth via F-actin-mediated vesicle trafficking (23). Such a role for the inositol lipids in actin-mediated vesicle trafficking was supported by the work of Preuss et al. (24). They showed that mutants in PI4Kbeta1 and PI4Kbeta2 or treatment with latrunculin (24) disrupted trafficking of specialized RabA4b-associated Golgi vesicles in root hairs. Blocking turnover of PtdIns(4,5)P2 and increasing PtdIns(4,5)P2 pools by a mutation in inositol lipid phosphatases also affected actin filament orientation and cell wall biosynthesis in inflorescent stems of Arabidopsis (25). Although all of these data strongly support a role for PtdIns(4,5)P2 regulating actin cytoskeleton and vesicle trafficking in plants, the underlying mechanism is not known. Because the major family of plant PIPKs have a very different structure to the PIPKs of animals and yeast, it is important to understand how they are regulated and to identify their interacting partners.

In this work our approach was to identify proteins that interact specifically with AtPIPK1 and AtPIPK10 and to determine whether one of the PIPKs would associate with F-actin. We show that Arabidopsis PIPK1 directly interacts with actin and recruits PI4Kbeta1, suggesting a plant-specific mechanism for influencing cytoskeletal dynamics and lipid signaling. Because AtPIPK1 has an N-terminal MORN domain with the potential for tight membrane adhesion, the presence of AtPIPK1 on F-actin would provide a discrete pool of PtdIns(4,5,)P2 for actin-mediated vesicle trafficking and/or fusion.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Production of GST Fusion Proteins in Escherichia coli—GST-AtPIPK1 and GST-AtPIPK10 were cloned and expressed in E. coli as previously described (4, 5). AtPI4Kbeta1 was PCR amplified using Pfx DNA polymerase with forward primer 5'-CACCATGCCGATGGGACGCTTT-3' and reverse primer 5'-CTCACACTCTTCCATTTAAGACCCGTTGGTA-3'. The resulting PCR product was subcloned into the pENTR/SD/D-TOPO destination vectors (Invitrogen) and then into pDEST15 for expression of GST-AtPI4Kbeta1 proteins in E. coli. The inactive form of AtPIPK1, AtPIPK1(K468A), was mutated in the ATP binding site using QuikChange XL site-directed mutagenesis kit (Stratagene, La Jolla, CA). The ATP binding site was identified by sequence homology to that of the Human PtdInsP kinase, HsPIPK1{alpha}, and mutated as described (26). The mutated construct was amplified with forward primer 5'-CTCAAGATGATAGATTTATGATCGCAACGGTGAAGAAATCAGAAGTCAAG-3' and reverse primer 5'-CTTGACTTCTGATTTCTTCACCGTTGCGATCATAAATCTATCATCTTGAG-3'.

The resulting PCR product was subcloned into the pENTR/SD/D-TOPO destination vectors (Invitrogen) and then into pDEST15 for expression of GST-AtPIPK1(K468A). Truncations of AtPIPK1, MORN, {Delta}MORN, {Delta}MORN/{Delta}L, {Delta}MORN/{Delta}C, and {Delta}MORN/{Delta}L/{Delta}C were produced as previously described (27). All GST fusion proteins were produced and purified as previously described (5). Protein concentration was determined using the Bio-Rad protein assay reagent with bovine serum albumin as a standard. Purified recombinant protein bound to the glutathione-Sepharose beads was stored at 4 °C until use in protein binding or activity assays.

Plant Material—The recombinant binary plasmid pK7WGF2-HsPIPK1{alpha} was transformed into Agrobacterium tumefaciens EHA105 by the freeze-thaw method. For stable transformation, NT-1 cells were transformed using A. tumefaciens-mediated gene transfer following the protocol of Perera et al. (4). Cells were subcultured weekly into 25 ml of NT-1 culture medium containing 50 µgml–1 kanamycin as described by Perera et al. (4). NT-1 cells expressing {Delta}MORN were produced as previously described (27).

Protein Pulldown Assays—Protein pulldown assays were performed with purified recombinant protein incubated with precleared Triton-solubilized Arabidopsis membrane fractions for 2 h at 4 °C with continuous mixing in 30 mM beta-cyclodextrin, phosphate-buffered saline (0.1 M KH2PO4, 0.1 M K2HPO4, 135 mM NaCl, and 2.7 mM KCl, pH 7.3), and final concentrations of 3 mM ATP or 0.5 mM GTP where indicated. Triton-solubilized Arabidopsis membranes were prepared by incubating a 40,000 x g pellet isolated as described previously in 1% (v/v) Triton X-100 10 min at 4 °C then centrifuging at 10,000 x g for 10 min to obtain a Triton-solubilized supernatant (5). Membrane fractions were precleared by incubating the solubilized membranes with purified GST immobilized on glutathione-Sepharose beads. The cleared supernatant was used for protein-protein interactions. After incubation with the membrane proteins, the beads were washed extensively with phosphate-buffered saline.

Direct interactions of proteins were investigated using purified recombinant proteins either coupled to beads, released from the beads with reduced glutathione (Novagen, manufacturers instructions), or cleaved from the GST tag with thrombin (Novagen, manufacturers instructions) in phosphate-buffered saline or where indicated by using purified native protein. After washing unbound proteins from the beads, the bound proteins and beads were either added directly into 4x SDS-PAGE sample buffer, heated at 100 °C for 5 min for separation by SDS-PAGE, or were used immediately for lipid kinase activity analysis.

Mass Spectrometry—After SDS-PAGE, the gels were stained with Coomassie Brilliant Blue, and stained bands were selected for excision. Digestion was carried out with bands excised from 10% polyacrylamide gel stained with Coomassie in 20 ml of 100 mM ammonium bicarbonate buffer, pH 8.0, 1 mM CaCl2. 1 ml of modified trypsin (sequencing grade, Promega) solution (2 mg/ml in 50 mM acetic acid) was added to yield a final enzyme protein ratio of 1:10. After incubation at 37 °C for 2 h, 2 ml of 1 N HCl was added to bring the mixture to pH 3.0 and inactivate the trypsin. One-half of the sample (10 ml) was moved to another tube and mixed with 2 ml of 0.1 M Tris-(2-carboxyethyl)phosphin (Pierce) in 0.1 M citrate buffer, pH 3.0. The mixture was incubated at 37 °C for 30 min for reduction of the disulfide bond-containing tryptic peptides. 1-ml aliquots of reduced or non-reduced tryptic peptide mixtures were used for MALDI-MS analysis without further purification. MALDI time-of-flight MS analyses were carried out using a Voyager-DE STR mass spectrometer (Applied Biosystems Inc.) equipped with a pulsed UV nitrogen laser (337 nm, 3-ns pulse) and a dual microchannel plate detector. For molecular mass determination of peptides, spectra were acquired at linear-delayed extraction (DE) mode, acceleration voltage set at 25 kV, grid voltage at 95% of the acceleration voltage, delay time at 320 ns, and low mass gate set at 1000 Da. The mass to charge ratio was calibrated with the molecular mass of a mixture of proteins (mass, 5,734.58–16,952.56). For analysis of tryptic peptides, the spectra were acquired at reflectron-DE mode with acceleration voltage set to 20 kV, grid voltage at 72% of the acceleration voltage, delay time at 200 ns, and low mass gate at 250 Da. The mass to charge ratio was calibrated with the mass of a mixture of standard peptides (mass, 904.46–5,734.58). Saturated {alpha}-cyano-4-hydroxycinnamic acid in 70% acetonitrile containing 0.1% trifluoroacetic acid was used as the matrix for analysis of tryptic peptides, and saturated sinapinic acid in 50% acetonitrile containing 0.1% trifluoroacetic acid was used as the matrix for protein analysis. 1 µl of a solution of reduced or non-reduced tryptic peptide mixture was applied on the MALDI plate followed by 1 µl of saturated matrix solution. Spectra were recorded after evaporation of the solvent and processed using Data Explorer software for data collection and analysis. Predicted masses were calculated by the ExPASy Peptide Mass program.

Immunoblotting Blotting—After separation by SDS-PAGE, proteins were transferred to polyvinylidene difluoride membranes by electroblotting. Membranes were blocked in 5% (w/v) powdered milk in Tris-buffered saline. Blots were incubated with the primary antibody for 1 h followed by incubation in the secondary antibody for 1 h. Secondary antibodies were coupled to horseradish peroxidase or to IRDye800 (Pierce). For antibodies coupled to horseradish peroxidase, immunoreactivity was visualized by incubating the blot in SuperSignal West Pico Chemiluminescent substrate (Pierce) and exposure to x-ray film. For the fluorescently labeled secondary antibodies, immunoreactivity was detected with Odyssey infrared imaging system (Licor, Lincoln NE) according to the manufacturer's instructions. After immunoreactivity detection, total protein was visualized by staining the blots with Amido Black (Sigma).

Lipid Kinase Assays—PtdInsP kinase activity and phosphatidylinositol kinase activity was assayed in duplicate as described by (5) with a final reaction volume of 50 µl. Each assay contained 10 µg of purified recombinant protein on glutathione-Sepharose beads that had been incubated with solubilized Arabidopsis membrane fraction or with purified proteins and washed once with 50 mM Tris, pH 7.5. Lipid substrate was prepared using PtdIns4P or PtdIns (porcine brain; Avanti%20Polar%20Lipids">Avanti Polar Lipids, Alabaster, AL) from 1 or 5 mg/ml stocks, respectively. Lipids were dried under an N2 atmosphere and solubilized for use in the lipid kinase assays in the presence of cyclodextrins as described previously (5). The lipid kinase assay was performed as described (5). Lipid extraction was performed using an acid solvent system. Extracted lipids were separated by TLC on silica gel plates (LK5D; Whatman, Clifton, NJ) using a CHCl3:MeOH: NH4OH:water (90:90:7:22, v/v) solvent system. The 32P-labeled phospholipids were quantified with a Bioscan System 500 imaging scanner.

Actin Polymerization—Polymerization buffer contained 20 mM PIPES, 2 mM EGTA, 2 mM MgCl2, 1 mM ATP, 50 mM KCl, pH 6.5 (28). Buffer was added to 30 µg of protein from the depolymerized actin fraction from Arabidopsis membranes, 5 µg of plasma membrane isolated from NT-1 cells as described previously (29), or to 3 µg of pure actin containing purified recombinant proteins eluted from glutathione-Sepharose beads with reduced glutathione. Polymerization reactions were incubated for 1 h at 25 °C, then the F-actin was pelleted by centrifugation. For centrifugation at 20,000 x g for 1 h at 4 °C, polymerization volume was 30 µl. For centrifugation at 100,000 x g for 30 min, polymerization volume was 100 µl (28). Latrunculin treatments were performed by adding 10 µM latrunculin B to 2 gof 4-day-old NT-1 cells for 1 h (30). An equal volume of Me2SO was used as a control. Cells were harvested, and the plasma membrane was isolated as previously described (29).

Insect Cell Protein Production—Serum-free Spodoptera frugiperda (Sf9) cells were obtained from Invitrogen and maintained at 28 °C at a concentration of 2.5 x 105 to 5 x 106 cells ml–1 in Sf-900 II insect cell serum-free medium (Invitrogen). The expression system used was the Bac-to-Bac baculovirus expression system (Invitrogen). Recombinant baculoviruses were generated from the recombinant expression vectors according to the manufacturer's recommendations. Optimal production of AtPI4Kbeta1 was 2 days after infection, and optimal production of AtPI4K{alpha}1 (31) and AtPI4K{gamma}7({Delta}N/{Delta}C) was on the third day after infection.3 All assays were performed using cells optimally producing the respective polypeptide. Infected cells were then harvested and lysed as previously described (31). The cleared lysate was analyzed for protein concentration by the Bradford method (Bio-Rad) and used for protein-protein interactions.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Cyclodextrin Enhanced Recovery of Proteins in Pulldown Reactions—Our first goal was to identify proteins that interacted specifically with AtPIPK1. For this purpose, constructs were developed to produce GST fusion proteins of AtPIPK1 and AtPIPK10 in E. coli to pulldown-interacting proteins from Arabidopsis cell fractions. Fig. 1 shows the production of the recombinant GST-tagged proteins in E. coli as detected by a GST antibody (Fig. 1A) and with antibodies raised against AtPIPK1 or AtPIPK10 (Fig. 1B). Although the antibodies raised against full-length AtPIPK1 and AtPIPK10 readily detected the recombinant E. coli-expressed proteins, these antibodies were unable to detect PtdInsP kinases from an Arabidopsis cell fraction (data not shown), suggesting that the antibodies were not robust and/or that the protein levels were very low. For this reason, we used GST antibodies or activity assays to monitor the enzymes.

Because PtdInsP 5-kinase activity was associated with F-actin as well as plasma membranes from plants (18, 29, 32), to increase the recovery of potential interacting proteins, cells were homogenized using a buffer developed to enhance the recovery of F-actin with the membranes (33). Recovery of interacting proteins was compared using a 40,000 x g pellet and the soluble fraction from 4-day-old Arabidopsis cells grown in suspension culture. As described under "Experimental Procedures," all fractions isolated from the suspension culture cells were pre-cleared by incubation with purified GST immobilized on glutathione-Sepharose beads to reduce the number of GST binding proteins. No interacting proteins were evident based on Coomassie and silver staining when the 40,000 x g supernatant was used for the pulldown assays with either GST-AtPIPK1 or GST-AtPIPK10 (data not shown); however, several proteins were evident if the Triton X-100-solubilized membrane fraction was used. Although there were interacting proteins present for both isoforms from the solubilized membrane fraction, there was not enough protein present on the stained gels to identify the proteins by mass spectrometry.


Figure 1
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  		FIGURE 1.
Detection of recombinant proteins. Purified recombinant proteins were detected by Western blotting. A, an anti-GST antibody was used to detect recombinant purified GST, GST-AtPIPK10, and GST-AtPIPK1. B, anti-PIPK1 was used to detect purified recombinant GST-PIPK1 (left panel) and anti-PIPK10 was used to detect GST-AtPIPK10 (right panel). C, beta-cyclodextrin (betaCD) enhances the recovery of interacting membrane-associated proteins. Purified recombinant GST-AtPIPK1 bound to Sepharose beads was incubated with Triton-solubilized Arabidopsis membranes that had been pre-cleared with GST and incubated in the presence and absence of 30 mM beta-cyclodextrin. The bound proteins were separated by SDS-PAGE and stained with Coomassie Brilliant Blue. The stained gel shown reveals differences in the relative amount of protein recovered. There were no detectable proteins interacting with the glutathione-Sepharose beads in the presence or absence of beta-cyclodextrin (data not shown). These experiments were repeated at least twice with similar results. The asterisk indicates the migration of GST-AtPIPK1, and the plus symbol (+) indicates the migration of GST-AtPIPK10.

 
To enhance the recovery of the solubilized proteins, we developed a protocol that involved adding cyclodextrins to the pulldown reaction mixture. Cyclodextrins have been used to aid in protein refolding and in sequestering lipids (34, 35). {alpha}-, beta-, and {gamma}-cyclodextrins (0–50 mM) were added after pre-clearing the Triton-solubilized membrane proteins to see which if any would enhance the recovery of interacting proteins. {gamma}-Cyclodextrin did not improve the interaction of the Arabidopsis proteins with the recombinant proteins at any of the concentrations tested, and {alpha}-cyclodextrin enhanced the interactions of a few of the proteins from the membranes with the recombinant proteins (data not shown); however, when 30 mM beta-cyclodextrin was added, there was a 6-fold increase in the recovery of the peptides detected (Fig. 1C). One possible explanation for the enhanced protein recovery is that the cyclodextrin helps the recombinant protein fold properly to allow for interaction with the Arabidopsis peptides (35). In addition, cyclodextrin will sequester the Triton or lipids present in the membrane fraction and thereby may enhance the interaction of the protein partners during the pulldown reaction. Because beta-cyclodextrin enhanced recovery and did not appear to affect the distribution or number of the proteins bands, it was used for all subsequent pulldown experiments using microsomal proteins unless indicated otherwise.

AtPIPK1 and AtPIPK10 Recovered Different Proteins Based on Mass Spectrometry Analysis—Using the optimized pull-down conditions for experiments, we were able to recover sufficient amounts of interacting peptides to identify them by mass spectrometry. To eliminate any false positives, we selected only for Coomassie-stained bands that were differentially present when GTP or ATP were added during the pulldown assays.

Peptide sequences were analyzed by GRAMS software and ExPASy Peptide Mass Program and then identified by comparison to the Arabidopsis data base. The results showed that AtPIPK1 and AtPIPK10 each interacted with a different subset of proteins (Table 1). Proteins that were recovered in the AtPIPK1 pulldown assays included Rop5, known to activate PtdInsP kinase activity in plants (19), Arf1Ad, also an activator of PtdInsP kinase activity and involved in vesicle trafficking (36), profilin 5, which can bind to PtdIns(4,5)P2 (37, 38), AtPI4Kbeta1, which binds to RabA4b (39), and phospholipase C1. AtPI4Kbeta1 was recovered with AtPIPK1 but not AtPIPK10. To study this interaction in more detail, we used purified recombinant proteins.


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  		TABLE 1
Proteins identified by mass spectrometry

 
AtPI4Kbeta1 Interacts Directly with AtPIPK1—To determine whether the PI4Ks and PIPKs would interact directly in vitro, recombinant AtPIPK1 and AtPIPK10 that had been cleaved from the GST tag with thrombin were incubated with GST-AtPI4Kbeta1 bound to the Sepharose beads. After the incubation, peptides bound to the GST-AtPI4Kbeta1 were separated by SDS-PAGE and stained with Coomassie (Fig. 2A). The stained gel indicated that AtPIPK1 interacted directly with AtPI4Kbeta1in what appears to be a 1:1 ratio based on recovery of Coomassie-stained proteins, but AtPIPK10 did not interact (Fig. 2A). The results were confirmed by assaying for PtdInsP 5-kinase activity, which is more sensitive than Coomassie staining. For the activity assay, the glutathione-Sepharose beads containing the purified recombinant GST-AtPI4Kbeta1 and either cleaved AtPIPK1 or AtPIPK10 were incubated with PtdIns4P and [{gamma}-32P]ATP to determine whether any PtdInsP kinase activity was present (Fig. 2B). As we found with the Coomassie-stained gels (Fig. 2A), only AtPIPK1 could be detected when GST-AtPI4Kbeta1 was used as bait.

To determine whether other PI4Ks might interact with AtPIPK1 or if the interaction with AtPI4Kbeta1 was specific, potential interactions between AtPIPK1 and AtPI4K{alpha}1, another member of the Arabidopsis type III family of PI4Ks, and the catalytic region of AtPI4K{gamma}7, a member of the type II PI4Ks (1), were investigated. Truncated AtPI4K{gamma}7(AtPI4K{gamma}7-{Delta}N/{Delta}C, amino acids 123–456) was used because we were not able to produce the full-length protein in either the E. coli or the Sf9 expression system.3 The interactions of AtPIPK1 and AtPIPK10 with the insect cell lysates producing AtPIKbeta1, AtPIK{alpha}1, and AtPI4K{gamma}7-{Delta}N/{Delta}C (supplemental Fig. 1A) were analyzed both by assaying for PtdIns 4-kinase activity associated with the PtdInsP kinases (supplemental Fig. 1B) and by immunoblotting with antibodies for the His tag on the recombinant PtdIns 4-kinases (Fig. 2C). The results indicated that AtPIPK1 interacts only with AtPI4Kbeta1 and that none of these PI4Ks interacted with AtPIPK10. Another member of the type II family of PtdIns kinases, AtPI4K{gamma}4, was tested for interaction with PtdInsP kinases. When full-length, purified recombinant GST-AtPI4K{gamma}43 was incubated with solubilized Arabidopsis microsomes, no PtdInsP kinase activity was recovered, indicating that AtPI4K{gamma}4 did not interact with any of the PtdInsP kinases present in the membrane fraction (data not shown).

Because AtPIPK1 would use the product of AtPI4Kbeta1, PtdIns4P, as a substrate, we thought that the interaction of AtPI4Kbeta1 and AtPIPK1 would require AtPIPK1 to be functional. To investigate the impact of lipid kinase activity on the interacting partners, a kinase inactive form of AtPIPK1 was generated using site-directed mutagenesis to mutate the ATP binding site in the catalytic region from Lys-468 to Ala. The recombinant mutated protein, AtPIPK1-K468A, had no lipid kinase activity in in vitro assays (Fig. 3A). AtPI4Kbeta1 was recovered equally well in pulldown assays with AtPIPK1 and an inactive mutant, AtPIPK1-K468A. These data indicate that AtPIPK1 does not need to be active to bind to AtPI4Kbeta1 (Fig. 3B).


Figure 2
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  		FIGURE 2.
AtPIPK1 and GST-AtPI4Kbeta1 interact directly. GST-AtPI4Kbeta1 was incubated with purified recombinant AtPIPK1 and AtPIPK10 that had been cleaved from the GST tag. A, the interacting proteins were separated by SDS-PAGE and visualized with Coomassie. The location of GST-AtPI4Kbeta1, AtPIPK1 (lane 1), and AtPIPK10 (lane 2) are indicated. B, the interaction of GST-AtPI4Kbeta1 with the PtdInsP kinases was confirmed by assaying for PtdInsP kinase activity in the presence of PtdIns4P and [{gamma}-32P]ATP. The products were separated by TLC and visualized using autoradiography. AtPIPK1 was recovered (lane 1), whereas AtPIPK10 did not interact with GST-AtPI4Kbeta1(lane 2). Lanes 1 and 2 are lanes from the same TLC plate, and the migration of the standards was the same. C, the interaction of purified recombinant AtPIPK1 and AtPIPK10 with AtPI4Ks expressed in insect cells also was monitored with an antibody to the His-tagged AtPI4K proteins. These experiments were repeated at least twice with similar results.

 


Figure 3
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  		FIGURE 3.
GST-AtPIPK1(K468A) does not produce PtdIns(4,5)P2 and binds AtPI4Kbeta1. A, purified recombinant GST-AtPIPK1 and GST-AtPIPK1(K468A) were assayed for PtdInsP kinase activity with the addition of PtdIns4P and [{gamma}-32P]ATP. The products were separated by TLC and imaged using autoradiography. GST-AtPIPK1(K468A) had no detectable PtdInsP kinase activity. The panels are lanes from the same TLC plate, and the migration of the standards was the same. B, AtPI4Kbeta1 interacts with the inactive GST-AtPIPK1(K468A). Purified recombinant GST-AtPIPK1(K468A) was incubated with solubilized Arabidopsis membranes. The interaction of AtPI4Kbeta1 with the inactive PtdInsP kinase was monitored with an antibody that recognizes type III PtdIns kinases. C, GST-AtPI4Kbeta1 interacts with endogenous Arabidopsis PtdInsP kinases. Purified recombinant GST-AtPI4Kbeta1 was incubated with solubilized Arabidopsis membranes and assayed for PtdInsP kinase activity with the addition of PtdIns4P and [{gamma}-32P]ATP (lane 1). The products were separated by TLC and visualized by autoradiography. Lane 2, GST-AtPI4Kbeta1 alone assayed with PtdIns as a substrate. The panels are lanes from the same TLC plate, and the migration of the standards is indicated. These experiments were repeated at least twice with similar results.

 


Figure 4
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  		FIGURE 4.
AtPIPK1 interacts with F-actin and recruits AtPI4Kbeta1 to F-actin. Purified recombinant GST, GST-AtPIPK10, and GST-AtPIPK1 were incubated with a depolymerized actin fraction from Arabidopsis membranes under conditions favorable for actin polymerization. Proteins in the F-actin pellet (P) and supernatant (S) were separated by SDS-PAGE then probed with an anti-GST antibody. A, only GST-AtPIPK1 was found in the F-actin pellet. B, GST-AtPI4Kbeta1 was incubated as in A, alone, or with GST-AtPIPK1. GST-AtPI4Kbeta1 was not found in the F-actin pellet unless GST-AtPIPK1 was added. C, the MORN region of AtPIPK1 does not interact with the Arabidopsis F-actin or with PtdIns kinases. Recombinant GST-AtPIPK1, GST-MORN, and GST-{Delta}MORN (AtPIPK1 minus N-terminal MORN domain) were incubated as in A. Interacting proteins were assayed for PtdIns kinase activity with the addition of PtdIns and [{gamma}-32P]ATP. The products were separated by TLC and visualized by autoradiography. The panels are lanes from the same TLC plate, and the migration of the standards was the same. D, purified recombinant GST-AtPIPK1, GST-MORN, and GST-{Delta}MORN were incubated with a depolymerized Arabidopsis actin fraction as in A, and GST-tagged proteins were detected with a GST antibody. GST-AtPIPK1 and GST-{Delta}MORN were found in the F-actin pellet, whereas GST-MORN was not. These experiments were repeated at least twice with similar results.

 
When assaying for lipid kinase activity using E. coli-produced recombinant proteins, we noted the unexpected production of phosphatidic acid (PtdOH). We suspected that the activity resulted from diacylglycerol kinase and diacylglycerol that bound GST. This hypothesis was confirmed by showing that a diacylglycerol kinase and diacylglycerol from E. coli were both recovered with GST and were not associated with thrombin-cleaved AtPIPK1 (supplemental Fig. 2).

The interaction between AtPI4Kbeta1 and AtPIPKs was confirmed in reciprocal pulldown assays using purified, recombinant AtPI4Kbeta1as the bait and solubilized Arabidopsis membranes. To determine whether PIPKs were present, the interacting peptides were incubated with PtdIns4P and [{gamma}-32P]ATP. PtdInsP2 was extracted and separated by TLC, and the product was visualized by autoradiography (Fig. 3C).

AtPIPK1 Interacts with Actin and Recruits AtPI4Kbeta1 to an F-actin Fraction—Because many others have indicated a connection between PtdInsP2 production and the actin cytoskeleton in plants (40) and because we had previously shown that PtdInsP kinase activity was associated with F-actin (18), we asked whether there was a direct link between F-actin and AtPIPK1 using actin polymerization experiments. Purified GST, GST-AtPIPK1, GST-AtPIPK10, and GST-AtPI4Kbeta1 were eluted from the glutathione-Sepharose beads with reduced glutathione. The eluted proteins were added to actin solubilized from Arabidopsis membranes and then incubated under actinpolymerizing conditions. The F-actin fraction, which contained the associated Arabidopsis actin-binding proteins such as eEF1A, was pelleted by centrifugation at 20,000 x g to recover large filaments and cables; the proteins in the supernatant and pellet were separated by SDS-PAGE, and the GST-tagged proteins were detected by immunoblotting. The immunoblots revealed that GST-AtPIPK1 was recovered with the F-actin fraction, but GST and GST-AtPIPK10 were not (Fig. 4A). When GST-AtPIPK1 and GST-AtPI4Kbeta1 were added together, they were also found in the F-actin pellet, but GST-AtPI4Kbeta1 alone was not (Fig. 4B). These results indicate that AtPIPK1 interacts selectively with the F-actin fraction and that AtPIPK1 will recruit AtPI4Kbeta1.

AtPIPK1 contains a MORN domain that is unique to plant lipid kinases. The N-terminal MORN domain contains the seven putative MORN motifs similar to those found in junctophilins (6), MORN1 of Toxoplasma gondii (41) and the chloroplast fission protein ARC3 (42). With this unique domain structure, it was possible that the MORN domain mediated the protein-protein interactions of AtPIPK1 with its interacting partners. To test this hypothesis, recombinant peptides consisting of the N-terminal 251 amino acids of AtPIPK1 (MORN domain) and the remaining 501 amino acid C-terminal catalytic region ({Delta}MORN) were used to investigate the interactions of AtPIPK1 with AtPI4Kbeta1 and actin. The MORN domain did not interact with AtPI4Kbeta1 (Fig. 4C) and was not found in an F-actin pellet (Fig. 4D). However, {Delta}MORN was in the F-actin pellet and recruited PtdIns 4-kinase activity from Arabidopsis membrane fractions. These results indicate that the N-terminal MORN domain is neither essential nor sufficient for the interaction of AtPI4Kbeta1 or F-actin with AtPIPK1.

The Predicted Linker Region of AtPIPK1 Is Essential for Actin Binding—The predicted linker region (amino acids 252–435) between the MORN domain and the catalytic region is enriched in proline residues. To test the hypothesis that the enrichment of proline residues would allow the linker region to interact with actin, we made truncations of AtPIPK1 with GST fused to the N terminus (Fig. 5A) and investigated their ability to bind to F-actin made from purified chicken muscle actin.

Full-length GST-AtPIPK1, GST-{Delta}MORN (amino acids 252–752), and GST-{Delta}MORN/{Delta}C (amino acids 252–697), all peptides containing the linker region, were found in the F-actin pellet (Fig. 5B). Although GST-MORN (amino acids 1–251), GST-{Delta}MORN/{Delta}L (amino acids 436–752), and GST-{Delta}MORN/{Delta}L/{Delta}C (amino acids 436–697), all peptides missing the linker region were not found in the F-actin pellet. These experiments were performed in a small volume to favor complete actin polymerization (28). These results support the hypothesis that the linker region is the site of AtPIPK1 interaction with F-actin and demonstrate that AtPIPK1 interacts directly with purified F-actin.

All of the AtPIPK1 truncations missing the linker region are inactive in vitro (27). To ensure that the lack of interaction with F-actin was not merely because of the lack of PtdInsP kinase activity, the inactive AtPIPK1-K468A was tested. AtPIPK1-K468A was recovered with an F-actin pellet (Fig. 5C), indicating that kinase function was not essential for direct actin binding and validating the requirement of the linker region.

Our data are in contrast to that describing mammalian PIPK. The mammalian PIPKs are recruited to F-actin through a scaffold of actin-binding proteins (10, 16). Because binding can vary under different conditions, we compared the actin binding of HsPIPK1{alpha} to AtPIPK1 using the same conditions. As shown in Fig. 5C, HsPIPK1{alpha} did not directly bind chicken muscle F-actin. These data confirm that AtPIPK1 has a different and direct mechanism for interacting with the actin cytoskeleton.

To characterize actin binding further, we investigated the binding of AtPIPK1 to G-actin. For these experiments, purified recombinant AtPIPK1 was immobilized on glutathione-Sepharose beads, and increasing concentrations of pure chicken muscle actin were added for the pulldown reactions (Fig. 6A). The binding results indicated that G-actin interacts directly with AtPIPK1 and the interaction was saturated when the ratio of AtPIPK1 to G-actin was higher than 1:1. The propensity for AtPIPK1 to interact with more than one molecule of actin would explain the preferential association of AtPIPK1 with F-actin and led to the hypothesis that AtPIPK1 might facilitate F-actin formation. To test this, we compared F-actin polymerization in the presence and absence of AtPIPK1 and eEF1A, an actin-bundling protein (43). For these experiments, commercial chicken muscle actin was first precleared by centrifuging at 100,000 x g under non-polymerizing conditions. The polymerization was performed in a volume that did not favor complete actin polymerization to assess the effect of AtPIPK1 (28). Either AtPIPK1 or eEF1A was added under polymerizing conditions, and the F-actin was pelleted at 100,000 x g. With actin alone, actin was detected approximately equally in the supernatant and pellet (Fig. 6B). When eEF1A was added, as anticipated, actin bundles formed, and all the actin was recovered in the pellet. When AtPIPK1 was added, there was more actin in the pellet than supernatant. When GST was added to the polymerization, the results were similar to that of actin alone (data not shown). It is difficult to quantify immunoblots; however, as summarized in Fig. 6B, replicate experiments showed similar differences in the relative actin recovered in the pellet and supernatant if AtPIPK1 was present. The data make a compelling argument that AtPIPK1 not only binds actin but also facilitates the recovery of F-actin.


Figure 5
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  		FIGURE 5.
AtPIPK1 interacts with F-actin through a predicted linker region. A, recombinant GST-AtPIPK1, GST-MORN, GST-{Delta}MORN, GST-{Delta}MORN/{Delta}L, GST-{Delta}MORN/{Delta}C, and GST-{Delta}MORN/{Delta}L/{Delta}C were incubated with pure actin under polymerizing conditions. A, a linear representation of AtPIPK1 indicating the truncations used for these studies. The F-actin pellet was recovered with a 20,000 x g centrifugation. The pellet (P) and supernatant (S) were separated by SDS-PAGE. The GST-fused proteins associated with the fractions were detected with an anti-GST antibody (top panel of each set), and actin was detected with an anti-actin antibody (bottom panel of each set) using the same blot. Peptides containing the predicted linker region, GST-AtPIPK1,GST-{Delta}MORN, and GST-{Delta}MORN/{Delta}C were found associated with the F-actin pellet. B, GST-AtPIPK1(K468A) was recovered in the F-actin pellet, indicating that the activity of the kinase is not necessary for association with F-actin. Top panel, AtPIPK1(K468A); bottom panel, actin. C, HsPIPK1{alpha} was not recovered in the F-actin pellet, indicating that the mammalian kinase does not directly associate with F-actin. Top panel, GST-HsPIPK1{alpha}; lower panel, actin.

 


Figure 6
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  		FIGURE 6.
AtPIPK1 binds G-actin and enhances the recovery of F-actin. A, purified recombinant GST-AtPIPK1 was incubated with increasing molar ratios of pure chicken muscle G-actin. The interaction of actin and GST-AtPIPK1 was monitored with an antibody to actin (top panel). Equal loading of GST-AtPIPK1 in each lane was confirmed by staining the blot with Amido Black (bottom panel). B, the ability of AtPIPK1 to enhance the recovery of F-actin was tested. Pure actin was incubated under polymerizing conditions in the presence and absence of AtPIPK1. F-actin was recovered by 100,000 x g centrifugation, and the pellet (P) and supernatant (S) were separated by SDS-PAGE. Actin present in the pellet and supernatant was detected by immunoblotting. The images from two independent experiments were scanned, and the relative density was quantified using the Image FX (Bio-Rad). The percent actin recovered is reported as the amount of actin recovered based on comparison to protein standards on the same gel relative to the amount of actin added.

 
Plant PIPKs Interact with F-actin in Vivo—PtdInsP kinase activity is primarily associated with the plasma membranes of plant cells (29, 32). In vivo interaction of plant PIPKs with membrane-associated F-actin was confirmed using wild-type tobacco NT-1 cells and NT-1 cells expressing {Delta}MORN. Cells were treated with 10 µM latrunculin B for 1 h to depolymerize the actin cytoskeleton or Me2SO as a control. Plasma membranes were isolated by aqueous-two phase partitioning, and the associated cytoskeleton was recovered after two cycles of Triton X-100 solubilization followed by the repolymerization of F-actin. PtdInsP kinase activity was measured in both the F-actin pellet and the soluble fraction. In the wild-type cells and the NT-1 cells expressing the {Delta}MORN fragment, most of the PtdInsP kinase activity was associated with the F-actin pellet, whereas a small amount of activity was present with the solubilized fraction (Fig. 7A). However, when the cells were treated for 1 h with latrunculin B to disrupt the cytoskeleton (supplemental Fig. 3), there was little membrane-associated F-actin, and the endogenous PtdInsP kinase activity from the wild-type and {Delta}MORN cells was found in the solubilized fraction (Fig. 7A). There was not a significant loss in the total plasma membrane PtdInsP kinase activity as a result of the latrunculin treatment. These data reveal that most of the plasma membrane PIPK is normally bound to membrane-associated actin filaments and that the plant PIPKs remain associated with the plasma membrane even if there is little F-actin present. It is not surprising that the lipid kinases would bind membrane lipids and other membrane-associated proteins in addition to actin.

To further compare AtPIPK1 binding to plasma membrane F-actin, we used a synthetic system where we expressed HsPIPK1{alpha} in tobacco cells. As shown in Fig. 5C, HsPIPK1{alpha} did not bind directly to F-actin in vitro. If specific mammalian actin-binding proteins are essential for mammalian PIPKs to interact with actin, we would not anticipate that HsPIPK1{alpha} would bind F-actin in the NT-1 cells.

To assess the contribution of the HsPIPK1{alpha} to the PtdInsP kinase activity associated with the plasma membranes of the HsPIPK1{alpha} cells, we took advantage of the differences in the kinetic properties of HsPIPK1{alpha} and the plant PIPKs (4). The Km of AtPIPK1 is 3-fold less than that of HsPIPK1{alpha}, and when assayed at low substrate concentration (50 µM PtdIns4P), membranes from the wild-type cells had very little activity (Fig. 7B). The specific activity increased significantly at 125 µM substrate as expected from the kinetic analysis. With both substrate concentrations, most of the endogenous PtdInsP kinase activity was recovered in the F-actin fraction (Fig. 7B). With membranes from the HsPIPK1{alpha} cell lines, not only was the PtdInsP kinase activity much greater but also almost all the activity was recovered in the soluble fraction (Fig. 7C). The activity in the soluble fraction was about the same at both substrate concentrations, which is consistent with the lower Km of HsPIPK1{alpha}. The small amount of enzyme activity associated with F-actin in the HsPIPK1{alpha} cell lines is likely the endogenous PtdInsP kinase activity as it increased ~2-fold with increased substrate. These data not only show differences in the human and plant enzymes in vivo, but they confirm previous data that the HsPIPK1{alpha} binds actin through a scaffold of actin binding proteins and show that orthologous proteins are not present in the tobacco cells.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Our goal was to identify AtPIPK1-interacting proteins and to determine whether AtPIPK1 was associated with F-actin. Our approach was to compare peptide bands from solubilized Arabidopsis microsomes that were selectively recovered with AtPIPK1 and AtPIPK10 in pulldown assay experiments. One of the challenges in using mass spectrometry to identify proteins in the pulldown experiments is the limited amount of protein recovered. By adding 30 mM beta-cyclodextrin to the pull down during incubation, the amount of interacting proteins recovered was increased dramatically. Cyclodextrins have been shown to aid in protein folding and lipid sequestration (35, 44). The reason that beta-cyclodextrin enhances the protein-protein interactions could be that the cyclodextrin sequesters lipids from the solubilized membranes and removes any barrier for the proteins to interact or allows the proteins to maintain an appropriate conformation for interaction. Using this technique, we identified PI4Kbeta1 and several other interacting proteins.


Figure 7
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  		FIGURE 7.
Plasma membrane-associated PtdInsP kinases are found with an F-actin fraction. A, plasma membranes were isolated from wild-type (WT) NT-1 cells and NT-1 cells expressing {Delta}MORN that had been treated with 10µM latrunculin B (LatB) or mock-treated with an equal volume of Me2SO for 1 h. The plasma membrane was solubilized, and the F-actin pellet was recovered. The F-actin fraction (white bars) and remaining solubilized fraction (gray bars) were assayed for PtdInsP kinase activity. Most of the PtdInsP kinase activity is associated with F-actin under normal growth conditions. After latrunculin treatment, there was little membrane-associated actin, and most of the membrane-associated PtdInsP kinase activity remained in the solubilized fraction. Note that the plasma membrane enzyme activity per mg of protein was approximately the same whether cells were treated with latrunculin or not. B and C, the plant kinase is predominantly associated with the plasma membrane F-actin fraction, whereas the human kinase is associated with the solubilized fraction. Plasma membranes were isolated from wild-type NT-1 cells and NT-1 cells expressing HsPIPK1{alpha}, and the F-actin pellet was recovered. The F-actin fraction (white bars) and remaining solubilized fraction (gray bars) were assayed for PtdInsP kinase activity. Note the different scales on the y axes in B and C. The HsPIPK1{alpha} kinase is much more active than the endogenous plant kinases, and activity of the HsPIPK1{alpha} saturates at 50 µM PtdIns4P.

To confirm the activity of the plant kinase in the actin fraction of the HsPIPK1{alpha} cell lines, the assay was done using 2 substrate concentrations (50 or 125 µM PtdIns4P). The activity in the F-actin fraction increased 2-fold in both cell lines, indicating the presence of the plant PIPK.

 
The most unusual finding came as we began to investigate the AtPIPK1 actin scaffold. Unlike what has been reported in mammalian cells (16), AtPIPK1 bound F-actin directly, and binding did not require a functional kinase. Through using a series of truncations we showed that the predicted linker region, a proline-enriched region between the MORN domains and the catalytic domain, was necessary for the direct interaction of AtPIPK1 with F-actin. Neither AtPIPK10 nor HsPIPK1{alpha} bound actin directly, and neither have high similarity to AtPIPK1 in the predicted linker region.

Comparisons of AtPIPK1 and HsPIPK1{alpha} actin binding both in vitro and in vivo emphasize the different mechanisms of regulation in plants and humans. None of the tobacco cell proteins fulfilled the role of the animal actin-binding proteins sufficiently for HsPIPK1{alpha} to be associated with F-actin isolated from the plant membranes. By taking advantage of these differences, one could use this heterologous eukaryotic system for investigating the human PIPK-actin scaffold.

AtPIPK1 bound G-actin as well as F-actin, and the actin binding did not saturate at a 1:1 ratio of G-actin to AtPIPK1. Furthermore, AtPIPK1 increased the recovery of F-actin under conditions that did not favor complete filament formation. These data suggested that there might be multiple actin binding sites. There was no increase in the recovery of F-actin with a 20,000 centrifugation unless eEF1A was present, indicating that AtPIPK1 does not bundle actin (data not shown), but it might facilitate actin branching or nucleation. Further kinetic studies of binding await the identification of the key amino acids within the linker region that are essential for binding.

The data revealed not only a direct interaction of AtPIPK1 with actin but that AtPIPK1 would recruit AtPI4Kbeta1 to the actin, thus initiating an actin scaffold. This result was not expected because AtPI4Kbeta1 alone did not localize to F-actin. As had been shown previously (45), AtPI4K{alpha}1, another type III PtdIns 4-kinase, is found in an F-actin fraction polymerized from Sf9 cell lysates, whereas AtPI4Kbeta1 was normally not detected. This is consistent with the data presented here since the binding partner of AtPI4Kbeta1, a plant PIPK, was not present in the Sf9 system to recruit AtPI4Kbeta1 to F-actin. The interaction of AtPI4Kbeta1 and AtPIPK1 is intriguing because it suggests a mechanism for supplying PtdIns4P to AtPIPK1. AtPI4Kbeta1, in contrast to AtPI4K{alpha}, is product activated (31). AtPIPK1 is also product activated (27). Therefore, the AtPI4Kbeta1-AtPIPK1 complex would be in a feed forward mode and could produce microdomains of PtdIns(4,5)P2.

Previous work showed that PtdOH-activation of AtPIPK1 (4) was mediated through the predicted linker region between the MORN domains and the catalytic domain (27). It was hypothesized that a PtdOH-mediated conformational change through the linker region was essential for activation. Selected isoforms of phospholipase D interact directly with actin, and activity increases when F-actin is present (46, 47). The interaction of phospholipase D and AtPIPK1 could localize the proteins together and provide a stimulus for AtPIPK1 as phospholipase D produces PtdOH. Because AtPIPK1 interaction with the cytoskeleton is through the predicted linker region, it is possible that a conformational change in the linker region upon PtdOH stimulation causes a conformational change in AtPIPK1 and facilitates its binding to the membrane where enriched microdomains of PtdIns(4,5)P2 would be generated.

Targeted PtdIns(4,5)P2 biosynthesis has been shown to be critical for pollen tube growth (19, 20, 39) and normal root development (22, 39). Preuss et al. (39) recently showed that AtPI4Kbeta1 binds to RabA4b and a subset of F-actin-associated Golgi vesicles in root hair cells. Taken together with the data presented here, the data could describe a dynamic process associated with selective actin-mediated vesicle trafficking where vesicles containing AtPI4Kbeta1 can be recruited to F-actin by AtPIPK1 and would supply PtdIns4P for the production of a pool of PtdIns(4,5)P2.

Many have shown that PtdIns(4,5)P2 levels can affect the actin cytoskeleton and that PtdIns(4,5)P2 binds to plant actin-binding proteins (19, 25, 37, 38). The assumption is that the inositol lipids are associated with the membrane and regulate actin dynamics by binding to the actin binding proteins. We showed that AtPIPK1 can initiate an actin scaffold and that lipid kinase function was not essential for AtPIPK1 to bind actin. It is likely that other actin binding proteins will compete with AtPIPK binding and that binding will vary with the physiology of the cell.

Our in vivo data also reveal that plant PIPKs associate with the membrane even when F-actin is disrupted by latrunculin treatment. Although membrane-associated actin is not essential for the lipid kinases to bind the membrane, it will surely affect the in vivo dynamics and would be expected to direct the generation of microdomains enriched in PtdInsP2.


   FOOTNOTES
 
* This work was supported in part by funding from the North Carolina Agricultural Research Service and by the National Science Foundation (to W. F. B.) and by the NIEHS, National Institutes of Health (to K. B. T.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1–3. Back

1 To whom correspondence should be addressed: Plant Biology, Box 7649, NC State University, Raleigh NC 27695-7649. Tel.: 919-515-3496; Fax: 919-515-3436; E-mail: wendy_boss{at}ncsu.edu.

2 The abbreviations used are: PIPK, phosphatidylinositol phosphate kinase; eEF1A, eukaryotic elongation factor 1A; MORN, membrane occupation and recognition nexus; PtdIns4P, phosphatidylinositol-4-phosphate; PtdIns(4,5)P2, phosphatidylinositol-4,5-bisphosphate; PI4K, phosphatidylinositol kinase; PtdOH, phosphatidic acid; MALDI, matrix-assisted laser desorption ionization time; PLC, phospholipase C; GST, glutathione S-transferase; MS, mass spectroscopy; PIPES, 1,4-piperazinediethanesulfonic acid. Back

3 R. M. Galvão and W. F. Boss, unpublished results. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Richard Anderson of the University of Wisconsin for the gift of HsPIPK1 {alpha} cDNA and Dr. Linda Hanley-Bowdoin for use of the Nikon microscope.



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 ABSTRACT
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 RESULTS
 DISCUSSION
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