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A conditional form of Bruton's tyrosine kinase is sufficient to activate multiple downstream signaling pathways via PLC Gamma 2 in B cells.

Tomlinson MG, Woods DB, McMahon M, Wahl MI, Witte ON, Kurosaki T, Bolen JB, Johnston JA - BMC Immunol. (2001)

Bottom Line: 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 PLCgamma2.In DT40 B cells deficient for PLCgamma2, Btk:ER activation failed to induce the signaling events described above with the consequence that the cells failed to undergo apoptosis.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: DNAX Research Institute of Molecular and Cellular Biology, Palo Alto, California 94304, USA. miket@itsa.ucsf.edu

ABSTRACT

Background: 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-gamma2 (PLCgamma2) 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 PLCgamma2. To address such issues we have generated Btk:ER, a conditionally active form of the kinase, and expressed it in the PLCgamma2-deficient DT40 B cell line.

Results: Activation of Btk:ER was sufficient to induce multiple B cell signaling pathways in PLCgamma2-sufficient DT40 cells. These included tyrosine phosphorylation of PLCgamma2, 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 PLCgamma2, Btk:ER activation failed to induce the signaling events described above with the consequence that the cells failed to undergo apoptosis.

Conclusions: These data suggest that Btk:ER regulates downstream signaling pathways primarily via PLCgamma2 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.

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Btk:ER-induced ERK and JNK activation, and apoptosis, are dependent on PLCγ2. A, Btk:ER-induced ERK MAPK activation is dependent on PLCγ2. Parental and PLCγ2-deficient cells expressing Btk:ER were treated for 60-min with vehicle control (0.1% ethanol), 4-HT (1 μM) and/or anti-μ mAb M4 (4 μg/ml), or for 15-min with PMA (100 ng/ml) as a positive control. ERK activation was measured by immune complex kinase assay as described in the legend for Fig 3A. B, Btk:ER-induced JNK phosphorylation is dependent on PLCγ2. Parental and PLCγ2-deficient cells expressing Btk:ER were treated for 30-min with vehicle control (0.1% ethanol), 4-HT (1 μM) and/or anti-μ mAb M4 (4 μg/ml), or for 10-min with PMA (100 ng/ml) and ionomycin (250 ng/ml) as a positive control. JNK phosphorylation was measured by immunblotting with anti-active JNK phosphospecific antiserum as described in the legend for Fig 3B. C, Btk:ER-induced apoptosis is dependent on PLCγ2. Apoptosis of parental and PLCγ2-deficient cells expressing Btk:ER was measured by TUNEL assay as described in the legend for Fig 3C. PMA (100 ng/ml) and ionomycin (250 ng/ml) were used as a positive control. These data are representative of three independent stable clones.
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Figure 5: Btk:ER-induced ERK and JNK activation, and apoptosis, are dependent on PLCγ2. A, Btk:ER-induced ERK MAPK activation is dependent on PLCγ2. Parental and PLCγ2-deficient cells expressing Btk:ER were treated for 60-min with vehicle control (0.1% ethanol), 4-HT (1 μM) and/or anti-μ mAb M4 (4 μg/ml), or for 15-min with PMA (100 ng/ml) as a positive control. ERK activation was measured by immune complex kinase assay as described in the legend for Fig 3A. B, Btk:ER-induced JNK phosphorylation is dependent on PLCγ2. Parental and PLCγ2-deficient cells expressing Btk:ER were treated for 30-min with vehicle control (0.1% ethanol), 4-HT (1 μM) and/or anti-μ mAb M4 (4 μg/ml), or for 10-min with PMA (100 ng/ml) and ionomycin (250 ng/ml) as a positive control. JNK phosphorylation was measured by immunblotting with anti-active JNK phosphospecific antiserum as described in the legend for Fig 3B. C, Btk:ER-induced apoptosis is dependent on PLCγ2. Apoptosis of parental and PLCγ2-deficient cells expressing Btk:ER was measured by TUNEL assay as described in the legend for Fig 3C. PMA (100 ng/ml) and ionomycin (250 ng/ml) were used as a positive control. These data are representative of three independent stable clones.

Mentions: In Fig 5 we have tested whether PLCγ2 is required for Btk:ER-induced ERK and JNK activation, and apoptosis. Two different BCR signaling pathways can result in ERK activation in DT40 cells, namely a PLCγ2-independent Grb2/Sos pathway and a PLCγ2-dependent pathway that requires protein kinase C [38]. In Fig 5A we found that Btk:ER activated ERK MAPK in parental but not in PLCγ2-deficient cells (lanes 2 and 7). Thus Btk:ER appears to induce ERK activation through its capacity to activate PLCγ2, and not through a Grb2/Sos pathway. In response to a 60-min treatment with anti-μ, ERK activation was detected in parental but not in PLCγ2-deficient cells (Fig 5A, lanes 3-4 and 8-9), but control PMA treatment activated ERK in both cell types (Fig 5A, lanes 5 and 10). Transient ERK activation, 5 to 10-min after anti-μ treatment, can be detected in PLCγ2-deficient cells [13]. These kinetics are identical to those observed in Btk-deficient cells [13]. Together with our Btk:ER data, this suggests that Btk:ER controls sustained ERK activation in B cells by regulating PLCγ2 activity.


A conditional form of Bruton's tyrosine kinase is sufficient to activate multiple downstream signaling pathways via PLC Gamma 2 in B cells.

Tomlinson MG, Woods DB, McMahon M, Wahl MI, Witte ON, Kurosaki T, Bolen JB, Johnston JA - BMC Immunol. (2001)

Btk:ER-induced ERK and JNK activation, and apoptosis, are dependent on PLCγ2. A, Btk:ER-induced ERK MAPK activation is dependent on PLCγ2. Parental and PLCγ2-deficient cells expressing Btk:ER were treated for 60-min with vehicle control (0.1% ethanol), 4-HT (1 μM) and/or anti-μ mAb M4 (4 μg/ml), or for 15-min with PMA (100 ng/ml) as a positive control. ERK activation was measured by immune complex kinase assay as described in the legend for Fig 3A. B, Btk:ER-induced JNK phosphorylation is dependent on PLCγ2. Parental and PLCγ2-deficient cells expressing Btk:ER were treated for 30-min with vehicle control (0.1% ethanol), 4-HT (1 μM) and/or anti-μ mAb M4 (4 μg/ml), or for 10-min with PMA (100 ng/ml) and ionomycin (250 ng/ml) as a positive control. JNK phosphorylation was measured by immunblotting with anti-active JNK phosphospecific antiserum as described in the legend for Fig 3B. C, Btk:ER-induced apoptosis is dependent on PLCγ2. Apoptosis of parental and PLCγ2-deficient cells expressing Btk:ER was measured by TUNEL assay as described in the legend for Fig 3C. PMA (100 ng/ml) and ionomycin (250 ng/ml) were used as a positive control. These data are representative of three independent stable clones.
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Figure 5: Btk:ER-induced ERK and JNK activation, and apoptosis, are dependent on PLCγ2. A, Btk:ER-induced ERK MAPK activation is dependent on PLCγ2. Parental and PLCγ2-deficient cells expressing Btk:ER were treated for 60-min with vehicle control (0.1% ethanol), 4-HT (1 μM) and/or anti-μ mAb M4 (4 μg/ml), or for 15-min with PMA (100 ng/ml) as a positive control. ERK activation was measured by immune complex kinase assay as described in the legend for Fig 3A. B, Btk:ER-induced JNK phosphorylation is dependent on PLCγ2. Parental and PLCγ2-deficient cells expressing Btk:ER were treated for 30-min with vehicle control (0.1% ethanol), 4-HT (1 μM) and/or anti-μ mAb M4 (4 μg/ml), or for 10-min with PMA (100 ng/ml) and ionomycin (250 ng/ml) as a positive control. JNK phosphorylation was measured by immunblotting with anti-active JNK phosphospecific antiserum as described in the legend for Fig 3B. C, Btk:ER-induced apoptosis is dependent on PLCγ2. Apoptosis of parental and PLCγ2-deficient cells expressing Btk:ER was measured by TUNEL assay as described in the legend for Fig 3C. PMA (100 ng/ml) and ionomycin (250 ng/ml) were used as a positive control. These data are representative of three independent stable clones.
Mentions: In Fig 5 we have tested whether PLCγ2 is required for Btk:ER-induced ERK and JNK activation, and apoptosis. Two different BCR signaling pathways can result in ERK activation in DT40 cells, namely a PLCγ2-independent Grb2/Sos pathway and a PLCγ2-dependent pathway that requires protein kinase C [38]. In Fig 5A we found that Btk:ER activated ERK MAPK in parental but not in PLCγ2-deficient cells (lanes 2 and 7). Thus Btk:ER appears to induce ERK activation through its capacity to activate PLCγ2, and not through a Grb2/Sos pathway. In response to a 60-min treatment with anti-μ, ERK activation was detected in parental but not in PLCγ2-deficient cells (Fig 5A, lanes 3-4 and 8-9), but control PMA treatment activated ERK in both cell types (Fig 5A, lanes 5 and 10). Transient ERK activation, 5 to 10-min after anti-μ treatment, can be detected in PLCγ2-deficient cells [13]. These kinetics are identical to those observed in Btk-deficient cells [13]. Together with our Btk:ER data, this suggests that Btk:ER controls sustained ERK activation in B cells by regulating PLCγ2 activity.

Bottom Line: 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 PLCgamma2.In DT40 B cells deficient for PLCgamma2, Btk:ER activation failed to induce the signaling events described above with the consequence that the cells failed to undergo apoptosis.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: DNAX Research Institute of Molecular and Cellular Biology, Palo Alto, California 94304, USA. miket@itsa.ucsf.edu

ABSTRACT

Background: 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-gamma2 (PLCgamma2) 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 PLCgamma2. To address such issues we have generated Btk:ER, a conditionally active form of the kinase, and expressed it in the PLCgamma2-deficient DT40 B cell line.

Results: Activation of Btk:ER was sufficient to induce multiple B cell signaling pathways in PLCgamma2-sufficient DT40 cells. These included tyrosine phosphorylation of PLCgamma2, 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 PLCgamma2, Btk:ER activation failed to induce the signaling events described above with the consequence that the cells failed to undergo apoptosis.

Conclusions: These data suggest that Btk:ER regulates downstream signaling pathways primarily via PLCgamma2 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.

Show MeSH
Related in: MedlinePlus