A Comparative Analysis of the Phosphoinositide Binding Specificity of Pleckstrin Homology Domains*

(Received for publication, March 28, 1997, and in revised form, May 30, 1997)

Lucia E. Rameh Dagger §, Ann-kristin Arvidsson Dagger , Kermit L. Carraway III Dagger , Anthony D. Couvillon Dagger , Gary Rathbun Dagger , Anne Crompton par , Barbara VanRenterghem **Dagger Dagger , Michael P. Czech **, Kodimangalam S. Ravichandran §§, Steven J. Burakoff ¶¶, Da-Sheng Wang ||, Ching-Shih Chen || and Lewis C. Cantley Dagger

From the Dagger  Department of Cell Biology, Harvard Medical School and Division of Signal Transduction, Beth Israel Hospital, Boston, Massachusetts 02115, par  Onyx Pharmaceuticals, Richmond, California 94806, the ** Program in Molecular Medicine and Department of Biochemistry and Molecular Biology, University of Massachusetts Medical Center, Worcester, Massachusetts 01605, the ¶¶ Division of Pediatric Oncology, Dana-Farber Cancer Institute and Department of Pediatrics, Harvard Medical School, Boston, Massachusetts 02115, the || Division of Medical Chemistry Pharmaceutics, College of Pharmacy, University of Kentucky, Lexington, Kentucky 50536-0082, and the §§ Beirne B. Carter Center for Immunology Research and Department of Microbiology, University of Virginia, Charlottesville, Virginia 22908

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

Pleckstrin homology (PH) and phosphotyrosine binding (PTB) domains are structurally related regulatory modules that are present in a variety of proteins involved in signal transduction, such as kinases, phospholipases, GTP exchange proteins, and adapter proteins. Initially these domains were shown to mediate protein-protein interactions, but more recently they were also found to bind phosphoinositides. Most studies to date have focused on binding of PH domains to phosphatidylinositol (PtdIns)-4-P and PtdIns-4,5-P2 and have not considered the lipid products of phosphoinositide 3-kinase: PtdIns-3-P, PtdIns-3,4-P2, and PtdIns-3,4,5-P3. Here we have compared the phosphoinositide specificity of six different PH domains and the Shc PTB domain using all five phosphoinositides. We show that the Bruton's tyrosine kinase PH domain binds to PtdIns-3,4,5-P3 with higher affinity than to PtdIns-4,5-P2, PtdIns-3,4-P2 or inositol 1,3,4,5-tetrakisphosphate (Ins-1,3,4,5-P4). This selectivity is decreased by the xid mutation (R28C). Selective binding of PtdIns-3,4,5-P3 over PtdIns-4,5-P2 or PtdIns-3,4-P2 was also observed for the amino-terminal PH domain of T lymphoma invasion and metastasis protein (Tiam-1), the PH domains of Son-of-sevenless (Sos) and, to a lesser extent, the PH domain of the beta -adrenergic receptor kinase. The oxysterol binding protein and beta -spectrin PH domains bound PtdIns-3,4,5-P3 and PtdIns-4,5-P2 with similar affinities. PtdIns-3,4,5-P3 and PtdIns-4,5-P2 also bound to the PTB domain of Shc with similar affinities and lipid binding was competed with phosphotyrosine (Tyr(P)-containing peptides. These results indicate that distinct PH domains select for different phosphoinositides.


INTRODUCTION

Proteins involved in signal transduction are often composed of regulatory modules such as PH1 domains, Src homology 2 domains, and PTB domains (1, 2). It is believed that these well defined units can, alone or in combination, determine the subcellular localization of a protein by mediating protein/protein interactions and also, as recently described, protein/lipid interactions (3-5).

PH domains were first identified as protein regions that share similarities with pleckstrin, the major protein kinase C substrate in platelets (6). They contain approximately 120 amino acids and are found in several proteins involved in signaling, such as protein kinases (Btk, Akt, and beta ark), phospholipases (PLC), and proteins that act as exchange factors or GTPase-activating proteins for small G-proteins (Ras-GRF, Dbl, SOS, Ras-GAP, Tiam-1) (7). Despite their relatively low sequence homology, the three-dimensional structures of the PH domains are highly conserved (8-15). Interestingly, the recently described PTB domains of Shc and IRS-1 have structural similarity to PH domains and may be thought of as a subclass of PH domains with an additional region that allows binding to tyrosine-phosphorylated proteins (5, 16).

The physiological relevance of phosphoinositide binding to PH domains is still not completely clear. Several PH domains have now been shown to bind with relatively high affinity to either PtdIns-4,5-P2 or Ins-1,4,5-P3 (3, 5, 17-24). In the case of the phospholipase C-delta PH domain, considerable evidence is provided that PtdIns-4,5-P2 binding to this domain recruits it to the membrane where substrates reside and that the product of PtdIns-4,5-P2 hydrolysis, Ins-1,4,5-P3 binds to the PH domain to dissociate it from the membrane as a feedback regulatory mechanism (15). In most of the studies referenced above, the possibility that the various PH domains might bind to products of PI 3-kinase was not investigated. This is an important question since unlike PtdIns-4,5-P2 and PtdIns-4-P, which are constitutively produced in cells, PtdIns-3,4-P2 and PtdIns-3,4,5-P3 are nominally absent in quiescent cells and only appear in response to cell stimulation (25). Thus, these lipids could provide a mechanism for regulated recruitment of proteins to the membrane. However, even in stimulated cells the levels of PtdIns-3,4-P2 and PtdIns-3,4,5-P3 are far below the levels of PtdIns-4-P and PtdIns-4,5-P2. Therefore, in order for a protein to be regulated by one of these lipids, it must have a strong selectivity for the D-3 lipids compared with the affinity for PtdIns-4-P or PtdIns-4,5-P2. While this manuscript was in review, two papers have appeared indicating that the PH domain of the Akt/PKB protein-Ser/Thr kinase selectively binds to and is activated by PtdIns-3,4-P2 (26, 27). Another paper appeared showing that the PH domain of the Btk protein-Tyr kinase selectively binds to PtdIns-3,4,5-P3 (28) and another paper appeared showing that the Btk PH domain selectively binds to Ins-1,3,4,5-P4 (29).

In this study we have compared the ability of six different PH domains and the Shc PTB domain to bind to five different phosphoinositides as well as to inositol phosphates. We find that a subgroup of the PH domains investigated has a relatively high selectivity for binding to PtdIns-3,4,5-P3, while others bind PtdIns-4,5-P2 and PtdIns-3,4,5-P3 with comparable affinity. All of the domains investigated here bound PtdIns-3,4-P2 poorly compared with PtdIns-4,5-P2 or PtdIns-3,4,5-P3, indicating specificity to the binding. These results indicate that distinct PH domains have evolved selectivity for different phosphoinositides to provide discriminatory regulation.


MATERIALS AND METHODS

GST Fusion Proteins

pGEX vectors containing the cDNA sequences encoding the mouse Btk PH domain (amino acids -6 to 217), the Shc-PTB domain (amino acids 17-207), the beta ark, OSBP, beta -spectrin, mSos1 (amino acids 456-569), and Tiam-1 N-terminal PH domains were expressed in Escherichia coli as GST fusion proteins by isopropyl-beta -D-thiogalactopyranoside induction (5, 30). Bacterial lysates (prepared as described in Ref. 31) were incubated with glutathione-Sepharose beads for 2 h at 4 °C and washed several times with 30 mM Hepes, pH 7.0, 100 mM NaCl, 1 mM EDTA (HNE) containing 0.5% Nonidet P-40, followed by HNE, without detergent. Point mutations within the Btk PH domain (R28C) were introduced using the Transformer site-directed mutagenesis kit (CLONTECH) and confirmed by DNA sequencing.

PtdIns-3,4,5-P3 Direct Binding Assay

Synthetic water-soluble 3H-labeled dioctanoyl-PtdIns-3,4,5-P3 (5 × 107 cpm/µmol) (32) was incubated with Sepharose beads containing approximately 800 nM of GST-Btk PH domain fusion protein or GST alone in HNE-0.02% Nonidet P-40 buffer. After 1 h at room temperature, the beads were separated from the supernatant by centrifugation. The supernatant was then collected, mixed with scintillation liquid, and counted on a Beckman counter. The amount of [3H]PtdIns-3,4,5-P3 bound to the Btk PH domain was calculated by subtracting the amount of free 3H present in the GST-Btk PH domain supernatant from the amount of free 3H present in the control GST supernatant under identical conditions. The beads containing GST alone retained less than 3% of the total counts. A nonlinear least squares fit to the data (Kaleidagraph) was determined using the equation [bound]= Bmax × [free]/(KD + [free]), where [bound] is the concentration of [3H]C8PtdIns-3,4,5-P3 bound, [free] is the concentration of [3H]C8PtdIns-3,4,5-P3 free in solution, Bmax is the saturation binding, and KD is the dissociation constant.

Lipid Competition Assay

[32P]3'-Phosphoinositides were prepared by incubating 200 µM of a crude brain phosphoinositide mixture (Sigma; contains 60% PtdIns and phosphatidylserine, 15-20% PtdIns-4-P, and 15-20% PtdIns-4,5-P2] with purified PI 3-kinase as described previously (33). After 30 min at room temperature, the reaction was stopped with 10 mM EDTA, and the lipids were isolated by chloroform:methanol extraction as described (34). Sonicated 32P-labeled phosphoinositides (prepared as described above) or synthetic water-soluble 3H-labeled C8PtdIns-3,4,5-P3 (6500 cpm; 0.93 µM) were incubated with competitor lipids (unlabeled PtdIns-4,5-P2 (Avanti) or unlabeled synthetic PtdIns-3,4,5-P3 (32)) and 15 µl of Sepharose beads containing approximately 2-20 µg of the indicated GST fusion protein in the HNE buffer containing 0.02% Nonidet P-40. The incubation was carried out for 1 h at room temperature, after which the beads were washed twice with 1 ml of HNE-0.5% Nonidet P-40 (2-3 min total). The 32P-labeled lipids that remained associated with the beads were extracted and resolved by thin layer chromatography (TLC) using 1-propanol:2 M acetic acid (65:35, v/v). The radioactivity migrating with PtdIns-3,4,5-P3 was quantified using a Bio-Rad phosphorimager. When 3H-labeled PtdIns-3,4,5-P3 was used as a tracer, the washed beads were mixed with scintillation liquid, and the radioactivity associated with them was measured in a Beckman scintillation counter. The data were plotted as a percentage of the control with no additional competitive lipid added. A nonlinear least squares fit to the data was performed using the equation % bound = 100 - n × L/(KI(app) L), where n indicates the percent specific binding, L indicates the concentration of unlabeled lipid added, and KI(app) is the apparent competitive dissociation constant for the unlabeled lipid. This equation assumes simple competitive binding and that the competitive lipid is in excess over the PH domain concentration (L ~ Ltotal). The apparent competitive dissociation constant relates to true KI by the equation KI(app) = KI (1 + L*/KD*), where L* and KD* are the free concentration and dissociation constant of the radiolabeled lipid, respectively. Thus, KI(app) is an overestimation of the true KI but approaches the true KI at low concentrations of L* and low concentrations of the PH domain where L ~ Ltotal. Since the same concentration of PH domain and radiolabeled lipid are used for a given set of PtdIns-4,5-P2 and PtdIns-3,4,5-P3 competition experiments, the ratios of the apparent dissociation constants measured equal the ratios of the true dissociation constants: e.g. KI(app)(PtdIns-4,5-P2)/KI(app)(PtdIns-3,4,5-P3) KI(PtdIns-4,5-P2)/KI(PtdIns-3,4,5-P3).

For the peptide competition for lipid binding to the Shc PTB domain, the tyrosine-phosphorylated peptides SCFTNQGpYFF (from the interleukin-2 receptor) and RENEpYMPMAPQIH (from the polyomavirus middle T protein) were used (pY indicates phosphotyrosine).

Lipid Selectivity

[32P]Phosphoinositides were prepared using a 200 µM solution of presonicated crude brain phosphoinositides (Sigma) and a mixture of purified PI 3-kinase, PtdIns 4-kinase, and PtdIns-4-P 5-kinase as described (33). Dried lipids were resuspended in HNE buffer, sonicated, and added to 40 µl of Sepharose beads containing ~5 µg of the Btk PH domain GST fusion protein in HNE-0.02% Nonidet P-40. Samples were incubated and washed as described above. Lipids that remained associated with the beads were extracted, deacylated, and analyzed by HPLC as described (34). The radioactivity associated with each selected lipid was then divided by the radioactivity associated with the same lipid in the starting mixture to determine the fraction of each lipid that bound to a given domain. On average, 8% of the total radioactivity added remained associated with the wild type Btk PH domain beads and 4% with the R28C mutant. The GST beads alone bound only 0.5% of the total lipids, such that the radioactivity associated with each lipid was often undetectable. To compare the relative selectivity of wild type and mutant Btk PH domain for PtdIns-3,4,5-P3, the data were normalized such that if all the lipids bound equally well, each would have a selectivity of 1 (4).

Protein Competition Assay

Phosphorylated EGF receptor intracellular domain, including the cytoplasmic tail (TKD 61, a gift from Dr. R. Cerione from Cornell University, Ithaca, NY), was prepared by infecting SF9 cells with baculovirus carrying the TKD 61 sequence. The cells were lysed in a hypotonic solution containing 30 mM Hepes, pH 7.0, 1 mM EDTA, by passing the cell suspension through a 23-gauge needle. The lysate was cleared by centrifugation at 15,000 rpm for 10 min and incubated with 10 mM MgCl2 and 1 mM ATP at 37 °C for 1 h. GST-Shc-PTB-containing beads were incubated with different concentrations of sonicated PtdIns-4,5-P2 or PtdIns-3,4,5-P3 for 1 h at room temperature in the presence of HNE containing 0.02% Nonidet P-40. Phosphorylated TKD 61 was added to the lipid/beads mixture and incubated for 20 min at room temperature. The beads were washed two times with HNE-0.5% Nonidet P-40, resuspended in an equal volume of 2 × Laemmli buffer and incubated for 10 min at 100 °C. Proteins were resolved by 7.5% SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose membrane, and blotted using anti-Tyr(P) antibody (4G10). Blots were developed using the chemiluminescence system (ECL from Amersham Corp.).


RESULTS

Wild Type Btk-PH Domain Preferentially Binds to PtdIns-3,4,5-P3

To avoid the complexity of interpreting protein binding to lipid bilayers and micelles, a water-soluble form of PtdIns-3,4,5-P3 (dioctanoyl-PtdIns-3,4,5-P3, abbreviated as C8PtdIns-3,4,5-P3) was used to investigate PH domain binding characteristics. Various concentrations of water soluble 3H-labeled C8PtdIns-3,4,5-P3 were incubated with bacterially expressed GST-Btk PH domain fusion protein immobilized on beads. Fig. 1A is a plot of the amount of the ligand bound versus free. The data fitted a hyperbolic curve indicating a KD of approximately 800 nM, and the stoichiometry was extrapolated to approximately 0.9 mol of C8PtdIns-3,4,5-P3 bound per mol of PH domain.


Fig. 1. Direct binding of PtdIns-3,4,5-P3 to the Btk PH domain. A, approximately 800 nM of the Btk PH domain was incubated with various concentrations of [3H]C8PtdIns-3,4,5-P3, and the amount of radioactivity bound versus free was determined as described under "Materials and Methods." The line drawn represents a best fit to the data assuming a single class of binding sites (KD = 800 nM; maximal binding 740 nM or 0.92 mol/mol). B, displacement of [3H]C8PtdIns-3,4,5-P3 (0.64 µM total) binding to the Btk PH domain (0.2 µM) by various concentrations of water-soluble C8PtdIns-3,4,5-P3 or Ins-1,3,4,5-P4. The lines through the data represent best fits assuming competitive binding (KI(app) for C8PtdIns-3,4,5-P3 = 4 µM; KI(app) for Ins-1,3,4,5-P4 = 150 µM; maximal displacement = 95%). Bars represent the range from two experiments.
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To determine the importance of the diacylglycerol moiety for binding, we compared the abilities of Ins-1,3,4,5-P4 (the head group of PtdIns-3,4,5-P3) and unlabeled C8PtdIns-3,4,5-P3 to displace [3H]C8PtdIns-3,4,5-P3 from the Btk PH domain. The results in Fig. 1B indicate that very high concentrations of Ins-1,3,4,5-P4 (>100 µM) are required to displace the [3H]C8PtdIns-3,4,5-P3. In contrast, unlabeled C8PtdIns-3,4,5-P3 caused 50% displacement at approximately 4 µM. As expected the KI(app) for C8PtdIns-3,4,5-P3 is somewhat higher than the KD determined by direct binding (0.8 µM), since the KI(app) is affected by the total concentration of the PH domain and the concentration of [3H]C8PtdIns-3,4,5-P3 used in the experiment (see "Materials and Methods"). These results indicate that PtdIns-3,4,5-P3 is capable of directly binding to the PH domain of Btk with approximately 40 times higher affinity than Ins-1,3,4,5-P4. Hence, the diacylglycerol moiety plays a significant role in the interaction of PtdIns-3,4,5-P3 with the Btk PH domain.

To measure the relative affinity of the Btk PH domain for phosphoinositides with long chain fatty acids, Nonidet P-40 micelles containing PtdIns-4,5-P2 or a synthetic dipalmitoyl version of PtdIns-3,4,5-P3 (C16PtdIns-3,4,5-P3) were used to compete for [3H]C8PtdIns-3,4,5-P3 binding to this domain (Fig. 2A). A least squares fit to the data yields a KI(app) of 0.7 µM for binding of C16PtdIns-3,4,5-P3. Thus, C16PtdIns-3,4,5-P3 appears to have a 5-6-fold higher affinity than C8PtdIns-3,4,5-P3 (Fig. 2A versus Fig. 1B). However, interpretation of this result is complicated by the fact that C16PtdIns-3,4,5-P3 forms a micelle. Fig. 2A also shows that C16PtdIns-3,4,5-P3 has a higher affinity for the Btk PH domain than does PtdIns-4,5-P2, as judged by the concentrations of the two lipids required to displace [3H]C8PtdIns-3,4,5-P3. The ratio of these two constants (8 µM/0.7 µM = 11) indicates that the Btk PH domain has an order of magnitude selectivity for PtdIns-3,4,5-P3 over PtdIns-4,5-P2. Addition of physiological concentrations of calcium and magnesium did not significantly affect binding of C8PtdIns-3,4,5-P3, C16PtdIns-3,4,5-P3, or PtdIns-4,5-P2 (not shown).


Fig. 2. The relative affinities of PtdIns-4,5-P2 and PtdIns-3,4,5-P3 for the Btk-PH domain. Various concentrations of sonicated unlabeled PtdIns-4,5-P2 or dipalmitoyl-PtdIns-3,4,5-P3 were used to compete [3H]C8PtdIns-3,4,5-P3 (A) or [32P]PtdIns-3,4,5-P3 (B) binding to the Btk PH domain. The amount of [3H]PtdIns-3,4,5-P3 bound was quantified by scintillation counts (A) while the [32P]PtdIns-3,4,5-P3 bound was extracted, separated by TLC, and quantified by a phosphorimager (B). The results were normalized to the amount of PtdIns-3,4,5-P3 bound in the absence of unlabeled lipids. Error bars represent the range from two experiments. The plots shown are representative of three experiments. The lines through the data represent best fits assuming competitive binding. The apparent dissociation constants are discussed in the text.
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Since the [3H]C8PtdIns-3,4,5-P3 utilized as a probe in Fig. 2A is water-soluble and not a natural form of PtdIns-3,4,5-P3, we also investigated binding using [32P]PtdIns-3,4,5-P3 produced enzymatically from natural lipids as the probe. In this experiment both the radiolabeled probe and the competing lipid are in micelles. The results presented in Fig. 2B again show that C16PtdIns-3,4,5-P3 has about a 1 order of magnitude higher affinity for the BTK PH domain than does PtdIns-4,5-P2. The apparent KI values determined in this experiment are higher than those determined in Fig. 2A due to differences in PH domain concentration, probe concentration, and probe affinity, but the ratio of the constants (21 µM/2.5 µM = 8.4) is similar to that found in Fig. 2A. High concentrations of PtdIns (20 µM), PtdIns-3,4-P2 (20 µM), Ins-1,4,5-P3 (1 mM), and Ins-1,3,4-P3 (1 mM) were unable to displace [32P]PtdIns-3,4,5-P3 (not shown).

As a third assessment of the selectivity of the Btk PH domain, we added this domain to a mixture of radiolabeled versions of all five phosphoinositides sonicated into the same vesicle containing carrier PtdIns and phosphatidylserine and determined which lipids were preferentially retained following stringent washing with Nonidet P-40. This experiment indicates which lipid has the slowest off rate. Consistent with the competitive binding experiments, [32P]PtdIns-3,4,5-P3 was preferentially retained (Fig. 3A). The fact that both PtdIns-4,5-P2 and PtdIns-3,4-P2 bound poorly compared with PtdIns-3,4,5-P3 indicates that phosphates at both the 3 and 5 positions are critical for high affinity binding. An Arg to Cys mutation at position 28 in the PH domain of Btk (BtkR28C) causes agammaglobulinemia. In agreement with other studies (28, 29), we find that this mutation dramatically reduces the selectivity for PtdIns-3,4,5-P3 over the other lipids by both direct binding experiments (Fig. 3B) and competition experiments (not shown).


Fig. 3. Lipid selectivity by Btk-PH domains. Wild type Btk PH domain (A) or the R28C mutant (B) were used to select phosphorylated inositides from a mixture containing 32P-labeled PtdIns-3-P, PtdIns-4-P, PtdIns-3,4-P2, PtdIns-4,5-P2, and PtdIns-3,4,5-P3, as described. The lipids that remained associated with the PH domains after washing were extracted, deacylated, and analyzed by HPLC. The radioactivity associated with each selected lipid was then divided by the radioactivity associated with the same lipid in the starting mixture to determine the fraction of each lipid that bound to a given domain. To compare the relative selectivity of wild type and mutant Btk PH domain for PtdIns-3,4,5-P3, the data were normalized such that if all the lipids bound equally well, each would have a selectivity of 1. Error bars represent the standard error from three experiments.
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PH domains from Sos and Tiam-1 Also Bind Preferentially to PtdIns-3,4,5-P3

We investigated the selectivity of additional PH domains using the competitive binding assays. Both the PH domain of Sos and the N-terminal PH domain of Tiam-1 bound PtdIns-3,4,5-P3 with high affinity and specificity. Using the [3H]C8PtdIns-3,4,5-P3 displacement assay, both of these PH domains had apparent KI values for C16PtdIns-3,4,5-P3 of less than 1 micromolar (Fig. 4, A and B). As discussed above, these measurements are underestimates of the true affinities for C16PtdIns-3,4,5-P3. In these experiments, the Sos PH domain had about a 5-fold selectivity for PtdIns-3,4,5-P3 over PtdIns-4,5-P2 (ratio of apparent KI values = 2 µM/0.4 µM). The Tiam-1 N-terminal PH domain had about a 3-fold selectivity for PtdIns-3,4,5-P3 over PtdIns-4,5-P2 (ratio of apparent KI values = 1.5 µM/0.5 µM).


Fig. 4. The relative affinities of PtdIns-4,5-P2, PtdIns-3,4-P2, and PtdIns-3,4,5-P3 for the Sos and Tiam-1 N-terminal PH domains. Competition of each of the lipids for [3H]C8PtdIns-3,4,5-P3 (A and B) and [32P]PtdIns-3,4,5-P3 (C and D) was performed as described for Fig. 2, A and B, respectively. Bars indicate the range from two distinct experiments (for some points the bars are smaller than the squares or circles). The PtdIns-3,4-P2 competition (triangles) is from a single experiment. The lines through the data represent best fits assuming competition at a single class of sites. The competitive binding constants are discussed in the text.
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The selectivities of these two domains were also investigated using displacement of micellar [32P]PtdIns-3,4,5-P3 (Fig. 4, C and D). Although the apparent KI values determined by this assay were higher due to the different probes and conditions, these assays confirmed that both PH domains preferentially bound C16PtdIns-3,4,5-P3 over PtdIns-4,5-P2 (Sos PH domain selectivity, 16 µM/7 µM = 2.3-fold: Tiam-1 N-terminal PH domain selectivity, 15 µM/4 µM = 3.75-fold). Thus two different types of binding experiments indicate that both of these domains have between 2-5-fold selectivities for binding to PtdIns-3,4,5-P3 compared with PtdIns-4,5-P2. Both direct binding experiments (not shown) and competition experiments (Fig. 4, A and B) indicated that PtdIns-3,4-P2 binds more weakly than either PtdIns-3,4,5-P3 or PtdIns-4,5-P2 to these two PH domains. Thus, like the Btk PH domain, the Tiam-1 and Sos PH domains have specificity for PtdIns-4,5-P2 over PtdIns-3,4-P2, but have highest affinity for PtdIns-3,4,5-P3.

PH Domains of beta ark, OSBP, and beta -Spectrin Bind PtdIns-3,4,5-P3 and PtdIns-4,5-P2 with Similar Affinities

The PH domains of beta ark, OSBP, and beta -spectrin were also analyzed. The percentage of [32P]PtdIns-3,4,5-P3 that was competed by 10 µM PtdIns-4,5-P2 was compared with the percentage that was competed by 10 µM PtdIns-3,4,5-P3, to give an estimation of the relative affinity of the different PH domains for these two lipids. The results are summarized in Table I. In contrast to the Btk PH domain, the OSBP and the beta -spectrin PH domains bind to PtdIns-4,5-P2 and PtdIns-3,4,5-P3 with similar affinities. The beta ark PH domain showed a somewhat higher affinity for PtdIns-3,4,5-P3 than for PtdIns-4,5-P2, both in this experiment and in direct binding experiments (not shown). However, this domain bound PtdIns-3,4,5-P3 more weakly than did the Btk, Tiam-1 and Sos PH domains.

Table I. Displacement of [32P]PtdIns-3,4,5-P3 binding to PH domains by unlabeled PtdIns-4,5-P2 and C16PtdIns-3,4,5-P3. Competition was performed as described under "Materials and Methods."


PH domain % competition PtdIns-4,5-P2 (10 µM) % competition C16PtdIns-3,4,5-P3 (10 µM)

Btk 26  ± 2a 72  ± 4a
N-terminal Tiam 31  ± 10a 67  ± 5a
Sos 35  ± 7a 51  ± 2a
 beta ark 9  ± 9 32  ± 13
OSBP 18  ± 2 24  ± 8
 beta -Spectrin 18  ± 3 24  ± 10

a Indicates results obtained from Fig. 2B and Fig. 4, C and D.

PtdIns-3,4,5-P3 and PtdIns-4,5-P2 Bind to the Shc-PTB Domain with Similar Affinities

Recently, it was shown that the Shc-PTB is able to bind to liposomes containing PtdIns-4,5-P2 (5). To determine whether this domain also binds PtdIns-3,4,5-P3, 32P-labeled PtdIns-3,4,5-P3 was incubated with GST-Shc-PTB immobilized on beads, in the presence or absence of Tyr(P)-containing peptides. After washing the beads with Nonidet P-40, approximately 20% of the total PtdIns-3,4,5-P3 (Fig. 5) remained associated with the beads. In the presence of 10 µM of a peptide containing the sequence NQGpY, that binds with high affinity to the Shc-PTB domain Tyr(P)-binding pocket (35), PtdIns-3,4,5-P3 binding was inhibited by 85%. A control phosphopeptide (10 µM) had little effect on PtdIns-3,4,5-P3 binding. These results suggest that, like PtdIns-4,5-P2, PtdIns-3,4,5-P3 can directly bind to the Shc-PTB domain, and this interaction can be competed with a phosphopeptide that binds with high affinity to this domain.


Fig. 5. PtdIns-3,4,5-P3 binding to the Shc-PTB domain. Beads containing the GST-Shc-PTB domain were incubated with [32P]PtdIns-3,4,5-P3 in the presence of buffer (lane 1), 10 µM tyrosine-phosphorylated peptide (NQGpY) (lane 3), 10 µM tyrosine-phosphorylated control peptide (pYMPM) (lane 4) or 20 mM phenyl phosphate (lane 2). The lipid that remained associated with the washed beads were extracted and separated by TLC. PIP3, PtdIns-3,4,5-P3.
[View Larger Version of this Image (28K GIF file)]

To compare the relative affinities of the different phosphoinositides for the Shc-PTB domain, PtdIns, PtdIns-4,5-P2, or PtdIns-3,4,5-P3 was incubated with GST-Shc-PTB beads, together with [32P]PtdIns-3,4,5-P3, in an experiment analogous to that depicted in Fig. 2B. The results presented in Fig. 6 indicate that PtdIns does not displace [32P]PtdIns-3,4,5-P3 binding, even at 40 µM, while PtdIns-4,5-P2 and C16PtdIns-3,4,5-P3 compete for [32P]PtdIns-3,4,5-P3 binding to the Shc-PTB domain with similar affinities (apparent KI of 25 µM for both lipids).


Fig. 6. The relative affinities of PtdIns, PtdIns-4,5-P2, and C16PtdIns-3,4,5-P3 for the Shc-PTB domain. Competition of each of the lipids for [32P]PtdIns-3,4,5-P3 was performed as described for Fig. 2B. The lines through the data represent least square fits assuming competition at a single class of sites.
[View Larger Version of this Image (16K GIF file)]

Phosphoinositide Binding to the Shc-PTB Domain Competes with Protein Binding

The ability of the tyrosine-phosphorylated peptide to block phosphoinositide binding to the Shc-PTB domain suggests that the lipid binding and Tyr(P) binding are competitive. To determine whether phosphoinositides and tyrosine-phosphorylated proteins are able to simultaneously bind to the Shc-PTB domain, the ability of PtdIns-4,5-P2 and PtdIns-3,4,5-P3 to compete with the tyrosine-phosphorylated EGF receptor for Shc-PTB domain binding was examined. Tyrosine-phosphorylated EGF receptor intracellular domain was incubated with GST-Shc-PTB beads in the presence of different concentrations of PtdIns-4,5-P2 or PtdIns-3,4,5-P3. The EGF receptor that remained associated with the Shc-PTB domain, after an extensive wash, was separated by SDS-polyacrylamide gel electrophoresis and blotted with anti-Tyr(P) antibody (Fig. 7). PtdIns-4,5-P2 and PtdIns-3,4,5-P3 were equally able to compete with the tyrosine phosphorylated EGF receptor for binding to the Shc-PTB domain. 50% competition was observed in the presence of 10-20 µM of each of these lipids, consistent with their abilities to displace [32P]PtdIns-3,4,5-P3 (Fig. 5). PtdIns and PtdIns-3,4-P2 had no effect on EGF receptor binding at concentrations of up to 80 µM (not shown), indicating that the binding is specific for PtdIns-4,5-P2 over PtdIns-3,4-P2.


Fig. 7. Phosphoinositide competition for Shc-PTB binding to tyrosine-phosphorylated EGF receptor. Sepharose beads containing approximately 1 µM of GST-Shc-PTB domain were incubated with different concentrations of sonicated PtdIns-4,5-P2 or PtdIns-3,4,5-P3 and tyrosine-phosphorylated EGF receptor TKD 61. The beads were washed and associated proteins analyzed by blotting with anti-Tyr(P) antibodies.
[View Larger Version of this Image (20K GIF file)]

The data shown in Figs. 5 and 7 suggest that PtdIns-4,5-P2 may play an important role in Shc-PTB domain association with membranes or tyrosine-phosphorylated proteins, but that conversion of PtdIns-4,5-P2 to PtdIns-3,4,5-P3 by PI 3-kinase is unlikely to alter these interactions.


DISCUSSION

The results presented here demonstrate that different PH domains have specificity for different phosphoinositides. The domains that we have investigated fall into two major categories, those with high affinity and specificity for PtdIns-3,4,5-P3 over PtdIns-4,5-P2 (Btk, Tiam-1 N-terminal, Sos) and those that bind PtdIns-3,4,5-P3 relatively weakly and have only slight or no selectivity for PtdIns-3,4,5-P3 over PtdIns-4,5-P2 (beta ark, OSBP and spectrin PH domains and Shc PTB domain). All of these domains had selectivity for PtdIns-4,5-P2 over PtdIns-3,4-P2 (Fig. 4 and data not shown) indicating that phosphate at the D-5 position is important for binding. In contrast, results from this laboratory (26) and another laboratory (27) have demonstrated that the Akt/PKB PH domain has high affinity and selectivity for PtdIns-3,4-P2 over PtdIns-4,5-P2. Thus, different PH domains have evolved the ability to bind to distinct phosphoinositides, presumably to allow regulation by distinct extracellular signals.

The specificity of the Btk PH domain for PtdIns-3,4,5-P3 may be of physiological importance. Using a water soluble C8PtdIns-3,4,5-P3 we were able to show that this domain binds monomeric PtdIns-3,4,5-P3 at one mol/mol with a KD less than 1 µM. Competitive binding experiments demonstrated that C16PtdIns-3,4,5-P3 has a 5-6-fold higher affinity than C8PtdIns-3,4,5-P3 and that Ins-1,3,4,5-P4 (the head group of PtdIns-3,4,5-P3) has a 40-fold lower affinity than C8-PtdIns-3,4,5-P3. These results demonstrate that this domain can bind with high affinity to a monomeric lipid and that the diacylglycerol moiety significantly contributes to the binding affinity of the monomer. Similarly, the dynamin PH domain was shown to bind to detergent-solublized PtdIns-4,5-P2 and glycerophosphorylinositol 4,5-bisphosphate, but to have relatively low affinity for PtdIns-4,5-P2 contained in lipid vesicles or to inositol 1,4,5-P3 (36). The apparent higher affinity of the Btk PH domain for C16PtdIns-3,4,5-P3 over C8PtdIns-3,4,5-P3 could be explained by additional contact with the longer chain fatty acid or to a preference for binding micellar lipid over monomeric lipid, due to partitioning into the micelle.

Three different approaches (Fig. 2, A and B, and Fig. 3) indicate that the Btk PH domain has about a 1 order of magnitude higher affinity for PtdIns-3,4,5-P3 than for PtdIns-4,5-P2. These results are in agreement with another study in which PtdIns-3,4,5-P3 liposome binding to the Btk PH domain was detected by the BIAcore biosensor (28). In that study, PtdIns-4,5-P2 liposomes failed to bind. Since with the BIAcore technique it is difficult to detect binding of ligands with KD values higher than 1 µM, the results are consistent with our finding that PtdIns-3,4,5-P3 binds with a KD of less than 1 µM, while PtdIns-4,5-P2 binds with a KD of about 8 µM. Salim et al. (28) did not investigate binding of inositol phosphates. Another laboratory measured direct binding of inositol phosphates to the Btk PH domain and found that Ins-1,3,4,5-P4 bound more tightly than Ins-1,4,5-P3 or other inositol polyphosphates examined (29). This result is in agreement with our observation that Ins-1,3,4,5-P4 is more effective than Ins-1,4,5-P3 or Ins-1,3,4-P3 in competing for PtdIns-3,4,5-P3 binding. However, the competitive binding constant we observed is much higher than the KD reported by Fukuda et al. (29) (40 nM), and we found that C8PtdIns-3,4,5-P3 bound with a 40-fold higher affinity than Ins-1,3,4,5-P4, suggesting that the natural ligand is the lipid rather than the inositol phosphate. Fukuda et al. (29) did not examine binding to phosphoinositides.

A point mutation in the Btk PH domain that causes agammaglobulinemia (R28C) was found to decrease the affinity for PtdIns-3,4,5-P3 (Fig. 3 and data not shown). This result is in agreement with the results obtained by Salim et al. (28) and suggests that binding to PtdIns-3,4,5-P3 may regulate the function of the Btk protein-Tyr kinase in vivo by affecting its location or activity.

In addition to Btk, several other PH domains selectively bind PtdIns-3,4,5-P3 (Group I in Table II). Included are three PH domains of G-protein exchange factors, Sos, Tiam-1 (N-terminal), and GRP-1. Sos activates Ras, Tiam-1 activates Rac, and Grp-1 is predicted to be an exchange factor for adenosine diphosphate ribosylation factor-1 (ARF-1) family members (37). The results we obtained with Sos do not agree with a previous finding that this domain preferentially bound to liposomes containing PtdIns-4,5-P2 compared with liposomes containing PtdIns-3,4,5-P3 (38). The previous study may be complicated by the fact that PtdIns-3,4,5-P3 perturbs liposome structures.2 The direct binding and competitive binding experiments with radiolabeled lipids clearly indicate a small but significant preference for PtdIns-3,4,5-P3. Our results suggest that local conversion of PtdIns-4,5-P2 to PtdIns-3,4,5-P3 by PI 3-kinase may enhance recruitment of Sos and/or Tiam-1 to the region of the membrane where PI 3-kinase is activated. Although these domains bind PtdIns-3,4,5-P3 with KD values that are submicromolar, they are less selective for PtdIns-3,4,5-P3 over PtdIns-4,5-P2 than is the Btk PH domain. Thus, it is likely that PtdIns-4,5-P2 also plays a role in recruitment of these proteins to the membrane. Deletion of the Sos PH domain was shown to interfere with its exchange activity in vivo (39). Whether phosphoinositide binding to the PH domains of these exchange factors stimulate their exchange activities remains to be determined. Tiam-1 also has a PH domain C-terminal of the exchange domain (40). Deletion of the N-terminal PH domain of Tiam-1 abolished membrane localization while deletion of the C-terminal PH domain did not affect localization (41). We found that the C-terminal PH domain bound relatively weakly to phosphoinositides (not shown). However, it might contribute to membrane binding or activation once Tiam-1 is located at the membrane via the N-terminal PH domain.

Table II. Phosphoinositide binding regions of PH domains

Alignment of various PH domain sequences comprising the region between the beta 1 and beta 2 strands. The positively charged residues are shown in blue. The Arg-28 of Btk is indicated by an asterisk, and the basic residues in the PLCdelta and spectrin PH domain that contact the phosphates of bound Ins-1,4,5-P3 are indicated by an exclamation point. The beta 1 and beta 2 borders are based on the PLCdelta crystal structure (14), and the alignments are based on overall homology with highly conserved regions of the PH domain family members.


Grp-1 (general receptor for phosphoinositides-1) was discovered because of its ability to bind to PtdIns-3,4,5-P3 in a expression cloning assay (37). GRP-1 is a member of a family of proteins with N-terminal Sec7 homology domains and C-terminal PH domains. The PH domain was shown to have high affinity and high selectivity for PtdIns-3,4,5-P3 (37). One member of this family, ARF nucleotide binding site opener, was shown to act as an exchange factor for ARF-1 and this activity was stimulated by phosphoinositides in vitro (24). Whether PtdIns-3,4,5-P3 regulates ARF family members in vivo remains to be determined.

We found another group of PH domains that bind PtdIns-3,4,5-P3 relatively weakly and have little or no preference for PtdIns-3,4,5-P3 over PtdIns-4,5-P2. These include PH domains of beta ark, spectrin, and OSBP (Table II) and the PTB domain of Shc. These domains do have specificity for PtdIns-4,5-P2 over PtdIns-3,4-P2, suggesting that the lipid binding is relevant for function. Indeed, PtdIns-4,5-P2 has been shown to act synergistically with beta gamma subunits in activation of beta ark (20). Since PtdIns-4,5-P2 is more abundant than PtdIns-3,4,5-P3 in vivo (25), the results indicate that these proteins are not regulated in vivo by products of PI 3-kinase. The PH domain of phospholipase C-delta appears to fit into this group, since it binds PtdIns-4,5-P2 and PtdIns-3,4,5-P3 with similar affinities and binds PtdIns-3,4,-P2 more weakly (22).

The Akt/PKB PH domain makes up a third category. In contrast to the other PH domains that we have investigated, this domain preferentially binds PtdIns-3,4-P2 (26, 27). In addition, PtdIns-3,4-P2 activates the protein-Ser/Thr kinase activity of Akt, while PtdIns-4,5-P2 has no effect, and PtdIns-3,4,5-P3 causes inhibition.

A final group of PH domains that we investigated bound very poorly to phosphoinositides under the assay conditions that we used (not shown). This group includes the C-terminal Tiam-1 PH domain (discussed above), the dynamin PH domain, and the IRS-1 PTB domain. A previous study indicated that the dynamin PH domain could bind with high affinity to PtdIns-4-P and PtdIns-4,5-P2 only when these lipids were presented in a detergent-solublized form (36). There are several possible interpretations for these results. The proteins that contain these domains may utilize weak phosphoinositide binding at PH domains in conjunction with lipid or protein interaction at other domains for membrane localization. These domains may function exclusively as protein interaction domains or they may bind to vesicles with a specific curvature or to lipids distinct from phosphoinositides. A trivial explanation is that these domains may fail to fold properly in bacteria.

Based on the crystal structure of the PLCdelta PH domain bound to Ins-1,4,5-P3 (14), residues from the beta 1-beta 2 loop and the beta 3-beta 4 loop of PH domains are predicted to interact with the phosphate moieties of bound phosphoinositides. In Table II the PH domains are divided into four groups based on phosphoinositide selectivity. This table also includes the sequences at the predicted beta 1-beta 2 loops of the PH domains studied here, aligned according to the crystal structure of PLCdelta (14). The exclamation marks indicate the basic residues of PLCdelta (14) and spectrin (42) from this region that are involved in phosphate binding. The Arg-28 residue of Btk is indicated by an asterisk. The analogous residue in PLCdelta (R40) coordinates the D-5 phosphate of Ins-1,4,5-P3. The effects of the R28C mutation described here and elsewhere (28, 29) suggest that this residue may coordinate the D-3 phosphate of PtdIns-3,4,5-P3. Recently published studies of the Akt PH domain are also in agreement with this idea (26). It is likely that other residues in the beta 1-beta 2 loop (as well as the beta 3-beta 4 loop) contribute to selectivity in phosphoinositide binding.

In general those PH domains with clusters of basic residues in the beta 1-beta 2 region tend to have higher affinity (and selectivity) for PtdIns-3,4,5-P3. Table II is color-coded so that basic residues are in blue and acidic residues in red. The Btk, Tiam-1 N-terminal, and GRP-1 PH domains have 8, 11, and 6 basic residues, respectively, in the beta 1-beta 2 region. Most of the other domains with lower affinity for PtdIns-3,4,5-P3 have 5 or fewer basic residues. Residues at the beta 3-beta 4 and beta 5-beta 6 loops were also shown to make contacts with Ins-1,4,5-P3 (14, 42); however, the number of positive residues in these regions does not correlate with high affinity binding to PtdIns-3,4,5-P3 (not shown). Although the spectrin PH domain has a large beta 1-beta 2 loop with many basic and acidic residues, this protein has a Val at the position analogous to Arg-28 of Btk. Compared with Btk it binds relatively weakly to phosphoinositides (and inositol phosphates). The structure of the spectrin PH·Ins-1,4,5-P3 complex indicates a very different binding mode from that of PLCdelta (42).

Finally, it is interesting that phosphoinositide binding to the Shc-PTB domain competes with phosphotyrosine-containing protein binding. This result is analogous to our previous finding that PtdIns-3,4,5-P3 binding to Src homology 2 domains is competitive with Tyr(P) peptides(4). These results suggest that modulation of Tyr(P) binding to adapter domains by phosphoinositides may be a general phenomenon in signaling. However, the Src homology 2 domains investigated had high selectivity for PtdIns-3,4,5-P3, while the Shc-PTB domain did not discriminate between PtdIns-4,5-P2 and PtdIns-3,4,5-P3.


FOOTNOTES

*   This work was supported by National Institutes of Health Grants GM41890 and GM36624 (to L. C. C.) and GM 53448 (to C.-S. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§   Supported by The Medical Foundation of the Charles King Trust, Boston.
   Supported by the Swedish Cancer Society.
Dagger Dagger    Supported by a postdoctoral fellowship grant from the Juvenile Diabetes Foundation International.
1   The abbreviations used are: PH, pleckstrin homology; PTB, phosphotyrosine binding; PLC, phospholipase; GST, glutathione S-transferase; HPLC, high performance liquid chromatography; EGF, epidermal growth factor; PtdIns, phosphatidylinositol; Ins, inositol; OSBP, oxysterol-binding protein.
2   A. D. Couvillon, C. L. Carpenter and P. Janmey, unpublished results.

ACKNOWLEDGEMENTS

We thank Dr. R. Lefkowitz and Dr. K. Touhara for providing the GST-beta ark and GST-OSBP constructs, Dr. Gerry Shaw for the GST-beta -spectrin construct, Dr. Jeremy Thorner for the purified PtdIns 4-kinase, Brian Duckworth for the purified PtdIns-4-P 5-kinase, and Dr. R. Cerione for the Baculovirus expression vector encoding the EGF cytoplasmic tail.


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