Function of Bruton’s Tyrosine Kinase during B Cell Development Is Partially Independent of Its Catalytic Activity

Abstract

The Tec family member Bruton’s tyrosine kinase (Btk) is a cytoplasmic protein tyrosine kinase that transduces signals from the pre-B and B cell receptor (BCR). Btk is involved in pre-B cell maturation by regulating IL-7 responsiveness, cell surface phenotype changes, and the activation of λ L chain gene rearrangements. In mature B cells, Btk is essential for BCR-mediated proliferation and survival. Upon BCR stimulation, Btk is transphosphorylated at position Y551, which promotes its catalytic activity and subsequently results in autophosphorylation at position Y223 in the Src homology 3 domain. To address the significance of Y223 autophosphorylation and the requirement of enzymatic activity for Btk function in vivo, we generated transgenic mice that express the autophosphorylation site mutant Y223F and the kinase-inactive mutant K430R, respectively. We found that Y223 autophosphorylation was not required for the regulation of IL-7 responsiveness and cell surface phenotype changes in differentiating pre-B cells, or for peripheral B cell differentiation. However, expression of the Y223F-Btk transgene could not fully rescue the reduction of λ L chain usage in Btk-deficient mice. In contrast, transgenic expression of kinase-inactive K430R-Btk completely reconstituted λ usage in Btk-deficient mice, but the defective modulation of pre-B cell surface markers, peripheral B cell survival, and BCR-mediated NF-κB induction were partially corrected. From these findings, we conclude that: 1) autophosphorylation at position Y223 is not essential for Btk function in vivo, except for regulation of λ L chain usage, and 2) during B cell development, Btk partially acts as an adapter molecule, independent of its catalytic activity.

Btk and phospholipase Cγ2 can function independently during B cell development

“…1 also demonstrates independent roles for Btk and PLCc2 in pre-BCR down-regulation and development beyond the pre-B stage. Some functions of Btk in pre-B cells do not require its catalytic activity, including control of Igk expression [6] and suppression of pre-B malignancies [30]. These are likely candidates for PLCc2-independent actions of Btk since a major mode of PLCc2 activation is phosphorylation by Btk [26,27].…”

“…3). Thus, normal Igk usage in newly generated B cell populations requires the expression of Btk and PLCc2 but not Btk kinase activity [6], suggesting a role for the PIP5K pathway [5].…”

“…Btk then phosphorylates and activates PLCc2, resulting in changes in intracellular Ca 2+ and the activation of protein kinase Cb. Btk can also mediate BCR-stimulated Ca 2+ flux [5] and B cell development [6] independently of its catalytic activity. Btk acts as an adaptor to recruit and activate phosphatidylinositol 4-phosphate 5-kinase (PIP5K) [5], which produces the substrate for PLCc2.…”

Constitutive activation of Bruton's tyrosine kinase induces the formation of autoreactive IgM plasma cells

“…2C) and micro-architecture of the spleen (data not shown). Moreover, we have previously found that Y223 phosphorylation is not essential for Btk function in vivo: Y223F-Btk can fully correct the features of the Btk-deficient phenotype, including pre-B-cell B-1 cell development, serum IgM levels and TI-II responses [27].The complex phenotype of mice with constitutively activated Btk largely resembles that of other Tg or knock-out mice with increased BCR signaling [12][13][14][15][16][17][18][19]. These mice also contain fewer follicular B cells, increased numbers of B-1 B cells, together with B-cell hyperresponsiveness and autoimmunity.…”

“…The pleckstrin homology domain mutant E41K-Btk displayed robust transformation potential in a soft-agar assay, increased membrane localization and phosphorylation in quiescent cells, independent of PI3K activity [26]. This capacity was augmented by mutation of the main autophosphorylation site in the SH3 domain, Y223F, although the role of Y223 phosphorylation for Btk function in vivo remains unclear [22,27]. We have previously reported that expression of Tg E41K-Btk throughout the B-cell lineage resulted in an almost complete deletion of immature B cells in the BM, irrespective of the presence of the endogenous intact Btk gene [28].…”