WDR26 Functions as a Scaffolding Protein to Promote Gβγ-mediated Phospholipase C β2 (PLCβ2) Activation in Leukocytes*

Background: It remains unclear how Gβγ regulates diverse effectors in cells. Results: WDR26 exists in oligomers. It simultaneously binds both Gβγ and PLCβ2 to enhance PLCβ2 membrane translocation and activation by Gβγ in leukocytes. Conclusion: WDR26 functions as a scaffolding protein to promote Gβγ-mediated PLCβ2 activation. Significance: These findings uncover a novel mechanism of regulating Gβγ signaling through a scaffolding protein. We have recently identified WDR26 as a novel WD40 repeat protein that binds Gβγ and promotes Gβγ signaling during leukocyte migration. Here, we have determined the mechanism by which WDR26 enhances Gβγ-mediated phospholipase C β2 (PLCβ2) activation in leukocytes. We show that WDR26 not only directly bound Gβγ but also PLCβ2. The binding sites of WDR26 and PLCβ2 on Gβ1γ2 were overlapping but not identical. WDR26 used the same domains for binding Gβγ and PLCβ but still formed a signaling complex with Gβγ and PLCβ2 probably due to the fact that WDR26 formed a higher order oligomer through its Lis homology and C-terminal to LisH (LisH-CTLH) and WD40 domains. Additional studies indicated that the formation of higher order oligomers was required for WDR26 to promote PLCβ2 interaction with and activation by Gβγ. Moreover, WDR26 was required for PLCβ2 translocation from the cytosol to the membrane in polarized leukocytes, and the translocation of PLCβ2 was sufficient to cause partial activation of PLCβ2. Collectively, our data indicate that WDR26 functions as a scaffolding protein to promote PLCβ2 membrane translocation and interaction with Gβγ, thereby enhancing PLCβ2 activation in leukocytes. These findings have identified a novel mechanism of regulating Gβγ signaling through a scaffolding protein.

protein interactions and protein dimerization. The binding of WDR26 to G␤␥ involves the G␣ contact surface of G␤␥ that interacts with many known G␤␥ effectors (16). It is not yet clear how WDR26 binding to G␤␥ leads to enhanced G␤␥ signaling.
Here, we provide evidence that WDR26 exists in a higher order oligomer and simultaneously binds both G␤␥ and PLC␤2. It promotes PLC␤2 membrane translocation and functions as a scaffolding protein to bring PLC␤2 in close proximity to G␤␥ for activation. Thus, our findings have uncovered a novel mechanism of regulating G␤␥ signaling through a scaffolding protein.
Cell Culture-Jurkat T cells stably expressing FLAG-WDR26 and the HL60 cell line (ATCC) were grown in RPMI 1640 medium (Invitrogen) containing 10% fetal bovine serum (FBS) as described previously (3,16). HEK293 cells (ATCC) were maintained in DMEM (Invitrogen) supplemented with 10% FBS. Differentiation of HL60 cells into human neutrophil-like cells was induced by adding 1.3% DMSO to the growth medium and incubating for 7 days.
Purification of WDR26 from E. coli-MBP, MBP-WDR26, and its mutants were expressed in E. coli BL21 cells and purified using amylose resin (New England Biolabs) (16,21). MBP was removed from WDR26 by digestion with 3C protease at 4°C overnight followed by gel filtration chromatography.
Immunoprecipitation and Western Blotting Analysis-To coimmunoprecipitate FLAG-WDR26 with endogenous G␤␥ and PLC␤2, Jurkat T cells were first serum-starved for 4 -6 h and then stimulated with SDF1␣ (50 nM) for the indicated times. In some cases, cells were pretreated with pertussis toxin (0.2 g/ml) overnight prior to stimulation by SDF1␣. After SDF1␣ stimulation, 1 mM dithiobis(succinimidyl propionate) (Pierce) was added to cross-link the proteins for 40 min at room temperature followed by the addition of 50 mM Tris-HCl (pH 7.4) to quench the unreacted dithiobis(succinimidyl propionate). After washing twice with PBS (pH 7.4), cells were lysed in radioimmune precipitation assay buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Nonidet P-40, 0.1% SDS, 1% deoxycholate, 1 mM EDTA) containing protease inhibitors. Immunoprecipitation was performed as described previously (16). Protein complexes were resolved by SDS-PAGE and analyzed by Western blotting analysis using the Odyssey imaging system (LI-COR Biosciences).
To co-immunoprecipitate FLAG-WDR26 and its mutants with PLC␤2 following their expression in HEK293 cells, cell lysates were prepared from the transfected cells without dithiobis(succinimidyl propionate)-mediated protein cross-linking. FLAG-tagged proteins were immunoprecipitated as described above except that modified radioimmune precipitation assay buffer containing 1% Nonidet P-40 but no SDS and deoxycholate was used. Similar co-immunoprecipitation experiments were carried out to determine the interaction of WDR26 with its mutants.
Protein Binding Assays-To determine its binding to PLC␤2, 0.2 M FLAG-WDR26 was first immunoprecipitated from the Sf9 cell lysates in radioimmune precipitation assay buffer using the anti-FLAG M2 antibody (Sigma) conjugated to Dynabeads protein G. The beads containing FLAG-WDR26 were washed extensively (five times) with radioimmune precipitation assay buffer to remove lipids and other proteins associated with WDR26 and beads and then incubated with increasing concentrations of PLC␤2 in buffer (50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, 0.2% Nonidet P-40) for 4 h at 4°C. Protein complexes were precipitated using a magnetic stand and subjected to SDS-PAGE and immunoblot analysis.
To confirm the direct protein-protein interaction between WDR26 and PLC␤2, 0.2 M purified His 6 -PLC␤2 was incubated with increasing concentrations of purified WDR26 in buffer (20 mM HEPES (pH 8.0), 200 mM NaCl, 1 mM DTT, 5 mM imidazole, 0.2% Nonidet P-40) at 4°C for 2 h. Protein complexes were precipitated by incubation with Ni-NTA resin at 4°C for 2 h followed by washing three times with high salt buffer (20 mM HEPES (pH 8.0), 500 mM NaCl, 1 mM DTT, 0.2% Nonidet P-40) and twice with binding buffer and then subjected to SDS-PAGE and immunoblot analysis.
To determine the effects of WDR26 and its mutants on the G␤ 1 ␥ 2 and PLC␤2 interaction, 0.2 M purified FLAG-G␤ 1 ␥ 2 was incubated with 0.5 M PLC␤2 and increasing concentrations of purified WDR26 (0 -2 M) or its mutant (0 -1 M). The protein complexes were then immunoprecipitated with the anti-FLAG M2 antibody conjugated to Dynabeads protein G as described above.
The binding of G␤ 1 ␥ 2 and G␤ 1 W99A␥ 2 to FLAG-WDR26 was determined as described previously (16). To determine the effects of M119, gallein, and M119B on G␤ 1 ␥ 2 binding to FLAG-WDR26, M119, gallein, and M119B were first incubated with G␤ 1 ␥ 2 for 1 h prior to the addition of FLAG-WDR26. To determine the effects of M119 and M119B on PLC␤2 binding to FLAG-WDR26, M119 and M119B were first incubated with precipitated FLAG-WDR26 for 1 h prior to the addition of PLC␤2.
Polarization of dHL60 Cells-Polarization of dHL60 cells to a uniform concentration of fMLP was performed in a Lab-Tek TM II 8-well chamber slide precoated with fibronectin (100 ng/ml). 5 ϫ 10 4 dHL60 cells in 0.15 ml of Hanks' balanced salt solution (Invitrogen) supplemented with 1% glucose and 1% human serum albumin were seeded onto each well and incubated at 37°C for 10 min. After washing twice with Hanks' balanced salt solution to remove unattached cells, cells were stimulated with buffer or 100 nM fMLP for 3 min at room temperature and then fixed with 4% paraformaldehyde and 0.05% glutaraldehyde (Sigma) for 10 min.
Measurement of Total Inositol Phosphate (IP) Turnover-G␤␥-mediated PLC␤2 activation was determined in HEK293 cells as described previously (21). Briefly, 1 day post-transfection, cells were labeled for 48 h with myo-[ 3 H]inositol (2 Ci/ ml) in inositol-free DMEM containing 1% dialyzed FBS. After serum starvation for 4 h, 10 mM LiCl was added to the cells to initiate IP accumulation for 40 min. Total IPs were separated by AG 1-X8 columns and expressed as percentage of total [ 3 H]inositol incorporated into the intact cells.
Statistical Analysis-Data were expressed as mean Ϯ S.E. Statistical comparisons between two groups were analyzed by two-tailed Student's t test (p Ͻ 0.05 was considered significant).

WDR26 Forms a Complex with G␤␥ and PLC␤2-We
showed previously that WDR26 interacts with endogenous G␤␥ in Jurkat T cells stimulated with SDF1␣, which activates the chemokine receptor CXCR4 (16). Given that WDR26 is required for G␤␥-mediated PLC activation in these cells, we asked whether PLC␤2 was co-precipitated with WDR26 and G␤␥ in a complex. Immunoprecipitation analyses of FLAG-WDR26 from Jurkat T cells stably expressing this protein indicated that WDR26 associated with endogenous PLC␤2 in both stimulated and unstimulated cells (Fig. 1A, left panel). In contrast, as we showed previously, WDR26 only formed a complex with G␤␥, but not G␣ i , in stimulated cells, and the interaction of WDR26 with G␤␥ decreased over time with prolonged SDF1␣ stimulation (16). Pretreatment of cells with pertussis toxin to uncouple receptors from G i/o proteins abolished the interaction of WDR26 with G␤␥ but had no effect on the interaction of WDR26 with PLC␤2 ( Fig. 1A, left panel). These findings suggest that WDR26 is constitutively associated with PLC␤2.
To determine whether WDR26 directly binds PLC␤2, we performed binding assays in vitro using purified proteins. As shown in Fig. 1B, upper panel, FLAG-WDR26 bound PLC␤2 in a dose-dependent manner with a binding affinity of 0.906 Ϯ 0.002 M. Reciprocal pulldown of His-PLC␤2 also showed that PLC␤2 bound WDR26 in a dose-dependent manner with a binding affinity of 0.44 Ϯ 0.13 M (Fig. 1B, lower panel). The binding affinity of WDR26 with PLC␤2 is comparable with that with G␤␥ (ϳ0.5 M) (16). Because G␤␥ also binds PLC␤2 with a similar affinity (ϳ1 M) (22), this raises a question of whether WDR26, G␤␥, and PLC␤2 can form a trimeric complex. To test this, we examined the binding of G␤ 1 ␥ 2 (0.2 M) to a constant amount of PLC␤2 (0.5 M) in the presence of increasing concentrations of WDR26 (0 -2 M). As shown in Fig. 1C, WDR26 was co-precipitated with G␤ 1 ␥ 2 and PLC␤2 in a complex. Moreover, the presence of WDR26 enhanced the binding of PLC␤2 to G␤ 1 ␥ 2 in a dose-dependent manner. The enhanced binding peaked at 0.5 M WDR26 and became less obvious at higher concentrations of WDR26. Similarly, co-expression with WDR26 also promoted G␤␥ interaction with PLC␤2 in HEK293 cells (Fig. 1E). These findings indicate that at optimal concentrations WDR26 is able to form a complex containing both PLC␤2 and G␤ 1 ␥ 2 , thereby enhancing PLC␤2 interaction with G␤ 1 ␥ 2 . In contrast, excess WDR26 may bind to PLC␤2 and G␤ 1 ␥ 2 individually and is therefore unable to increase the binding of PLC␤2 to G␤ 1 ␥ 2 .
WDR26 and PLC␤2 Bind to an Overlapping, but Not Identical, Site on G␤ 1 ␥ 2 -WDR26 binds to the G␣ contact surface on G␤ 1 ␥ 2 , which is known to interact with diverse effectors including PLC␤2 (16). To understand the molecular basis for the abil- ity of WDR26 to form a complex with PLC␤2 and G␤ 1 ␥ 2 , we further characterized the binding sites of WDR26 on G␤ 1 ␥ 2 . Initially, we used small molecules, M119 and gallein, for competition binding assays because they bind to a "hot spot" on G␤ 1 ␥ 2 that interacts with multiple effectors including PLC␤2 (24,25). M119, but not its inactive analog, M119B, inhibited WDR26 binding to G␤ 1 ␥ 2 at a concentration range that is known to inhibit PLC␤2 binding to G␤ 1 ␥ 2 ( Fig. 2A, upper panel, and B). Similar results were obtained with gallein (data not shown). In contrast, M119 had no effect on WDR26 interaction with PLC␤2 ( Fig. 2A, lower panel), suggesting that M119 does not bind to WDR26. These data indicate that WDR26 and PLC␤2 share a common binding site on G␤ 1 ␥ 2 .
To further characterize specific residues on G␤␥ required for binding WDR26 and PLC␤2, we evaluated WDR26 binding to a G␤␥ mutant defective in binding to and activating PLC␤2, G␤ 1 W99A␥ 2 (23,26). The binding of WDR26 to G␤ 1 W99A␥ 2 was comparable with that of the wild-type G␤ 1 ␥ 2 (Fig. 2B), suggesting that G␤␥ likely uses distinct residues within the hot spot for binding WDR26 and PLC␤2.
WDR26 Uses the Same Domains for Binding G␤ 1 ␥ 2 and PLC␤2-We showed previously that the C-terminal fragment of WDR26 consisting of the LisH-CTLH and WD40 domains is involved in binding G␤␥ (16). To identify the binding sites of PLC␤2 on WDR26, the interaction of PLC␤2 with a series of WDR26 deletion mutants was tested (Fig. 3). Most of the mutants can be readily detected in the immunoprecipitates, but only a small amount of WDR123-231 was detected in the precipitates (Fig. 3). This is probably due to its lower level of expression or low efficiency of retention to the blotting membrane because of its small size (molecular mass, ϳ10 kDa). As compared with the full-length WDR26, mutants WDR123- 231, WDR123-661, and WDR232-661 retained the capacity to bind PLC␤2, whereas WDR1-122 failed to bind PLC␤2 (Fig.  3). These data indicate that the LisH-CTLH and WD40 domains of WDR26 are involved in binding PLC␤2. Interestingly, the WDR26 mutants that bound PLC␤2 also interact with G␤␥ (16), suggesting that PLC␤2 and G␤␥ may share overlapping binding sites on WDR26. WDR26 Exists in Oligomers-Given that WDR26 and PLC␤2 share overlapping binding sites on G␤␥ and that the same domains of WDR26 are involved in binding PLC␤2 and G␤␥, we were surprised to find they still formed a complex. Based on this observation, we questioned whether WDR26 forms a homodimer or a larger oligomer. Gel filtration analysis of purified WDR26 with a Superdex 200 column showed that WDR26 eluted in several fractions with peaks corresponding to ϳ160, 370, and larger than 600 kDa, the separation limit of the column (Fig. 4A). The fractions with peaks larger than 370 kDa appeared to be predominant (Fig. 4A). Given that the monomer of WDR26 is about 75 kDa, these results indicate that WDR26 exist in a mixture of multimers with higher order oligomers larger than a pentamer (ϳ375 kDa) being predominant (Ͼ90%) (Fig. 4A). The formation of WDR26 oligomers was unlikely simply due to protein aggregation because they were still detected when gel filtration was performed with WDR26 tagged with the MBP, which enhances the solubility of fusion proteins (data not shown). To verify these findings in mammalian cells, we co-transfected FLAG-and myc-tagged WDR26 in HEK293 cells and then performed co-immunoprecipitation assays with anti-FLAG antibody. Myc-WDR26 was co-immunoprecipitated with FLAG-WDR26 (Fig. 4B), indicating that they formed oligomers.
ToidentifythestructuralelementsrequiredforWDR26oligomerization, we evaluated the interaction of WDR26 deletion mutants with the full-length WDR26. The N-terminal fragment WDR1-122 did not bind WDR26, but the C-terminal fragment WDR123-661 retained the same binding to WDR26 as the full-length WDR26. Deletion of either the LisH-CTLH domain (WDR232-661) or WD40 domain (WDR123-231 and WDR1-231) from WDR123-661 impaired its binding to WDR26, indicating that both domains are involved in WDR26 oligomerization (Fig. 4C). Interestingly, gel filtration analysis of purified WDR123-661 showed that unlike the full-length WDR26, which formed predominantly larger oligomers, it existed primarily in a dimer (ϳ135 kDa) or trimer (ϳ189 kDa) (Fig. 4D). These findings suggest that the N-terminal fragment WDR1-122 is required for stabilization of WDR26 in the form of higher order oligomers.
WDR26 Enhances G␤␥-mediated PLC␤2 Activation-To determine whether the ternary complex formed by WDR26, G␤␥, and PLC␤2 is signaling-competent, we tested G␤␥-stimulated PLC␤2 activity in the presence of increasing concentrations of WDR26. WDR26 did not alter the basal activity of PLC␤2 but increased G␤␥-stimulated PLC␤2 activity with a bell-shaped curve (Fig. 5A). The concentrations of WDR26 required for enhancing PLC␤2 activity were consistent with those required for increasing PLC␤2 binding to G␤␥ (Fig. 1, C  and D), indicating that WDR26 enhanced PLC␤2 activation primarily through promoting PLC␤2 interaction with G␤␥. In support of this notion, we found that WDR26 was unable to rescue PLC␤2 activation by G␤ 1 W99A␥ 2 , which is deficient in binding PLC␤2 (Fig. 5B). Additionally, the WDR123-661 mutant inhibited the interaction between G␤ 1 ␥ 2 and PLC␤2 and consequently blocked G␤ 1 ␥ 2 -mediated PLC␤2 activation in a dose-dependent manner (Fig. 5, A and C).
To verify the findings in intact cells, we co-transfected WDR26 with PLC␤2 and different concentrations of G␤ 1 ␥ 2 in HEK293 cells and then analyzed IP accumulation. G␤␥ alone was able to activate PLC␤2 in a dose-dependent manner (Fig.  5D). Co-transfection with WDR26 significantly enhanced the potency of G␤ 1 ␥ 2 in activating PLC␤2, particularly with lower concentrations of G␤ 1 ␥ 2 (0.4 and 0.8 g) (Fig. 5D). WDR26 alone did not affect the basal activity of PLC␤2, nor did it affect the expression of PLC␤2 and G␤ 1 ␥ 2 (Fig. 5D). In contrast, cotransfection with WDR123-661 inhibited G␤ 1 ␥ 2 -mediated PLC␤2 activation (Fig. 5E). Together, these findings indicate that WDR26 enhanced PLC␤2 activation by G␤ 1 ␥ 2 both in vitro and in intact cells.
WDR26 Promotes PLC␤2 Membrane Translocation-We showed previously that stimulation of Jurkat T and dHL60 cells with chemoattractants leads to WDR26 translocation from the cytosol to the plasma membrane (16). Immunostaining analysis showed that in unstimulated dHL60 cells the cellular localiza- tion of both WDR26 (16) and PLC␤2 was difficult to be determined by first glance as these cells have a large nucleus and a small fraction of cytosol. However, colocalization analysis of WDR26 and PLC␤2 with the cortical actin showed that they were located within the cortical actin, indicating that they were predominantly localized in the cytosol (Fig. 6A) (16). The cytosolic localization of WDR26 and PLC␤2 was confirmed by cell fractionation analysis (data not shown). Upon stimulation with fMLP, dHL60 cells became polarized, generating an F-actinenriched lamellipodial membrane protrusion (Fig. 6A). As with WDR26 (16), PLC␤2 accumulated within the membrane protrusion (Fig. 6A). This accumulation was not an artifact of the increase in plasma membrane surface at the cell's protrusion because polarized cells labeled with the lipid membrane probe CM-DiI exhibited uniform CM-DiI fluorescence across the cell (Fig. 6A). Given that WDR26 bound PLC␤2, we asked whether WDR26 is required for PLC␤2 translocation to the membrane protrusion. Transient transfection of dHL60 cells with an siRNA against WDR26 led to about 50 -60% reduction in the level of WDR26 expression (Fig. 6C). The down-regulation of WDR26 did not affect the response of dHL60 to fMLP-stimulated F-actin polarization, nor did it affect the cytosolic localization of PLC␤2 in unstimulated cells (Fig. 6, B and C). However, PLC␤2 translocation to the membrane protrusion was significantly inhibited in fMLP-stimulated dHL60 cells (Fig. 6,  B and C). These findings indicate that WDR26 regulates the membrane translocation of PLC␤2.
We showed previously that down-regulation of WDR26 in dHL60 cells abolished fMLP-stimulated Ca 2ϩ signaling, which is known to be mediated by PLC␤2/3 (16). To determine whether WDR26-regulated PLC␤2 translocation contributed to PLC␤2 activity, we first tested whether membrane translocation alone is sufficient for PLC␤2 activation. To render PLC␤2 membrane localization, we attached a myristoylation sequence to its N terminus. We then transfected different concentration of the wild-type PLC␤2 and myr-PLC␤2 in HEK293 cells and compared their basal activities in generating total IP. As expected, confocal microcopy analysis indicated that PLC␤2 was expressed in the cytosol, whereas myr-PLC␤2 was located primarily in the plasma membranes (Fig. 7A). Overexpression of either the wild-type or myr-PLC␤2 caused an expression level-dependent increase in IP accumulation (r 2 ϭ 0.84 and 0.71 for the wild-type and myr-PLC␤2, respectively, p Ͻ 0.01) (Fig.  7E). At a similar expression level, myr-PLC␤2 displayed a higher activity in mediating IP production than its wild-type counterpart (Fig. 7E). Based on the slope of their dose-response curves, the basal activity of myr-PLC␤2 is estimated to be ϳ2-fold higher than that of the wild-type PLC␤2 (0.212 Ϯ 0.077 versus 0.083 Ϯ 0.025, p Ͻ 0.05, n ϭ 3). Notably, co-transfection of myr-PLC␤2 with G␤ 1 ␥ 2 caused a further increase in IP production (Fig. 7F), suggesting that membrane translocation alone is insufficient for maximum activation of PLC␤2.
To provide evidence that WDR26-mediated PLC␤2 translocation contributes to its activation, we generated myr-WDR26 and co-expressed it with PLC␤2. As expected, the wild-type WDR26 alone was expressed primarily in the cytosol, whereas myr-WDR26 was expressed in the plasma membrane (Fig. 7B). Co-expression of myr-WDR26 but not the wild-type WDR26 with PLC␤2 resulted in a significant amount of PLC␤2 localized in the plasma membrane (Fig. 7C). Corresponding to their membrane localization, co-expression with the wild-type WDR26 had no effects on the basal activity of PLC␤2, whereas co-expression with myr-WDR26 led to PLC␤2 activity (Fig.  7G).

DISCUSSION
In this study, we have demonstrated that the G␤␥-binding protein WDR26 serves as a scaffolding protein to promote G␤␥-mediated PLC␤2 activation by regulating PLC␤2 membrane translocation and interaction with G␤␥. PLC␤2 is a cytosolic protein (12,27). To hydrolyze its membrane-localized substrate, phosphatidylinositol 4,5-bisphosphate, PLC␤2 must first be translocated to the membrane. Indeed, agonist-stimulated PLC␤2 translocation has been demonstrated in both neu- trophils and macrophages (12,28). However, the underlying mechanisms for PLC␤2 translocation have not yet been defined. Although PLC␤2 directly binds G␤␥, which is localized in the membrane, it is not clear whether interaction with G␤␥ itself is sufficient for translocation. Our data indicate that PLC␤2 membrane translocation critically depends on its interaction with WDR26. As with G␤␥, PLC␤2 directly binds WDR26. However, unlike G␤␥, PLC␤2 binding to WDR26 does not require the activation of G protein-coupled receptors, sug-gesting that WDR26 and PLC␤2 likely exist in a preformed complex in the cytosol of unstimulated cells. In support of this notion, both endogenous WDR26 and PLC␤2 are predominantly located in the cytosol of unstimulated dHL60 cells. Moreover, they are both translocated to the membrane protrusion by stimulation with the chemoattractant fMLP. The role of WDR26 in facilitating PLC␤2 membrane translocation is further demonstrated by the findings that partially suppressing WDR26 in dHL60 cells inhibited PLC␤2 translocation but had no significant effects on actin polymerization and cell polarization, suggesting that PLC␤2 translocation is not simply the result of cell polarization but rather its interaction with WDR26. In line with these findings, co-expressing PLC␤2 with the membrane-localized WDR26 in HEK293 cells results in a significant enhancement of PLC␤2 membrane localization. It is not clear from our studies how WDR26 translocation is regulated. Our previous work suggests that WDR26 is not simply anchored to the membrane protrusion by its interaction with G␤␥ because the concentration gradient of WDR26 from the leading to trailing edge in a polarized leukocyte is significantly steeper than that of G␤␥ (16). However, WDR26 translocation is sensitive to pertussis toxin treatment, suggesting that activated G␣ i/o or G␤␥ signaling is required for the translocation (16). Membrane translocation has been shown to be sufficient to cause activation of a number of enzymes including the G␤␥ effector PI3K␥ (11). It is not yet clear whether membrane translocation is sufficient for PLC␤2 activation. Recent studies of Rac-dependent activation of PLC␤2 have suggested that Rac1 may activate PLC␤2 by inducing membrane translocation because there is no significant conformational difference between the structures of free and Rac1-bound PLC␤2 (29). Our findings that targeting PLC␤2 to the membrane either by attaching a myristoylation sequence to its N terminus or by co-expression with myristoylated WDR26 enhances its basal activity to hydrolyze phosphatidylinositol 4,5-bisphosphate have provided direct evidence to support the role of membrane translocation in PLC␤2 activation. However, it remains unclear how membrane localization causes PLC␤2 activation. Previous work by Sondek and co-workers (30) has identified a linker region between the X and Y domains of the PLC␤2 catalytic domain that is folded back to occlude its catalytic site. This inhibitory linker consists of a high density of negatively charged residues and was proposed to autoinhibit PLC␤2. Upon PLC␤2 recruitment to negatively charged substrate membranes, the inhibitory linker is displaced from the active site by electrostatic repellence, leading to PLC␤2 activation. This hypothesis was supported by the findings that deletion of the linker region is sufficient to cause PLC␤2 activation in the absence of any stimuli (30). However, the PLC␤2 mutant with the linker deletion exhibited up to a 20-fold increase in basal activity, which is significantly larger than what we observed with the membranetargeted PLC␤2 (ϳ2-fold). Although the observed different activities of the PLC␤2 deletion mutant and membrane-targeted PLC␤2 could be due to different assay conditions used by the two laboratories, these findings may also suggest that the increased PLC␤2 activity by translocation cannot be simply attributed to the displacement of the inhibitory linker.  D). HEK293 cells were transiently transfected with the indicated constructs and stained with a rabbit anti-PLC␤2 and/or mouse anti-FLAG. Representative images are shown. The graphs in the right panel of C and D show the distribution of fluorescence intensity of PLC␤2 and WDR26 along the lines drawn across the cells. Bar, 5 m. E, total IPs in HEK293 cells transfected with increasing concentrations of PLC␤2 or myr-PLC␤2. The levels of protein expression were quantified by Western blotting and expressed as -fold increases over that in cells transfected with the smallest amount of plasmids. IP accumulation is expressed as -fold increases over that in cells expressing the lowest level of proteins (n ϭ 3). F, total IP accumulation in HEK293 cells transfected with G␤ 1 ␥ 2 (0.4 g), myr-PLC␤2 (0.2 g), or G␤ 1 ␥ 2 (0.4 g) plus myr-PLC␤2 (0.2 g). Data are expressed as -fold increases of IPs over that generated by cells expressing myr-PLC␤2 alone. *, p Ͻ 0.05 indicates significance versus myr-PLC␤2 alone (n ϭ 4). G, total IP accumulation in HEK293 cells transfected with PLC␤2 (0.1 g) alone or PLC␤2 together with the indicated concentration (g) of WDR26 or myr-WDR26. Data are expressed as -fold increases of IPs over that generated by cells expressing PLC␤2 alone. *, p Ͻ 0.05 indicates significance versus PLC␤2 alone (n ϭ 3). Representative images in the right panel show the level of protein expression. Error bars represent S.E.
Although membrane translocation is likely the first step of PLC␤2 activation in vivo, it has been shown previously that G␤␥ can activate PLC␤2 independently of its translocation in vitro (14,15). These findings indicate that once arriving at the membrane PLC␤2 is further activated by direct interaction with G␤␥. Interestingly, under such a condition, G␤␥-mediated PLC␤2 activation can be further enhanced by WDR26. The effect of WDR26 on PLC␤2 activation is concentration-dependent and correlated to its ability to enhance G␤␥ interaction with PLC␤2. Given that WDR26 interacts with both G␤␥ and PLC␤2, these findings indicate that at optimal concentrations WDR26 forms a complex with G␤␥ and PLC␤2 that promotes G␤␥-mediated PLC␤2 activation. Intriguingly, the binding sites of WDR26 on G␤␥ appear to involve a contact surface on G␤␥ that is required for binding and activation of many effectors including PLC␤2. Moreover, the binding of G␤␥ and PLC␤2 to WDR26 involves similar domains of WDR26. These findings raise the question of how WDR26, G␤␥, and PLC␤2 can be assembled into a complex. Previous work has found that G␤␥ contains multiple binding sites for PLC␤2. In addition to the G␣ contact surface, the N-terminal coiled coil domain and the outer surface of the G␤ are also involved in binding PLC␤2 (22,31). In the absence of other constraints, G␤␥ activates PLC␤2 primarily through the G␣ t contact region (26). However, in the presence of one G␤␥-interacting protein, AGS8, G␤␥ binds to PLC␤2 through an alternative site located at the N terminus of G␤ (23). Consequently, AGS8, G␤␥, and PLC␤2 can co-exist in a complex. Moreover, AGS8 rescues PLC␤2 binding and activation by an inactive G␤ that contains a mutation (G␤ 1 W99A) in the effector contact surface. Our data indicate that WDR26 may not use the same mechanism to form a complex with G␤␥ and PLC␤2 because unlike AGS8, which only binds G␤␥, WDR26 interacts with both G␤␥ and PLC␤2. Moreover, WDR26 was unable to restore the ability of G␤ 1 W99A␥ 2 to activate PLC␤2, suggesting that unlike AGS8 WDR26 does not direct G␤␥ to use the alternative site for binding and activation of PLC␤2. Rather, our findings that WDR26 exists in oligomers suggest that WDR26 may use different monomers to bind G␤␥ and PLC␤2, thereby bringing G␤␥ and PLC␤2 in close proximity for enhanced interaction and activation. In this sense, WDR26 serves as a scaffolding protein to assemble G␤␥ and PLC␤2 in a complex.
The interactions of WDR26 with G␤␥ and PLC␤2 are likely to be transient in cells because we cannot isolate WDR26, G␤␥, and PLC␤2 as a stable complex by gel filtration chromatography, nor can we accurately determine the stoichiometric relationship of their bindings by binding assays involving extensive washing steps (data not shown). Interestingly, the ability of WDR26 to enhance G␤␥ and PLC␤2 interaction and activation appears to depend on its formation of oligomers larger than a pentamer because the N-terminal truncation mutant of WDR26, WDR123-661, which predominantly exists in dimers and trimers, fails to promote PLC␤2 binding and activation by G␤␥. Rather, WDR123-661 exhibits a dominant negative activity of inhibiting G␤␥ and PLC␤2 interaction and activation both in vitro and in vivo.
In summary, our studies have uncovered a novel mechanism of regulating G␤␥ signaling by a WD40 repeat protein that serves as a scaffolding protein to promote the interaction and activation of PLC␤2 by G␤␥. Our data suggest that in unstimulated cells WDR26 forms a complex with PLC␤2 in the cytosol. Upon G protein-coupled receptor-mediated G protein activation, WDR26 facilitates the recruitment of PLC␤2 to the plasma membrane. Once on the membrane, WDR26 binds to G␤␥, thereby bringing PLC␤2 in close proximity to G␤␥ for interaction and activation. Given that WDR26 is ubiquitously expressed and is required for the efficient activation of other G␤␥ effectors such as PI3K␥ (16), such a mechanism of regulation by WDR26 may extend beyond PLC␤2 (32). In addition to PLC␤2, G␤␥ can also stimulate PLC␤3. Although the expression of PLC␤2 is limited to leukocytes, PLC␤3 is ubiquitously expressed. It will be interesting to determine whether WDR26 can also bind PLC␤3 and regulate G␤␥-mediated PLC␤3 activation in other cell types. Moreover, our previous work has already demonstrated that the regulation of G␤␥ signaling by WDR26 is critical for leukocyte migration (16). Recent work has shown that G␤␥ signaling is also involved in numerous pathological conditions such as heart failure and tumor growth and metastasis (5,33). It would be interesting to determine whether aberrant regulation of G␤␥ signaling by WDR26 contributes to these pathological conditions.