Abstract
BackgroundBruton's tyrosine kinase (Btk) is essential for B cell development and function. Mutations of Btk elicit X-linked agammaglobulinemia in humans and X-linked immunodeficiency in the mouse. Btk has been proposed to participate in B cell antigen receptor-induced signaling events leading to activation of phospholipase C-γ2 (PLCγ2) and calcium mobilization. However it is unclear whether Btk activation is alone sufficient for these signaling events, and whether Btk can activate additional pathways that do not involve PLCγ2. To address such issues we have generated Btk:ER, a conditionally active form of the kinase, and expressed it in the PLCγ2-deficient DT40 B cell line.ResultsActivation of Btk:ER was sufficient to induce multiple B cell signaling pathways in PLCγ2-sufficient DT40 cells. These included tyrosine phosphorylation of PLCγ2, mobilization of intracellular calcium, activation of extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK) mitogen-activated protein kinase (MAPK) pathways, and apoptosis. In DT40 B cells deficient for PLCγ2, Btk:ER activation failed to induce the signaling events described above with the consequence that the cells failed to undergo apoptosis.ConclusionsThese data suggest that Btk:ER regulates downstream signaling pathways primarily via PLCγ2 in B cells. While it is not known whether activated Btk:ER precisely mimics activated Btk, this conditional system will likely facilitate the dissection of the role of Btk and its family members in a variety of biological processes in many different cell types.
Highlights
Bruton's tyrosine kinase (Btk) is essential for B cell development and function
We show that Btk:estrogen receptor (ER) activation alone is sufficient to activate multiple downstream signaling pathways in B cells, including calcium mobilization, extracellular signal-regulated kinase (ERK) and Jun NH2terminal kinase (JNK) mitogen-activated protein kinase (MAPK), and apoptosis
Apoptosis was detected in parental and phospholipase C-γ2 (PLCγ2)-deficient cells in response to the control PMA and ionomycin treatment (Fig 5C). These data suggest that Btk:ER induces apoptosis in DT40 B cells by a PLCγ2-dependent mechanism
Summary
Bruton's tyrosine kinase (Btk) is essential for B cell development and function. Mutations of Btk elicit X-linked agammaglobulinemia in humans and X-linked immunodeficiency in the mouse. Btk has been proposed to participate in B cell antigen receptor-induced signaling events leading to activation of phospholipase C-γ2 (PLCγ2) and calcium mobilization. It is unclear whether Btk activation is alone sufficient for these signaling events, and whether Btk can activate additional pathways that do not involve PLCγ2. To address such issues we have generated Btk:ER, a conditionally active form of the kinase, and expressed it in the PLCγ2-deficient DT40 B cell line. Mutations in Bruton's tyrosine kinase (Btk) are responsible for the human disease termed X-linked agammaglobulinemia (XLA) (reviewed in reference [1]). Btk was originally identified in B cells but is known to be expressed in most leukocytes with the exception of T cells and NK cells
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