Bruton's tyrosine kinase is essential for hydrogen peroxide-induced calcium signaling.
Using Btk-deficient DT40 cells and the transfectants expressing wild-type Btk or Btk mutants in either kinase (Arg(525) to Gln), Src homology 2 (SH2, Arg(307) to Ala), or pleckstrin homology (PH, Arg(28) to Cys) domains, we investigated the roles and structure-function relationships of Btk in hydrogen peroxide-induced calcium mobilization. Our genetic evidence showed that Btk deficiency resulted in a significant reduction in hydrogen peroxide-induced calcium response. This impaired calcium signaling is correlated with the complete elimination of IP3 production and the significantly reduced tyrosine phosphorylation of PLCgamma2 in Btk-deficient DT40 cells. All of these defects were fully restored by the expression of wild-type Btk in Btk-deficient DT40 cells. The data from the point mutation study revealed that a defect at any one of the three functional domains would prevent a full recovery of Btk-mediated hydrogen peroxide-induced intracellular calcium mobilization. However, mutation at either the SH2 or PH domain did not affect the hydrogen peroxide-induced activation of Btk. Mutation at the SH2 domain abrogates both IP3 generation and calcium release, while the mutant with the nonfunctional PH domain can partially activate PLCgamma2 and catalyze IP3 production but fails to produce significant calcium mobilization. Thus, these observations suggest that Btk-dependent tyrosine phosphorylation of PLCgamma2 is required but not sufficient for hydrogen peroxide-induced calcium mobilization. Furthermore, hydrogen peroxide stimulates a Syk-, but not Btk-, dependent tyrosine phosphorylation of B cell linker protein BLNK. The overall results, together with those reported earlier [Qin et al. (2000) Proc. Natl. Acad. Sci. U.S.A. 97, 7118], are consistent with the notion that functional SH2 and PH domains are required for Btk to form a complex with PLCgamma2 through BLNK in order to position the Btk, PLCgamma2, and phosphatidylinositol 4,5-bisphosphate in close proximity for efficient activation of PLCgamma2 and to maximize its catalytic efficiency for IP3 production.
Qin
S
S
Laboratory of Biochemistry, National Heart, Lung, and Blood Institute, National Institutes of Health, Building 50, Room 2134, Bethesda, Maryland 20892-8012, USA.
Chock
P B
PB
D048868:Adaptor Proteins, Signal Transducing; D000077329:Agammaglobulinaemia Tyrosine Kinase; D000361:Agammaglobulinemia / Q000201:enzymology / Q000235:genetics; D000818:Animals; D001402:B-Lymphocytes / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D001798:Blood Proteins / Q000235:genetics; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects / Q000235:genetics; D002352:Carrier Proteins / Q000378:metabolism; D020134:Catalytic Domain / Q000235:genetics; D002460:Cell Line; D002645:Chickens; D004789:Enzyme Activation / Q000187:drug effects / Q000235:genetics; D004792:Enzyme Precursors / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D015544:Inositol 1,4,5-Trisphosphate / Q000096:biosynthesis; D007424:Intracellular Fluid / Q000201:enzymology / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D051966:Phospholipase C gamma; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000172:deficiency / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D000072377:Syk Kinase; D014162:Transfection; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism; D018909:src Homology Domains / Q000235:genetics
D048868:Adaptor Proteins, Signal Transducing; C113939:B cell linker protein; D001798:Blood Proteins; D002352:Carrier Proteins; D004792:Enzyme Precursors; D047908:Intracellular Signaling Peptides and Proteins; D007527:Isoenzymes; D010750:Phosphoproteins; C039077:platelet protein P47; D014443:Tyrosine; D015544:Inositol 1,4,5-Trisphosphate; D006861:Hydrogen Peroxide; D011505:Protein-Tyrosine Kinases; D000077329:Agammaglobulinaemia Tyrosine Kinase; C000625949:BTK protein, human; C000606896:SYK protein, human; D000072377:Syk Kinase; D010738:Type C Phospholipases; D051966:Phospholipase C gamma; D002118:Calcium