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Phosphatidylinositol 3-Kinase-dependent Membrane Association of the Bruton's Tyrosine Kinase Pleckstrin Homology Domain Visualized in Single Living Cells

1999; Elsevier BV; Volume: 274; Issue: 16 Linguagem: Inglês

10.1074/jbc.274.16.10983

1083-351X

Péter Várnai, Kristina I. Rother, Tamás Balla,

Platelet Disorders and Treatments

Phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3) has been proposed to act as a second messenger to recruit regulatory proteins to the plasma membrane via their pleckstrin homology (PH) domains. The PH domain of Bruton's tyrosine kinase (Btk), which is mutated in the human disease X-linked agammaglobulinemia, has been shown to interact with PI(3,4,5)P3 in vitro. In this study, a fusion protein containing the PH domain of Btk and the enhanced green fluorescent protein (BtkPH-GFP) was constructed and utilized to study the ability of this PH domain to interact with membrane inositol phospholipids inside living cells. The localization of expressed BtkPH-GFP in quiescent NIH 3T3 cells was indistinguishable from that of GFP alone, both being cytosolic as assessed by confocal microscopy. In NIH 3T3 cells coexpressing BtkPH-GFP and the epidermal growth factor receptor, activation of epidermal growth factor or endogenous platelet-derived growth factor receptors caused a rapid (<3 min) translocation of the cytosolic fluorescence to ruffle-like membrane structures. This response was not observed in cells expressing GFP only and was completely inhibited by treatment with the PI 3-kinase inhibitors wortmannin and LY 292004. Membrane-targeted PI 3-kinase also caused membrane localization of BtkPH-GFP that was slowly reversed by wortmannin. When the R28C mutation of the Btk PH domain, which causes X-linked agammaglobulinemia, was introduced into the fluorescent construct, no translocation was observed after stimulation. In contrast, the E41K mutation, which confers transforming activity to native Btk, caused significant membrane localization of BtkPH-GFP with characteristics indicating its possible binding to PI(4,5)P2. This mutant, but not wild-type BtkPH-GFP, interfered with agonist-induced PI(4,5)P2 hydrolysis in COS-7 cells. These results show in intact cells that the PH domain of Btk binds selectively to 3-phosphorylated lipids after activation of PI 3-kinase enzymes and that losing such binding ability or specificity results in gross abnormalities in the function of the enzyme. Therefore, the interaction with PI(3,4,5)P3 is likely to be an important determinant of the physiological regulation of Btk and can be utilized to visualize the dynamics and spatiotemporal organization of changes in this phospholipid in living cells. Phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3) has been proposed to act as a second messenger to recruit regulatory proteins to the plasma membrane via their pleckstrin homology (PH) domains. The PH domain of Bruton's tyrosine kinase (Btk), which is mutated in the human disease X-linked agammaglobulinemia, has been shown to interact with PI(3,4,5)P3 in vitro. In this study, a fusion protein containing the PH domain of Btk and the enhanced green fluorescent protein (BtkPH-GFP) was constructed and utilized to study the ability of this PH domain to interact with membrane inositol phospholipids inside living cells. The localization of expressed BtkPH-GFP in quiescent NIH 3T3 cells was indistinguishable from that of GFP alone, both being cytosolic as assessed by confocal microscopy. In NIH 3T3 cells coexpressing BtkPH-GFP and the epidermal growth factor receptor, activation of epidermal growth factor or endogenous platelet-derived growth factor receptors caused a rapid (<3 min) translocation of the cytosolic fluorescence to ruffle-like membrane structures. This response was not observed in cells expressing GFP only and was completely inhibited by treatment with the PI 3-kinase inhibitors wortmannin and LY 292004. Membrane-targeted PI 3-kinase also caused membrane localization of BtkPH-GFP that was slowly reversed by wortmannin. When the R28C mutation of the Btk PH domain, which causes X-linked agammaglobulinemia, was introduced into the fluorescent construct, no translocation was observed after stimulation. In contrast, the E41K mutation, which confers transforming activity to native Btk, caused significant membrane localization of BtkPH-GFP with characteristics indicating its possible binding to PI(4,5)P2. This mutant, but not wild-type BtkPH-GFP, interfered with agonist-induced PI(4,5)P2 hydrolysis in COS-7 cells. These results show in intact cells that the PH domain of Btk binds selectively to 3-phosphorylated lipids after activation of PI 3-kinase enzymes and that losing such binding ability or specificity results in gross abnormalities in the function of the enzyme. Therefore, the interaction with PI(3,4,5)P3 is likely to be an important determinant of the physiological regulation of Btk and can be utilized to visualize the dynamics and spatiotemporal organization of changes in this phospholipid in living cells. Phosphoinositides are important precursor molecules that generate multiple second messengers in stimulated cells. Phospholipase C (PLC) 1The abbreviations used are: PLC, phospholipase C; PI, phosphatidylinositol; PH, pleckstrin homology; Btk, Bruton's tyrosine kinase; GFP, enhanced green fluorescent protein; EGF, epidermal growth factor; PDGF, platelet-derived growth factor; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid; PKC, protein kinase C; GST, glutathione S-transferase.1The abbreviations used are: PLC, phospholipase C; PI, phosphatidylinositol; PH, pleckstrin homology; Btk, Bruton's tyrosine kinase; GFP, enhanced green fluorescent protein; EGF, epidermal growth factor; PDGF, platelet-derived growth factor; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid; PKC, protein kinase C; GST, glutathione S-transferase.-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) yields the water-soluble messenger molecule inositol 1,4,5-trisphosphate, which mobilizes intracellular Ca2+, and the hydrophobic moiety diacylglycerol, which activates protein kinase C isozymes (1Berridge M.J. Irvine R.F. Nature. 1984; 312: 315-321Crossref PubMed Scopus (4218) Google Scholar,2Nishizuka Y. Nature. 1984; 308: 693-698Crossref PubMed Scopus (5735) Google Scholar). In addition, PI(4,5)P2 can be phosphorylated by PI 3-kinase(s) to form phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3), a lipid product found only in stimulated cells (3Jackson T.R. Stephens L.R. Hawkins P.T. J. Biol. Chem. 1992; 267: 16627-16636Abstract Full Text PDF PubMed Google Scholar, 4Franke T.F. Kaplan D.R. Cantley L.C. Toker A. Science. 1997; 275: 665-668Crossref PubMed Scopus (1290) Google Scholar). The multiplicity and divergent regulation of PI 3-kinases by receptor and non-receptor tyrosine kinases as well as by GTP-binding proteins (5Toker A. Cantley L.C. Nature. 1997; 387: 673-676Crossref PubMed Scopus (1216) Google Scholar) and the resistance of PI(3,4,5)P3 to hydrolysis by any known phospholipase C have led to the proposal that 3-phosphorylated inositides, in particular PI(3,4,5)P3, serve important regulatory functions (5Toker A. Cantley L.C. Nature. 1997; 387: 673-676Crossref PubMed Scopus (1216) Google Scholar). The pleckstrin homology (PH) domains of several regulatory proteins have been shown to bind PI(3,4,5)P3 in vitro(6Salim K. Bottomley M.J. Querfurth E. Zvelebil M.J. Gout I. Scaife R. Margolis R.L. Gigg R. Smith C.I.E. Driscoll P.C. Waterfield M.D. Panayotou G. EMBO J. 1996; 15: 6241-6250Crossref PubMed Scopus (487) Google Scholar, 7Chen R.-H. Corbalan-Garcia S. Bar-Sagi D. EMBO J. 1997; 16: 1351-1359Crossref PubMed Scopus (114) Google Scholar, 8Rameh L.E. Arvidsson A. Carraway III, K.L. Couvillon A.D. Rathbun G. Crompton A. VanRenterghem B. Czech M.P. Ravichandran K.S. Burakoff S.J. Wang D.S. Chen C.-S. Cantley L.C. J. Biol. Chem. 1997; 272: 22059-22066Crossref PubMed Scopus (423) Google Scholar, 9Klarlund J.K. Guilherme A. Holik J.J. Virbasius J.V. Chawla A. Czech M.P. Science. 1997; 275: 1927-1930Crossref PubMed Scopus (368) Google Scholar, 10Kavran J.M. Klein D.E. Lee A. Falasca M. Isakoff S.J. Skolnik E.Y. Lemmon M.A. J. Biol. Chem. 1998; 273: 30497-30508Abstract Full Text Full Text PDF PubMed Scopus (375) Google Scholar). One of these proteins is Bruton's tyrosine kinase (Btk), a member of the Tec family of non-receptor tyrosine kinases (11Rawlings D.J. Witte O.N. Semin. Immunol. 1995; 7: 237-246Crossref PubMed Scopus (113) Google Scholar), mutations of which are associated with the human disease X-linked agammaglobulinemia and its murine equivalent, X-linked immunodeficiency (12Tsukada S. Saffran D.C. Rawlings D.J. Parolini O. Allen R.C. Klisak I. Sparkes R.S. Kubagawa H. Mohandas T. Quan S. Cell. 1993; 72: 279-290Abstract Full Text PDF PubMed Scopus (1124) Google Scholar, 13Rawlings D.J. Saffran D.C. Tsukada S. Largaespada D.A. Grimaldi J.C. Cohen L. Mohr R.N. Bazan J.F. Howard M. Copeland N.G. Jenkins N.A. Witte O.N. Science. 1993; 261: 358-361Crossref PubMed Scopus (770) Google Scholar). Although Btk also contains a protein kinase as well as SH2 and SH3 domains, its PH domain alone has been shown to be sufficient to bind PI(3,4,5)P3 selectively in vitro (6Salim K. Bottomley M.J. Querfurth E. Zvelebil M.J. Gout I. Scaife R. Margolis R.L. Gigg R. Smith C.I.E. Driscoll P.C. Waterfield M.D. Panayotou G. EMBO J. 1996; 15: 6241-6250Crossref PubMed Scopus (487) Google Scholar, 8Rameh L.E. Arvidsson A. Carraway III, K.L. Couvillon A.D. Rathbun G. Crompton A. VanRenterghem B. Czech M.P. Ravichandran K.S. Burakoff S.J. Wang D.S. Chen C.-S. Cantley L.C. J. Biol. Chem. 1997; 272: 22059-22066Crossref PubMed Scopus (423) Google Scholar). Many of the Btk mutations that cause the B-cell defect that leads to X-linked agammaglobulinemia in humans (14Vihinen M. Iwata T. Kinnon C. Kwan S.P. Ochs H.D. Vorechovsky I. Smith C.I. Nucleic Acids Res. 1996; 24: 160-165Crossref PubMed Scopus (30) Google Scholar) are within the Btk PH domain of the protein, and one of these, the R28C substitution, is responsible for X-linked immunodeficiency in mice (13Rawlings D.J. Saffran D.C. Tsukada S. Largaespada D.A. Grimaldi J.C. Cohen L. Mohr R.N. Bazan J.F. Howard M. Copeland N.G. Jenkins N.A. Witte O.N. Science. 1993; 261: 358-361Crossref PubMed Scopus (770) Google Scholar). The latter mutation has also been shown to abolish the binding of Btk to inositol lipidsin vitro (6Salim K. Bottomley M.J. Querfurth E. Zvelebil M.J. Gout I. Scaife R. Margolis R.L. Gigg R. Smith C.I.E. Driscoll P.C. Waterfield M.D. Panayotou G. EMBO J. 1996; 15: 6241-6250Crossref PubMed Scopus (487) Google Scholar, 8Rameh L.E. Arvidsson A. Carraway III, K.L. Couvillon A.D. Rathbun G. Crompton A. VanRenterghem B. Czech M.P. Ravichandran K.S. Burakoff S.J. Wang D.S. Chen C.-S. Cantley L.C. J. Biol. Chem. 1997; 272: 22059-22066Crossref PubMed Scopus (423) Google Scholar). In addition, a transforming mutant of Btk (Btk*, E41K) has been reported to show increased membrane association, which further indicates that PH domain-mediated binding of Btk to cell membrane(s) is critical for its activation (15Li T. Tsukada S. Satterthwaite A. Havlik M.H. Park H. Takatsu K. Witte O.N. Immunity. 1995; 2: 451-460Abstract Full Text PDF PubMed Scopus (151) Google Scholar). This study was designed to investigate whether the isolated PH domain of Btk is sufficient to interact with membrane phosphoinositides within intact living cells with similar specificity to that described in vitro and whether this interaction can localize the protein to the membrane without additional binding motifs. Expression of the Btk PH domain fused to the enhanced green fluorescent protein (BtkPH-GFP) has demonstrated that PI 3-kinase activation recruits these molecules to the plasma membrane, suggesting that they specifically recognize 3-phosphorylated inositol lipids without binding to PI(4,5)P2. This methodology also allows visualization of dynamic changes in 3-phosphorylated inositides in single living cells. myo-[3H]Inositol (80 Ci/mmol) was purchased from Amersham Pharmacia Biotech. EGF (recombinant human) and PDGF-AB (recombinant human) were obtained from Life Technologies, Inc. Ionomycin, LY 294002, bisindolylmaleimide I, and BAPTA were purchased from Calbiochem, and wortmannin was a gift from Kyowa Hakko Laboratories (Tokyo, Japan). All other chemicals were of high pressure liquid chromatography or analytical grade. The PH domains of PLCδ1(amino acids 1–170) and of Bruton's tyrosine kinase (amino acids 1–177) were amplified with the Advantage Klentaq polymerase mixture (CLONTECH) from human cDNAs (Marathon cDNA from brain and K562 leukemia cells, CLONTECH) with the following primer pairs: PLCδ, 5′-GGCATGGACTCGGGCCGGGACTTCCTG-3′ and 5′-AAGATCTTCCGGGCATAGCTGTCG-3′; Btk, 5′-CCAAGTCCTGGCATCTCAATGCATCTG-3′ and 5′-TGGAGACTGGTGCTGCTGCTGGCTC-3′. The amplified products were subcloned into the pGEM-Easy T/A cloning vector (Promega) and sequenced with dideoxy sequencing (Thermosequenase, Amersham Pharmacia Biotech). A second amplification reaction was performed from these plasmids with nested primers that contained restriction sites for appropriate cloning into the pEGFP-N1 plasmid (CLONTECH) to preserve the reading frame. Plasmids were transfected into COS-7 or NIH 3T3 cells and analyzed by SDS-polyacrylamide gel electrophoresis followed by Western blotting for the presence of the GFP fusion constructs using a polyclonal antibody against GFP (CLONTECH). Mutations were created with the QuickChangeTM mutagenesis kit (Stratagene). All mutations were confirmed by dideoxy sequencing, and the expression of the protein was confirmed by Western blotting. Cells were plated onto poly-l-lysine-coated 30-mm diameter circular glass coverslips at a density of 5 × 104 cells/dish and cultured for 3 days before transfection with plasmid DNAs (1 μg/ml) using the LipofectAMINE reagent (10 μg/ml; Life Technologies, Inc.) and Opti-MEM (Life Technologies, Inc.). Forty-eight 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, and 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 of prewarmed buffer after removing 0.5 ml of medium from the cells. Cells were examined in an inverted microscope under a 40× oil immersion objective (Nikon Inc.) and a Bio-Rad laser confocal microscope system (MRC-1024) with Lasersharp acquisition software (Bio-Rad). Inositol phosphates were analyzed in COS-7 cells transfected with the AT1a angiotensin II receptor together with selected PH domain-GFP fusion constructs after labeling withmyo-[3H]inositol for 24 h as described previously (16Hunyady L. Baukal A.J. Balla T. Catt K.J. J. Biol. Chem. 1994; 269: 24798-24804Abstract Full Text PDF PubMed Google Scholar, 17Balla T. Sim S.S. Baukal A.J. Rhee S.G. Catt K.J. Mol. Biol. Cell. 1994; 5: 17-27Crossref PubMed Scopus (32) Google Scholar). 3H-Labeled inositol phosphates were analyzed with Dowex minicolumns. To follow the localization of isolated PH domains within intact cells, they were fused to GFP and expressed in NIH 3T3 cells. As shown in Fig. 1 A (left panel), expressed GFP (without any attached sequence) was found to be cytosolic and was also present in the nucleus. Addition of the Btk PH domain (amino acids 1–177) to the N terminus of GFP had no effect on its localization in quiescent cells (after serum deprivation) and was indistinguishable from GFP alone (Fig. 1 A, center panel). In contrast, fusion of the PLCδ PH domain to GFP caused prominent membrane localization of the construct due to its interaction with membrane PI(4,5)P2 (18Stauffer T.P. Ahn S. Meyer T. Curr. Biol. 1998; 8: 343-346Abstract Full Text Full Text PDF PubMed Google Scholar) (Fig. 1 A,right panel). These results suggest that, unlike the PH domain of PLCδ, the PH domain of Btk does not possess high enough affinity to interact with PI(4,5)P2 so that it would be sufficient for its membrane targeting. Next we examined whether stimulation of cells with growth factors that are known to activate PI 3-kinases and to increase formation of PI(3,4,5)P3 causes any change in the distribution of the BtkPH-GFP protein. NIH 3T3 cells were transfected with the BtkPH-GFP construct alone or together with the cDNA encoding the human EGF receptor and were stimulated either with PDGF (25–100 ng/ml) (to activate the endogenous PDGF receptor) or with EGF (100 ng/ml) after serum starvation. As shown in Figs. 1 B and2, stimulation of either receptor caused a translocation of the cytosolic fluorescence to the plasma membrane. The decrease in cytosolic fluorescence was most obvious when compared with the bright nuclear signal that did not change in intensity following stimulation in the NIH 3T3 cells. This was in contrast with our previous finding in the same cell type, where the nuclear intensity of the PLCδ PH domain-GFP fusion protein slowly followed the cytoplasmic changes, although with a slight delay (19Várnai P. Balla T. J. Cell Biol. 1998; 143: 501-510Crossref PubMed Scopus (657) Google Scholar). The redistribution of fluorescence was clearly demonstrated by comparing line intensity histograms calculated at selected "cross-sections" of the cell. Dividing the fluorescence intensity of the plasma membrane (I pm) by that of the cytosol (I cyt) yields a ratio that can be used as an index of membrane localization (Fig. 1 B, right panels). To determine whether a similar translocation of BtkPH-GFP takes place in response to stimulation of other receptors that activate PI 3-kinases, NIH 3T3 cells were also stimulated with EGF (via expressed EGF receptors). EGF stimulation also evoked a translocation response, and we could not detect a notable difference in the localization of the fluorescent signal whether PDGF or EGF was used as the agonist (Fig. 2,A and B). To examine the effects of insulin, another known activator of PI 3-kinases, immortalized hepatocytes (20Rother K.I. Imai Y. Caruso M. Beguinot F. Formisano P. Accili D. J. Biol. Chem. 1998; 273: 17491-17497Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar) were transfected with the BtkPH-GFP construct and subjected to a 6–12-h serum deprivation. Addition of insulin (100 nm) to such cells also stimulated the translocation of the fluorescent signal to the plasma membrane. Interestingly, in this case, localization of the construct to intracellular vesicular structures that moved toward the plasma membrane was also observed. It is also noteworthy that, in the hepatocytes, the nuclear signal intensity decreased in parallel with the insulin-induced membrane localization (Fig.2 C). To determine whether the Btk PH domain binds to PI(3,4,5)P3, the lipid product of PI 3-kinase, we used the PI 3-kinase inhibitors wortmannin and LY 294002 to prevent formation of the lipid in growth factor-stimulated cells. Addition of wortmannin or LY 294002 to cells after EGF or PDGF stimulation caused dissociation of BtkPH-GFP from the plasma membrane (Fig. 3, A andC). This finding suggested that the Btk PH domain localizes to the membrane by a mechanism that requires the sustained activity of PI 3-kinase(s) and supports the notion that PI(3,4,5)P3 is the membrane component to which the PH domain of Btk binds in living cells. These results also show that PI(3,4,5)P3 is actively dephosphorylated during stimulation since, after blocking its increased synthesis, its level rapidly decreases as indicated by the dissociation of BtkPH-GFP from the plasma membrane. The finding that the maintenance of PI(3,4,5)P3 levels requires the sustained phosphorylation of PI(4,5)P2 by PI 3-kinases prompted us to investigate whether decreasing the level of PI(4,5)P2 has an indirect effect on the localization of the Btk PH domain. Addition of ionomycin (which elevates intracellular [Ca2+] and thereby induces hydrolysis of PI(4,5)P2) to EGF-stimulated cells caused the rapid release of the fluorescence from the membranes back to the cytosol (data not shown). Since PI(3,4,5)P3 is not hydrolyzed directly by any known PLC, these findings are consistent with the sensitivity of PI(3,4,5)P3 levels both to PI 3-kinase activity and to changes in the level of its substrate, PI(4,5)P2. Next we tested whether cellular production of 3-phosphorylated inositides without receptor stimulation is sufficient to cause the translocation of the Btk PH domain construct to the plasma membrane. For this, we cotransfected NIH 3T3 cells with a membrane-targeted form of PI 3-kinase γ (PI3Kγ-CAAX) (21Bondeva T. Pirola L. Bulgarelli-Leva G. Rubio I. Wetzker R. Wymann M.P. Science. 1998; 282: 293-296Crossref PubMed Scopus (299) Google Scholar) and BtkPH-GFP. Expression of this PI 3-kinase construct has been shown to cause production of PI(3,4,5)P3 and a high level of activation of the Akt protein kinase in COS-7 cells (21Bondeva T. Pirola L. Bulgarelli-Leva G. Rubio I. Wetzker R. Wymann M.P. Science. 1998; 282: 293-296Crossref PubMed Scopus (299) Google Scholar). As shown in Fig. 4 A, this manipulation yielded cells in which the fluorescent construct was localized to the membranes, but unlike after hormonal stimulation, this localization was not confined to the plasma membrane. In many cells, there were intracellular bright spots that, in some cases, formed "aggregates" with high fluorescent intensities (data not shown). Moreover, very little nuclear localization of fluorescence was observed in these cells. Addition of wortmannin (300 nm) caused the release of fluorescence from the plasma membrane to the cytosol, but it required a significantly longer time than in cases of acute stimulation by agonists. Also, the spotty intracellular signal appeared to be more resistant to wortmannin treatment. The C1 region of various protein kinase C (PKC) isozymes has been shown to interact with the Btk PH domain based on in vitro binding assay (22Yao L. Suzuki H. Ozawa K. Deng J. Lehel C. Fukamachi H. Anderson W.B. Kawakami Y. Kawakami T. J. Biol. Chem. 1997; 272: 13033-13039Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). This interaction was found to be inhibited by agents that bind either to the C1 domain of PKC (such as phorbol esters) or to the Btk PH domain (such as PI(4,5)P2). Moreover, recently, the PI 3-kinase-dependent kinase PDK-1 has been shown to phosphorylate and activate PKCζ (23Chou M.M. Hou W. Johnson J. Graham L.K. Lee M.H. Chen C.-S. Newton A.C. Schaffhausen B.S. Toker A. Curr. Biol. 1998; 8: 1069-1077Abstract Full Text Full Text PDF PubMed Google Scholar). Therefore, we examined the effect of phorbol esters and PKC inhibitors on the membrane translocation response of the Btk PH domain. Stimulation of NIH 3T3 cells with phorbol esters (12-O-tetradecanoylphorbol-13-acetate, 100 nm to 1 μm) had no effect on the distribution of BtkPH-GFP (Fig. 4 B). Moreover, stimulation with EGF (or PDGF) induced translocation of BtkPH-GFP in cells that either were pretreated with the PKC inhibitor bisindolylmaleimide I (Gö 6850, up to 500 nm, 10 min) (Fig. 4 B) or were treated with 100 nm phorbol 12-myristate 13-acetate for 12 h before PDGF stimulation (data not shown). Similarly, addition of the PKC inhibitor to PDGF-stimulated cells (after Btk PH domain translocation to the membrane had taken place) had no significant effect on the distribution of fluorescence, whereas subsequent addition of wortmannin rapidly released the fluorescence from the membranes (data not shown). (The PKC inhibitor applied at this concentration reversed the effects of phorbol 12-myristate 13-acetate on a variety of cell responses in our laboratory.) Although these data cannot rule out PKC being a regulator of the holoprotein Btk, they did not suggest an important role of PKC in the PI 3-kinase-mediated membrane targeting of the Btk PH domain. Several mutations of Btk that are responsible for X-linked agammaglobulinemia are located within the PH domain of the protein and impair its membrane association (11Rawlings D.J. Witte O.N. Semin. Immunol. 1995; 7: 237-246Crossref PubMed Scopus (113) Google Scholar). Therefore, we examined the ability of the best characterized mutant, R28C (which is unable to bind PI(3,4,5)P3 in vitro (6Salim K. Bottomley M.J. Querfurth E. Zvelebil M.J. Gout I. Scaife R. Margolis R.L. Gigg R. Smith C.I.E. Driscoll P.C. Waterfield M.D. Panayotou G. EMBO J. 1996; 15: 6241-6250Crossref PubMed Scopus (487) Google Scholar, 8Rameh L.E. Arvidsson A. Carraway III, K.L. Couvillon A.D. Rathbun G. Crompton A. VanRenterghem B. Czech M.P. Ravichandran K.S. Burakoff S.J. Wang D.S. Chen C.-S. Cantley L.C. J. Biol. Chem. 1997; 272: 22059-22066Crossref PubMed Scopus (423) Google Scholar)), to localize to the membrane in living cells in response to stimulation. As shown in Fig. 3 (B and C), no membrane localization of the R28C mutant of BtkPH-GFP was observed after PDGF or EGF stimulation in NIH 3T3 cells (or in COS-7 cells; data not shown). The E41K mutation, which causes transformation in NIH 3T3 cells and shows enhanced membrane association (15Li T. Tsukada S. Satterthwaite A. Havlik M.H. Park H. Takatsu K. Witte O.N. Immunity. 1995; 2: 451-460Abstract Full Text PDF PubMed Scopus (151) Google Scholar), was also introduced into the BtkPH-GFP construct, and its localization within intact cells was examined. As shown in Fig. 5 A, the E41K mutant displayed significant membrane localization even in quiescent NIH 3T3 cells and still displayed translocation in response to EGF (100 ng/ml). Application of wortmannin (300 nm) reversed the EGF-induced increase in membrane association, but did not reduce it below its initial level. Wortmannin also failed to affect the basal localization of the construct when added to cells that were not stimulated with EGF (data not shown). In view of the ability of the E41K mutant to associate with membranes of unstimulated cells, which contain only small amounts (if any) of PI(3,4,5)P3, we investigated the possibility that this mutant BtkPH-GFP has a diminished binding specificity for PI(3,4,5)P3 and hence is also able to bind to PI(4,5)P2 that is present in membranes of unstimulated cells. As shown in Fig. 5 A (panels d–h), PI(4,5)P2 breakdown evoked by the addition of the Ca2+ ionophore ionomycin caused a complete release of the membrane-bound fluorescence into the cytosol, whereas subsequent chelation of Ca2+ by BAPTA caused a reappearance of the signal at the plasma membrane. The effect of ionomycin was inhibited by preincubation with 10 mm neomycin (to inhibit PLC; data not shown). The relocalization of fluorescence after Ca2+chelation was not prevented by 300 nm wortmannin, but was abolished by the addition of 100 μm quercetin, which inhibits PI(4,5)P2 resynthesis (19Várnai P. Balla T. J. Cell Biol. 1998; 143: 501-510Crossref PubMed Scopus (657) Google Scholar) (Fig. 5 B). These changes were very similar to those observed with the PLCδ PH domain-GFP construct (which binds to PI(4,5)P2 (18Stauffer T.P. Ahn S. Meyer T. Curr. Biol. 1998; 8: 343-346Abstract Full Text Full Text PDF PubMed Google Scholar, 19Várnai P. Balla T. J. Cell Biol. 1998; 143: 501-510Crossref PubMed Scopus (657) Google Scholar,24Ferguson K.M. Lemmon M.A. Schlessinger J. Sigler P.B. Cell. 1995; 83: 1037-1046Abstract Full Text PDF PubMed Scopus (522) Google Scholar)) and are consistent with the assumption that the E41K mutation impairs the ability of the Btk PH domain to discriminate between PI(3,4,5)P3 and PI(4,5)P2 effectively. To further investigate whether the E41K mutant Btk PH domain is able to bind to membrane PI(4,5)P2within the intact cell, we examined the ability of this construct to interfere with agonist-induced PI(4,5)P2 hydrolysis. As shown earlier for the PLCδ PH domain (25Rhee S.G. Bae Y.S. J. Biol. Chem. 1997; 272: 15045-15048Crossref PubMed Scopus (808) Google Scholar) and for the pleckstrin PH domain (26Abrams C.S. Wu H. Zhao W. Belmonte E. White D. Brass L.F. J. Biol. Chem. 1995; 270: 14485-14492Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar) (both of which bind to PI(4,5)P2), expression of these molecules inhibited receptor-mediated inositol phosphate production, presumably by interfering with PLC-mediated hydrolysis of PI(4,5)P2. As shown in Fig.6, neither the wild-type Btk PH domain nor the R28C mutant had a significant effect on angiotensin II-stimulated inositol phosphate production in COS-7 cells expressing the AT1a receptor together with the GFP construct of interest. In contrast, E41K mutant BtkPH-GFP showed a significant inhibitory effect, although this inhibition was not as strong as that caused by expression of PLCδ PH domain-GFP (Fig. 6). The important role of the Btk PH domain was originally recognized after identifying and analyzing mutations that cause X-linked agammaglobulinemia in humans (14Vihinen M. Iwata T. Kinnon C. Kwan S.P. Ochs H.D. Vorechovsky I. Smith C.I. Nucleic Acids Res. 1996; 24: 160-165Crossref PubMed Scopus (30) Google Scholar). Several (although not all) of these mutations are within the PH domain of Btk. Some mutations cause a folding defect, and others affect its function, presumably interfering with the binding characteristics of this module (27Hyvönen M. Saraste M. EMBO J. 1997; 16: 3396-3404Crossref PubMed Scopus (189) Google Scholar). The ability of Btk to interact with membranes appears to be the key regulatory element in determining the function(s) of the kinase, and several lines of evidence suggest that the PH domain is a critical region of the molecule for membrane association (27Hyvönen M. Saraste M. EMBO J. 1997; 16: 3396-3404Crossref PubMed Scopus (189) Google Scholar). Although βγ-subunits of heterotrimeric G-proteins (28Tsukada S. Simon M. Witte O. Katz A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11256-11260Crossref PubMed Scopus (236) Google Scholar) and various PKC isozymes (29Yao L. Kawakami Y. Kawakami T. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9175-9179Crossref PubMed Scopus (307) Google Scholar)