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The WD40 Repeat Protein WDR26 Binds Gβγ and Promotes Gβγ-dependent Signal Transduction and Leukocyte Migration*

Background: How Gβγ regulates leukocyte migration through numerous signaling partners remains elusive. Results: WDR26 binds Gβγ and is required for Gβγ signaling and leukocyte migration. Conclusion: WDR26 is a novel Gβγ-binding partner that promotes Gβγ signaling and leukocyte migration. Significance: Elucidating the signaling mechanisms of Gβγ is crucial for understanding its key role in many important cellular processes. The Gβγ subunits of heterotrimeric G proteins transmit signals to control many cellular processes, including leukocyte migration. Gβγ signaling may regulate and be regulated by numerous signaling partners. Here, we reveal that WDR26, a member of the WD40 repeat protein family, directly bound free Gβγ in vitro, and formed a complex with endogenous Gβγ in Jurkat T cells stimulated by the chemokine SDF1α. Suppression of WDR26 by siRNAs selectively inhibited Gβγ-dependent phospholipase Cβ and PI3K activation, and attenuated chemotaxis in Jurkat T cells and differentiated HL60 cells in vitro and Jurkat T cell homing to lymphoid tissues in scid mice. Similarly, disruption of the WDR26/Gβγ interaction via expression of a WDR26 deletion mutant impaired Gβγ signaling and Jurkat T cell migration, indicating that the function of WDR26 depends on its binding to Gβγ. Additional data show that WDR26 also controlled RACK1, a negative regulator, in binding Gβγ and inhibiting leukocyte migration. Collectively, these experiments identify WDR26 as a novel Gβγ-binding protein that is required for the efficacy of Gβγ signaling and leukocyte migration.

G protein-coupled receptors (GPCRs) 3 comprise a large family of cell surface proteins that play an important role in many physiological functions, such as the sense of sight, smell, and taste, metabolism, neuronal activity, and cardiovascular homeostasis (1). GPCRs transmit extracellular signals through het-erotrimeric G proteins, which are comprised of G␣ and G␤␥ subunits (1,2). Both the G␣ and G␤␥ subunits can activate downstream effectors to relay signals from GPCRs.
G␤␥ subunits play a prominent role in several cellular processes, including leukocyte migration (3)(4)(5). Leukocyte migration is directed by chemoattractants including bacterial by-products such as the formyl peptide, N-formyl-methionineleucine-phenylalanine (fMLP), and a superfamily of chemotactic cytokines, chemokines, such as SDF1␣ and C5␣. These chemoattractants act on GPCRs that primarily couple to pertussis toxin (PTX)-sensitive G i/o proteins. Previous work has shown that chemoattractants predominantly transmit chemotactic signals through G␤␥ subunits liberated from the activated G i/o proteins (6 -9). Free G␤␥ activates diverse effectors including PI3K␥ (10 -15), PLC␤ 2/3 (16), and guanine nucleotide exchange factors for Rac and Cdc42 (17)(18)(19)(20)(21)(22) to direct leukocyte polarization and chemotaxis. However, although the critical role of G␤␥ signaling and its downstream effectors in leukocyte migration has been well established, our understanding of how the activation of various G␤␥ effectors is regulated spatially and temporally for precise control of directional cell migration remains limited (4,23).
G␤ is a prototype of the WD40 repeat-containing protein family (24). These proteins are characterized by the presence of several repeats consisting of between 40 and 60 amino acids with two internal conserved dipeptide sequences, glycine-histidine (GH) and tryptophan-aspartic acid (WD) (24 -27). These repeats form a circular bladed ␤-propeller structure, which defines multiple binding surfaces for diverse interacting proteins. Not surprisingly, many WD40 proteins primarily function as adaptor or scaffolding proteins to orchestrate the cellular localization and formation of signaling complexes (25).
Using G␤1 as bait in a yeast two-hybrid screen, several WD40 repeat proteins were unexpectedly identified as binding partners of G␤␥ (24,28). The G␤-interacting regions identified in this screen all reside in the WD40 region of these proteins, suggesting that the WD40 motif itself may mediate the interactions with G␤␥. We have previously characterized one of these interacting proteins, RACK1, and found that RACK1 binds to a side surface of G␤␥ that overlaps with the binding sites of select G␤␥ effectors, such as PI3K␥ and PLC␤ 2 (29 -31). Consequently, binding of RACK1 to G␤␥ leads to selective inhibition of G␤␥ signaling and impaired leukocyte migration (31). These findings indicate that RACK1 functions as a negative regulator that may confer signaling specificity of G␤␥.
WDR26 is another WD40-containing protein that was identified as a binding partner of G␤␥ in the yeast two-hybrid screen (28). It is ubiquitously expressed and has been shown to protect neuronal cells from hydrogen peroxide-induced cell death (32,33). Overexpression of the C-terminal fragment of WDR26 inhibited MEKK1-mediated transcription activities in COS7 cells (33), but promoted proliferation of a rat cardiomyoblast cell line (34). However, the molecular mechanisms underlying the activity of WDR26 are unknown. A recent proteomic analysis has identified WDR26 as one of many WD40 proteins associated with the cullin 4 (CUL4)-DNA damage-binding protein 1 (DDB1) ubiquitin E3 ligase complex, but whether WDR26 is involved in ubiquitin-mediated protein degradation has not yet been investigated (35).
In this study, we show that WDR26 binds free G␤␥ both in vitro and in intact cells. Inhibition of WDR26 by siRNAs impaired G␤␥-mediated signal transduction and leukocyte migration in vitro and in vivo. By overexpressing a WDR26 fragment to perturb the G␤␥/WDR26 interaction, we further show that WDR26 mediates G␤␥ signaling and leukocyte migration by acting on G␤␥. Taken together, our data establish for the first time that WDR26 functions as a novel binding partner of G␤␥ that promotes G␤␥-dependent signal transduction and leukocyte migration.
Transfection-Transient transfection of HEK293 cells was performed using Polyjet DNA in vitro transfection reagent (Sig-Nagen). Transient transfection of Jurkat T and dHL 60 cells was achieved by using the Neon transfection system (Invitrogen) according to the manufacturer's protocol. For transfection of Jurkat T cells, 1 ϫ 10 6 and 1 ϫ 10 7 cells were used for 10 and 100 l of electroporation tips, using the electroporation parameters, 1325 voltage/10 ms/4 pulse and 1350 voltage/10 ms/4 pulse, respectively. Transfection of dHL60 cells (1 ϫ 10 6 ) was performed 5 days post-differentiation using 10 l of electroporation tips and the electroporation parameters 1500 voltage/25 ms/1 pulse. Up to 80 -90 and 90 -100% transfection efficiency for plasmids and oligonucleotides, respectively, could be obtained as judged by the percentage of GFP-or fluorescencepositive cells 1-day post-transfection of plasmids encoding enhanced green fluorescent protein or FITC-labeled oligonucleotides. Cells were harvested for assays 48 -62 h post-transfection.
Jurkat T cells stably expressing FLAG-WDR26 were generated after transduction with lentiviruses encoding the gene and selection with increasing concentrations of puromycin (0.2-10 g/ml) for 4 -6 weeks. Surviving cells were pooled and maintained in 1 g/ml of puromysin.
Generation of Lentiviruses and Baculoviruses-Lentiviruses encoding FLAG-WDR26 were generated by transfecting HEK293FT cells with the pLenti-CMV-DEST vectors encoding FLAG-WDR26 together with the packaging vectors pMDL, pRSV, and pVSV using the Polyjet DNA in vitro transfection reagent (SignaGen) (36). The supernatant of culture media containing lentivirus was collected on days 2 and 3 post-transfection. Lentivirus was concentrated by ultracentrifugation (47,000 ϫ g for 2 h) and resuspended in 0.2 ml of DMEM. Baculoviruses encoding FLAG-WDR26 were generated by using the Bac-To-Bac baculovirus expression system (Invitrogen) as described (30,37).
Immunoprecipitation and Western Blotting Analysis-To coimmunoprecipitate FLAG-G␤1␥2 with WDR26 and its mutants following their expression in HEK293 cells, cell lysates were prepared using modified RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 1 mM EDTA) and protease inhibitors. FLAG-G␤1␥2 was then immunoprecipitated using anti-FLAG M2 magnetic beads (Sigma). Protein complexes were resolved by SDS-PAGE and analyzed by Western blotting analysis.
To co-immunoprecipitate FLAG-WDR26 with endogenous G␤␥, Jurkat T cells were first serum-starved for 4 -6 h and then stimulated with SDF1␣ for the indicated times. FLAG-WDR26 was immunoprecipitated from cell lysates and analyzed for forming a complex with G␤␥ as described above.
Expression of various proteins was examined by Western blotting analysis using an Odyssey infrared imaging system (LI-COR Biosciences). To determine SDF1␣ (50 nM) and OKT3mediated phosphorylation of ERK1/2 and AKT, Jurkat T cells were serum-starved for 4 -6 h before stimulation. Flow Cytometry-The expression level of CXCR4 in siRNA-treated Jurkat T cells was determined by labeling with the phycoerythrin-conjugated mouse anti-CXCR4 antibody (R & D Systems, Inc.), followed by flow cytometry analysis. Briefly, 48 -62 h post-transfection, cells were washed with saline and then incubated with control IgG or phycoerythrin-conjugated mouse anti-human CXCR4 antibody (5 g/ml) for 45 min at 4°C. After washing with saline 3 times, cells were analyzed by flow cytometry (BD FACSCalibur).
Immunofluorescence Staining-To induce Jurkat T cell polarization, Jurkat T cells were suspended in serum-free RPMI containing 0.1% BSA and stimulated with control or SDF1␣conjugated sulfate latex beads for 5 min at 37°C as described previously (31,41). Polarization of dHL60 cells was performed by first placing cells onto fibronectin (100 g/ml)-coated coverslips for 10 min at 37°C. After unbound cells were washed away, the coverslips were assembled in a Zigmond chamber (42). An fMLP concentration gradient was established by filling one side of the chamber with Hanks' balanced salt solution (Invitrogen) containing 10 mM HEPES (pH 7.2) and 1% gelatin, and another side with the same buffer containing 100 nM fMLP. Cells were incubated at 37°C for 15 min before fixation. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.5% Triton X-100 for 5 min. Cells were stained with rabbit anti-WDR26 (1:1000) or anti-G␤ (1:400) at room temperature for 1 h, followed by incubation with the secondary antibody Alexa 488-conjugated anti-rabbit IgG and Alexa 568-conjugated phalloidin (1:200). Slides were visualized with a LSM510 Meta inverted confocal microscope (Carl Zeiss, Jena, Germany) with an argon/krypton laser and a Plan Apo 40 ϫ 1.3 NA oil immersion lens (31). Images were acquired with LSM5 Image software (Carl Zeiss) and processed with Adobe Photoshop (San Jose, CA).
Cell Migration Assay-Transwell migration of Jurkat T cells was determined using a quantitative approach as described previously (29,31). To measure chemotaxis of dHL60 cells in the Dunn chamber by time-lapse microscopy, cells were first seeded onto a coverslip pre-coated with 100 g/ml of fibronectin for 10 -20 min at 37°C. The coverslip was then assembled with the Dunn chamber (43). The chemoattractant gradient was established by filling the inner well with modified Hanks' balanced salt solution (Invitrogen) containing 1% human serum albumin and the outer well with the same solution containing 100 nM fMLP. Images were taken every 30 s for 40 min at room temperature using an inverted microscope (Leica DMI6000B) equipped with a motorized stage. A Leica ϫ10, NA 0.3 Fluor DIC objective was used for imaging. Cell trajectories were tracked using Metamorph software (Molecular Devices, Sunnyvale, CA). To exclude undifferentiated cells or immobile cells resulting from deleterious effects of transient transfection, only cells moving in any directions for at least 10 m in 30 min were included in the analysis. The migration speed was calculated as the total distance of cell movement divided by time, whereas the chemotaxis index was calculated as net distance of migration toward the direction of the fMLP source divided by the total distance moved (44). The directional change was calculated based on the average of each angle formed by the line drawn through the centroid of the cell parallel to the direction of the fMLP gradient and the line from the starting point of the cell to the next ending point for the whole period of cell migration (44).
Cell Homing in Vivo-Jurkat T cells treated with PTX or transiently transfected with a control or WDR26 siRNA were labeled with the fluorescence dye, carboxyfluorescein succinimidyl ester (Invitrogen). To normalize variations between different assays, untreated Jurkat T cells were labeled with 5-(and -6)-(((4chloromethyl)benzoyl)amino)tetramethylrhodamine (Invitrogen), and mixed with an equal number of carboxyfluorescein succinimidyl ester-labeled cells (1 ϫ 10 7 ) in 200 l of saline and injected intravenously into the tail vein of a severe combined immunodeficiency (SCID) mouse (45,46), based on an Institutional Animal Care and Use Committee-approved protocol at the University of Iowa. 3 h post-injection, cells were isolated from the bone marrow and spleen of the mouse and analyzed by flow cytometry (BD FACSCalibur) to identify the number of fluorescence-labeled cells that home to these tissues (45,46). Data are expressed as the ratio of carboxyfluorescein succinimidyl esterlabeled cells to 5-(and -6)-(((4-chloromethyl)benzoyl)amino)tetramethylrhodamine-labeled untreated cells.
Measurement of Cytosolic Ca 2ϩ Concentration-The cytosolic concentration of Ca 2ϩ ([Ca 2ϩ ] i ) in Jurkat T and dHL60 cells was measured as previously described (36). Briefly, cells were serum-starved for 4 -6 h and loaded with 4 M Fura 2/AM at room temperature for 40 min. The basal and agonist-stimulated changes in [Ca 2ϩ ] i were monitored at dual excitation wavelengths 340 and 380 nm and single emission wavelength 510 nm using a Biotek synergy 4 microplate reader (36). At the end of experiment, cells were first permeabilized with 2.5% Triton X-100 and then treated with 20 mM EGTA to obtain the fluorescent signal in the presence and absence of saturating amounts of Ca 2ϩ . The cytosolic free Ca 2ϩ concentration was calculated as described (47).
Measurement of Cyclic AMP-To measure SDF1␣-mediated inhibition of cAMP production in Jurkat T cells, cells (1.5 ϫ 10 6 ) were preincubated with 250 M 3-isobutyl-1-methylxanthine for 20 min and then SDF1␣ (50 nM) for 10 min before stimulation with 10 M forskolin for 20 min (29). cAMP levels were determined using the cAMP immunoassay system (Cell Biolabs, Inc.) according to the manufacturer's instructions.
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).

RESULTS
WDR26 Binds Free G␤␥-To verify the result from the yeast two-hybrid screen showing that WDR26 binds G␤1␥2, we per-formed in vitro binding assays using FLAG-tagged full-length WDR26 immunoprecipitated from Sf9 cells. As shown in Fig.  1A, WDR26 binds G␤1␥2 in a dose-dependent manner, but did not bind GDP-bound G␣ i . Moreover, WDR26 binding to G␤1␥2 was blocked by the presence of GDP-G␣ i , suggesting that WDR26 interacts with free G␤␥ but not G␤␥ in a heterotrimeric complex with G␣ (Fig. 1A). To more accurately estimate the binding affinity, we used a fluorescence-based approach to monitor the binding of MBP-conjugated WDR26 to G␤1␥2 labeled with an environmentally sensitive fluorescent probe, M8. As shown in Fig. 1B, the addition of MBP-WDR26 to M8-labeled G␤1␥2 dose-dependently enhanced the intensity of fluorescence, presumably due to the increased hydrophobicity around the M8 fluorescent probe on G␤␥ upon WDR26 binding. MBP alone had little effect. Based on this assay, the binding affinity for the WDR26/G␤1␥2 interaction was estimated to be about 1 M, which is comparable with that of G␤1␥2 with other known binding partners including RACK1 (ϳ0.5 M) and PLC␤ 2 (ϳ1 M).
Database searches of conserved protein domains revealed that in addition to the WD40 repeats, WDR26 also contains a Lis-homology (LisH) and C-terminal to LisH (CTLH) domains at its N-terminal segment (Fig. 1C). To identify the binding sites  1-661)) or the indicated WDR26 deletion mutants. Cell lysates were immunoprecipitated (IP) with an anti-FLAG antibody. The presence of proteins in the immunoprecipitates (pellets) and lysates was blotted (IB) for FLAG and RFP. Arrows indicate RFP-tagged WDR26 and its mutants. Schematic of the WDR26 structure and its mutants is shown in the right panel. DECEMBER 23, 2011 • VOLUME 286 • NUMBER 51 of G␤␥ on WDR26, we constructed a series of deletion mutants of WDR26 conjugated with RFP and then expressed them together with FLAG-G␤1␥2 in HEK293 cells (Fig. 1C). Co-immunoprecipitation assays indicate that the G␤␥-binding sites on WDR26 are located at the C terminus of WDR26 consisting of not only the WD40 domain but also the LisH-CTLH domain (Fig. 1C).

WDR26 Promotes G␤␥ Signaling and Leukocyte Migration
WDR26 Interacts with Endogenous G␤␥ in Leukocytes-To test whether WDR26 interacts with endogenous G␤␥ in cells, we stably expressed FLAG-tagged WDR26 in Jurkat T cells, because the commercially available antibodies for WDR26 and G␤ are not effective for immunoprecipitation. Co-immunoprecipitation analyses indicate that WDR26 had little association with G␤␥ in unstimulated cells (Fig. 2, A and B). However, upon stimulation of Jurkat T cells with SDF1␣, which activates the endogenous receptor CXCR4, WDR26 formed a complex with G␤␥ within 5 min (Fig. 2, A and B). The association of WDR26 with G␤␥ was decreased after prolonged stimulation, and was abolished by pre-treatment with PTX, indicating that G i/o proteins are involved. G␣ i/o and RACK1 were not detected in the WDR26 precipitates from either stimulated or unstimulated cells ( Fig. 2A), indicating that WDR26 selectively interacts with free G␤␥ released from the activated heterotrimeric G proteins.
We then determined the cellular localization of WDR26 and G␤␥. Jurkat T cells treated with control beads were rounded and displayed little F-actin staining. In these cells, WDR26 was localized in the cytosol, whereas G␤␥ was primarily distributed in the plasma membrane (Fig. 3, A and B). Stimulation with SDF1␣-conjugated beads induced cell polarization and F-actin formation around the beads. In these cells, a substantial amount of G␤␥ and WDR26 was found to be located at the plasma membrane where F-actin formed (Fig. 3, A and B), suggesting that SDF1␣ may induce WDR26 relocation and colocalization with G␤␥ at the membrane. The translocation of WDR26 from the cytosol to the membrane was more evident in differentiated HL60 cells, a human neutrophil-like cell line that exhibits robust polarization in response to fMLP stimulation (supplemental Fig. S1). The polarized dHL60 cells displayed a well defined leading and trailing edge. In contrast to its uniform distribution in the cytosol of unstimulated cells, WDR26 was primarily detected at the leading edge of the polarized dHL60 cells where F-actin was located (supplemental Fig. S1). As we observed previously (31), G␤␥ was distributed across the cell membrane of the polarized cells with a slightly increased accumulation at the leading edge (data not shown). These findings suggest that interaction with G␤␥ is not solely responsible for membrane translocation of WDR26. However, pretreatment of dHL60 cells with PTX to block G␤␥ activation inhibited both fMLP-stimulated dHL60 cell polarization and WDR26 translocation (supplemental Fig. S1), suggesting that G␤␥ signaling may be required for WDR26 translocation.
WDR26 Is Required for Chemotaxis of Leukocytes-Chemoattractants such as SDF1␣ and fMLP transduce chemotactic signals for leukocyte migration primarily via G␤␥ released from the activated G i/o proteins (3). Given that WDR26 specifically associates with G␤␥, we asked whether WDR26 plays a role in regulating G␤␥-mediated chemotaxis. We first evaluated the effect of WDR26 inhibition on Jurkat T cell migration. Transient transfection of siRNAs targeting two distinct regions of WDR26 led to specific inhibition of WDR26 expression, without affecting the expression of G␤␥ or other proteins including the receptor CXCR4, PLC␤ 2 , PI3K␥, and RACK1 (Fig. 4, A-C). Down-regulation of WDR26 impaired the transwell migration of Jurkat T cells induced by SDF1␣, but had no effect on the random migration of cells in the absence of SDF1␣ stimulation, suggesting that WDR26 specifically affects directional cell migration (Fig. 4D). In contrast, inhibition of CUL4, the ubiquitin ligase that binds WDR26 (35), did not affect cell migration (Fig. 4D), suggesting that the inhibitory effect of WDR26 suppression on chemotaxis is unlikely due to its impaired association with CUL4, and subsequent effects on ubiquitin-mediated protein degradation.
To determine whether WDR26 regulates leukocyte migration via its binding to G␤␥, we transiently expressed in Jurkat T cells an RFP-tagged C-terminal fragment of WDR26 (WDR(123-661)) (Fig. 4E), which contains the G␤␥-binding sites (Fig. 1C) and presumably competes with the full-length WDR26 for G␤␥ binding. As a control, we also expressed RFP and an RFP-tagged N-terminal fragment of WDR26 (WDR(1-122)) ( Fig. 4E) that did not bind G␤␥ (Fig. 1C). Notably, as compared with cells expressing RFP, Jurkat T cells expressing WDR(123-661) but not WDR(1-122) exhibited a reduced response to SDF1␣-stimulated chemotaxis (Fig. 4F), supporting the role of WDR26 interaction with G␤␥ in mediating cell migration. To further establish the functional significance of WDR26 in leukocyte trafficking in vivo, we adopted an in vivo homing assay in scid mice (45,46). In this assay, Jurkat T cells were fluorescence-labeled and then injected into the mice by tail vein. The extent of cells homing to the lymphoid tissues, including spleen and bone marrow, was then evaluated 3 h post-injection. Previous work indicates that the homing of leukocytes to the lymphoid tissues relies on the activation of chemokine receptors expressed in leukocytes (45,46). Consistent with this observation, pretreatment of cells with PTX to uncouple G i/o proteins from their cognate receptors led to impaired Jurkat T cell homing (Fig. 4G). Down-regulation of WDR26 had the similar inhibitory effect as PTX on the homing response (Fig. 4G). Together, these data indicate that WDR26 plays a critical role in chemotaxis of Jurkat T cell both in vitro and in vivo.
To determine whether WDR26 also plays a key role in the migratory response of other types of leukocytes, we extended our studies to dHL60 cells, which exhibit a robust chemotaxis response to fMLP stimulation, therefore enabling the migratory process to be monitored by the time-lapse microscopy. The chemotaxis of dHL60 cells was induced by a shallow and linear gradient of fMLP established in a Dunn chamber. As shown in Fig. 5, A-C, and supplemental Videos 1-2, although the control siRNA-treated cells preferentially migrated up the fMLP gradient in relatively straight paths, the WDR26-deficient cells moved in random directions with zigzag paths and took more turns in a short period. Supporting this, analysis of chemotaxis parameters indicates that as compared with the control cells, WDR26-deficient cells exhibited a decrease in migration speed and chemotaxis index (a measurement of how straight the cells move toward the higher concentration of the chemoattractant gradient), and an increase in the degrees of directional changes during the course of migration along the chemoattractant gradient (Fig. 5D). These findings indicate that inhibition of WDR26 impaired both the migratory ability and the directional sensing of dHL60 cells. Because fMLP-induced HL60 migration is also dependent on G␤␥-mediated signaling (48), these findings indicate that WDR26 plays a critical role in regulating G␤␥-dependent leukocyte migration.
WDR26 Controls Ability of RACK1 to Bind G␤␥ and Inhibit Leukocyte Migration-We showed previously that RACK1 binds G␤␥ and negatively regulates G␤␥-mediated leukocyte migration (31). Given that WDR26 and RACK1 are both expressed in leukocytes, and inhibition of WDR26 and RACK1 had opposite effects on leukocyte migration, it raises the possibility that WDR26 serves to counter-regulate the ability of RACK1 to bind G␤␥ and inhibit G␤␥-mediated leukocyte migration. To test this, we determined the effect of WDR26 suppression on RACK1 binding to G␤␥. As we reported previously (31), co-immunoprecipitation studies show that the association of RACK1 to G␤␥ was significantly increased after stimulation of Jurkat T cells with SDF1␣ for 30 min (Fig. 6, A and B). This is in contrast to WDR26, whose binding to G␤␥ became evident within 5 min of SDF1␣ stimulation, and decreased after 30 min of SDF1␣ stimulation (Fig. 2, A and B). Moreover, significantly less G␤␥ was co-immunoprecipitated with RACK1. This is probably due to the fact that the anti-RACK1 antibody immunoprecipitated only about 50% of total RACK1 from the lysates (data not shown), and/or a smaller fraction of RACK1 binding to G␤␥. Notably, inhibition of WDR26 abolished the association of RACK1 with G␤␥ induced by SDF1␣ stimulation (Fig. 6, A and B), suggesting that WDR26 controls RACK1  DECEMBER 23, 2011 • VOLUME 286 • NUMBER 51 interaction with G␤␥, rather than simply competes with RACK1 for G␤␥ binding. To determine whether WDR26 functions upstream of RACK1 to regulate its ability to inhibit G␤␥mediated leukocyte migration, we evaluated the effect of suppressing WDR26 and RACK1 either alone or simultaneously on Jurkat T cell migration. Although silencing either RACK1 or WDR26 alone led to a similar degree of change in Jurkat T cell migration, simultaneous suppression of RACK1 and WDR26 did not restore normal cell migration. Cells with deficiency in both WDR26 and RACK1 exhibited a similar degree of decrease in migration as the WDR26-deficient cells (Fig. 6C), indicating that WDR26 is essential for leukocyte migration. Collectively, these data suggest that WDR26 likely functions upstream of RACK1 in regulating G␤␥-mediated leukocyte migration.

WDR26 Promotes G␤␥ Signaling and Leukocyte Migration
WDR26 Promotes G␤␥-dependent Signal Transduction-G␤␥ activates a diverse set of downstream effectors, including PLC␤ and PI3K to regulate leukocyte migration (3). To determine whether WDR26 contributes to leukocyte migration by modulating G␤␥ signaling, we evaluated the effect of WDR26 inhibition on SDF1␣-stimulated Ca 2ϩ signaling, and AKT and ERK1/2 phosphorylation in Jurkat T cells, which are known to be mediated by G␤␥ (8, 10, 31). Suppressing WDR26 significantly reduced SDF1␣-stimulated Ca 2ϩ signaling and AKT phosphorylation, but had little effects on ERK1/2 phosphorylation (Fig. 7, A-D), although the activation of these signaling pathways was equally sensitive to PTX-mediated inhibition (data not shown), suggesting the involvement of G␤␥. Similar results were found in dHL60 cells transfected with WDR26 siRNAs (supplemental Fig. S2). The effect of WDR26 inhibition is specific to G␤␥ signaling, because it affected neither G␣ imediated inhibition of cAMP accumulation nor the increase in Ca 2ϩ signaling stimulated by OKT3 through the T-cell receptor (Fig. 7, A and E). Together, these findings indicate that WDR26 promotes the activation of selective signaling pathways mediated by G␤␥ in leukocytes.

DISCUSSION
In this study, we have demonstrated that WDR26 serves as a novel binding protein of G␤␥ that promotes G␤␥-dependent signal transduction and leukocyte migration. Data from both in vitro and in vivo binding assays support the specific binding of WDR26 to free G␤␥ but not G␤␥ in the heterotrimers or to G␣ subunits. Such interaction likely involves subcellular relocation of WDR26 because in quiescent cells, WDR26 primarily localizes to the cytosol, whereas G␤␥ is located in the plasma membrane. As demonstrated by confocal microscopic and co-immunoprecipitation studies, activation of G␤␥ leads to WDR26 translocation from the cytosol to the plasma membrane and also an enhanced interaction between G␤␥ and WDR26. Interestingly, in the polarized human neutrophil-like dHL60 cells, WDR26 accumulates primarily at the leading edge of cells, whereas G␤␥ is distributed only in a shallow gradient from the leading edge to the trailing edge (31). These findings suggest that the recruitment of WDR26 to the leading edge of the polarized cells is unlikely to be simply anchored through G␤␥. Rather, it may also involve G␤␥-dependent signaling because blocking G␤␥ activation with PTX inhibits WDR26 translocation.
The functional importance of WDR26 interaction with G␤␥ is demonstrated by the findings that suppressing WDR26 impaired SDF1␣-induced Jurkat T cell migration in vitro and also the homing of these cells to lymphoid tissues in vivo. Moreover, the chemotactic response of dHL60 cells to a shallow gradient of fMLP was significantly suppressed by WDR26 inhibition. SDF1␣ and fMLP transmit chemotactic signals through distinct GPCRs, CXCR4 and formyl peptide receptors, but both receptors primarily couple to PTX-sensitive G i/o proteins and signal via G␤␥. Given that WDR26 interacts with G␤␥, these findings suggest that WDR26 likely regulates leukocyte migration by acting on G␤␥ rather than on receptors.  DECEMBER 23, 2011 • VOLUME 286 • NUMBER 51 G␤␥ transmits chemotactic signals for leukocyte migration through several downstream effectors, including PI3K␥ and PLC␤ 2/3 (3,49). Our data indicate that WDR26 regulates leukocyte migration by selectively promoting G␤␥-dependent signal transduction. This is evident from the data showing that down-regulation of WDR26 alleviated SDF1␣or fMLP-stimulated Ca 2ϩ signaling and AKT phosphorylation in Jurkat T cells or dHL60 cells. Moreover, suppressing WDR26 affects neither TCR-stimulated Ca 2ϩ signaling nor a G␣ i/o -mediated decrease in cAMP production or ERK1/2 activation, indicating that WDR26 is required for the activation of specific G␤␥ effectors. The ability of WDR26 to regulate G␤␥ signaling and leukocyte migration likely depends on its binding to G␤␥, because Jurkat T cell migration is inhibited by the WDR26 deletion mutant (WDR(123-661)) that binds G␤␥, but not by the mutant (WDR(1-122)) that fails to bind G␤␥. In addition to PI3K and PLC␤, it has been shown that G␤␥ may activate other signaling pathways including those mediated by PLA 2 and p38 MAPKs to regulate leukocyte migration (50 -54). It remains to be determined whether WDR26 is also required for the activation of these pathways by G␤␥. Moreover, it will be interesting to determine whether WDR26 also links G␤␥ to other unknown pathways that control leukocyte migration. In this sense, WDR26 may function as an effector rather than simply a regulator of G␤␥.

WDR26 Promotes G␤␥ Signaling and Leukocyte Migration
It is well established that PI3K␥ and PLC␤ 2/3 can be activated by G␤␥ via protein-protein interactions in vitro (4), but how they are activated by G␤␥ in cells and how the activation is regulated remain to be resolved. We showed previously that G␤␥-mediated PI3K␥ and PLC␤ activation is negatively regulated by RACK1, which competes with PI3K␥ and PLC␤ for binding G␤␥ (31). Given that the function of WDR26 in regulating G␤␥ is opposite to that of RACK1, WDR26 may simply counter-regulate the activity of RACK1 in G␤␥ signaling. However, we found that suppressing WDR26 did not increase RACK1 binding to G␤␥, but, rather, abolished RACK1 binding to G␤␥. Moreover, down-regulation of RACK1 could not rescue the migratory defect of Jurkat T cells with deficiency in WDR26, indicating that WDR26 functions upstream of RACK1. These findings suggest a possible scenario that, in response to chemoattractants, WDR26 and RACK1 may alternately interact with G␤␥ to fine-tune G␤␥ signaling for leukocyte migration. In this scenario, chemoattractants stimulate the binding of WDR26 to G␤␥ to promote G␤␥mediated signal transduction for chemotaxis. The increased G␤␥ signaling may in turn result in the recruitment of RACK1 to bind FIGURE 6. WDR26 controls the ability of RACK1 to bind G␤␥ and inhibit chemotaxis. A and B, Jurkat T cells transiently transfected with a control (siCT) or WDR26 siRNA (siWDR26) were stimulated with SDF1␣ (50 nM) for the indicated time. Cell lysates were immunoprecipitated (IP) with mouse IgG control or a mouse anti-RACK1 antibody. The presence of G␤␥, WDR26, and RACK1 in the precipitates (pellets) and lysates (2.5% of total) was detected with specific antibodies. Representative blots are shown in A, and quantitative data from three to four different experiments are shown in B. The amount of G␤␥ binding to RACK1 is expressed as a percentage of total G␤␥ in the lysates, with the background binding to mouse IgG substracted. C, chemotaxis was determined in Jurkat T cells transiently transfected with a control siRNA (siCT), or siRNAs against WDR26 (siWDR26) or RACK1 (siRACK1) either individually or in combination (siRϩsiW) (n ϭ 3). Chemotaxis was induced by SDF1␣ (0, 1, and 10 nM) for 3 h. * and #, p Ͻ 0.05 indicate significance versus siCT and siRACK1, respectively. G␤␥, which possibly functions in a negative feedback loop to decrease or terminate G␤␥-mediated signal transduction. Further studies to uncover the molecular basis for the regulation of WDR26 and RACK1 interaction with G␤␥ will be important for revealing the mechanisms by which WDR26 and RACK1 are involved in the precise regulation of G␤␥ signaling for leukocyte migration.
WDR26 was recently identified as one of several WD40 proteins associated with the CUL4-DDB 1 ubiquitin E3 ligase complex (35). These WD40 proteins have been proposed to act as adaptors that direct the CUL4-DDB1 ubiquitin ligase to specific substrates, thereby regulating ubiquitin-dependent proteolysis (55,56). It is conceivable that WDR26 may regulate PI3K␥ and PLC␤ activation by controlling the expression level of a critical regulator through CUL4-DDB1 ubiquitin ligase. However, our data indicate that this is unlikely because inhibition of WDR26 does not affect the expression level of numerous proteins we examined, ranging from the receptor to effectors and regulators. Moreover, silencing CUL4 could not recapitulate the inhibitory effect of WDR26 suppression on Jurkat T cell che-motaxis, suggesting that the activity of CUL4 is not involved in leukocyte chemotaxis.
WDR26 contains a conserved WD40 repeat domain that is present in a large family of proteins confined primarily to eukaryotic organisms (25,26). The WD40 domain in these proteins has no intrinsic enzymatic activity but often functions as an adaptor/ scaffold to orchestrate protein-protein interactions for the regulation of the specificity, efficacy, and localization of multiple signaling pathways. Given that the WD40 domain of WDR26 is involved in binding G␤␥, it will be interesting to determine in future studies whether WDR26 utilizes the WD40 domain as a scaffold to form a macroprotein complex with G␤␥ and its effectors, thereby promoting G␤␥-mediated signal transduction.
Our data demonstrate that WDR26 plays a critical role in regulating G␤␥ signaling and leukocyte migration. In addition to regulating leukocyte function, G␤␥ controls a variety of physiological processes, including heart rate and neuronal excitability (4). Excessive G␤␥ signaling has been implicated in diverse pathological conditions, such as heart failure (57, 58), inflammation (59), and tumor cell growth/metastasis (36, 60, 61). Given that WDR26 is ubiquitously expressed (33), future studies will be important to determine whether WDR26 contributes to the regulation of G␤␥ signaling in other normal cellular processes and if its dysregulation contributes to aberrant G␤␥ signaling and disease.