A sensitized genetic system for the analysis of murine B lymphocyte signal transduction pathways dependent on Bruton's tyrosine kinase

Anne B. Satterthwaite, Fiona Willis, Prim Kanchanastit, David Fruman, Lewis C. Cantley, Cheryl D. Helgason, R. Keith Humphries, Clifford A. Lowell, Melvin Simon, Michael Leitges, Alexander Tarakhovsky, Thomas F. Tedder, Ralf Lesche, Hong Wu, Owen N. Witte

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Abstract

Modifier screens have been powerful genetic tools to define signaling pathways in lower organisms. The identification of modifier loci in mice has begun to allow a similar dissection of mammalian signaling pathways. Transgenic mice (Btk(lo)) expressing 2.5% of endogenous levels of Bruton's tyrosine kinase (Btk) have B cell functional responses between those of wild- type and Btk-(/)- mice. We asked whether reduced dosage or complete deficiency of genes previously implicated as Btk regulators would modify the Btk(lo) phenotype. We used two independent assays of Btk-dependent B cell function. Proliferative response to B cell antigen receptor cross-linking in vitro was chosen as an example of a relatively simple, well-defined signaling system. In vivo response to type II T-independent antigens (TI-II) measures complex interactions among multiple cell types over time and may identify additional Btk pathways. All modifiers identified differentially affected these two assays, indicating that Btk mediates these processes via distinct mechanisms. Loss of Lyn, PTEN (phosphatase and tensin homolog), or SH2- containing inositol phosphatase suppressed the Btk(lo) phenotype in vitro but not in vivo, whereas CD19 and the p85α form of phosphoinositide 3-kinase behaved as Btk(lo) enhancers in vivo but not in vitro. Effects of Lyn, PTEN, or p85α haploinsufficiency were observed. Haploinsufficiency or complete deficiency of protein kinase C β, Fyn, CD22, Gαq, or Gα11 had no detectable effect on the function of Btk(lo) B cells. A transgenic system creating a reduction in dosage of Btk can therefore be used to identify modifier loci that affect B cell responses and quantitatively rank their contribution to Btk-mediated processes.

Original languageEnglish (US)
Pages (from-to)6687-6692
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume97
Issue number12
DOIs
StatePublished - Jun 6 2000

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Satterthwaite, A. B., Willis, F., Kanchanastit, P., Fruman, D., Cantley, L. C., Helgason, C. D., Humphries, R. K., Lowell, C. A., Simon, M., Leitges, M., Tarakhovsky, A., Tedder, T. F., Lesche, R., Wu, H., & Witte, O. N. (2000). A sensitized genetic system for the analysis of murine B lymphocyte signal transduction pathways dependent on Bruton's tyrosine kinase. Proceedings of the National Academy of Sciences of the United States of America, 97(12), 6687-6692. https://doi.org/10.1073/pnas.110146697