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Bruton's tyrosine kinase cooperates with the B cell linker protein SLP-65 as a tumor suppressor in Pre-B cells.

Kersseboom R, Middendorp S, Dingjan GM, Dahlenborg K, Reth M, Jumaa H, Hendriks RW - J. Exp. Med. (2003)

Bottom Line: Expression of the pre-B cell receptor (pre-BCR) leads to activation of the adaptor molecule SLP-65 and the cytoplasmic kinase Btk.Our recent findings of defective SLP-65 expression in approximately 50% of childhood pre-B acute lymphoblastic leukemias and spontaneous pre-B cell lymphoma development in SLP-65-/- mice demonstrate that SLP-65 acts as a tumor suppressor.To investigate cooperation between Btk and SLP-65, we characterized the pre-B cell compartment in single and double mutant mice, and found that the two proteins have a synergistic role in the developmental progression of large cycling into small resting pre-B cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, Erasmus MC Rotterdam, PO Box 1738, NL-3000 DR Rotterdam, Netherlands.

ABSTRACT
Expression of the pre-B cell receptor (pre-BCR) leads to activation of the adaptor molecule SLP-65 and the cytoplasmic kinase Btk. Mice deficient for one of these signaling proteins have an incomplete block in B cell development at the stage of large cycling pre-BCR+CD43+ pre-B cells. Our recent findings of defective SLP-65 expression in approximately 50% of childhood pre-B acute lymphoblastic leukemias and spontaneous pre-B cell lymphoma development in SLP-65-/- mice demonstrate that SLP-65 acts as a tumor suppressor. To investigate cooperation between Btk and SLP-65, we characterized the pre-B cell compartment in single and double mutant mice, and found that the two proteins have a synergistic role in the developmental progression of large cycling into small resting pre-B cells. We show that Btk/SLP-65 double mutant mice have a dramatically increased pre-B cell tumor incidence ( approximately 75% at 16 wk of age), as compared with SLP-65 single deficient mice (<10%). These findings demonstrate that Btk cooperates with SLP-65 as a tumor suppressor in pre-B cells. Furthermore, transgenic low-level expression of a constitutive active form of Btk, the E41K-Y223F mutant, prevented tumor formation in Btk/SLP-65 double mutant mice, indicating that constitutive active Btk can substitute for SLP-65 as a tumor suppressor.

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Low-level BtkAct expression can partially substitute for the absence of Btk and SLP-65. (A) Western blotting analysis of Btk expression in WT and BtkAct B cells from BM and spleen. Membrane was reblotted with anti-Erk. (B) Protein tyrosine phosphorylation in extracts of untreated and anti-IgM stimulated WT or BtkAct splenic B cells, analyzed by immunoblotting with a phosphotyrosine (pY)-specific antibody. Membrane was reblotted with anti-Erk. (C) Serum concentrations of IgM and IgG3 in the indicated mutant mouse strains. Mice were 2 mo of ages and each symbol represents an individual animal. (D) Flow cytometric analysis of surface IgM/IgD expression on total lymphoid cells in the spleen of the indicated mice. (E) Expression profiles of B220 and IgM on total lymphoid cells in the BM of the indicated mice (top). The B220+IgM- pro-/pre-B cell fractions were gated and analyzed for CD43/FSC and cytoplasmic SLC and μ H chain (bottom). Data are displayed as dot plots and the percentages of cells within the indicated quadrants or gates are given. Data shown are representative of four mice examined within each group.
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fig4: Low-level BtkAct expression can partially substitute for the absence of Btk and SLP-65. (A) Western blotting analysis of Btk expression in WT and BtkAct B cells from BM and spleen. Membrane was reblotted with anti-Erk. (B) Protein tyrosine phosphorylation in extracts of untreated and anti-IgM stimulated WT or BtkAct splenic B cells, analyzed by immunoblotting with a phosphotyrosine (pY)-specific antibody. Membrane was reblotted with anti-Erk. (C) Serum concentrations of IgM and IgG3 in the indicated mutant mouse strains. Mice were 2 mo of ages and each symbol represents an individual animal. (D) Flow cytometric analysis of surface IgM/IgD expression on total lymphoid cells in the spleen of the indicated mice. (E) Expression profiles of B220 and IgM on total lymphoid cells in the BM of the indicated mice (top). The B220+IgM- pro-/pre-B cell fractions were gated and analyzed for CD43/FSC and cytoplasmic SLC and μ H chain (bottom). Data are displayed as dot plots and the percentages of cells within the indicated quadrants or gates are given. Data shown are representative of four mice examined within each group.

Mentions: The finding that Btk apparently cooperated with SLP-65 as a tumor suppressor prompted us to investigate whether transgenic expression of a constitutive active Btk mutant could prevent tumor development in SLP-65-deficient mice. The PH domain gain-of-function mutant E41K shows increased membrane localization and phosphorylation in quiescent cells, independent of PI3K activity and induces transformation of 3T3 fibroblasts in soft agar cultures (26, 27). This capacity is augmented by mutation of the main autophosphorylation site in the Btk SH3 domain (Y223F; reference 28). In Ramos B cells expression of E41K-Btk enhances the sustained increase in intracellular [Ca2+] after BCR cross-linking (29). Thus, the E41K mutant and the E41K-Y223F double mutant represent activated forms of Btk. When E41K-Btk was expressed at physiological levels in transgenic mice under the control of the B cell–specific CD19 promoter, B cell development was arrested at the immature B cells in the BM (probably because the E41K-Btk mutant mimics BCR occupancy by auto-antigens), while residual B cells were efficiently driven into IgM plasma cell differentiation (21). We recently generated a panel of 7 independent E41K-Btk (n = 3) or E41K-Y223F-Btk (n = 4) transgenic mouse lines, which were crossed onto the Btk background. Expression of the two different mutants resulted in parallel phenotypes, whereby the deletion at the immature B cell stage was dose-dependent (unpublished data). From this panel we selected a low-copy E41K-Y223F-Btk transgenic mouse strain (BtkAct), in which the extent of the B cell arrest was limited, while the finding of enhanced protein tyrosine phosphorylation in splenic B cells and significantly increased serum IgM levels provided evidence for the constitutive active nature of BtkAct in vivo (see below; Fig. 4, A–C).


Bruton's tyrosine kinase cooperates with the B cell linker protein SLP-65 as a tumor suppressor in Pre-B cells.

Kersseboom R, Middendorp S, Dingjan GM, Dahlenborg K, Reth M, Jumaa H, Hendriks RW - J. Exp. Med. (2003)

Low-level BtkAct expression can partially substitute for the absence of Btk and SLP-65. (A) Western blotting analysis of Btk expression in WT and BtkAct B cells from BM and spleen. Membrane was reblotted with anti-Erk. (B) Protein tyrosine phosphorylation in extracts of untreated and anti-IgM stimulated WT or BtkAct splenic B cells, analyzed by immunoblotting with a phosphotyrosine (pY)-specific antibody. Membrane was reblotted with anti-Erk. (C) Serum concentrations of IgM and IgG3 in the indicated mutant mouse strains. Mice were 2 mo of ages and each symbol represents an individual animal. (D) Flow cytometric analysis of surface IgM/IgD expression on total lymphoid cells in the spleen of the indicated mice. (E) Expression profiles of B220 and IgM on total lymphoid cells in the BM of the indicated mice (top). The B220+IgM- pro-/pre-B cell fractions were gated and analyzed for CD43/FSC and cytoplasmic SLC and μ H chain (bottom). Data are displayed as dot plots and the percentages of cells within the indicated quadrants or gates are given. Data shown are representative of four mice examined within each group.
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Related In: Results  -  Collection

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fig4: Low-level BtkAct expression can partially substitute for the absence of Btk and SLP-65. (A) Western blotting analysis of Btk expression in WT and BtkAct B cells from BM and spleen. Membrane was reblotted with anti-Erk. (B) Protein tyrosine phosphorylation in extracts of untreated and anti-IgM stimulated WT or BtkAct splenic B cells, analyzed by immunoblotting with a phosphotyrosine (pY)-specific antibody. Membrane was reblotted with anti-Erk. (C) Serum concentrations of IgM and IgG3 in the indicated mutant mouse strains. Mice were 2 mo of ages and each symbol represents an individual animal. (D) Flow cytometric analysis of surface IgM/IgD expression on total lymphoid cells in the spleen of the indicated mice. (E) Expression profiles of B220 and IgM on total lymphoid cells in the BM of the indicated mice (top). The B220+IgM- pro-/pre-B cell fractions were gated and analyzed for CD43/FSC and cytoplasmic SLC and μ H chain (bottom). Data are displayed as dot plots and the percentages of cells within the indicated quadrants or gates are given. Data shown are representative of four mice examined within each group.
Mentions: The finding that Btk apparently cooperated with SLP-65 as a tumor suppressor prompted us to investigate whether transgenic expression of a constitutive active Btk mutant could prevent tumor development in SLP-65-deficient mice. The PH domain gain-of-function mutant E41K shows increased membrane localization and phosphorylation in quiescent cells, independent of PI3K activity and induces transformation of 3T3 fibroblasts in soft agar cultures (26, 27). This capacity is augmented by mutation of the main autophosphorylation site in the Btk SH3 domain (Y223F; reference 28). In Ramos B cells expression of E41K-Btk enhances the sustained increase in intracellular [Ca2+] after BCR cross-linking (29). Thus, the E41K mutant and the E41K-Y223F double mutant represent activated forms of Btk. When E41K-Btk was expressed at physiological levels in transgenic mice under the control of the B cell–specific CD19 promoter, B cell development was arrested at the immature B cells in the BM (probably because the E41K-Btk mutant mimics BCR occupancy by auto-antigens), while residual B cells were efficiently driven into IgM plasma cell differentiation (21). We recently generated a panel of 7 independent E41K-Btk (n = 3) or E41K-Y223F-Btk (n = 4) transgenic mouse lines, which were crossed onto the Btk background. Expression of the two different mutants resulted in parallel phenotypes, whereby the deletion at the immature B cell stage was dose-dependent (unpublished data). From this panel we selected a low-copy E41K-Y223F-Btk transgenic mouse strain (BtkAct), in which the extent of the B cell arrest was limited, while the finding of enhanced protein tyrosine phosphorylation in splenic B cells and significantly increased serum IgM levels provided evidence for the constitutive active nature of BtkAct in vivo (see below; Fig. 4, A–C).

Bottom Line: Expression of the pre-B cell receptor (pre-BCR) leads to activation of the adaptor molecule SLP-65 and the cytoplasmic kinase Btk.Our recent findings of defective SLP-65 expression in approximately 50% of childhood pre-B acute lymphoblastic leukemias and spontaneous pre-B cell lymphoma development in SLP-65-/- mice demonstrate that SLP-65 acts as a tumor suppressor.To investigate cooperation between Btk and SLP-65, we characterized the pre-B cell compartment in single and double mutant mice, and found that the two proteins have a synergistic role in the developmental progression of large cycling into small resting pre-B cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, Erasmus MC Rotterdam, PO Box 1738, NL-3000 DR Rotterdam, Netherlands.

ABSTRACT
Expression of the pre-B cell receptor (pre-BCR) leads to activation of the adaptor molecule SLP-65 and the cytoplasmic kinase Btk. Mice deficient for one of these signaling proteins have an incomplete block in B cell development at the stage of large cycling pre-BCR+CD43+ pre-B cells. Our recent findings of defective SLP-65 expression in approximately 50% of childhood pre-B acute lymphoblastic leukemias and spontaneous pre-B cell lymphoma development in SLP-65-/- mice demonstrate that SLP-65 acts as a tumor suppressor. To investigate cooperation between Btk and SLP-65, we characterized the pre-B cell compartment in single and double mutant mice, and found that the two proteins have a synergistic role in the developmental progression of large cycling into small resting pre-B cells. We show that Btk/SLP-65 double mutant mice have a dramatically increased pre-B cell tumor incidence ( approximately 75% at 16 wk of age), as compared with SLP-65 single deficient mice (<10%). These findings demonstrate that Btk cooperates with SLP-65 as a tumor suppressor in pre-B cells. Furthermore, transgenic low-level expression of a constitutive active form of Btk, the E41K-Y223F mutant, prevented tumor formation in Btk/SLP-65 double mutant mice, indicating that constitutive active Btk can substitute for SLP-65 as a tumor suppressor.

Show MeSH
Related in: MedlinePlus