Regulation of oxidative stress-induced calcium release by phosphatidylinositol 3-kinase and Bruton’s tyrosine kinase in B cells Suofu Qin, Earl R. Stadtman, and P. Boon Chock* Laboratory of Biochemistry, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892-0342 Contributed by Earl R. Stadtman, May 2, 2000 Hydrogen peroxide stimulates a tyrosine kinase-dependent cal- is necessary but not sufficient for the full activation of PLC␥2. cium release from intracellular stores, which is assumed to be Furthermore, Rameth et al. demonstrated that mutated PDGF achieved through the activation of phospholipase C␥2 (PLC␥2) receptor, which is capable of phosphorylating PLC␥ with similar 2ϩ via a tyrosine phosphorylation mechanism in B cells. Here we efficiency as the wild-type, can stimulate an increase in [Ca ]i, ␥ ϩ show that H2O2 induces both tyrosine phosphorylation on PLC 2 but fails to achieve full-scale Ca2 mobilization (12). and the activation of phosphatidylinositol 3-kinase (PI3K) in Phosphatidylinositol 3,4,5-trisphosphate (PIP3) is a phos- B cells, and that the phosphatidylinositol 3-kinase inhibitor, phorylated product of PIP2 at the D3 position catalyzed by Wortmannin, partially inhibited the H2O2-induced calcium re- phosphatidylinositol 3-kinase (PI3K), which is activated by lease without affecting tyrosine phosphorylation on PLC␥2. ligation of a variety of receptors (13, 14). Bae et al. recently Overexpression of human Bruton’s tyrosine kinase (Btk), which reported that PIP3 specifically activates PLC␥ isozymes in vitro was activated by H2O2, almost completely overcame the inhibi- by interacting with their SH2 domains (3). Furthermore, the tion of calcium release by Wortmannin. The reversal of Wort- expression of an activated catalytic subunit of PI3K in COS mannin’s inhibition by enhancing Btk concentration seemed cells resulted in an increase in IP3 formation, whereas platelet- unique to the H2O2-mediated effect, because Btk failed to derived growth factor-induced PLC␥ activation in NIH 3T3 overcome the inhibition of Wortmannin on B cell receptor- cells was markedly inhibited by the PI3K-specific inhibitor triggered calcium mobilization. Immunoblot analysis revealed LY294002. Thus, receptors coupled to PI3K may activate that Btk formed stable complexes with several tyrosine-phos- ␥ ␥ ␥ PLC indirectly in the absence of PLC -tyrosine phosphory- phorylated proteins, including PLC 2, only in Btk-overexpressed lation through the generation of PIP3 (3, 15). Furthermore, the cells on H2O2 stimulation. Together, our data are consistent with ͞ PI3K inhibitors, Wortmannin or LY294002, have been shown the notion that PIP3 and or a high concentration of Btk target to inhibit Fc␥ receptor-dependent activation of PLC␥, and this the activated PLC␥2 to its substrate site for maximal catalytic inhibition can be restored by the preincubation with exogenous efficiency. PIP3 without affecting the tyrosine phosphorylation of PLC␥2 (16). In platelet-derived growth factor signaling, Wortmannin alcium mobilization is a crucial event for many types of cells blocks the activation of PLC␥1 by inhibiting its membrane Cin response to agonists binding to their receptors. This targeting by PIP3 with no effect on tyrosine phosphorylation process is achieved through the activation of phospholipase C of PLC␥1 (17). Genetic analyses of PLC␥ activation in immu- (PLC) to catalyze the hydrolysis of phosphatidylinositol 4,5 noreceptor signaling reported by several groups (18–20) show bisphosphate (PIP2) to diacylglycerol and inositol 1,4,5- that PIP3 interacts with the PH domain of the Tec family trisphosphate (IP3), which activates protein kinase C and ele- tyrosine kinases, thereby promoting their membrane targeting 2ϩ vates intracellular calcium ([Ca ]i), respectively (1). There are and activation. Tec family kinases are known to phosphorylate three classes of known PLC isozymes, ␤, ␥, and ␦ (2). PLC␤ is ␥ ͞ ␥ PLC and lead to its activation. In addition, Tec kinase PIP3 activated by a G protein-mediated process, whereas PLC can be complexes have been suggested to function as adaptors to bring activated by a protein tyrosine kinase (PTK)-dependent pathway ␥ the activated PLC within proximity of PIP2, independent of (3). Both receptor- (4, 5) and nonreceptor-type PTKs (6, 7) have ␥ Tec kinase activity (6, 20). been shown to activate PLC . In B cells, including chicken DT40 Reactive oxygen species (ROS) have emerged as physiological B cells, crosslinking of antigen receptors results in Syk- and͞or ␥ mediators of cellular responses. The production of ROS has been (Bruton’s tyrosine kinase) Btk-dependent induction of PLC 2- detected in a variety of cells stimulated with cytokines (21, 22), mediated hydrolysis of PIP2 to IP3, which induces intracellular ϩ peptide growth factor (23, 24), and agonists of receptors con- Ca2 mobilization (8, 9). This sequential model is supported by taining seven transmembrane spans (25). When exogenous H O Takata et al. (10), who showed that PLC␥2-deficient DT40 cells 2 2 ϩ is applied to cells as one form of ROS, it leads to an increase in exhibit no Ca2 mobilization after B cell receptor ligation. tyrosine-phosphorylated proteins that might derive from the Tyrosine phosphorylation is critical for the activity of PLC␥, activation of nonreceptor- and receptor-type PTKs (26–31) because point mutation of the specific tyrosine residues of PLC␥ to phenylalanine abolishes the IP3 production mediated by the platelet-derived growth factor, epidermal growth factor, and Abbreviations: Btk, Bruton’s tyrosine kinase; PI3K, phosphatidylinositol 3-kinase; PLC, nerve growth factor (11). Mutation of the phosphotyrosine- phospholipase C; PIP2, phosphatidylinositol 4,5-bisphosphate; PIP3, phosphatidylinositol binding motif within the SH2 of PLC␥2 also prevents tyrosine 3,4,5-trisphosphate; IP3, inositol 1,4,5-trisphosphate; PTK, protein tyrosine kinase; BCR, ␥ B-cell antigen receptor. phosphorylation of PLC 2 and subsequent IP3 generation due to B cell receptor activation (10). However, the tyrosine phosphor- *To whom reprint requests should be addressed at: Laboratory of Biochemistry, National ␥ Heart, Lung, and Blood Institute, National Institutes of Health, Building 3, Room 204, 3 ylation level of PLC is not well correlated with IP3 production Center Drive, MSC-0342, Bethesda, MD 20892-0342. E-mail: [email protected]. and Ca2ϩ mobilization in PTK-deficient cells. For example, in ␥ The publication costs of this article were defrayed in part by page charge payment. This Btk-deficient DT40 cells, tyrosine phosphorylation of PLC 2is article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. dramatically reduced but still significantly detectable on B cell §1734 solely to indicate this fact. 2ϩ receptor activation, yet IP3 production and Ca mobilization Article published online before print: Proc. Natl. Acad. Sci. USA, 10.1073͞pnas.130198197. are completely lost (6), suggesting that tyrosine phosphorylation Article and publication date are at www.pnas.org͞cgi͞doi͞10.1073͞pnas.130198197 7118–7123 ͉ PNAS ͉ June 20, 2000 ͉ vol. 97 ͉ no. 13 Downloaded by guest on September 25, 2021 and͞or inhibition of protein tyrosine phosphatases (32, 33). a short centrifugation step. Lysates were clarified by centrifu- 2ϩ ϫ Furthermore, H2O2-stimulated Ca mobilization and tyrosine gation at 16,000 g for 15 min at 4°C. phosphorylation patterns in lymphocytes are similar to those observed following antigen receptor activation (34, 35). Wien- Immunoblot Analysis. Cell extracts or immunoprecipitates were ands et al. (36) also reported that the B cell receptor complex is resolved on SDS͞PAGE, transferred electrophoretically onto required for oxidative stress signaling. Hydrogen peroxide- poly(vinylidene difluoride) membranes and then immunoblotted treated B cells (37, 38) have been shown to exhibit PTK- with the indicated antibodies. Immunoreactive proteins were 2ϩ dependent IP3 generation and Ca release. Comparative stud- visualized by enhanced chemiluminescence. ies by Qin et al. (37), by using Syk- and Lyn-negative DT40 cells, 2ϩ revealed that Syk and Lyn regulate Ca mobilization and IP3 PI3K Assay. The cell extracts from treated or untreated cells were production in B cells in response to oxidative stress, most likely incubated with antiphosphotyrosine antibody 4G10 for 30 min through tyrosine phosphorylation of PLC␥2. In this study, we followed by further incubation with protein A-agarose for 1 h. show that tyrosine phosphorylation of PLC␥2 is apparently The immunoprecipitates were washed three times with lysis ⅐ reduced but still significantly detectable in H2O2-treated Syk- buffer, twice in buffer containing 10 mM Tris HCl, pH 7.5, 100 negative DT40 cells, yet IP3 production was abolished, raising the mM NaCl, and 1 mM EDTA, and once in PI3K assay buffer (20 ⅐ ͞ ͞ ͞ question that tyrosine phosphorylation is a primary event but not mM Tris HCl, pH 7.5 100 mM NaCl 10 mM MgCl2 0.1 mM sufficient to fully activate PLC␥2. Taking into account recent EGTA͞100 mM vanadate͞20 mM ATP͞200 mM adenosine). progress in immunoreceptor-mediated PLC␥ activation, we in- After the last wash was removed, 10 ␮l of sonicated PI substrate vestigated the functions of PI3K and Bruton’s tyrosine kinase (1 mg͞ml in 10 mM Hepes, pH 7.5, 3 ϫ 20 s) was added to each (Btk), one member of the Tec family PTKs expressed in B cells, sample, and the samples were incubated for 10 min on ice. 2ϩ in H2O2-induced Ca mobilization. Our data