G Protein βγ Subunits Act on the Catalytic Domain to Stimulate Bruton's Agammaglobulinemia Tyrosine Kinase

G proteins are critical cellular signal transducers for a variety of cell surface receptors. Both α and βγ subunits of G proteins are able to transduce receptor signals. Several direct effect molecules for Gβγ subunits have been reported; yet the biochemical mechanism by which Gβγ executes its modulatory role is not well understood. We have shown that Gβγ could directly increase the kinase activity of Bruton's tyrosine kinase (Btk) whose defects are responsible for X chromosome-linked agammaglobulinemia in patients. The well characterized interaction of Gβγ with the PH (pleckstrin homology)/TH (Tec-homology) module of Btk was proposed to be the underlying activation mechanism. Here we show that Gβγ also interacts with the catalytic domain of Btk leading to increased kinase activity. Furthermore, we showed that the PH/TH module is required for Gβγ-induced membrane translocation of Btk. The membrane anchorage is also dependent on the interaction of Btk with phosphatidylinositol 3,4,5-trisphosphate, the product of phosphoinositide 3-kinase. These data support a dual role for Gβγ in the activation of Btk signaling function, namely membrane translocation and direct regulation of Btk catalytic activity.

G proteins are critical cellular signal transducers for a variety of cell surface receptors. Both ␣ and ␤␥ subunits of G proteins are able to transduce receptor signals. Several direct effect molecules for G␤␥ subunits have been reported; yet the biochemical mechanism by which G␤␥ executes its modulatory role is not well understood. We have shown that G␤␥ could directly increase the kinase activity of Bruton's tyrosine kinase (Btk) whose defects are responsible for X chromosomelinked agammaglobulinemia in patients. The well characterized interaction of G␤␥ with the PH (pleckstrin homology)/TH (Tec-homology) module of Btk was proposed to be the underlying activation mechanism. Here we show that G␤␥ also interacts with the catalytic domain of Btk leading to increased kinase activity. Furthermore, we showed that the PH/TH module is required for G␤␥-induced membrane translocation of Btk. The membrane anchorage is also dependent on the interaction of Btk with phosphatidylinositol 3,4,5-trisphosphate, the product of phosphoinositide 3-kinase. These data support a dual role for G␤␥ in the activation of Btk signaling function, namely membrane translocation and direct regulation of Btk catalytic activity.
Heterotrimeric (␣␤␥) GTP-binding regulatory proteins (G proteins) transduce signals from cell surface receptors across the membrane to the inside of cells (1). G proteins pass these extracellular signals to downstream effector molecules by directly interacting with these effectors. Both ␣ and ␤␥ subunits are able to interact with downstream effectors and actively participate in signal transduction (2). G␤␥ subunits have been demonstrated to interact with several proteins in the yeast mating pathway (3), G protein-gated potassium channels (4), certain isotypes of adenylyl cyclases (in the presence of G␣ s ) (5), certain isotypes of phospholipase C-␤ (6 -8), G protein-coupled receptor kinases (9), phosphoinositide 3-kinase-␥ (10), and Btk 1 (Bruton's tyrosine kinase) (11). However, the biochemical mechanism by which G␤␥ activates these effectors is not well understood.
We have previously shown that G␤␥ could increase the kinase activity of Btk-family tyrosine kinases (11). Btk kinase was the first tyrosine kinase shown to be directly regulated by G proteins (11)(12)(13). The Btk family tyrosine kinases include Btk/Atk, Tec, Itk/Tsk, and Bmx/Etk (14). Defects in Btk are responsible for X chromosome-linked agammaglobulinemia in humans and X chromosome-linked immunodeficiency in mouse. G␤␥ subunits were shown to bind directly to the PH (pleckstrin homology) domain and its adjacent BM (Btk motif) domain (within the Tec-homology (TH) domain) of Btk (15,16). This interaction was assumed to be responsible for the G␤␥ activation of Btk. Here we show that G␤␥ can stimulate a purified recombinant Btk lacking the PH/TH module as well as full-length Btk, demonstrating that the effect of G␤␥ on Btk kinase activity is actually mediated by an alternate domain. In vitro binding studies further show that G␤␥ can bind to both the PH/TH module and the catalytic domain, providing an activation mechanism for Btk. In addition, G␤␥ could stimulate the membrane translocation of full-length Btk, but not Btk⌬PHTH. The membrane anchorage is also dependent on Btk interaction with phosphatidylinositol 3,4,5-trisphosphate since a Btk mutant defective in interaction with phosphatidylinositol 3,4,5-trisphosphate was not able to be translocated to the membrane. These data support a model whereby G␤␥ can regulate Btk signaling by increasing its membrane localization and directly stimulating its catalytic activity.

EXPERIMENTAL PROCEDURES
Protein Purification-Sf9 cells were coinfected with baculoviruses containing cDNAs for G␤ 1 and G␥ 2 with a His 6 tag (17). Cells were harvested after 72 h and resuspended in buffer I (40 mM Tris, pH 8.0, 300 mM NaCl, 10 mM MgCl 2 , 10 mM 2-mercaptoethanol). Cells were lysed by sonication and Genapol was added to 0.1%. After addition of AlF 4 Ϫ , the lysates were gently agitated for 1 h at 4°C. Ni-NTA beads were added and incubated for 4 h. After extensive washes in buffer I, G␤␥ was eluted three times in buffer I with 500 mM imidazole. After concentration and desalting into storage buffer (40 mM Tris, pH 8.0, 50 mM NaCl, 10 mM 2-mercaptoethanol, 10 mM MgCl 2 , 0.1% Genapol), G␤␥ was flash frozen at Ϫ80°C.
In Vitro Binding Assay-GST pull-down assay was done as previously described (18). Two micrograms each of G␤␥ and GST-Btk fusion proteins were combined as indicated in the figures in 300 l of binding buffer (40 mM Tris, pH 8.0, 50 mM NaCl, 10 mM 2-mercaptoethanol, 10 mM MgCl 2 ) and incubated at 4°C for 2 h. Complexes were washed extensively in buffer with 100 mM NaCl and run on SDS-PAGE. The gel was transferred and Western blotted with antibody against G␤ (Santa Cruz Biotechnology).
Immunocomplex Kinase Assay-Btk immunocomplex kinase assay was done as described (19). Whole-cell extract of HEK-293 cells trans-* This work is supported by grants from the National Institutes of Health, the American Cancer Society, and the Dorothy Rodbell Cohen Foundation for Sarcoma Research. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
fected with Btk-GFP (green fluorescent protein) or myr-Btk⌬PHTH-GFP (the first 15 amino acid residues of c-Src was fused to the Nterminal of Btk⌬PHTH-GFP) with or without G␤␥, were pre-cleared with 20 l of protein A agarose beads, and Btk was immunoprecipitated with 1 g of polyclonal anti-GFP antibody (Santa Cruz Biotechnology). After washing three times with IP buffer (150 mM NaCl, 10 mM Tris, pH 7.4, 1 mM EDTA, 0.1% SDS, 1% sodium deoxycholate, 1% Triton X-100, 0.5% Nonidet P-40) and three times with kinase buffer (50 mM Hepes, pH 7.4, 10 mM MnCl 2 ), 5 g of peptide substrate and 10 Ci of [␥-32 P]ATP were added, and the mixture was incubated at 30°C for 30 min. After SDS/PAGE, the gel was autoradiographed and quantified. Fold of stimulation by G␤␥ was normalized by the expressed Btk, and mutant proteins detected by Western blot with anti-Btk antibody.

G␤␥ Stimulates the Catalytic Activity of Both Btk and
Btk⌬PHTH-We set out to study the biochemical mechanism by which G␤␥ activates Btk. We previously showed that G␤␥ subunits could stimulate Btk-family kinase activity (11). Also, there were two reports of the direct interaction of G␤␥ with the PH domain and a portion of the adjacent sequences of the TH domain of Btk (15,16). In light of these binding data, we investigated whether this interaction was responsible for stimulation of the kinase activity of Btk by G␤␥. Previously, utilizing purified recombinant Btk kinases, we have shown that deletion of the PH/TH module has little effect on the intrinsic catalytic activity of Btk on peptide substrates (21). This PH/TH module does, however, mediate protein substrate recognition, as purified Btk lacking this domain (Btk⌬PHTH) was unable to phosphorylate larger protein substrates but was able to phosphorylate peptide substrates (21).
If G␤␥ binding to the PH/TH module of Btk was responsible for the increased kinase activity of Btk, this model would predict that G␤␥ should increase the kinase activity of full-length Btk but not the PHTH-truncated Btk (Btk⌬PHTH). As shown in Fig. 1, we found that G␤␥ could increase the kinase activity of both Btk and Btk⌬PHTH. Purified Btk (lanes 1-6) or Btk⌬PHTH (lanes 8 -13) were assayed in the absence or presence of increasing concentrations of G␤␥ subunits. The kinase activity of Btk and Btk⌬PHTH was measured by the phosphorylation of a peptide substrate and autophosphorylation. G␤␥ increased both the phosphorylation of the peptide substrate as well as the autophosphorylation of both Btk and Btk⌬PHTH with similar kinetics. These data indicate that the interaction of G␤␥ with the PH/TH module is not essential for G␤␥ activation of Btk and that, in addition to the PH/TH module, G␤␥ must have other contact site(s) on Btk.
G␤␥ Directly Binds to Both the PH/TH Module and the Catalytic Domain of Btk-To identify the additional interacting site(s) of G␤␥ on Btk, we purified GST fusion proteins of different domains of Btk: GST-PHTH, GST-SH3SH2, and GST-CAT (for catalytic domain) ( Fig. 2A). Purified G␤␥ was incubated with these GST fusion proteins and GST alone (as a negative control). Bound G␤␥ was precipitated with glutathione-agarose beads and Western blotted with an anti-G␤ antibody (Fig. 2B). While GST and GST-SH3SH2 did not precipitate G␤, GST-PHTH and GST-CAT did (Fig. 2B). Similar experiments using cell extracts expressing G␤␥ as a source for G proteins yielded identical results (data not shown). These data demonstrate that G␤␥ has two contact sits on Btk: the PH/TH module and the catalytic domain. Since G␤␥ could increase the kinase activity of Btk⌬PHTH, the interaction with the catalytic domain is likely responsible for the G␤␥ activation of Btk.
G␤␥ Could Translocate Btk, but Not Btk⌬PHTH, to the Membrane-Btk is a cytoplasmic tyrosine kinase. It was proposed that membrane translocation is accompanied by activation (22)(23)(24)(25)(26). To investigate the activation mechanism further, we used fluorescence microscopy to analyze the subcellular distribution of Btk and Btk⌬PHTH after stimulation G␤␥ (Fig. 3). We fused the GFP to Btk and Btk⌬PHTH to monitor the distribution of these proteins in living cells. GFP alone was used as negative control; with or without serum, GFP is uniformly distributed throughout the cells (Fig. 3A). The PH/BM domain of Btk alone (PHBM-GFP) was used as positive control (20); addition of serum, which contains a variety of growth factors (some that act through G protein-coupled receptors), caused PHBM-GFP to be redistributed from cytoplasm to plasma membrane (Fig. 3B). While Btk-GFP was uniformly distributed throughout cells in the absence of serum, co-expression of G␤␥ or addition of serum led to predominantly plasma membrane localization (Fig. 3C). This indicates that after G␤␥ stimulation, Btk is indeed translocated from cytosol to plasma membrane. On the other hand, G␤␥ or serum had no effect on the subcellular distribution of Btk⌬PHTH-GFP, which is mainly in the cytosol (Fig. 3D). Thus, G␤␥ could translocate Btk, but not Btk⌬PHTH, to the plasma membrane.
G␤␥ Induced Btk Membrane Translocation in Cells through PI 3-Kinase-Since G␤␥ could not translocate Btk⌬PHTH to the membrane, the PH/TH module must be essential for this translocation event. Because the PH/TH module can bind to G␤␥ directly and G␤␥ is membrane-associated, it is possible that this direct interaction is responsible for Btk membrane anchorage. Also, the PH domain of Btk can bind phosphatidylinositol 3,4,5-trisphosphate (PIP 3 ), the product of phosphoinositide 3-kinase (PI 3-kinase) (27). To test whether the membrane anchorage of Btk-GFP after G␤␥ stimulation is due to the PH/TH module binding to G␤␥ or binding to PIP 3 , we investigated the response of Btk(R28C)-GFP after G␤␥ stimulation (Fig. 4). Mutation of arginine to cysteine at residue 28 within the PH domain reduces the binding to PIP 3 , but not to G␤␥ (16,28). This mutation also causes X chromosome-linked agammaglobulinemia in patients (29). As shown in Fig. 4A, co-expressing Btk(R28C)-GFP and G␤␥ did not lead to subcellular redistribution of Btk(R28C)-GFP, implying that the PIP 3 -PH interaction is essential for Btk membrane translocation. A prediction of this model would be that G␤␥-induced Btk membrane translocation is PI 3-kinase-dependent. To test this, we examined the Btk-GFP subcellular distribution after G␤␥ stimulation in the presence of a PI 3-kinase inhibitor, LY294002. As shown in Fig. 4B, treatment with LY294002 blocked G␤␥-induced Btk-GFP membrane translocation. A role for PI 3-kinase in Btk activation had been observed for other types of receptors (22)(23)(24)(25)(26). Therefore, G␤␥-induced Btk membrane translocation depends on PI 3-kinase activation by G␤␥ and the generation of PIP 3 .
To test whether constitutive membrane localization is sufficient to activate Btk, we examined the kinase activity of Btk-GFP and myr-Btk⌬PHTH-GFP, in which the myristoylation signal sequence from c-Src (amino acid residues 1-15) was fused to the N terminus of Btk⌬PHTH-GFP (Fig. 5A). The membrane localization of myr-Btk⌬PHTH-GFP was verified by GFP localization on the cell membrane (Fig. 5B). G␤␥ increased the kinase activity of Btk-GFP (by ϳ3to 4-fold) (Fig. 5C). Btk-GFP had been shown to have similar level of kinase activ-  3. Role of PHTH in mediating membrane anchoring. A, GFP alone was used as negative control; with or without serum, GFP is uniformly distributed throughout HEK-293 cells. B, the PH/BM domain of Btk alone (PHBM-GFP) was used as positive control; addition of serum caused PHBM-GFP to be redistributed from cytoplasm to plasma membrane. C, while Btk-GFP is uniformly distributed throughout cells in the absence of serum, addition of serum or co-expression of G␤␥ led to predominantly plasma membrane localization. D, serum or G␤␥ had no effect on the subcellular distribution of Btk⌬PHTH-GFP, which is mainly in the cytosol. Data are representative of three similar experiments. ity with Btk (26). More importantly, the basal kinase activity of myr-Btk⌬PHTH-GFP (in the absence of G␤␥) was higher than that of Btk-GFP (Fig. 5C). Co-expression of G␤␥ further increased the kinase activity of myr-Btk⌬PHTH-GFP moderately (by ϳ1.5-fold) (Fig. 5C). These data imply that constitutive membrane localization could partially activate Btk and that G␤␥ could further increase the kinase activity of membranelocalized Btk. DISCUSSION ⌻he data shown here reveal a possible activation mechanism by which G␤␥ regulates a tyrosine kinase. Here we show that G␤␥ can bind to not only the PH/TH module but also the catalytic domain of Btk. Given that G␤␥ could activate Btk⌬PHTH, it is unlikely that the interaction of G␤␥ with the PH/TH module transmits allosteric information to the catalytic domain, leading to increased kinase activity of full-length Btk. Rather, the direct contact of G␤␥ with the catalytic domain is responsible for the activation. Based on our data, we proposed an activation model in which G␤␥ plays a dual role in both membrane translocation and direct activation of Btk (Fig. 6). Activation of G protein-coupled receptors, such as chemokine receptors in lymphocytes, leads to the release of G␤␥ subunits from heterotrimeric G proteins. G␤␥ then directly or indirectly activates a PI 3-kinase, which produces PIP 3 . Btk is anchored to the membrane through binding to PIP 3 with its PH/TH module. G␤␥ then directly activates Btk by directly causing a conformational change in the catalytic domain of Btk, leading to increased kinase activity. Activated Btk could modulate various biological responses such as actin cytoskeletal reorganization, gene expression, calcium mobilization, apoptosis, and cell differentiation (14, 29 -31) (Fig. 6). Since addition of the myristoylation signal sequence of c-Src to the N terminus of Btk⌬PHTH increased myr-Btk⌬PHTH kinase activity in cells in the absence of G␤␥, constitutive membrane localization can partially activate Btk in cells.
The direct interaction of G␤␥ with the catalytic domain of Btk and the subsequent activation of Btk kinase activity might have general implications for the activation mechanism of other G␤␥ effectors by G␤␥. G␤␥ can interact with the Nterminal PH domain of PLC-␤2. It has recently been proposed that this binding might lead to the activation of PLC-␤2 (32). However, it was also reported that G␤␥ could interact with the catalytic core of PLC-␤2 (33). Although no direct biochemical data are available on a G␤␥ effect on a PH-deleted PLC-␤2, it is possible that, similar to Btk, the interaction with the catalytic core rather than the PH domain is responsible for the activation of PLC-␤2 by G␤␥. This model would be consistent with an observation that transfer of the PH domain of PLC-␤2 to PLC-␤1 did not confer the G␤␥ regulation on PLC-␤1 (34). Furthermore, G␤␥ has been shown to interact with the catalytic core of adenylyl cyclase type II (35,36). Moreover, the crystal structure of the p21-activated kinase shows that the G␤␥-binding motif within the catalytic domain of p21-activated kinase forms an ␣ helix, which packs against the inter-lobe hinge, preventing the small lobe from moving into its active state orientation (37). Although no data yet has shown direct stimulation of purified p21-activated kinase activity by purified G␤␥, this could provide a structural model for G␤␥ activation of protein kinases. Therefore, it could be a general mechanism that G␤␥ binds directly with the catalytic domain (or core) of its effector proteins, to induce conformational changes in the active site, leading to increased activity. FIG. 6. An activation model of Btk by G␤␥ in cells. G␤␥ plays dual roles: membrane translocation and direct activation of Btk. Activation of G protein-coupled receptors leads to the release of G␤␥ subunits from heterotrimeric G proteins. G␤␥ activates a PI 3-kinase, which produces PI3,4,5P 3 . Btk is anchored or translocated to the membrane through binding to PI3,4,5P 3 by its PH/TH module. G␤␥ then activates Btk by directly causing conformational changes of the catalytic domain, leading to increased kinase activity.