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

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... linemia 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 (11-13) . 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.