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Originally published In Press as doi:10.1074/jbc.M607079200 on October 12, 2006

J. Biol. Chem., Vol. 281, Issue 51, 39396-39406, December 22, 2006
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Structural and Membrane Binding Analysis of the Phox Homology Domain of Phosphoinositide 3-Kinase-C2{alpha}*Formula

Robert V. Stahelin{ddagger}, Dimitrios Karathanassis§1, Karol S. Bruzik, Michael D. Waterfield||, Jerónimo Bravo§2, Roger L. Williams§, and Wonhwa Cho{ddagger}3

From the Departments of {ddagger}Chemistry and Medicinal Chemistry and Pharmacognosy, University of Illinois, Chicago, Illinois 60607, §MRC Laboratory of Molecular Biology, Cambridge CB2 2QH, United Kingdom, and ||Ludwig Institute of Cancer Research, University College, London W1P 8BT, United Kingdom

Received for publication, July 25, 2006 , and in revised form, October 10, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Phox homology (PX) domains, which have been identified in a variety of proteins involved in cell signaling and membrane trafficking, have been shown to interact with phosphoinositides (PIs) with different affinities and specificities. To elucidate the structural origin of diverse PI specificities of PX domains, we determined the crystal structure of the PX domain from phosphoinositide 3-kinase C2{alpha} (PI3K-C2{alpha}), which binds phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2). To delineate the mechanism by which this PX domain interacts with membranes, we measured the membrane binding of the wild type domain and mutants by surface plasmon resonance and monolayer techniques. This PX domain contains a signature PI-binding site that is optimized for PtdIns(4,5)P2 binding. The membrane binding of the PX domain is initiated by nonspecific electrostatic interactions followed by the membrane penetration of hydrophobic residues. Membrane penetration is specifically enhanced by PtdIns(4,5)P2. Furthermore, the PX domain displayed significantly higher PtdIns(4,5)P2 membrane affinity and specificity when compared with the PI3K-C2{alpha} C2 domain, demonstrating that high affinity PtdIns(4,5)P2 binding was facilitated by the PX domain in full-length PI3K-C2{alpha}. Together, these studies provide new structural insight into the diverse PI specificities of PX domains and elucidate the mechanism by which the PI3K-C2{alpha} PX domain interacts with PtdIns(4,5)P2-containing membranes and thereby mediates the membrane recruitment of PI3K-C2{alpha}.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Numerous cellular processes, including cell signaling and membrane trafficking, involve complex arrays of protein-protein and lipid-protein interactions. Research in the past decade has revealed that a large number of cellular proteins reversibly translocate to specific subcellular locations to form lipid-protein interactions (1, 2). These proteins, collectively known as peripheral proteins, typically contain one or more modular domains specialized in lipid binding (3, 4). Common targets of many of these lipid-binding domains are phosphoinositides (PIs),4 which play a crucial role in cell signaling and membrane trafficking by serving as site-specific membrane signals to modulate the intracellular localization and/or biological activity of effector proteins (5-8). PIs are metabolized by kinases and phosphatases, which catalyze the phosphorylation and dephosphorylation of PI at the 3'-, 4'-, or 5'-position (9-12). PI 3-kinases (PI3Ks) are enzymes that catalyze the phosphorylation of phosphatidylinositol (PtdIns), phosphatidylinositol 4-phosphate (PtdIns4P), or phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) at the 3'-position of the inositol ring (13-15). PI3Ks are divided into three classes (class I, II, and III) based upon their structures and in vitro activities (16, 17). Class II PI3K includes PI3K-C2{alpha}, PI3K-C2beta, and PI3K-C2{gamma}. These enzymes phosphorylate PtdIns and PtdIns4P at the 3'-position in vitro (18-21) and act downstream of receptors for growth factors (22), chemokines (23), and integrins (24). Specifically, PI3K-C2{alpha} is activated by clathrin and has been shown to regulate clathrin-mediated membrane trafficking (25, 26) as well as being essential for ATP-dependent priming of neurosecretory granule exocytosis (27). Class II PI3Ks harbor two membrane-targeting modules near their C terminus, a Phox homology (PX) domain and a C2 domain. The PX domain of PI3K-C2{alpha} was reported to bind PtdIns(4,5)P2 (28), whereas the C2 domain was reported to contain a nuclear localization signal (29) and bind PtdIns(4,5)P2 and phosphatidylinositol-3,4-bisphosphate (PtdIns(3,4)P2) (30). The roles and mutual interactions of these two domains in the membrane targeting and activation of PI3K-C2{alpha} still remain unknown.

The PX domain is a structural module composed of 100-140 amino acids that was first identified in the p40phox and the p47phox subunits of NADPH oxidase (31) and has since been found in a variety of other proteins involved in membrane trafficking (e.g. Mvp1p, Vps5p, Bem1p, and Grd19p and sorting nexins) and cell signaling (e.g. phospholipase D, PI3K, cytokine-independent survival kinase, and five SH3 domains (FISH) adaptor) (32-34). Sequence comparisons of PX domains have shown that they contain several conserved regions, including a proline-rich stretch (PXXP) and a number of basic residues (32-34). Recently, PX domains have been shown to interact with different PIs and target the host proteins to different subcellular locations. Many PX domains, including those of Vam7p (35), sorting nexin 3 (36), and p40phox (37, 38), specifically interact with phosphatidylinositol 3-phosphate (PtdIns3P) in vitro and also target the host proteins to early endosomes in the cell. In addition, most of yeast PX domains were reported to bind PtdIns3P, albeit with varying affinities (39). Unlike these PX domains, the PX domain of PI3K-C2{alpha} interacts with PtdIns(4,5)P2 (28), whereas the p47phox PX domain preferentially interacts with PtdIns(3,4)P2 (37). Also, the PX domains of the yeast protein Bem1p (40) and phospholipase D1 (41) have specificity for PtdIns4P and phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3), respectively. Recently, the PX domain of Nox organizing protein 1 was reported to bind PtdIns4P, phosphatidylinositol 5-phosphate (PtdIns5P), and phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2) (42).

Recent structural and modeling studies of a variety of PX domains have provided insight into the stereospecific head group recognition and membrane-binding mechanisms of PX domains. For example, the crystal structure of the p40phox-PtdIns3P complex revealed that basic residues, Lys92 and Arg58, specifically form hydrogen bonds with the 1- and 3-phosphate of PtdIns3P, respectively, thereby achieving the stereospecific recognition of PtdIns3P (43). The crystal structure of cytokine-independent survival kinase-PX showed that this domain also has all the basic residues necessary for binding the 3-phosphate of PtdIns3P (44). The crystal structure of the PX domain of p47phox (45) revealed that this PX domain has two phospholipid-binding sites for PtdIns(3,4)P2 and phosphatidic acid (or phosphatidylserine), respectively. Similarly, a mutational and homology modeling study of the phospholipase D1 PX domain suggested that it also has two binding sites, one specifically binding PtdIns(3,4,5)P3 and the other interacting nonspecifically with anionic phospholipids (41). The crystal structures of the free and PtdIns3P-bound PX domain of yeast Grd19p protein showed the lipid-induced local conformational changes involving putative membrane-penetrating hydrophobic residues (46).

To gain a better understanding of differential PI specificities and membrane-binding mechanisms of PX domains, we determined the x-ray crystal structure of the PI3K-C2{alpha} PX domain that was reported to specifically bind PtdIns(4,5)P2 (28). We also measured the interaction of the PX domain and mutations with model membranes containing various PIs by surface plasmon resonance (SPR) and monolayer penetration analyses. In addition, we measured the PI-dependent membrane binding of the C2 domain and the full-length PI3K-C2{alpha}. Results from these studies provide new insight into how the PI3K-C2{alpha} PX domain specifically recognizes PtdIns(4,5)P2 and how the PX and C2 domains mediate the PtdIns(4,5)P2-dependent membrane targeting and activation of PI3K-C2{alpha}.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphate, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (POPS), and 1-palmitoyl-2-oleoyl-sn-glycero-phosphoethanolamine (POPE) were from Avanti%20Polar%20Lipids">Avanti Polar Lipids (Alabaster, AL). PtdIns3P, PtdIns4P, PtdIns5P, PtdIns(3,4)P2, PtdIns(3,5)P2, PtdIns(4,5)P2, and PtdIns(3,4,5)P3 were synthesized as described previously (47). Phospholipid concentrations were determined by a modified Bartlett analysis (48). The Liposofast microextruder and 100-nm polycarbonate filters were from Avestin (Ottawa, Ontario). Fatty acid-free bovine serum albumin was from Bayer, Inc. (Kankakee, IL). Restriction endonucleases and other enzymes for molecular biology were from New England Biolabs (Beverly, MA). CHAPS and octyl glucoside were from Sigma and Fisher, respectively. Pioneer L1 sensor chip was from Biacore AB (Piscataway, NJ).

Structure Determination—For the structure determination, DNA encoding the human PI3K-C2{alpha} PX domain (residues 1405-1541) was amplified by PCR from IMAGE clone 825260 and subsequently cloned with a C-terminal GSH6 affinity tag in pJL vector. The cloned PX domain was sequenced and showed two differences with respect to the only published human sequence of PI3K-C2{alpha} (accession code NP_002636 [GenBank] ). Residue 1450 is Arg in our sequence instead of Trp, and residue 1464 is an Asp instead of Val. Residue 1464 is critical for the lipid specificity of PX domains (see below). All of the PI3K-C2{alpha} sequences that have been reported have an Asp at the position equivalent to human PI3K-C2{alpha} residue 1464, and all of the PI3K-C2beta and PI3K-C2{gamma} sequences have a Glu at this position. Residue 1450 corresponds to a more variable region among PX domains in general, but it is an Arg in all other mammalian PI3K-C2 sequences. Consequently, it is likely that these differences with respect to the data base reflect errors in the data base entry.

The protein was expressed in Escherichia coli strain C41(DE3) and purified by Ni2+-affinity, heparin, and gel-filtration chromatography as described in the Supplemental Methods. The protein in gel-filtration buffer (20 mM Tris-HCl (pH 7.4 at 25 °C), 100 mM NaCl, and 5 mM dithiothreitol) was concentrated to 5 mg/ml. Crystals were obtained by hair seeding drops (3 µl of protein plus 3 µl of reservoir solution) that were incubated at 17 °C over a reservoir consisting of 1.7 M Li2SO4 and 0.05 M MES, pH 5.6. For the hair seeding, a hair from a poodle was passed through a drop with crystals and then passed through a series of six freshly set up drops with no crystals so that the number of nuclei would thereby be serially diluted. When passing through each of the 6 drops, a pattern of an "X" was traced through the drop with the hair. In most cases, the 1st drop of the series had a shower of small crystals along the path of the hair, and in the later drops in the series only a few crystals were formed, but these were much larger. Crystals were visible after 24 h and grew to full size within 1 week.

For diffraction data collection, crystals were cryoprotected in reservoir solution supplemented with 15% glycerol and were flash-frozen in a nitrogen stream at 100 K. A heavy metal derivative crystal was prepared by soaking a crystal in cryoprotectant supplemented with 1 mM ethylmercury thiosalicylate for 15 min. An initial, lower resolution, 2.9 Å native data set was also collected and used for initial single isomorphous replacement phasing. Tables 1, 2, 3 summarizes the data collection statistics. Images were processed with the program MOSFLM (49) and refined with SCALA (50). Three mercury sites were located with RANTAN and refined with AutoSHARP. After density modification with SOLOMON (51) and DM (52), an initial model was manually built in a 2.9-Å resolution electron density map using COOT (53) and O (54). A subsequent 2.6-Å data set was obtained for a native crystal, and this data set was used for refinement with REFMAC (55). There is one PX domain in the asymmetric unit. The Ramachandran analysis with the program PROCHECK (56) shows 88.8% of residues in the most probable regions and none in disallowed regions. The refinement statistics are given in Tables 1, 2, 3. A representative section of the experimental and refined electron densities are illustrated in supplemental Fig. 1. The coordinates have been deposited in the Protein Data Bank (accession number 2iwl).


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  		TABLE 1
Data collection statistics

 


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  		TABLE 2
Phasing statistics

 


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  		TABLE 3
Refinement statistics

 
Mutagenesis and Protein Expression—Mutagenesis of the PI3K-C2{alpha} PX domain was performed using the overlap extension PCR method (57). Constructs were subcloned into the pET21a vector containing a C-terminal hexahistidine tag and transformed into DH5{alpha} cells for plasmid isolation. After checking each construct for correct sequence, the plasmid was transformed into BL21(DE3) cells for protein expression. The PX domains used for monolayer and SPR measurements were expressed and purified as described previously (58) with minor variation, i.e. the domains purified by nickel-nitrilotriacetic acid column chromatography were further purified by gel filtration chromatography. Briefly, a 1-ml sample of each PX domain was loaded onto a Superdex 200 column (Amersham Biosciences) equilibrated and eluted with 20 mM Tris-HCl buffer, pH 7.4, containing 0.16 M KCl. Fractions corresponding to the PI3K-C2{alpha} PX peak were pooled and concentrated to 1 ml. The C2 domain (residue 1562-1679) was cloned into pET21a using NdeI and XhoI sites. For C2 domain expression, BL21(DE3) cells were grown at 37 °C in LB media containing 100 µg/ml ampicillin. Protein expression was induced when the absorbance at 600 nm reached 0.8 and continued for 4 h at 25 °C. Cells were pelleted by centrifugation, and the C2 domain was purified as described previously (59). The full-length PI3K-C2{alpha} was expressed in Sf9 cells, purified, and assayed as described previously (21). Protein concentration was determined by the bicinchoninic acid method (Pierce).

Monolayer Measurements—The penetration of PI3K-C2{alpha} and isolated domains into the phospholipid monolayers of different lipid compositions was measured in terms of the change in surface pressure ({pi}) using a 10-ml circular Teflon trough and a Wilhelmy plate connected to a Cahn microbalance as described previously (60).

SPR Measurements—All SPR measurements were performed at 23 °C in 10 mM HEPES, pH 7.4, containing 0.16 M KCl. A detailed protocol for coating the L1 sensor chip with lipid vesicles was reported previously (61, 62). Briefly, after washing the sensor chip surface with the buffer, 90 µl of vesicles composed of POPC/POPE/PI (78:20:2) (see Fig. 3) were injected at 5 µl/min to give a response of 5500 resonance units. Similarly, a control surface was coated with POPC/POPE (80:20) vesicles, to give the same resonance unit response as the active binding surface. Under our experimental conditions, no binding was detected to this control surface beyond the refractive index change for all proteins. Each lipid layer was washed with 10 µlof 50 mM NaOH three times at 100 µl/min. Typically, no decrease in lipid signal was seen after the first injection. Equilibrium (steady state) SPR measurements were performed with the flow rate of 5 µl/min to allow sufficient time for the R values of the association phase to reach saturating response values (Req) (62, 63). After sensorgrams were obtained for five different concentrations of each protein within a 10-fold range of Kd (see Table 4), each of the sensorgrams was corrected for refractive index change by subtracting the control surface response from it. Req values were then plotted versus protein concentrations (C), and the Kd value was determined by a nonlinear least squares analysis of the binding isotherm using an equation, Req = Rmax/(1 + Kd/C). Each data set was repeated three times to calculate average and standard deviation values.


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  		TABLE 4
Binding parameters for PI3K-C2{alpha} PX domain and mutants determined from SPR analysis Values represent the mean ± S.D. from three determinations. All measurements were performed in 10 mM HEPES, pH 7.4, containing 0.16 M KCl. Lipid vesicles were POPC/POPE/PtdIns(4,5)P2 (75:20:2).

 


Figure 1
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  		FIGURE 1.
PI3K-C2{alpha} PX domain overall structure. A, ribbon diagram of the overall fold of the PI3K-C2{alpha} PX domain colored from blue at the N terminus to red at the C terminus. B, superposition of the C{alpha} trace of the PI3K-C2{alpha} PX domain (red) on the p40phox PX domain (green). The disordered PPII/{alpha}2 loop in the PI3K-C2{alpha} PX domain is represented by the dashed line. Molecular illustrations were prepared using the program PyMOL.

 


Figure 2
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  		FIGURE 2.
Phosphoinositide-binding pocket of PI3K-C2{alpha}. A, solid surface of the PI3K-C2{alpha} PX domain showing the lipid-binding pocket. The PtdIns3P from the p40phox PX domain structure is superimposed on the surface. The presence of Thr1462 (magenta) and Asp1464 (red) in place of Arg58 and Arg60 from the p40phox PX domain preclude binding of a phosphoinositide head group with a 3-phosphate. Arg1503 (blue) is in the vicinity of the 4- and 5-OH groups of the phosphoinositide and is the principal ligand of the sulfate bound in the PI3K-C2{alpha} PX domain lipid-binding pocket. B, expanded view of the lipid-binding pocket with the p40phox PX domain (green) superimposed on the Grd19 PX domain (salmon) and the PI3K-C2{alpha} PX domain (white). Despite the variation in conformation in the PPII/{alpha}2 loop, the residue analogous to Lys92 of the p40phox PX domain is the 1-phosphate ligand for both Grd19 and p40phox. This suggests that the analogous residue in the PI3K-C2{alpha} PX domain, Arg1488, may also be the 1-phosphate ligand. The polyproline loop (PPII) of PI3K-C2{alpha} is highlighted as a yellow tube. The N- and C-terminal regions of the domains away from the PI-binding pocket have been removed for clarity. The dashed line joining the end of the PPII to helix {alpha}2 emphasizes the disordered region of the PI3K-C2{alpha} PI-binding pocket.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Description of the Overall Structure—Unlike p40phox, Vam7p, and Grd19p PX domains, PI3K-C2{alpha} has been reported to bind PtdIns(4,5)P2 (28). To elucidate the basis of stereospecific recognition of PtdIns(4,5)P2 by the PI3K-C2{alpha} PX domain, we determined its crystal structure by x-ray diffraction analysis.

The PI3K-C2{alpha} PX domain crystallized in space group P423 with a = 115.9 Å and one molecule per asymmetric unit. Residues 1405-1408, 1488-1497, and 1540-1541 are disordered. The fold of the PI3K-C2{alpha} PX domain consists of a three-stranded meander beta-sheet subdomain followed by a helical subdomain with four {alpha}-helices and one 310 helix (Fig. 1A). The beta-sheet is twisted by the bulge at residues 1430 and 1431 in strand beta1. This twist of the beta-sheet toward the helical subdomain enables the formation of a pocket bordered on one side by the beginning of strand beta2. A sulfate is bound in this pocket on the surface of the domain and interacts with Arg1503 of helix {alpha}3.

The PI3K-C2{alpha} PX domain superimposes closely on other PX domains such as the PX domain from p40phox (Fig. 1B). However, a loop immediately following the PXXP motif at residues 1483-1486 is disordered in the PI3K-C2{alpha} PX domain (shown as dashed line in Fig. 1, A and B). This disorder gives the lipid-binding pocket a much more open appearance compared with the analogous pocket of p40phox. The core of the beta-sheet subdomain is nearly identical among PX domains. However, the beta1-beta2 turn in the PI3K-C2{alpha} PX domain has a cis-proline (Pro1438), accentuating the curvature of the concave face of the beta-sheet relative to other PX domains.

The comparison of the lipid-binding sites from p40phox (43) and PI3K-C2{alpha} PX domains immediately reveals why it would be impossible for PtdIns3P to bind the PI3K-C2{alpha} PX domain. Both negative selection against the 3-phosphate and loss of positive selection for the 3-phosphate contribute to the lipid specificity of the PI3K-C2{alpha} PX domain. Fig. 2, A and B, illustrate that positive selection is lost because the essential, conserved determinant of the 3-phosphate interaction, which is Arg58 in p40phox, has been replaced by Thr1462 in PI3K-C2{alpha}. Arg60 of the p40phox PX domain, which is one of the residues responsible for its 1-phosphate coordination, has been replaced in PI3K-C2{alpha} by Asp1464. The orientation of Asp1464 is such that it imposes a negative selection against 3-phosphate, as it would electrostatically repel a phosphate.

The PPII/{alpha}2 loop is disordered in the PX domain structure, preventing us from suggesting a detailed model of PtdIns(4,5)P2 binding to the PI3K-C2{alpha} PX domain. However, the bound sulfate molecule in the PI3K-C2{alpha} PX domain seems to indicate a plausible position of 4-phosphate binding. The sulfate in PI3K-C2{alpha} is hydrogen-bonded to Arg1503, which superimposes well with the p40phox residue (Arg105) that interacts with the 4- and 5-OH of PtdIns3P. In the PI3K-C2{alpha} PX domain structure, there is no clear indication for a 5-phosphate specificity determinant. However, a few candidate residues could be suggested as illustrated in Fig. 2B. In the p40phox and Grd19 PX domains, the backbone of the variable loop between PPII and {alpha}2 embraces the 4- and 5-OH of the bound phosphoinositide, and the side chain of a basic residue analogous to Arg1503 in helix {alpha}2 of PI3K-C2{alpha} interacts with these groups (Fig. 2B). Consequently, basic residues in the variable loop or at the beginning of helix {alpha}2 are likely possibilities as 5-phosphate ligands. Arg1488, Arg1493, and Lys1497 are such candidates. Another possibility might be Lys1440 from the beta1/beta2 loop. The fact that the variable PPII/{alpha}2 loop is disordered indicates an accented flexibility, suggesting that lipid binding may confer localized conformational changes enabling Lys1440, Arg1488, Arg1493, or Lys1497 to point toward the lipid-binding pocket. Indeed, studies of the Grd19 PX domain in the absence and presence of PtdIns3P show that the PPII/{alpha}2 loop closes in on the bound lipid (46). The variable PPII/{alpha}2 loops of p40phox and Grd19 also provide the 1-phosphate ligand (Fig. 2B).

Intramolecular interactions between polyproline regions and SH3 domains have been reported previously for several proteins, including p47phox (45). It appears that for such intramolecular interactions to occur in PX domains, a type II polyproline motif is required. The p40phox, Grd19, and PI3K-C2{alpha} PX domains all contain polyproline regions, which feature the PXXPXR(K) consensus, which corresponds to the type II SH3 ligand motif PXXPX+ (where + refers to a positively charged amino acid and X to any residue). In p40phox-PX the last residue of the motif, Lys92, participates in lipid recognition. The equivalent residue in PI3K-C2{alpha}-PX, Arg1488, is disordered, but it is possible that Arg1488 might become ordered on lipid binding, and it may be that this residue is the 1-phosphate ligand (Fig. 2B). In all three proteins, the prolines of the PXXP loop are buried. PI3K-C2{alpha} isoforms do not contain SH3 domains, and to date there has been no report of an interaction between PI3K-C2{alpha} and the SH3 domain from another protein. These observations suggest that perhaps type II SH3-ligand motifs in PX domains are not sufficient on their own to mediate interactions with SH3 domains.

Membrane Binding Properties—To determine the functional roles of the putative phospholipid-binding residues of PI3K-C2{alpha} PX, we measured the vesicle binding of wild type and a series of site-specific mutants by SPR and monolayer penetration analyses. We first measured the PI specificity of PI3K-C2{alpha} PX domain by SPR analysis. In these experiments, an active surface was coated with POPC/POPE/PI (75:20:5), whereas a control surface was coated with POPC/POPE (80:20). Fig. 3 shows the binding response changes for 500 nM of PI3K-C2{alpha} PX injected over various PI-containing membranes. Clearly, PI3K-C2{alpha} PX has specificity for PtdIns(4,5)P2-containing vesicles, as it exhibited no significant binding to all other membranes. Fig. 4A shows representative sensorgrams for equilibrium binding analysis of PI3K-C2{alpha} PX-POPC/POPE/PtdIns(4,5)P2 (78:20:2) vesicle binding, and Kd values determined from the curve fitting (Fig. 4B) are summarized in Table 4. The affinity of the PI3K-C2{alpha} PX domain for PX-POPC/POPE/PtdIns(4,5)P2 (78:20:2) vesicles (Kd = 25 nM) is higher than that of an archetypal PtdIns(4,5)P2-binding domain, the PH domain of phospholipase C{delta}1 (1-8 µM) (64, 65), and comparable with the ENTH domain of epsin 1 (66).


Figure 3
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  		FIGURE 3.
Phosphoinositide specificity of the PI3K-C2{alpha} PX domain. The PX domain (500 nM) was injected over a POPC/POPE/PI (78:20:2) surface to gauge affinity and specificity for different PIs, including PtdIns3P, PtdIns4P, PtdIns5P, PtdIns(3,4)P2, PtdIns(3,5)P2, PtdIns(4,5)P2, and PtdIns(3,4,5)P3. The SPR response curves for respective PI are shown after background correction for binding to the control surface coated with POPC/POPE (80:20). Binding to the control surface was minimal, and little evidence of nonspecific binding was evident at 500 nM of protein.

 


Figure 4
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  		FIGURE 4.
Equilibrium SPR binding analysis of PI3K-C2{alpha} PX. A, PI3K-C2{alpha} PX domain was injected at 2 µl/min at varying concentrations (2, 5, 10, 20, and 60 nM from bottom to top) over the POPC/POPE/PtdIns(4,5)P2 (78:20:2) surface, and Req values were measured. B, binding isotherm was generated from the Req (average of triplicate measurements) versus the concentration of the PI3K-C2{alpha} PX domain plot. A solid line represents a theoretical curve constructed from Rmax (=170 ± 2) and Kd values (=25 ± 4 nM) determined by nonlinear least squares analysis of the isotherm using the following equation: Req = Rmax/(1 + Kd/C). 10 mM HEPES buffer, pH 7.4, with 0.16 M KCl was used for all measurements.

 
We then measured the binding of PI3K-C2{alpha} PX mutants to POPC/POPE/PtdIns(4,5)P2 (78:20:2) vesicles. In agreement with the structural analysis, the mutation of the conserved Arg1503 (i.e. R1503A) abolished binding to PtdIns(4,5)P2-containing membranes, corroborating the notion that this conserved Arg residue is essential for recognition of PtdIns(4,5)P2 and may interact with the 4-phosphate. Mutations of two other basic residues, Arg1488 and Arg1493, that are candidates for 5-phosphate recognition in the PtdIns(4,5)P2-binding pocket of PI3K-C2{alpha} PX (R1488A and R1493A) reduced the vesicle affinity of the PX domain by 7- and 23-fold, respectively, whereas mutations of other neighboring residues (K1440A and K1497A) had little effect. This indicates that Arg1488 and Arg1493, in addition to Arg1503, may be involved in specific PtdIns(4,5)P2 recognition. Gross misfolding was precluded for all mutations as they were expressed at the same level as wild type and bound POPC/POPS (5:5) vesicles with the same affinity as wild type (data not shown).

Recently, it has been shown that hydrophobic residues surrounding the PI-binding pockets of FYVE (67, 68), PX (46, 58), and ENTH (66, 69) domains can penetrate the membrane following PI binding. We thus measured the effects of mutations of hydrophobic residues (Val1490 and Leu1491) surrounding the PI-binding pocket of the PI3K-C2{alpha} PX domain to see if they are involved in membrane penetration. V1490A and L1491A exhibited a 7- and 5-fold lower membrane affinity than the wild type, respectively. Interestingly, mutation of Leu1479, which is located on the same molecular surface with Val1490 and Leu1491 but distant from the PI-binding pocket, had little effect on binding to PtdIns(4,5)P2-containing vesicles. This indicates that only hydrophobic residues adjacent to the PI-binding pocket specifically penetrate the membrane (see below).

Membrane penetration properties of PI3K-C2{alpha} PX domain and mutants were further characterized by lipid monolayer penetration measurements. Recent studies have shown that PIs can specifically induce the membrane penetration of FYVE (68), PX (58), and ENTH (66, 69) domains. To determine whether or not PtdIns(4,5)P2 can also elicit the membrane penetration of the PI3K-C2{alpha} PX domain, we first measured the penetration of the PX domain to monolayers with different lipid compositions (see Fig. 5). The PI3K-C2{alpha} PX domain cannot penetrate the POPC/POPE (80:20) monolayer with the surface pressure above 26 dynes/cm; however, when 2 mol % PtdIns(4,5)P2 was included in the monolayer (i.e. POPC/POPE/PtdIns(4,5)P2 (78:20:2)), the domain penetrated the monolayer with the surface pressure of up to 33 dynes/cm. Because the surface pressure of biological membranes has been estimated to be 30-35 dynes/cm (70-72), these data suggest that PtdIns(4,5)P2 can induce the penetration of the PI3K-C2{alpha} PX domain into cell membranes under physiological conditions. The specific nature of this penetration was confirmed by two additional measurements. First, neither 2 mol % PtdIns(3,4)2 nor 2 mol % PtdIns(3,4,5)P3 nor 20 mol % phosphatidylserine in the monolayer significantly influenced the penetration behaviors of PI3K-C2{alpha} PX. Second, mutations of residues involved in specific PtdIns(4,5)P2 recognition (i.e. R1503A and R1488A) greatly reduced the penetration of the PI3K-C2{alpha} PX domain to the POPC/POPE/PtdIns(4,5)P2 (78:20:2) monolayer, whereas mutations that had no effect on Kd values, K1440A and L1479A, did not cause reduction in monolayer penetration (Fig. 6A). Finally, V1490A and L1491A had low monolayer penetration into the POPC/POPE/PtdIns(4,5)P2 (78:20:2) monolayer (Fig. 6B), confirming the notion that these hydrophobic residues are directly involved in the membrane penetration of the PI3K-C2{alpha} PX domain. In contrast, the mutation of Leu1479 located at the other end of the membrane-binding surface had little effect on the monolayer penetration (Fig. 6B), showing that this part of PI3K-C2{alpha} PX does not significantly participate in membrane penetration.


Figure 5
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  		FIGURE 5.
Monolayer penetration analysis of PI3K-C2{alpha} PX domain. {Delta}{pi} was measured as a function of {pi}0 for wild type PI3K-C2{alpha} PX domain with POPC/POPE (80:20) ({circ}), POPC/POPE/PtdIns(4,5)P2 (78:20:2) (•), POPC/POPE/POPS (60:20:20) ({square}), POPC/POPE/PtdIns(3,4)P2 (78:20:2) ({Delta}), and POPC/POPE/PtdIns(3,4,5)P3 (78:20:2) ({blacksquare}) monolayers. The subphase was 10 mM HEPES buffer, pH 7.4 containing 0.16 M KCl for all experiments. Data represent the average of duplicate measurements.

 
PtdIns(4,5)P2 Binding of C2 Domain Versus PX Domain— The recent study of the C2 domain of PI3K-C2{alpha} indicated that it had affinity for PIs, particularly PtdIns(4,5)P2 and PtdIns(3,4)P2 (30). This finding raises a question as to whether the PX domain or the C2 domain plays a major role in PI-mediated membrane binding of PI3K-C2{alpha}. To compare the PI specificities and affinities of the PX and C2 domains, we measured the binding of the C2 domain to PI-containing vesicles by SPR analysis and also monitored their binding to PI-containing lipid monolayers.

We first measured the PI specificity of PI3K-C2{alpha} C2 domain by SPR analysis using the active surface coated with POPC/POPE/PI (75:20:5) vesicles. A control surface was coated with POPC/POPE (80:20) vesicles. Fig. 7 shows the binding response changes for 500 nM of the PI3K-C2{alpha} C2 domain injected over various PI-containing membranes. In agreement with the previous report (30), the PI3K-C2{alpha} C2 domain binds PtdIns(3,4)P2 and PtdIns(4,5)P2; however, unlike the PX domain, the C2 domain can significantly bind other PIs and consequently shows low PI selectivity. In fact, quantitative analysis of the binding data (see Table 5) showed that the C2 domain could not effectively distinguish among various PIs. Perhaps more importantly, the affinity of the C2 domain for POPC/POPE/PtdIns(4,5)P2 (78:20:2) (Kd ~ 490 nM) was about 20-fold lower than that of the PX domain (Kd ~25 nM) under the same conditions. We also measured the binding of the PX and C2 domains as a function of PtdIns(4,5)P2 concentration (i.e. using POPC/POPE/PtdIns(4,5)P2 (80-x:20:x) vesicles). As shown in Fig. 8, the PX domain had higher affinity than the C2 domain throughout the PtdIns(4,5)P2 concentration range used, and the difference was the most dramatic at the physiologically relevant PtdIns(4,5)P2 concentration (i.e. <5 mol %). Also, the PtdIns(4,5)P2 concentration for half-maximal binding of the PX domain (~2 mol %) was significantly lower than that of the C2 domain (~7 mol %), and the latter is consistent with the reported value (30). Collectively, these results indicate that the PX domain would play a dominant role in PtdIns(4,5)P2-mediated membrane binding of PI3K-C2{alpha}.


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  		TABLE 5
Binding affinities of PI3K-C2{alpha} C2 and PX domains for different lipid vesicles Values represent the mean and standard deviation from three determinations. All measurements were performed in 10 mM HEPES, pH 7.4, containing 0.16 M KCl.

 


Figure 6
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  		FIGURE 6.
Monolayer penetration analysis of PI3K-C2{alpha} PX domain mutations. A, {Delta}{pi} was measured as a function of {pi}0 for POPC/POPE/PtdIns(4,5)P2 (78:20:2) with wild type PI3K-C2{alpha} PX domain ({circ}), K1440A ({diamondsuit}), R1488A (•), K1497A ({blacktriangleup}), and R1503A ({blacksquare}). B, {Delta}{pi} was measured as a function of {pi}0 for POPC/POPE/PtdIns(4,5)P2 (78:20:2) with wild type PI3K-C2{alpha} PX domain ({circ}), L1479A ({square}), V1490A ({Delta}), and L1491A ({diamond}). The subphase was 10 mM HEPES buffer, pH 7.4 containing 0.16 M KCl for all experiments. Data represent the average of duplicate measurements.

 


Figure 7
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  		FIGURE 7.
Phosphoinositide specificity of the PI3K-C2{alpha} C2 domain. The C2 domain (500 nM) was injected over a POPC/POPE/PI (75:20:5) surface to estimate affinity and specificity for different PIs. The SPR response curves for respective PI are shown after background correction for binding to the control surface coated with POPC/POPE (80:20). Binding to the control surface was minimal at 500 nM of protein.

 
We then measured the monolayer penetration of the C2 domain to see if the domain shows significant membrane penetration and if PIs could induce the penetration of the C2 domain. Fig. 9A illustrates that the C2 domain has much lower monolayer penetrating power than the PX domain in the presence of PtdIns(4,5)P2 and the {pi}c value for the C2 domain was significantly lower than 30 dynes/cm. Also, unlike the case with the PX domain, PtdIns(4,5)P2 (or PtdIns(3,4)P2) enhanced the monolayer penetration of the C2 domain only slightly. This again indicates that the PX domain should be a main player in all aspects of membrane interactions of PI3K-C2{alpha}, including membrane penetration.

PtdIns(4,5)P2-mediated Membrane Binding of the Full-length PI3K-C2{alpha}—To verify the notion that the PX domain controls the membrane binding of PI3K-C2{alpha}, we prepared wild type and R1503A mutant (PtdIns(4,5)P2 binding-defective; see Table 4) of the full-length PI3K-C2{alpha} and compared their membrane binding properties with the isolated PX and C2 domains. R1503A was expressed as effectively as wild type in the Sf9 cells and had the basal activity comparable with that of wild type, precluding the gross misfolding of the mutant (data not shown). SPR analysis (see Table 5) shows that the full-length PI3K-C2{alpha} and the isolated PX domain have essentially the same affinity for PtdIns(4,5)P2-containing membranes (~20 nM), underscoring the predominant role of the PX domain in the membrane binding of PI3K-C2{alpha}. Interestingly, the R1503A mutant of the full-length protein had 25-fold lower affinity than the wild type, which was comparable with that of the isolated C2 domain. This suggests that the C2 domain supplements the function of the PX domain in PtdIns(4,5)P2-mediated membrane binding and that the two domains work additively rather than synergistically. Finally, we compared the monolayer penetration of the full-length PI3K-C2{alpha} and the isolated PX domain (see Fig. 9B). PI3K-C2{alpha} penetrated the monolayer in a PtdIns(4,5)P2-dependent manner similarly to the isolated PX domain, whereas the R1503A mutation abrogated the PtdIns(4,5)P2 dependent penetration, demonstrating that the PX domain is essential for the penetration of the full-length enzyme.


Figure 8
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  		FIGURE 8.
Membrane binding of the C2 and PX domains as a function of PtdIns(4,5)P2 concentration. Maximum binding response (Rmax) of PX and C2 domains for the surface coated with POPC/POPE/PtdIns(4,5)P2 (80-x:20:x) vesicles, where x is the concentration of PtdIns(4,5)P2. Data represent the average of triplicate measurements.

 


Figure 9
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  		FIGURE 9.
Monolayer penetration analysis of PI3K-C2{alpha} and its isolated domains. A, {Delta}{pi} was measured as a function of {pi}0 for the PI3K-C2{alpha} C2 domain with POPC/POPE (80:20) (•), POPC/POPE/PtdIns(4,5)P2 (78:20:2) ({diamondsuit}), and POPC/POPE/PtdIns(3,4)P2 (78:20:2) ({blacktriangleup}), and POPC/POPE/PtdIns(3,4,5)P3 (78:20:2) ({blacksquare}) monolayers. PI3K-C2{alpha} PX domain ({circ}) is also shown as a control. B, {Delta}{pi} was measured as a function of {pi}0 using a POPC/POPE/PtdIns(4,5)P2 (78:20:2) monolayer for the full-length PI3K-C2{alpha} (•), R1503A ({blacksquare}), and PI3K-C2{alpha} PX domain ({blacktriangleup}). The penetration of the full-length PI3K-C2{alpha} into a POPC/POPE (80:20) monolayer is also shown as a control ({circ}). The subphase was 10 mM HEPES buffer, pH 7.4, containing 0.16 M KCl for all experiments. Data represent the average of duplicate measurements.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
This study addresses two important questions: how does the PX domain of PI3K-C2{alpha} specifically recognize PtdIns(4,5)P2 and how does the PX domain mediate the membrane binding and activation of PI3K-C2{alpha}? PX domains are similar to PH domains in that they have highly variable PI specificities and affinities. High resolution structures of several PX domains that bind the 3'-phosphate have been determined as either free proteins or complexes with PI (43-46). However, the structural information for the PX domains that specifically bind non-3'-phosphate-PI has not been available.

The crystal structure of the PI3K-C2{alpha} PX domain clearly accounts for its inability to bind PtdIns3P that most PX domains prefer. A canonical 3'-phosphate ligand (i.e. Arg58 for p40phox PX) is substituted for by Thr, and an acidic residue, Asp1464, replaces a 1'-phosphate ligand (i.e. Arg60 in p40phox PX). This negative selection would not allow PtdIns3P to favorably interact with the PI-binding pocket of the PI3K-C2{alpha} PX domain. Because of the disorder in the PPII/{alpha}2 loop of the PI3K-C2{alpha} PX domain, there is no direct structural information on how the domain achieves stereospecific recognition of the 4'- and 5'-phosphates of PtdIns(4,5)P2. However, the location of a bound sulfate ion, sequence alignment, and our mutational analysis suggest that three basic residues, Arg1488, Arg1493, and Arg1503, are involved in PtdIns(4,5)P2 binding. Presumably, Arg1503 interacts with the 4'-phosphate, whereas Arg1493 contacts the 5'-phosphate. Arg1488 may be involved in binding to either the 5'-phosphate or the 1'-phosphate.

Our previous studies on the FYVE (68), PX (58), and ENTH (66) domains have indicated that PI binding specifically induces the membrane penetration of surface hydrophobic residues surrounding the PI-binding pocket, presumably by causing local conformational changes of proteins and by neutralizing the positive electrostatic potential surrounding hydrophobic loop residues that interferes with the membrane penetration of domains. Based on these previous studies, we proposed a membrane-binding mechanism for PI-binding domains in which initial membrane adsorption of the domain driven by nonspecific electrostatic interactions between the cationic protein surface and the anionic membrane surface is followed by specific PI-triggered membrane penetration of the domain. This study indicates that PtdIns(4,5)P2-mediated membrane binding of the PI3K-C2{alpha} PX domain follows essentially the same mechanism. Our monolayer data clearly indicate that PtdIns(4,5)P2 binding by the PI3K-C2{alpha} PX domain is not a consequence of but a prerequisite for the penetration of the domain into the monolayer, the packing density of which is comparable with that of cellular membranes and large unilamellar vesicles (70-72). The penetration of hydrophobic residues (Val1490 and Leu1491) located in the loop adjacent to the PtdIns(4,5)P2-binding pocket of PI3K-C2{alpha} PX is a specific PtdIns(4,5)P2-dependent process, because it was abrogated either by removal of PtdIns(4,5)P2 in the monolayer or by mutation of the PtdIns(4,5)P2-coordinating residues, Arg1488, Arg1493, or Arg1503.

This study provides no direct information as to whether or not PtdIns(4,5)P2 binding causes conformational changes of the PI3K-C2{alpha} PX domain. Structural studies of other PX domains have indicated, however, that PI binding causes local conformational changes. For example, a recent NMR study on the PX domain of Vam7p revealed that residues in its putative membrane binding loops underwent large changes in chemical shift when dodecylphosphocholine micelles were added to the PX domain-PtdIns3P complex (35). Also, crystal structures of the Grd19p PX domain in the absence and presence of a bound PtdIns3P showed conformational differences in the putative membrane attachment loop (46). This, in conjunction with the PI3K-C2{alpha} PX domain structure showing high flexibility of the PPII/{alpha}2 loop, suggests that PtdIns(4,5)P2 binding may also induce the conformational change and thereby facilitate the membrane penetration of hydrophobic side chains.

On the basis of qualitative vesicle pelleting assay, a recent study indicated that the C2 domain of PI3K-C2{alpha} has high affinity and specificity for PtdIns(4,5)P2 and PtdIns(3,4)P2 (30). Our quantitative SPR measurements confirm that the C2 domain of PI3K-C2{alpha} can indeed bind PtdIns(4,5)P2 and PtdIns(3,4)P2; however, the C2 domain lacks PI specificity and has about 20-fold lower affinity for PtdIns(4,5)P2-containing vesicles than the PX domain. The promiscuity and lower affinity of the C2 domain for PIs has been reported for other C2 domains from synaptotagmin I (73), JFC1 (74), and Rsp5p (75). Also, unlike the PX domain, the C2 domain of PI3K-C2{alpha} is not able to penetrate the lipid monolayer with the packing density comparable with that of cell membranes with or without PI. Furthermore, the isolated PX domain and the full-length PI3K-C2{alpha} have comparable affinity for PtdIns(4,5)P2-containing monolayers and bilayers. Thus, it is evident that the PX domain plays a more dominant role than the C2 domain in the PtdIns(4,5)P2-mediated membrane recruitment of PI3K-C2{alpha}. It should be noted, however, that promiscuous PI specificity of the C2 domain may allow this domain to play a more significant role in the membrane recruitment of PI3K-C2{alpha} in response to PIs other than PtdIns(4,5)P2. It is also possible that the membrane recruitment of PI3K-C2{alpha} is mediated by both the PX domain and the C2 domain, interacting with PtdIns(4,5)P2 and another PI, respectively. Interestingly, the C2 domain of PI3K-C2{alpha} harbors a nuclear localization sequence and associates with nuclear speckles (29). Further studies are needed to understand how the nuclear localization signal and the lipid-binding site of the C2 domain are related.

It is generally thought that specific subcellular localization of PI3K-C2{alpha} is critical for spatiotemporal regulation of the formation of 3'-phospho-PIs. Although this study clearly shows that the PX domain drives the binding of PI3K-C2{alpha} to PtdIns(4,5)P2-containing membranes in vitro, the role of the PX domain (and the C2 domain) in the subcellular localization of PI3K-C2{alpha} remains less clear. In particular, two recent reports indicate that protein-protein interactions play a primary role in subcellular localization of PI3K-C2{alpha}. Domin et al.(76) reported that PI3K-C2{alpha} was enriched in clathrin-coated vesicles and trans-Golgi network of various mammalian cells but that neither the PX domain nor the C2 domain was necessary for this observed subcellular localization. Also, Gaidarov et al. (25) reported that clathrin bound the N-terminal region of PI3K-C2{alpha} and thereby activated the enzyme. This apparent discrepancy between the in vitro membrane binding properties and subcellular localization of PI3K-C2{alpha} is not totally surprising, however, as similar observations have been made for other peripheral proteins. For example, a study of all PH domains from the yeast genome showed that PI affinity, while contributing to membrane binding, does not specify subcellular localization (77). As a matter of fact, accumulating evidence shows that the subcellular localization of many peripheral proteins, including those proteins that were known to be recruited to the membrane primarily by protein-protein interactions, is orchestrated by a combination of protein-protein and lipid-protein interactions, and that the relative contribution of two types of interactions varies among proteins (1, 78). It should also be noted that an interaction that appears to make a minor contribution in terms of the overall energetics of subcellular targeting of a protein may play a pivotal role in regulating its subcellular localization and activities (1, 78).

For PI3K-C2{alpha}, protein-protein interactions involving its N-terminal region may play a major role in subcellular localization, but the membrane interactions by the PX domain (and the C2 domain) should also contribute significantly. PtdIns(4,5)P2 is present in significant concentrations in the plasma membrane (~1 mol %) and internal membranes and can be locally enriched (79). In particular, clathrin-coated pits in the plasma membrane are known to have a high local PtdIns(4,5)P2 concentration (80-83). The affinity of the PI3K-C2{alpha} PX domain for PtdIns(4,5)P2 is similar to that of other PtdIns(4,5)P2-binding domains, including the ENTH domain of epsin1 (66), which were shown to recruit their respective host proteins to the PtdIns(4,5)P2-rich region in the plasma membrane. Thus, it is reasonable to postulate that the PX domain of PI3K-C2{alpha} should make a contribution to the binding of PI3K-C2{alpha} to PtdIns(4,5)P2-rich cell membranes, including clathrin-coated pits, clathrin-coated vesicles, and possibly the trans-Golgi network. Another salient feature of the PX domain to be considered in terms of enzyme activation is the PtdIns(4,5)P2-mediated membrane penetration. It has been shown that triggering and sustaining the catalytic activity of many lipid-dependent enzymes, such as protein kinases C (84, 85) and phospholipases (60), involves partial membrane penetration of their lipid-binding domains. Therefore, it is possible that the PtdIns(4,5)P2-mediated membrane penetration of the PX domain is a part of the activation process for PI3K-C2{alpha} and/or is necessary for the processive enzymatic action at the membrane. Additionally, the promiscuous C2 domain could also play a role of prolonging membrane residence of PI3K-C2{alpha}, whereas the local concentration of 3'-phospho-PIs increases as a result of PI3K-C2{alpha} action. Undoubtedly, further studies are necessary to fully understand the relative importance of protein-protein versus lipid-protein interactions in the subcellular localization and activation of PI3K-C2{alpha}.

In summary, this study elucidates the structural basis of specific PtdIns(4,5)P2 recognition by the PI3K-C2{alpha} PX domain and the mechanism by which the PX domain interacts with PtdIns(4,5)P2-containing membranes. This, in conjunction with our previous work on other PX domains, shows that PX domains achieve unique modes of ligand and membrane binding through the variation of a limited number of nonconserved residues. This work may serve as a framework with which to systematically study the mechanism of specific membrane recruitment and regulation of PI3K-C2{alpha} as well as to comprehensively study the effects of structural and functional diversity of PX domains on lipid-protein and protein-protein interactions during cell signaling and membrane trafficking.


   FOOTNOTES
 
The atomic coordinates and structure factors (code 2iwl) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

* This work was supported by National Institutes of Health Grant GM68849 (to W. C.) and by the Medical Research Council (to R. L. W.). 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 Methods and supplemental Fig. S1. Back

1 Present address: Research Center for Biomaterials S.A. 15, 16562 Glyfada-Athens, Greece. Back

2 Present address: Centro Nacional de Investigaciones Oncológicas, Melchor Fernández Almagro 3, E-28029 Madrid, Spain. Back

3 To whom correspondence should be addressed: Dept. of Chemistry (M/C 111), University of Illinois, 845 West Taylor St., Chicago, IL 60607-7061. Tel.: 312-996-4883; Fax: 312-996-0431; E-mail: wcho{at}uic.edu.

4 The abbreviations used are: PI, phosphoinositide; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; POPE, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine; POPS, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine; PtdIns3P, phosphatidylinositol 3-phosphate; PtdIns4P, phosphatidylinositol 4-phosphate; PtdIns5P, phosphatidylinositol 5-phosphate; PtdIns(3,4)P2, phosphatidylinositol 3,4-bisphosphate; PtdIns(3,5)P2, phosphatidylinositol 3,5-bisphosphate; PtdIns(4,5)P2, phosphatidylinositol 4,5-bisphosphate; PtdIns(3,4,5)P3, phosphatidylinositol 3,4,5-trisphosphate; PX, phox homology; PH, pleckstrin homology domain; SPR, surface plasmon resonance; PI3K, phosphoinositide 3-kinase; MES, 4-morpholineethanesulfonic acid; SH, Src homology. Back


   ACKNOWLEDGMENTS
 
The expert assistance of Joanne McCarthy with ESRF beamline ID14-2 is gratefully acknowledged. We thank Olga Perisic for assistance with the structural work and for critically reviewing the manuscript.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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