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

Open AccessPublished:April 16, 1999DOI:https://doi.org/10.1074/jbc.274.16.10983
      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)
      The 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 (
      • Berridge M.J.
      • Irvine R.F.
      ,
      • Nishizuka Y.
      ). 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 (
      • Jackson T.R.
      • Stephens L.R.
      • Hawkins P.T.
      ,
      • Franke T.F.
      • Kaplan D.R.
      • Cantley L.C.
      • Toker A.
      ). The multiplicity and divergent regulation of PI 3-kinases by receptor and non-receptor tyrosine kinases as well as by GTP-binding proteins (
      • Toker A.
      • Cantley L.C.
      ) 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 (
      • Toker A.
      • Cantley L.C.
      ).
      The pleckstrin homology (PH) domains of several regulatory proteins have been shown to bind PI(3,4,5)P3 in vitro(
      • Salim 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.
      ,
      • Chen R.-H.
      • Corbalan-Garcia S.
      • Bar-Sagi D.
      ,
      • Rameh 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.
      ,
      • Klarlund J.K.
      • Guilherme A.
      • Holik J.J.
      • Virbasius J.V.
      • Chawla A.
      • Czech M.P.
      ,
      • Kavran J.M.
      • Klein D.E.
      • Lee A.
      • Falasca M.
      • Isakoff S.J.
      • Skolnik E.Y.
      • Lemmon M.A.
      ). One of these proteins is Bruton's tyrosine kinase (Btk), a member of the Tec family of non-receptor tyrosine kinases (
      • Rawlings D.J.
      • Witte O.N.
      ), mutations of which are associated with the human disease X-linked agammaglobulinemia and its murine equivalent, X-linked immunodeficiency (
      • Tsukada S.
      • Saffran D.C.
      • Rawlings D.J.
      • Parolini O.
      • Allen R.C.
      • Klisak I.
      • Sparkes R.S.
      • Kubagawa H.
      • Mohandas T.
      • Quan S.
      ,
      • Rawlings 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.
      ). 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 (
      • Salim 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.
      ,
      • Rameh 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.
      ). Many of the Btk mutations that cause the B-cell defect that leads to X-linked agammaglobulinemia in humans (
      • Vihinen M.
      • Iwata T.
      • Kinnon C.
      • Kwan S.P.
      • Ochs H.D.
      • Vorechovsky I.
      • Smith C.I.
      ) are within the Btk PH domain of the protein, and one of these, the R28C substitution, is responsible for X-linked immunodeficiency in mice (
      • Rawlings 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.
      ). The latter mutation has also been shown to abolish the binding of Btk to inositol lipidsin vitro (
      • Salim 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.
      ,
      • Rameh 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.
      ). 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 (
      • Li T.
      • Tsukada S.
      • Satterthwaite A.
      • Havlik M.H.
      • Park H.
      • Takatsu K.
      • Witte O.N.
      ).
      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.

      DISCUSSION

      The important role of the Btk PH domain was originally recognized after identifying and analyzing mutations that cause X-linked agammaglobulinemia in humans (
      • Vihinen M.
      • Iwata T.
      • Kinnon C.
      • Kwan S.P.
      • Ochs H.D.
      • Vorechovsky I.
      • Smith C.I.
      ). 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 (
      • Hyvönen M.
      • Saraste M.
      ). 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 (
      • Hyvönen M.
      • Saraste M.
      ). Although βγ-subunits of heterotrimeric G-proteins (
      • Tsukada S.
      • Simon M.
      • Witte O.
      • Katz A.
      ) and various PKC isozymes (
      • Yao L.
      • Kawakami Y.
      • Kawakami T.
      ) have been shown to interact with the PH domain of Btk, more recent studies indicate that the 3-phosphorylated inositides, PI(3,4,5)P3 in particular, are its binding partners in the membrane. The isolated recombinant PH domain of Btk in the form of a GST fusion protein has been demonstrated to bind PI(3,4,5)P3 in a BIAcore assay system (
      • Salim 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.
      ) or by utilizing lipid micelles in vitro (
      • Rameh 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.
      ). Binding of the lipid to the PH domain of Btk was found to depend on the ionic composition (
      • Salim 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.
      ), and the fatty acid side chains of PI(3,4,5)P3 were also shown to be important for the interaction (
      • Rameh 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.
      ). In contrast, soluble inositol 1,3,4,5-tetrakisphosphate was found to bind to the Btk PH domain with high affinity in one report (
      • Fukuda M.
      • Kojima T.
      • Kabayama H.
      • Mikoshiba K.
      ). These in vitro studies also showed that the binding of the Btk PH domain to PI(4,5)P2 is much weaker, providing the specificity that would be required for its general regulation by 3-phosphorylated inositides. In all of these studies, the R28C mutant PH domain, which causes X-linked immunodeficiency in mice, was found to be unable to bind PI(3,4,5)P3.
      In this report, we provide evidence that the isolated PH domain of Btk fused to the fluorescent reporter molecule GFP exhibits agonist-dependent membrane association when expressed in a variety of cells. This method allowed analysis of the binding specificity as well as imaging of spatiotemporal changes in the membrane association of this protein module inside single living cells. Our results show that the Btk PH domain does not localize to membranes inside quiescent cells (suggesting that its binding to PI(4,5)P2 is probably too weak for such membrane targeting) and that the agonist-induced translocation of BtkPH-GFP to membranes is dependent on PI 3-kinase activation as well as on membrane PI(4,5)P2 levels. Moreover, the production of PI(3,4,5)P3 by membrane-targeted PI 3-kinase was sufficient to target the Btk PH domain to membranes. These results, together within vitro binding data (
      • Salim 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.
      ,
      • Rameh 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.
      ), further support the idea that the Btk PH domain binds to PI(3,4,5)P3 of the plasma membrane and that this mechanism is sufficient to regulate membrane localization of the protein.
      These results do not rule out the possibility that additional factors (proteins) participate in the membrane anchoring of the Btk PH domain, similarly to the protein anchors (RACK proteins) (
      • Mochly-Rosen D.
      • Gordon A.S.
      ) that stabilize the various motifs of PKC. Similarly, even though the Akt kinase has a PH domain that appears to be sufficient to localize this protein to 3-phosphorylated inositides (
      • Kontos C.D.
      • Stauffer T.P.
      • Yang W.P.
      • York J.D.
      • Huang L.
      • Blanar M.A.
      • Meyer T.
      • Peters K.G.
      ), its activation requires PDK-1, a kinase that also possesses a PH domain and that also is regulated by 3-phosphorylated lipids (
      • Stephens L.
      • Anderson K.
      • Stokoe D.
      • Erdjument-Bromage H.
      • Painter G.F.
      • Holmes A.B.
      • Gaffney P.R.J.
      • Reeses C.B.
      • McCormick F.
      • Tempst P.
      • Coadwell J.
      • Hawkins P.T.
      ). Although in vitro studies suggested that the C1 domain of various PKC isozymes can interact with the Btk PH domain (
      • Yao L.
      • Suzuki H.
      • Ozawa K.
      • Deng J.
      • Lehel C.
      • Fukamachi H.
      • Anderson W.B.
      • Kawakami Y.
      • Kawakami T.
      ), our studies in intact cell did not provide evidence for a role of PKC in the membrane targeting of the Btk PH domain. Based on the studies of Yao et al. (
      • Yao L.
      • Suzuki H.
      • Ozawa K.
      • Deng J.
      • Lehel C.
      • Fukamachi H.
      • Anderson W.B.
      • Kawakami Y.
      • Kawakami T.
      ), the association of the Btk PH domain with PKCs would be the strongest in quiescent cells, and either the binding of diacylglycerol (or phorbol 12-myristate 13-acetate) to PKC or the inositol lipid head group to the Btk PH domain (i.e. activation of the respective enzymes) would eliminate this association. Certainly, stimulation with phorbol 12-myristate 13-acetate, which has been widely demonstrated to induce membrane translocation of various PKC isozymes (
      • Meyer T.
      • Oancea E.
      ,
      • Shirai Y.
      • Kashiwagi K.
      • Yagi K.
      • Sakai N.
      • Saito N.
      ), failed to induce membrane translocation of the Btk PH domain in the present study, and a PKC inhibitor did not affect translocation induced by PDGF. The existence of additional factors contributing to the membrane localization of PH domains has been suggested by recent studies in which the cytohesin-1 PH domain was shown to require a small basic flanking sequence to effectively localize it to membranes and to exert a dominant-negative effect on cell adhesion (
      • Nagel W.
      • Schilcher P.
      • Zeitlmann L.
      • Kolonaus W.
      ). In this context, it is important to note that our Btk PH domain construct also contained the small adjacent Btk motif that had been found important for proper folding in bacteria (
      • Hyvönen M.
      • Saraste M.
      ). Clearly, more studies are needed to fully explore the complexity of inositol lipid-PH domain interactions that regulate Btk and other PI 3-kinase-regulated effectors.
      Introduction of the R28C mutation into the BtkPH-GFP construct prevented its membrane localization in response to stimulation, in agreement with the data regarding the diminished affinity of this mutant for PI(3,4,5)P3 (
      • Salim 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.
      ,
      • Rameh 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.
      ,
      • Fukuda M.
      • Kojima T.
      • Kabayama H.
      • Mikoshiba K.
      ). Arg-28 is located within the predicted inositide-binding pocket of the Btk PH domain and, based on structural alignments, corresponds to Arg-40 of the PLCδ PH domain. Mutation of this residue in the latter molecule (which makes contact with the 5-phosphate of PI(4,5)P2 (
      • Ferguson K.M.
      • Lemmon M.A.
      • Schlessinger J.
      • Sigler P.B.
      )) prevents its binding to PI(4,5)P2 and its membrane association (
      • Stauffer T.P.
      • Ahn S.
      • Meyer T.
      ,
      • Várnai P.
      • Balla T.
      ). Based on this analogy, it is expected that replacing the positively charged Arg-28 with a non-charged residue would result in the loss of binding affinity for PI(3,4,5)P3.
      Another mutation within the Btk PH domain, E41K (
      • Li T.
      • Tsukada S.
      • Satterthwaite A.
      • Havlik M.H.
      • Park H.
      • Takatsu K.
      • Witte O.N.
      ), was found to show increased membrane localization in quiescent cells and further translocation in response to EGF stimulation. Although there are a number of reasons why this mutant protein could bind to the membrane (including a higher affinity for PI(3,4,5)P3), its similar behavior compared with the PLCδ PH domain raised the possibility that it also binds to membrane PI(4,5)P2. Membrane association of the E41K mutant of BtkPH-GFP was not abolished by PI 3-kinase inhibitors, but showed correlation with PI(4,5)P2 levels after manipulations of the latter by Ca2+ ionophores, Ca2+ chelators, and inhibitors of PI(4,5)P2resynthesis. Also, it showed a significant inhibitory effect on agonist-induced PI(4,5)P2 hydrolysis, a feature of PH domains that bind PI(4,5)P2. Comparison of the crystal structure of PLCδ and Btk reveals that Glu-41 is located in a position that corresponds to a region of the PLCδ PH domain (Ser-55 and Arg-56) that makes important contacts with the phosphates at the 4- and 5-positions of the inositol ring in PI(4,5)P2 (
      • Ferguson K.M.
      • Lemmon M.A.
      • Schlessinger J.
      • Sigler P.B.
      ). An acidic amino acid (Glu-41) in this position could provide a significant repulsive force to prevent association of the mutant protein with PI(4,5)P2. Mutation of Glu-41 to Lys would therefore be expected to increase PI(4,5)P2 binding. Indeed, an analogous mutation within the PH domain of PLCδ (E54K) has been reported to enhance the catalytic activity of the enzyme, presumably by increasing its affinity to PI(4,5)P2 (
      • Bromann P.A.
      • Boetticher E.E.
      • Lomasney J.W.
      ). Such an affinity increase in the E41K mutant of Btk toward both PI(4,5)P2and PI(3,4,5)P3 could explain why the E41K substitution did not significantly affect the ability of Ins(1,4,5)P3 to displace Ins(1,3,4,5)P4 in the binding studies performed on the isolated BtkPH-GST fusion protein (
      • Fukuda M.
      • Kojima T.
      • Kabayama H.
      • Mikoshiba K.
      ).
      Members of the Tec tyrosine kinase family have been recognized recently as important modulators of Ca2+ influx pathways in B- and T-lymphocytes (
      • Scharenberg A.M.
      • Kinet J.-P.
      ) via a mechanism that amplifies inositol 1,4,5-trisphosphate formation after PLCγ activation (
      • Fluckiger A.-C.
      • Li Z.
      • Kato R.M.
      • Wahl M.I.
      • Ochs R.M.
      • Longnecker R.
      • Kinet J.-P.
      • Witte O.N.
      • Scharenberg A.M.
      • Rawlings D.J.
      ,
      • Liu K.-Q.
      • Bunnell S.C.
      • Gurniak C.B.
      • Berg L.J.
      ). This function of the kinases also relies upon the interaction of their PH domains with membrane PI(3,4,5)P3 (
      • Fluckiger A.-C.
      • Li Z.
      • Kato R.M.
      • Wahl M.I.
      • Ochs R.M.
      • Longnecker R.
      • Kinet J.-P.
      • Witte O.N.
      • Scharenberg A.M.
      • Rawlings D.J.
      ). However, it is also important to note that the present results only explore the aspect of Btk function from the standpoint of its PH domain and that additional interactions mediated by other domains of the molecule may greatly affect the overall localization of the holoprotein. Nevertheless, the ability of the isolated Btk PH domain to confer PI(3,4,5)P3-dependent membrane localization may also be utilized as a probe that can detect changes in the level of this lipid in single living cells with fine spatial resolution. Such a feature of other PH domain-GFP fusion constructs that can interact with PI(3,4,5)P3 has been recently demonstrated in insulin-stimulated adipocytes (
      • Venkateswarlu K.
      • Oatey P.B.
      • Tavare J.M.
      • Cullen P.J.
      ) and EGF-stimulated PC-12 cells (
      • Venkateswarlu K.
      • Gunn-Moore F.
      • Oatey P.B.
      • Tavare J.M.
      • Cullen P.J.
      ).
      In summary, we have shown that the isolated PH domain of Btk interacts with plasma membranes with characteristics that are consistent with its binding to membrane PI(3,4,5)P3. This interaction appears to be a fundamental aspect of the Btk protein that is severely compromised in a human disease and can now be monitored in single living cells. This methodology will also help to better understand the role of inositide phospholipids in membrane-protein interactions.

      Acknowledgments

      We thank Dr. Domenico Accili (NICHD, National Institutes of Health) for providing the immortalized hepatocytes and Drs. Tzvetanka Bondeva and Mathias Wymann for providing the PI3Kγ-CAAX construct. The skillful technical assistance of Yue Zhang is greatly appreciated.

      REFERENCES

        • Berridge M.J.
        • Irvine R.F.
        Nature. 1984; 312: 315-321
        • Nishizuka Y.
        Nature. 1984; 308: 693-698
        • Jackson T.R.
        • Stephens L.R.
        • Hawkins P.T.
        J. Biol. Chem. 1992; 267: 16627-16636
        • Franke T.F.
        • Kaplan D.R.
        • Cantley L.C.
        • Toker A.
        Science. 1997; 275: 665-668
        • Toker A.
        • Cantley L.C.
        Nature. 1997; 387: 673-676
        • Salim 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: 6241-6250
        • Chen R.-H.
        • Corbalan-Garcia S.
        • Bar-Sagi D.
        EMBO J. 1997; 16: 1351-1359
        • Rameh 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: 22059-22066
        • Klarlund J.K.
        • Guilherme A.
        • Holik J.J.
        • Virbasius J.V.
        • Chawla A.
        • Czech M.P.
        Science. 1997; 275: 1927-1930
        • Kavran J.M.
        • Klein D.E.
        • Lee A.
        • Falasca M.
        • Isakoff S.J.
        • Skolnik E.Y.
        • Lemmon M.A.
        J. Biol. Chem. 1998; 273: 30497-30508
        • Rawlings D.J.
        • Witte O.N.
        Semin. Immunol. 1995; 7: 237-246
        • Tsukada 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: 279-290
        • Rawlings 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: 358-361
        • Vihinen M.
        • Iwata T.
        • Kinnon C.
        • Kwan S.P.
        • Ochs H.D.
        • Vorechovsky I.
        • Smith C.I.
        Nucleic Acids Res. 1996; 24: 160-165
        • Li T.
        • Tsukada S.
        • Satterthwaite A.
        • Havlik M.H.
        • Park H.
        • Takatsu K.
        • Witte O.N.
        Immunity. 1995; 2: 451-460
        • Hunyady L.
        • Baukal A.J.
        • Balla T.
        • Catt K.J.
        J. Biol. Chem. 1994; 269: 24798-24804
        • Balla T.
        • Sim S.S.
        • Baukal A.J.
        • Rhee S.G.
        • Catt K.J.
        Mol. Biol. Cell. 1994; 5: 17-27
        • Stauffer T.P.
        • Ahn S.
        • Meyer T.
        Curr. Biol. 1998; 8: 343-346
        • Várnai P.
        • Balla T.
        J. Cell Biol. 1998; 143: 501-510
        • Rother K.I.
        • Imai Y.
        • Caruso M.
        • Beguinot F.
        • Formisano P.
        • Accili D.
        J. Biol. Chem. 1998; 273: 17491-17497
        • Bondeva T.
        • Pirola L.
        • Bulgarelli-Leva G.
        • Rubio I.
        • Wetzker R.
        • Wymann M.P.
        Science. 1998; 282: 293-296
        • Yao L.
        • Suzuki H.
        • Ozawa K.
        • Deng J.
        • Lehel C.
        • Fukamachi H.
        • Anderson W.B.
        • Kawakami Y.
        • Kawakami T.
        J. Biol. Chem. 1997; 272: 13033-13039
        • Chou 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: 1069-1077
        • Ferguson K.M.
        • Lemmon M.A.
        • Schlessinger J.
        • Sigler P.B.
        Cell. 1995; 83: 1037-1046
        • Rhee S.G.
        • Bae Y.S.
        J. Biol. Chem. 1997; 272: 15045-15048
        • Abrams C.S.
        • Wu H.
        • Zhao W.
        • Belmonte E.
        • White D.
        • Brass L.F.
        J. Biol. Chem. 1995; 270: 14485-14492
        • Hyvönen M.
        • Saraste M.
        EMBO J. 1997; 16: 3396-3404
        • Tsukada S.
        • Simon M.
        • Witte O.
        • Katz A.
        Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11256-11260
        • Yao L.
        • Kawakami Y.
        • Kawakami T.
        Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9175-9179
        • Fukuda M.
        • Kojima T.
        • Kabayama H.
        • Mikoshiba K.
        J. Biol. Chem. 1996; 271: 30303-30306
        • Mochly-Rosen D.
        • Gordon A.S.
        FASEB J. 1998; 12: 35-42
        • Kontos C.D.
        • Stauffer T.P.
        • Yang W.P.
        • York J.D.
        • Huang L.
        • Blanar M.A.
        • Meyer T.
        • Peters K.G.
        Mol. Cell. Biol. 1998; 18: 4131-4140
        • Stephens L.
        • Anderson K.
        • Stokoe D.
        • Erdjument-Bromage H.
        • Painter G.F.
        • Holmes A.B.
        • Gaffney P.R.J.
        • Reeses C.B.
        • McCormick F.
        • Tempst P.
        • Coadwell J.
        • Hawkins P.T.
        Science. 1998; 279: 710-714
        • Meyer T.
        • Oancea E.
        Cell. 1998; 95: 307-318
        • Shirai Y.
        • Kashiwagi K.
        • Yagi K.
        • Sakai N.
        • Saito N.
        J. Cell Biol. 1998; 143: 511-521
        • Nagel W.
        • Schilcher P.
        • Zeitlmann L.
        • Kolonaus W.
        Mol. Biol. Cell. 1998; 9: 1981-1994
        • Bromann P.A.
        • Boetticher E.E.
        • Lomasney J.W.
        J. Biol. Chem. 1997; 272: 16240-16246
        • Scharenberg A.M.
        • Kinet J.-P.
        Cell. 1998; 94: 5-8
        • Fluckiger A.-C.
        • Li Z.
        • Kato R.M.
        • Wahl M.I.
        • Ochs R.M.
        • Longnecker R.
        • Kinet J.-P.
        • Witte O.N.
        • Scharenberg A.M.
        • Rawlings D.J.
        EMBO J. 1998; 17: 1973-1985
        • Liu K.-Q.
        • Bunnell S.C.
        • Gurniak C.B.
        • Berg L.J.
        J. Exp. Med. 1998; 187: 1721-1727
        • Venkateswarlu K.
        • Oatey P.B.
        • Tavare J.M.
        • Cullen P.J.
        Curr. Biol. 1998; 8: 463-466
        • Venkateswarlu K.
        • Gunn-Moore F.
        • Oatey P.B.
        • Tavare J.M.
        • Cullen P.J.
        Biochem. J. 1998; 335: 139-146