1 Endocrinology and Reproduction Research Branch, National Institutes of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA
2 Department of Physiology, Faculty of Medicine, Semmelweis University, 1085 Budapest, Hungary
3 Department of Medical Chemistry, Faculty of Medicine, Semmelweis University, 1085 Budapest, Hungary
* Author for correspondence (e-mail: ballat{at}mail.nih.gov)
Accepted 2 August 2005
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Summary |
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Key words: PH domain, PI 3-kinase, PtdIns(3,4,5)P3, PIP3, Akt kinase, Cell adhesion, GFP
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Introduction |
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Several observations suggest that PH domains might function in a more complex manner. In many proteins, such as in dynamin, the PH domain alone does not have high enough affinity to phosphoinositides to determine solely the localization of the protein, yet the protein shows clear regulation by inositol lipids (Artalejo et al., 1997; Szaszak et al., 2002
; Yu et al., 2004
). Moreover, using in vitro assay systems, it has been shown that the PH domains of Btk and PLC
1 could transmit regulatory effects by the respective lipid, suggesting that PH domains might have conformational regulatory roles (Saito et al., 2001
; Lomasney et al., 1996
). Third, several PH domains are also capable of protein-protein interactions and in most cases the protein interaction surface does not overlap with the lipid-binding region within the PH domain (Carman et al., 2000
; Lodowski et al., 2003
). Also, in spite of their great success in providing invaluable spatial and temporal information on inositide dynamics, the PH-GFP fusion proteins have certain limitations, mainly that they do not always report on the entire lipid pool that they are supposed to recognize (Balla et al., 2000
; Irvine, 2004
).
These observations prompted us to evaluate the principles of interactions of PH domains with the plasma membrane within the intact cells. We have chosen four PH domains with known ability to interact with membrane phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P3, PIP3] to determine whether their overexpression would affect selected PIP3-regulated cellular functions in a similar manner. Our data show that the PH domains have distinctive inhibitory profiles on specific PIP3-regulated pathways indicating that, in addition to binding to inositol lipids, these small protein modules could sequester additional specific protein components of particular signaling pathways.
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Materials and Methods |
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DNA constructs
The PH domains of Btk and Akt, as wells as their mutants, have been described previously (Varnai et al., 1999; Servant et al., 2000
). The human EGF receptor cDNA was subcloned from the SPER-7 plasmid, kindly provided by A. Clark and I. Pastan, NIH, Bethesda, MD, USA (Clark et al., 1986
), into the pCDNA3.1() plasmid (Invitrogen) with XbaI and HindIII restriction enzymes. The ARNO (239-399) and GRP1 (267-399 and 245-399) PH domains were amplified from marathon-ready human brain cDNAs (Clontech). The amplified DNA encoding the GRP1 PH domain was cloned between the BglII/EcoRI sites and the ARNO PH domain between the EcoRI/BamHI sites of the pEGFP-C1 plasmid. Mutations within the PH domains were generated by the Quikclone mutagenesis kit (Stratagene). All constructs were confirmed by dideoxy sequencing. Recombinant PH domains were created by subcloning the exact same constructs used for the mammalian expression studies (including the mutants) into the pET-23b plasmids containing the 6xHis residues at the C-terminus.
Akt activity measurements
COS-7 cells were seeded onto 35 mm culture dishes and transfected with HA-Akt (Bondeva et al., 1998) and the indicated plasmid DNA constructs (1 µg each) at 60% confluency using the lipofectamine 2000 reagent. 24-36 hours after transfection, cells were lysed in 1 ml lysis buffer containing 50 mM Tris/Hcl, pH 7.5, 150 mM NaCl, 1 mM Na-pyrophosphate, 1% NP-40, 20 mM ß-glycerophosphate, 10% glycerol and protease inhibitors (0.1 mM Na3VO4, 25 mM NaF, 1 µg/ml aprotinin, 0.02 mM leupeptin, 0.02 mM pepstatin and 100 µM AEBMSF) and 1 mM DTT freshly added prior to use. Lysates were cleared by centrifugation and 200 µl was incubated with 1 µg of anti-HA monoclonal antibody (Covance) for 1-2 hours at 4°C before the addition of 15 µl protein G-sepharose prewashed in lysis buffer for an additional incubation at 30 minutes. Immunoprecipitates were washed twice with lysis buffer, once with 100 mM Tris/0.5 M LiCl pH 7.5, and finally with the kinase reaction buffer without ATP. Beads were then resuspended in 50 µl kinase buffer (20 mM Hepes, pH 7.5, 10 mM MgCl2, 1 mM DTT) containing 2 µCi
-[32P]ATP, 10 µM ATP and 30 µM crosstide peptide. Reactions were run for 30 minutes at 30°C and terminated by rapid centrifugation (9000 g/5 minutes) and transfer of 40 µl reaction product to a Whatman P81 phosphocellulose paper. Filters were washed four times with 75 mM phosphoric acid and once in acetone before drying and scintillation counting. Expression levels of the various components were tested in each experiment from the lysates by western blot analysis. In later experiments, endogenous Akt activation was measured by western blotting using the phospho-Akt (Ser473) antibody (Cell Signaling) and the data were analyzed with densitometry.
Transfection of cells for confocal microscopy
Cells were plated onto 25 mm diameter circular glass cover slips at a density of 3x105 cells/dish and were transfected the next day with plasmid DNAs (2 µg/ml) using the Lipofectamine 2000 reagent and OPTI-MEM (Life Technologies). 24-36 hours after transfection, cells were washed twice with a modified Krebs-Ringer buffer, containing 120 mM NaCl, 4.7 mM KCl, 1.2 mM CaCl2, 0.7 mM MgSO4, 10 mM glucose, 10 mM Na-Hepes, pH 7.4, and the coverslip was placed into a chamber that was mounted on a heated stage with the medium temperature kept at 33°C. Cells were incubated in 1 ml of the Krebs-Ringer buffer and stimuli were added in 0.5 ml prewarmed buffer after removing 0.5 ml medium from the cells. Cells were examined in an inverted microscope under a 40x oil-immersion objective (Nikon) and a BioRad laser confocal microscope system (MRC-1024) with the Lasersharp aquisition software (BioRad) or in a Zeiss LSM510 laser confocal microscope.
Cell attachment assay
COS-7 cells (2x106) were plated onto 10 cm culture dishes and transfected the next day using Lipofectamine 2000 (5 µg DNA /dish). After 24-36 hours, cells were removed from the plates by gentle trypsinization and divided into three aliquots. One aliquot was centrifuged and the cells were immediately lysed in Laemmli buffer. The other two aliquots were plated into 35 mm culture dishes and the cells were allowed to attach for 30 minutes. After this, the medium was removed and the cells were washed twice with 2 ml of ice-cold Dulbecco's phosphate-buffered saline (PBS) before lysis in the same volume of Laemmli buffer that was added to the first aliquot of cells. Samples were then sonicated (but not boiled) and loaded onto a 8-16% Tris/glicine gel and separated by SDS-PAGE. Electrophoresis gels were analyzed in a Storm 860 Phosphorimager (Molecular Dynamics) using the blue fluorescent laser for quantitation of the GFP fusion protein band in the gel. The fraction of GFP signal found in the attached cells relative to the total amount of cells seeded was calculated for each construct as well as GFP alone, which served as a control.
Cell spreading on fibronectin
Glass coverslips were coated with fibronectin (20 µg/ml) for 2 hours at 37°C, and washed twice with PBS to remove any unbound fibronectin. To block any remaining binding sites, coverslips were then incubated with 1 mg/ml fatty acid free bovine serum albumin (BSA) for 1 hour at 37°C. COS-7 cells expressing the different PH domain constructs (24 hours after transfection) were prepared for plating by trypsinization. After pelleting, cells were treated with 0.3 mg/ml soybean trypsin inhibitor in DMEM for 10 minutes at 37°C followed by incubation in 0.5% BSA in DMEM for an additional 1 hour to allow cell recovery. Cells were then plated onto the fibronectin-coated coverslips and fixed by 4% paraformaldehyde 10 minutes after plating. Fixed cells were permeabilized by 0.2% Triton X-100 in PBS for 5 minutes followed by phalloidin staining (0.1 µg/ml TRITC-phalloidin in PBS for 20 minutes). Cells overexpressing the GFP fusion proteins were identified by fluorescent microscopy without knowing the treatment regimen and were classified into three groups: unspread (adherent with no projections), partially spread (adherent with limited lamellipodia), and fully spread. The percent of fully spread cells was then calculated for each group of cells expressing the GFP fusion proteins.
Analysis of expression kinetics
To transfect COS-7 cells in 96-well plates (black with clear bottom) 1 µg DNA in 25 µl OPTI-MEM/well was mixed with 0.5 µl Lipofectamine-2000 in 25 µl OPTI-MEM/well and incubated for 30 minutes in the well. Freshly trypsinized COS-7 cells (4.5x104/100 µl OPTI-MEM/well) were then added to the wells and the plate was incubated in a CO2 incubator. After 6-10 hours, 150 µl/well DMEM containing 10% FBS was added. GFP fluorescence was measured at the indicated times using a plate reader (Ascent FL, ThermoLabsystems; or Mithras LB940, Berthold). To determine the PIP3-dependent component of the effects of the expressed PH domains on the balance of proliferation/apoptosis, the ratios of the fluorescence of cells expressing the wild-type PH domains were calculated using the mutant forms of the same domain incapable of lipid binding as a control at each time point for each PH domain.
Analysis of inositol phosphates
Inositol phosphates were analyzed from COS-7 cells transfected with cDNA encoding the human EGF receptor, together with selected PH-GFP fusion constructs as described previously (Varnai et al., 1999). One day after transfection, cells were labeled with myo-[3H]inositol for 24 hours and after 30 minutes stimulation in the presence of 10 mM LiCl, [3H]-labeled inositol phosphates were separated by Dowex minicolumns, and measured by liquid scintillation counting as described elsewhere (Hunyady et al., 1994
). The relatively small activation of PLC
by EGF in these cells did not permit direct analysis of the 1,4,5-isomer of InsP3 and the pooled InsP3 and InsP2 samples were used as an indicator of overall PLC activity.
Protein purification and InsP4 or PIP3 binding
Recombinant PH-GFP proteins were produced in BL-21 cells (Invitrogen). Overnight cultures were grown to OD600: 0.6-0.9 and induced with 200 µM isopropyl-ß-D-thiogalactopyranoside (IPTG) for 7 hours at room temperature. Proteins were purified from the bacterial lysates by Ni-NTA columns (Spin Kit, Qiagen) following the manufacturers instruction. InsP4 binding was performed as described previously (Varnai et al., 2002), using 1.1 nCi (1 nM) [3H]InsP4 and 200 ng proteins in the 50 µl incubation volume. Binding to PIP3-coated agarose beads (Echelon) was performed following the manufacturer's protocol with minor modifications. To elute the proteins bound to the beads, after centrifugation and washing, beads were incubated at room temperature for 30 minutes instead of boiling to preserve fluorescence. The amounts of bound and free proteins were assessed by phosphoimager analysis of the samples resolved by SDS PAGE.
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Results |
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Inhibition of Akt activation by the overexpressed PH domains
One of the best known and most studied downstream targets of PIP3 [and phosphatidylinositol (3,4)-bisphosphate, PtdIns(3,4)P2] is the serine/threonine kinase Akt. Therefore, first we decided to study the inhibitory effects of the expressed PH domains on Akt activation in COS-7 cells. In order to investigate the Akt response of only the transfected cells, an epitope-tagged Akt (HA-Akt) was transfected together with the selected PH-GFP constructs and also with a membrane-targeted form of phosphoinositide 3-kinase (PI3K
-CAAX) (Bondeva et al., 1998
), to generate PIP3. After 24 hours of transfection, Akt was immunoprecipitated from the cell lysates and its activity was determined using 32P-ATP and crosstide as substrates (Bondeva et al., 1998
). As shown in Fig. 2, membrane-targeted PI3K
strongly stimulated Akt activity and both Akt-PH and Btk-PH were able to inhibit this activation (P<0.05). By contrast, GRP1-PH and ARNO-PH had no effect. Mutant Btk-PH (R28C), which is unable to bind the lipid, was also without effect, indicating the need for lipid binding to exert an inhibitory effect.
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Inhibition of cell attachment and spreading by the various PH domains
To test a PIP3-regulated process that is linked to the function of the GRP1/ARNO family of GTP-binding protein exchange factors, we chose two simple assays that rely upon inside-out signaling. Cell adhesion and spreading have both been shown to be PIP3-regulated processes in which integrins and small GTP-binding proteins have been implicated (Kinashi et al., 1995; Heraud et al., 1998
; Yamboliev et al., 2001
; Hawadle et al., 2002
). COS-7 cells were transfected with the various GFP fusion constructs and were removed from the culture plates with Versene. Aliquots of the transfected cells were then reseeded onto small culture dishes and incubated for the indicated times (usually 30 minutes). Non-attached cells were then washed away and the attached cells were subjected to SDS page analysis along with the sample of the total amount of cells seeded, so that the fraction of transfected cells attached could be calculated after quantification of the GFP fluorescence in a Phosphorimager. This method allowed monitoring of the attachment efficiency of only the population of transfected cells. As shown in Fig. 3, the PH domains of GRP1 and ARNO were found to inhibit the attachment of cells (P<0.05), whereas the PH domains of Akt and Btk were without effect in this assay.
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Inhibition by PH domain overexpression of complex cellular responses
In subsequent experiments, we investigated the abilities of the various PH domains to influence more-complex cellular responses such as proliferation and apoptosis. These responses rely upon a number of signal transduction events and therefore are more difficult to interpret in terms of the specific biochemical event being targeted; nevertheless, they could reveal significant phenotypic changes caused by the expression of PH domains. Therefore, we studied how the expression of the fluorescent PH domain changes in time for the individual constructs. Although the expression kinetic of any given protein can vary as a result of multiple factors within the cells, a difference between the expression dynamics of wild-type and mutant forms (that do not bind phosphoinositides) of the PH domains would indicate the involvement of a PIP3-dependent process. For example, any positive effect of the PH domain protein on apoptosis would be expected to eliminate the transfected cells more rapidly and hence rapidly decrease the population of fluorescent cells after the construct is expressed. As shown in Fig. 6, the various constructs showed significant variations in their expression patterns. When the data were expressed as fluorescence of wild-type version relative to the mutant version of the same PH domain, the only remarkable difference was observed with the Akt-PH domain, which clearly showed a lipid-binding-dependent self-elimination.
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Mutagenesis of Akt-PH and GRP1-PH domains
The specific inhibitory effect of the PH domains on selected cellular responses raised the possibility that these domains might interact with protein-binding partners in addition to binding PIP3. The putative binding partners would have to interact with a surface on the PH domain that is distinct from that involved in lipid binding. It has been previously reported that Thr34 of the Akt-PH domain is phosphorylated by PKC and this phosphorylation prevents Akt membrane recruitment and activation (Powell et al., 2003
). This Thr residue is located on the surface of the Akt-PH domains that is not directly involved in PIP3 binding and which corresponds to the ß
-binding interface of the GRK2-PH domain (Lodowski et al., 2003
). Therefore, we generated a series of Thr34 mutants with various substitutions (T34A, T34S, T34P, T34D, T34L and T34F) within the Akt-PHGFP construct and determined their ability to bind PIP3, localize to membranes in intact cells, and exert inhibition on Akt activation. Of these mutants, T34A and T34S behaved as the wild-type version, whereas T34P showed no membrane localization or binding to lipids (not shown). However, T34D, T34L and T34F displayed quite interesting features. As shown in Fig. 7, these mutants all showed prominent membrane localization in stimulated COS-7 cells or in HEK 293 cells, and only T34F showed a somewhat impaired membrane recruitment (Fig. 7A). This was in good agreement with the PIP3 binding data, which showed that binding of these mutant recombinant proteins to PIP3 beads was only slightly impaired (Fig. 7B). However, in spite of their PIP3 binding and membrane localization, these mutants showed no inhibition on Akt activation compared with the R25C mutant that is unable to bind the lipids and localize to the membrane (Fig. 7C). Similarly, none of the Akt-PH mutants showed self-elimination in the assay monitoring the kinetics of expression of the domains as a function of time, in fact the T34F mutant showed better expression than the R25C mutant (panel D). These data clearly suggested that membrane localization is necessary but may not be sufficient for the construct to interfere with the signaling of the endogenous Akt protein, and the latter is related to a feature of the PH domain consistent with additional interaction(s).
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Similar experiments were designed with the GRP1-PH domain. Here, two mutants were generated at residues located on the same surface probed with the Akt-PH domain, away from the lipid-binding site. The two mutants, I307E and K340L, were chosen because of their size and side chain orientation. As shown in Fig. 8, these mutations failed to affect the binding of the domain to PIP3 beads (Fig. 8B); however, they did impair the abilities of the constructs to localize to the membranes of HEK 293 cells, although still showed localization in peroxyvanadate-stimulated COS-7 cells (Fig. 8A). Because of their somewhat impaired cellular localization, the InsP4-binding affinity of these constructs was also determined (Fig. 8C). These data showed no significant difference between the domains confirming that their PIP3/InsP4 affinities are not affected by the mutations. Importantly, neither mutant showed the inhibitory effect on cell spreading that was observed with the wild-type GRP1-PH domain, and their effect was less or equal to the R284C mutant unable to bind PIP3 or localize to the membrane. These data also indicated that the dominant-negative effect of the GRP1-PH domain requires interactions other than with the lipid PIP3.
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Discussion |
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The present experiments were designed to investigate whether PH domains that recognize the same phosphoinositide species, namely PIP3, exert similar inhibitory effect on distinct cellular processes known to be regulated by this critically important phosphoinositide. We reasoned that if PH domains solely interacted with the membrane lipids, their inhibitory potencies should follow an identical rank order determined by their relative lipid affinities regardless of the PIP3-dependent regulatory pathway examined. Our data clearly demonstrate that, of the selected PH domains with PIP3 recognition, some can quite specifically inhibit one PIP3-regulated process without significantly affecting another. For example, expression of the GRP1-PH domain was effective at inhibiting cell adhesion and spreading, but had no effect on Akt activation, whereas the Akt-PH domain behaved the opposite way, i.e. showing a strong inhibitory effect on Akt activation but no effect on the cell spreading process. These findings cannot be simply explained by sequestration of the lipids by the PH domains. In fact, inhibition by lipid sequestration may not be as easily achieved since production of the lipid can simply make up for the PH-domain-bound fraction in any given PIP3-mediated process. However, if PH domains also bind to protein partners that are important for a specific downstream signaling process, sequestration of this protein will not be easily compensated for, thereby explaining the selective inhibition of a particular pathway by the overexpressed PH domain.
Thus, mutations generated on a surface within the Akt-PH and GRP1-PH domains that is not directly involved in lipid binding can abrogate the inhibitory effects of the constructs without affecting lipid binding. This finding is also consistent with interaction of the domains with additional binding partners, most probably with other proteins. Also, a putative protein interaction with the PH domain might indirectly affect the membrane localization of the construct. This was seen more prominently with the GRP1-PH domain, which showed impaired membrane localization in spite of an unchanged lipid-binding affinity, whereas the selected Akt-PH domain mutants still localized to the membrane. It is worth pointing out that C2 ceramide has been reported to eliminate the binding of both Akt-PHGFP and GRP1-PHGFP after PDGF stimulation (Stratford et al., 2001), an affect attributed to the PKC
-mediated phosphorylation of T34 in the case of the Akt-PH domain (Powell et al., 2003
). In the present study, the T34D substitution failed to mimic the phosphorylation effect on membrane localization, but this could be due to the much larger charge of the phosphate residue in this position.
It is important to note that the selectivity of the PH domains was not absolute, for example, the Btk-PH also inhibited Akt activation and all but Btk-PH was able to inhibit EGF-induced InsP3 formation. Whether this reflects a similarity between the domains in regard to the protein interacting partner, or the multiple components through which InsP3 generation can be affected in the latter case, remains to be elucidated. It is also important to emphasize that, in the case of responses regulated by more complex regulatory networks, such as the growth of the transfected cells, multiple effects could determine the final outcome. For example, although Btk-PH inhibited Akt activation similarly to that of Akt-PH, the two domains affected the EGF-mediated InsP3 response differently, Akt-PH having an inhibitory effect, and Btk-PH having a strong stimulatory effect. As a result, only Akt-PH caused a selective elimination of the cells in which it was expressed; by contrast, in the case of Btk, the two opposing effects seem to have balanced one another and hence, Btk-PH had no prominent effect on cell elimination.
These findings have important implications regarding inositol lipid regulation of effectors containing PH domains. It is conceivable that, upon lipid binding, the PH domain undergoes a conformational change that will affect its interaction with a protein-binding partner. This partner could be either another regulatory protein or another domain within the same protein forming an intramolecular interaction. A conformational change upon binding of InsP4 to the Akt-PH has been described recently (Milburn et al., 2003) but not many other studies are available in which this question has been examined at the structural level. By contrast, there are several examples of PH domains regulating the functions of proteins; this has been observed even in vitro, where PIP3-mediated membrane recruitment cannot be responsible for the observed effects of the lipid. For example, Btk kinase activity in vitro was found to be stimulated by PIP3 (Saito et al., 2001
). Recently, the PH domain of the ELMO protein (which, together with Dock180, functions as a bipartite Rac guanine-nucleotide exchange factor) was shown to be essential for the binding and regulation of the Rac-Dock180 complex independent of membrane targeting (Lu et al., 2004
). Similarly, in several guanine nucleotide exchange factors with tandem Dbl-homology (DH) and PH domains, the PH domain may cooperate with the DH domain in promoting nucleotide exchange (Rossman et al., 2002
; Soisson et al., 1998
; Worthylake et al., 2004
), although this may not be regulated by inositides in vitro (Snyder et al., 2001
). Conversely, the inositol lipid binding of the nucleotide exchange factor Trio was found to be influenced by the binding of RhoG (Skowronek et al., 2004
), providing additional support to the idea that the lipid and protein binding of PH domains might regulate one another.
In summary, the present studies demonstrate that overexpression of isolated PH domains can act as inhibitors of PIP3-regulated cellular pathways and show a degree of specificity that was unexpected given their inositol lipid recognition properties. Selected mutations that do not affect their lipid recognition are able to prevent the inhibitory effects, suggesting that PH domains might also interact with proteins. PH domains, therefore, could serve as molecular switches regulated by phosphoinositides. This type of regulation could explain the parallel and specific control of multiple effectors by the membrane phospholipids. More studies will be needed to validate this concept and to identify the protein regulatory partners that participate in the process.
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Acknowledgments |
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