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Direct inhibition of Bruton's tyrosine kinase by IBtk, a Btk-binding protein

Abstract

Bruton's tyrosine kinase (Btk) is required for human and mouse B cell development. Btk deficiency causes X-linked agammaglobulinemia (XLA) in humans and X-linked immunodeficiency in mice. Unlike Src proteins, Btk lacks a negative regulatory domain at the COOH terminus and may rely on cytoplasmic Btk-binding proteins to regulates its kinase activity by trans-inhibitor mechanisms. Consistent with this possibility, IBtk, which we identified as an inhibitor of Btk, bound to the PH domain of Btk. IBtk downregulated Btk kinase activity, Btk-mediated calcium mobilization and nuclear factor-κB–driven transcription. These results define a potential mechanism for the regulation of Btk function in B cells.

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Figure 1: Sequence of human IBtk.
Figure 2: Tissue expression of IBtk mRNA.
Figure 3: In vitro and in vivo interaction of IBtk and Btk.
Figure 4: Inhibition of Btk kinase activity by IBtk.
Figure 5: IBtk down-regulates the intracellular Ca2+ fluxes in B cells after BCR cross-linking.
Figure 6: IBtk interferes with the NF-κB activation induced by BCR triggering in DT40 B cells.

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Acknowledgements

We thank S. J. Elledge for the B cell cDNA library in pACT; F. A. Uckun for the DT40 cells; P. M. Schwartzberg for pBS-ItkF P; M. Ghedina for pZ-STAT; and J. J. O'Shea, M. J. Lenardo and P. L. Schwartzberg for careful reading of the manuscript. Supported by grants from AIRC, ISS, Telethon and MURST.

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Correspondence to Giuseppe Scala.

Supplementary information

Web Figure 1.

IBtk does not associate with Itk Tec kinase. 293T cells were transfected with pCDNA-IBtk together with pBS-ItkF, a plasmid expressing a Itk-FLAG fusion protein. Cell lysates from transfected cells were subjected to immunoprecipitation with anti-IBtk antibody to peptide 1 followed by immunoblotting with anti-Itk mAb (Upstate). No detectable Itk was recovered from immunoprecipitated proteins (lane 1). Itk was detected by immunoblotting in the cell lysate (20 µg) (lane 2). Expression of IBtk in lysates (20 µg) from transfected cells was detected by immunoblotting (lower panel). (GIF 10 kb)

41590_2001_BFni1001939_MOESM2_ESM.gif

Web Figure 2. IBtk does not interfere with the NF-κB activation induced by PMA in DT40 B cells. DT40 WT cells were transfected with pNF-κB-Luc (5 µg) and pCDNA-IBtk together with pRLTK plasmid. 16 h after transfection, cell aliquots were treated either with M4 anti-IgM or with phorbol 12-myristate 13-acetate (PMA) (10 ng/ml). Cell extracts were assayed for luciferase activity as detailed in Methods. (GIF 20 kb)

41590_2001_BFni1001939_MOESM3_ESM.gif

Web Figure 3. IBtk interference with the induction of transcription factors. MC3 cells (a, b, c) and Jurkat cells (d) were transfected with a mixture of 10 µg of pCDNA-IBtk, 5 ˙g of luciferase reporter plasmids and 2 µg of pRLTK plasmid expressing Renilla luciferase gene. (a) pZSTAT-Luc carries the STAT binding site of the IGF2/IRF promoter that is responsive to IFN-γ51; (b) pC/EBP-Luc carries the interleukin-6 (IL-6)-responsive cis-binding site of IL-6 promoter52; (c) pE2F-Luc harbors the E2F responsive sequence of E2F gene, which is activated during cell cycle progression53. After 16 h, cells were (a) stimulated for 12 h with interferon-γ (10 ng/ml, Genzyme [AUTHOR: location?]), (b) stimulated for 12 h with IL-6 (50 ng/ml, Genzyme), or (c) left untreated. (d) Jurkat cells were transfected with pNFAT-Luc (5 µg, Clontech) and stimulated with anti-CD3 (5 µg, Pharmingen) followed by goat anti-mouse antibody (5 µg, Caltag) to cross-link the TCR. Transcriptional levels were not affected by the expression of IBtk. (GIF 24 kb)

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Liu, W., Quinto, I., Chen, X. et al. Direct inhibition of Bruton's tyrosine kinase by IBtk, a Btk-binding protein. Nat Immunol 2, 939–946 (2001). https://doi.org/10.1038/ni1001-939

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