Roles of Non-catalytic Subunits in Gβγ-induced Activation of Class I Phosphoinositide 3-Kinase Isoforms β and γ
1999; Elsevier BV; Volume: 274; Issue: 41 Linguagem: Inglês
10.1074/jbc.274.41.29311
ISSN1083-351X
AutoresUdo Maier, А. Г. Бабич, Bernd Nürnberg,
Tópico(s)Chronic Lymphocytic Leukemia Research
ResumoBy using purified preparations we show that nanomolar concentrations of Gβγ significantly stimulated lipid kinase activity of phosphatidylinositol 3-kinase (PI3K) β and PI3Kγ in the presence as well as in the absence of non-catalytic subunits such as p85α or p101. Concomitantly, Gβγ stimulated autophosphorylation of the catalytic subunit of PI3Kγ (EC50, 30 nm; stoichiometry ≥0.6 mol of Pi/mol of p110γ), which also occurred in the absence of p101. Surprisingly, we found that p101 affected the lipid substrate preference of PI3Kγ in its Gβγ-stimulated state. With phosphatidylinositol as substrate, p110γ but not p101/p110γ was significantly stimulated by Gβγ to form PI-3-phosphate (EC50, 20 nm). The opposite situation was found when PI-4,5-bisphosphate served as substrate. Gβγ efficiently and potently (EC50, 5 nm) activated the p101/p110γ heterodimer but negligibly stimulated the p110γ monomer to form PI-3,4,5-trisphosphate. However, this weak stimulatory effect on p110γ was overcome by excess concentrations of Gβγ (EC50, 100 nm). This finding is in accordance with the in vivo situation, where activated PI3K catalyzes the formation of PI-3,4,5-trisphosphate but not PI-3-phosphate. We conclude that p101 is responsible for PI-4,5-bisphosphate substrate selectivity of PI3Kγ by sensitizing p110γ toward Gβγ in the presence of PI-4,5-P2. By using purified preparations we show that nanomolar concentrations of Gβγ significantly stimulated lipid kinase activity of phosphatidylinositol 3-kinase (PI3K) β and PI3Kγ in the presence as well as in the absence of non-catalytic subunits such as p85α or p101. Concomitantly, Gβγ stimulated autophosphorylation of the catalytic subunit of PI3Kγ (EC50, 30 nm; stoichiometry ≥0.6 mol of Pi/mol of p110γ), which also occurred in the absence of p101. Surprisingly, we found that p101 affected the lipid substrate preference of PI3Kγ in its Gβγ-stimulated state. With phosphatidylinositol as substrate, p110γ but not p101/p110γ was significantly stimulated by Gβγ to form PI-3-phosphate (EC50, 20 nm). The opposite situation was found when PI-4,5-bisphosphate served as substrate. Gβγ efficiently and potently (EC50, 5 nm) activated the p101/p110γ heterodimer but negligibly stimulated the p110γ monomer to form PI-3,4,5-trisphosphate. However, this weak stimulatory effect on p110γ was overcome by excess concentrations of Gβγ (EC50, 100 nm). This finding is in accordance with the in vivo situation, where activated PI3K catalyzes the formation of PI-3,4,5-trisphosphate but not PI-3-phosphate. 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Activation of class I PI3Ks is observed in response to a wide array of cellular ligands including hormones, neurotransmitters, growth factors, and cytokines. Although an ever increasing number of cellular responses are elicited by these lipid kinases, a remarkable degree of specificity is maintained within pleiotropic PI3K-dependent signaling pathways, allowing the assignment of intracellular effects to extracellular stimuli. This surprising specificity is certainly due to a structural heterogeneity of class I PI3K isoforms in concert with different expression patterns as well as spatial and/or temporal compartmentation. Based on their tight association with non-catalytic binding proteins, catalytic subunits of class I PI3Ks are subdivided into class IA p85- or class IB p101-associated heterodimers. Until recently this grouping went strictly parallel with the isoform-specific regulation by different signaling pathways. Whereas class IA-isoforms (p110α, -β, and -δ) were assumed to be sensitive to tyrosine kinases, the only class IB member p110γ is activated by G-proteins. This structure-function relationship was challenged by reports detailing a synergistic activation of PI3Kβ and other so far unidentified isoforms by either regulator (24Kurosu H. Hazeki O. Kukimoto I. Honzawa S. Shibasaki M. Nakada M. Ui M. Katada T. Biochem. Biophys. Res. Commun. 1995; 216: 655-661Crossref PubMed Scopus (13) Google Scholar, 25Kurosu H. Maehama T. Okada T. Yamamoto T. Hoshino S. Fukui Y. Ui M. Hazeki O. Katada T. J. Biol. Chem. 1997; 272: 22426-24252Abstract Full Text Full Text PDF Scopus (234) Google Scholar, 26Tang X. Downes C.P. J. Biol. Chem. 1997; 272: 14193-14199Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). In line with their sensitivity toward tyrosine kinases, the catalytic p110 subunits of PI3Kα, -β, and -δ are associated with p85 adaptors, which are indispensable for activation (27Dhand R. Hara K. Hiles I. Bax B. Gout I. Panayotou G. Fry M.J. Yonezawa K. Kasuga M. Waterfield M.D. EMBO J. 1994; 13: 511-521Crossref PubMed Scopus (295) Google Scholar). These adaptors harbor various regions such as SH2-, SH3-, or proline-rich domains, which contribute to further selectivity within tyrosine kinase-dependent signaling pathways (18Fruman D.A. Meyers R.E. Cantley L.C. Annu. Rev. Biochem. 1998; 67: 481-507Crossref PubMed Scopus (1319) Google Scholar,28Shepherd P.R. Nave B.T. Rincon J. Nolte L.A. Bevan A.P. Siddle K. Zierath J.R. Wallberg-Henriksson H. J. Biol. Chem. 1997; 272: 19000-19007Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 29Yu J. Wjasow C. Backer J.M. J. Biol. 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Cell. 1997; 89: 105-114Abstract Full Text Full Text PDF PubMed Scopus (494) Google Scholar). Surprisingly, p101 shows no homology with any known protein. Nevertheless at first it was assumed to serve as an adaptor for Gβγ liberated by GPCRs. However, we and others (26Tang X. Downes C.P. J. Biol. Chem. 1997; 272: 14193-14199Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 31Stoyanov B. Volinia S. Hanck T. Rubio I. Loubtchenkov M. Malek D. Stoyanova S. Vanhaesebroeck B. Dhand R. Nürnberg B. Gierschik P. Seedorf K. Hsuan J.J. Waterfield M.D. Wetzker R. Science. 1995; 269: 690-693Crossref PubMed Scopus (642) Google Scholar, 33Leopoldt D. Hanck T. Exner T. Maier U. Wetzker R. Nürnberg B. J. Biol. Chem. 1998; 273: 7024-7029Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar) have found significant stimulation of the purified catalytic subunit of PI3Kγ by Gβγ in the absence of p101. Thus, the function of this p101 non-catalytic subunit of PI3Kγ has yet to be identified. Versatility in PI3K-dependent signaling appears to be accomplished by a second enzymatic function, i.e.protein-serine kinase activity, which is inherent to the class I PI3Ks (34Hunter T. Cell. 1995; 83: 1-4Abstract Full Text PDF PubMed Scopus (261) Google Scholar). Accordingly, first evidence emerged that cellular signaling bifurcates at the level of the PI3Kγ (35Bondeva T. Pirola L. Bulgarelli-Leva G. Rubio I. Wetzker R. Wymann M.P. Science. 1998; 282: 293-296Crossref PubMed Scopus (302) Google Scholar). Whereas lipid kinase activity of PI3Kγ stimulated protein kinase B, its protein kinase activity signaled to the extracellular signal-regulated kinase/mitogen-activated protein kinase pathway. In this context recent reports are of interest which suggested that the non-catalytic p101 subunit was crucial for supporting p110γ-induced protein kinase B and c-Jun amino-terminal kinase activation but had only little effect on extracellular signal-regulated kinase/mitogen-activated protein kinase activation (36Lopez-Ilasaca M. Crespo P. Pellici P.G. Gutkind J.S. Wetzker R. Science. 1998; 275: 394-397Crossref Scopus (629) Google Scholar, 37Murga C. Laguinge L. Wetzker R. Cuadrado A. Gutkind J.S. J. Biol. Chem. 1998; 273: 19080-19085Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar). Therefore, p101 may function as a regulatory subunit of PI3Kγ that differently modulates protein and lipid kinase activity of PI3Kγ. The intention of the present study was to examine the sensitivity of PI3Ks toward Gβγ. We therefore purified recombinant proteins including all four known class I PI3K isoforms. Monomeric and heterodimeric enzyme preparations were analyzed for the role of non-catalytic subunits as adaptors for Gβγ-induced stimulation of PI3Ks. Furthermore, this experimental approach was successfully applied to assign a novel functional role to the p101 subunit of PI3Kγ. Construction of recombinant baculoviruses for expression of PI3K subunits was described previously (31Stoyanov B. Volinia S. Hanck T. Rubio I. Loubtchenkov M. Malek D. Stoyanova S. Vanhaesebroeck B. Dhand R. Nürnberg B. Gierschik P. Seedorf K. Hsuan J.J. Waterfield M.D. Wetzker R. Science. 1995; 269: 690-693Crossref PubMed Scopus (642) Google Scholar, 33Leopoldt D. Hanck T. Exner T. Maier U. Wetzker R. Nürnberg B. J. Biol. Chem. 1998; 273: 7024-7029Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar, 38Vanhaesebroeck B. Welham M.J. Kotani K. Stein R. Warne P.H. Zvelebil M.J. Higashi K. Volinia S. Downward J. Waterfield M.D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4330-4335Crossref PubMed Scopus (373) Google Scholar). For protein expression, cells were incubated at a multiplicity of infection (m.o.i.) of 1 virus per cell. Subunits of heterodimeric PI3Ks were coexpressed at equal m.o.i. numbers in Sf9 cells and used for functional studies. After 48–60 h of infection cells were pelleted by centrifugation (1,000 ×g) and washed with phosphate-buffered saline twice. For purification of GST fusion proteins cells were resuspended in ice-cold buffer A containing 150 mm NaCl, 1 mm NaF, 1 mm EDTA, 50 mm Tris/HCl, pH 8.0, 10 mm dithiothreitol, 10 μg/ml each ofl-1-tosylamido-2-phenylethyl chloromethyl ketone, benzamidine, leupeptin, and 0.2 mm Pefabloc® SC (Roche Molecular Biochemicals). They were disrupted by N2cavitation (30 min at 4 °C, 25 bar) or by forcing the cell suspension through a 22-gauge needle (5 times) and subsequently through a 26-gauge needle (10 times). Nuclei and debris were discarded. The cytosolic fraction was separated from the particulate by centrifugation at 100,000 × g for 50 min. Cytosol was incubated 3–4 h with glutathione-Sepharose 4B beads (Amersham Pharmacia Biotech) prewashed with buffer A. The Sepharose-bound GST fusion proteins were stored at −20 °C in buffer B containing 50% glycerol, 1 mm EDTA, 40 mm Tris/HCl, pH 8.0, 1 mm dithiothreitol, and 1.57 mg/ml benzamidine. For enzymatic assays GST fusion proteins were freshly eluted with buffer C consisting of buffer A with 10 mm glutathione for 1 h at 4 °C. For purification of hexa-His-tagged PI3Kβ cells were disrupted using buffer D (20 mm HEPES, 150 mmNaCl, 10 mm β-mercaptoethanol) containing 10 μg/ml each of l-1-tosylamido-2-phenylethyl chloromethyl ketone, benzamidine, leupeptin, and 0.2 mm Pefabloc®SC (Roche Molecular Biochemicals) using the same procedure as described above. The cytosolic fraction was incubated 1–2 h with Ni2+-NTA-agarose (Qiagen, Hilden, Germany) prewashed with buffer D containing 20 mm imidazole. After extensive washing with buffer D, proteins were eluted with buffer D containing 150 mm imidazole. Purified proteins were quantified by Coomassie Blue staining following SDS-PAGE with bovine serum albumin as the standard. For coexpression experiments with recombinant subunits of PI3Kγ, i.e. p101, p110γ, and mutants thereof, equal m.o.i. numbers for all recombinant baculoviruses were used. Cell lysates were incubated with glutathione-Sepharose 4B, and eluted GST fusion proteins were analyzed for binding of p101. For coexpression experiments with recombinant Gβ1γ2, cell lysates were obtained by forcing the Sf9 cell suspension through needles as described above and subsequent incubation (30 min) with buffer A supplemented with 0,5% Lubrol PX. After incubation with glutathione-Sepharose 4B eluted proteins were analyzed for bound Gβγ. For isolation of bovine brain Gβγ, we employed standard techniques with modifications. Bovine brain G-proteins were purified to apparent homogeneity in the presence of aluminum fluoride. Isolation and final purification of Gβγ was achieved using a Mono Q (Amersham Pharmacia Biotech) fast protein liquid chromatography column (39Exner T. Jensen O.N. Mann M. Kleuss C. Nürnberg B. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1327-1332Crossref PubMed Scopus (39) Google Scholar). Gβγ complexes were identified by their immunoreactivity to specific antisera and quantified by the method of Lowry and by Coomassie Blue staining of Gβ following SDS-PAGE with bovine serum albumin as the standard. Contamination by pertussis toxin-sensitive (PT) Gα subunits was excluded by analysis of autoradiographic signals after PT-mediated [32P]ADP ribosylation with a BAS 1500 Fuji-Imager (Raytest, Straubenhardt, Germany). Purified Gβγ were highly concentrated and contained Lubrol PX (0.1%) and CHAPS (11 mm) as detergents and were stored at −70 °C until use. The doubly tyrosine-phosphorylated peptide used in this study, CGGY(P)MDMSKDESVDY(P)VPMLDM, was based on that of the human platelet-derived growth factor receptor (40Stephens L. Smrcka A. Cooke F.T. Jackson T.R. Sternweis P.C. Hawkins P.T. Cell. 1994; 77: 83-93Abstract Full Text PDF PubMed Scopus (520) Google Scholar) and supplied by Schering AG, Berlin. A non-phosphorylated peptide was used as a control and had no effect. Characterization of the monoclonal antibody against p110γ, antisera against p101, and Gβ subunits (AS 398) were detailed elsewhere (33Leopoldt D. Hanck T. Exner T. Maier U. Wetzker R. Nürnberg B. J. Biol. Chem. 1998; 273: 7024-7029Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar, 41Viard P. Exner T. Maier U. Mironneau J. Nürnberg B. Macrez N. FASEB J. 1999; 13: 685-694Crossref PubMed Scopus (108) Google Scholar). The polyclonal anti-GST antibody was purchased from Santa Cruz Biotechnology (Heidelberg, Germany). For detection of GST fusion proteins, p110γ, p101, or Gβ, proteins were fractionated by SDS-PAGE and transferred to nitrocellulose or polyvinylidene difluoride membranes (Millipore, Eschborn, Germany). Visualization of specific antisera was performed using the ECL chemiluminescence system (Amersham Pharmacia Biotech) or the CDP-Star chemiluminescence reagent (Tropix, Bedford, MA) according to the manufacturers' instructions. Lipid kinase activity was determined basically as detailed previously (33Leopoldt D. Hanck T. Exner T. Maier U. Wetzker R. Nürnberg B. J. Biol. Chem. 1998; 273: 7024-7029Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar, 41Viard P. Exner T. Maier U. Mironneau J. Nürnberg B. Macrez N. FASEB J. 1999; 13: 685-694Crossref PubMed Scopus (108) Google Scholar). In brief, the assays were conducted in a final volume of 50 μl containing 0.1% bovine serum albumin, 1 mm EGTA, 120 mm NaCl, 40 mm HEPES, pH 7.4, 1 mm dithiothreitol, 1 mm β-glycerophosphate (buffer E) as described with some modifications. Lipid vesicles (30 μl containing 320 μmphosphatidylethanolamine, 300 μm phosphatidylserine, 140 μm phosphatidylcholine, 30 μm sphingomyelin supplemented with either 300 μm PI or 40 μmPI-4,5-P2 in buffer E) were mixed with stimuli as indicated and incubated on ice for 10 min. For measuring the effects of Gβγ on PI3K activity, it was ensured that stimuli-containing vehicles did not suppress enzymatic activity. In addition, assay samples containing different amounts of Gβγ were adjusted to identical detergent concentrations, i.e. 0.0004% for Lubrol PX and 0.044% for CHAPS. Mg2+ at 10 and 7 mm for PI and PI-4,5-P2 as substrates, respectively, was added to lipids before sonification. For inhibition assays, kinase preparations were preincubated with wortmannin or 17-OH wortmannin (Schering AG) at 37 °C. Thereafter, the enzyme fraction (1–10 ng) was added, and the mixture was incubated for a further 10 min at 4 °C in a final volume of 40 μl. The assay was then started by adding 40 μmATP (1 μCi of [γ-32P]ATP, NEN Life Science Products) in 10 μl of the above assay buffer (30 °C). After 15 min the reaction was stopped with ice-cold 150 μl of 1 n HCl and placing tubes on ice. The lipids were extracted by vortexing samples with 500 μl of chloroform/methanol (1:1). After centrifugation the organic phase was washed twice with 200 μl of 1 n HCl. Subsequently, 40–80 μl of the organic phase were resolved on potassium oxalate-pretreated TLC plates (Whatman) with 35 ml of 2n acetic acid and 65 ml of n-propyl alcohol as the mobile phase. Dried TLC plates were exposed to Fuji imaging plates, and autoradiographic signals were quantitated with a BAS 1500 Fuji-Imager. The assays were performed as described for lipid kinase activity with some modifications. The assay volume was 25 μl (2–3 μCi of [γ-32P]ATP per tube) and usually contained 7 mm Mg2+. Lipid vesicles were devoid of PI3-kinase lipid substrates such as PI or PI-4,5-P2. The reaction was stopped with 25 μl of ice-cold 2× sample buffer according to Laemmli containing 10 mm ATP. Following separation on SDS-PAGE, proteins were transferred to nitrocellulose membranes. Dried membranes were exposed to Fuji imaging plates, and autoradiographic signals were quantitated with a BAS 1500 Fuji-Imager. Based on the observation that the structural subdivision of class I PI3Ks does not correlate with regulation by different signaling pathways, all four members were examined for their Gβγ sensitivity. Therefore we isolated heterodimeric enzymes following expression in Sf9 cells (Fig.1 A). As previously reported for p110α (42Yu J. Zhang Y. McIlroy J. Rordorf-Nikolic T. Orr G.A. Backer J.M. Mol. Cell. Biol. 1998; 18: 1379-1387Crossref PubMed Google Scholar), coexpression of p110α, -β, and -δ catalytic subunits together with p85 increased the amount of protein in Sf9 cytosolic fractions. In contrast, p101 did not enhance expression of p110γ but preserved the catalytic subunit from rapid degradation during storage. In initial experiments we found no difference in the enzymatic activities of PI3Kγ preparations, regardless whether the GST tag was fused to p110γ or p101. For convenience we used p101-GST/p110γ heterodimeric preparations throughout the study. The isolated heterodimers were assayed for their sensitivity toward purified bovine brain Gβγ and phosphotyrosyl peptides resembling an intracellular p85-binding region of the platelet-derived growth factor receptor (Fig. 1 B). As expected, PI3Kγ was solely activated by Gβγ, whereas PI3Kα only responded to phosphotyrosyl peptides. However, coincubation of PI3Kβ with both stimuli led to a remarkable synergistic activation of lipid kinase activity in accordance with previous reports (24Kurosu H. Hazeki O. Kukimoto I. Honzawa S. Shibasaki M. Nakada M. Ui M. Katada T. Biochem. Biophys. Res. Commun. 1995; 216: 655-661Crossref PubMed Scopus (13) Google Scholar). Moreover, unlike these published results, PI3Kβ was also significantly activated by Gβγ alone, suggesting that PI3Kβ represents an effector of Gβγ (see Fig.1 B). Interestingly, PI3Kδ like PI3Kα was only responsive to phosphotyrosyl peptides but not to Gβγ, although p110δ exhibits the highest degree of overall amino acid sequence identity to p110β. It should be mentioned that all stimulatory effects were observed in a concentration-dependent manner (data not shown). Since PI3Kβ and -γ were sensitive to Gβγ but complexed with different non-catalytic subunits, we next examined the influence of p85α or p101 on Gβγ-induced activation of p110β and p110γ lipid kinase activity, respectively (Fig.1 C). In initial experiments we have confirmed that p110β copurified with p85α but not with p101, whereas p110γ showed the opposite association pattern to the non-catalytic subunits (data not shown and Ref. 31Stoyanov B. Volinia S. Hanck T. Rubio I. Loubtchenkov M. Malek D. Stoyanova S. Vanhaesebroeck B. Dhand R. Nürnberg B. Gierschik P. Seedorf K. Hsuan J.J. Waterfield M.D. Wetzker R. Science. 1995; 269: 690-693Crossref PubMed Scopus (642) Google Scholar). In addition, we ascertained that the type of tag fused to p110γ, i.e. GST or polyhistidine, did not affect the enzymatic activity of the catalytic subunit. p110γ as well as p110β were significantly stimulated by Gβγ irrespective of non-catalytic subunits (see Fig. 1 C, upper panel). In contrast, sensitivity to phosphotyrosyl peptides was seen only when p110β was complexed with p85α but not when p110γ was associated with p101 (see Fig. 1 C, lower panel). Accordingly, in the absence of p85α, costimulation of p110β by Gβγ together with phosphotyrosyl peptides showed no synergistic effect (data not shown). These findings provide unequivocal evidence that structural elements necessary for Gβγ-induced stimulation of PI3K isoforms are located on the catalytic subunits of PI3Kβ and PI3Kγ. Hence, p101 does not function as a p110γ-specific adaptor that is necessary for activation by Gβγs. This obviously contrasts with the role of p85 for tyrosine kinase-induced activation of PI3Ks. As another putative parallel between class IA and IB PI3Ks, the p101-binding site on p110γ was hypothesized to lie within a region analogous to the p85-binding site on class IA members, i.e. the amino terminus (16Zvelebil M.J. Macdougall L. Leevers S. Volina S. Vanhaesebroeck B. Gout I. Panayotou G. Domin J. Stein R. Pages F. Koga H. Salim K. Linacre J. Das P. Panaretou C. Wetzker R. Waterfield M. Phil. Trans. R. Soc. Lond. Biol. 1996; 351: 217-223Crossref PubMed Scopus (89) Google Scholar, 43Stoyanova S. Bulgarelli-Leva G. Kirsch C. Hanck T. Klinger R. Wetzker R. Wymann M.P. Biochem. J. 1997; 324: 489-495Crossref PubMed Scopus (100) Google Scholar). To examine this assumption we studied the binding of p101 to a GST-fused full-length or an amino-terminally truncated construct of p110γ (Δ1–97) by copurification from Sf9 cells (Fig.2 A). The specificity of this approach was proven by expression of p101 with or without GST as a control. p101 was bound equally well by both p110γ constructs suggesting that the non-catalytic subunit associates with regions of p110γ outside the amino terminus. Nevertheless, as an indication that p101 may be still involved in Gβγ-induced activation of p110γ, we confirmed that p101 bound much stronger to Gβγ than p110γ (Fig.2 B and Ref. 32Stephens L.R. Eguinoa A. Erdjument-Bromage H. Lui M. Cooke F. Coadwell J. Smrcka A.S. Thelen M. Cadwallader K. Tempst P. Hawkins P.T. Cell. 1997; 89: 105-114Abstract Full Text Full Text PDF PubMed Scopus (494) Google Scholar). Previous studies by two different laboratories have resulted in an apparent discrepancy concerning the function of p101 in Gβγ-induced activation of p110γ (31Stoyanov B. Volinia S. Hanck T. Rubio I. Loubtchenkov M. Malek D. Stoyanova S. Vanhaesebroeck B. Dhand R. Nürnberg B. Gierschik P. Seedorf K. Hsuan J.J. Waterfield M.D. Wetzker R. Science. 1995; 269: 690-693Crossref PubMed Scopus (642) Google Scholar, 32Stephens L.R. Eguinoa A. Erdjument-Bromage H. Lui M. Cooke F. Coadwell J. Smrcka A.S. Thelen M. Cadwallader K. Tempst P. Hawkins P.T. Cell. 1997; 89: 105-114Abstract Full Text Full Text PDF PubMed Scopus (494) Google Scholar, 33Leopoldt D. Hanck T. Exner T. Maier U. Wetzker R. Nürnberg B. J. Biol. Chem. 1998; 273: 7024-7029Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar, 44Krugmann S. Hawkins P.T. Pryer N. Braselmann S. J. Biol. Chem. 1999; 274: 17152-17158Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). However, some experimental conditions varied which may have contrib
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