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Abstract
Defects in Bruton's tyrosine kinase (Btk) are responsible for X chromosome-linked agammaglobulinemia in patients. Mutations in each of the structural domains of Btk have been detected in patients, yet a mechanistic explanation for most of these mutant phenotypes is lacking. To understand the possible role of the unique pleckstrin homology and Tec homology (PHTH) module of Btk, we have compared the enzymatic properties of full-length Btk and a Btk mutant lacking the PHTH module (BtkDeltaPHTH). Here we show that Btk and BtkDeltaPHTH have similar basal catalytic activity but very different abilities to recognize protein substrates. Furthermore, the catalytic domain of Btk is inactive, in contrast to the catalytic domain of the prototypical Src tyrosine kinase that retains full catalytic ability. These data suggest that the PHTH module plays an important role in protein substrate recognition, that Btk and Src likely have different interdomain organizations and regulations, and that alterations in substrate recognition might play a role in X chromosome-linked agammaglobulinemia.
Highlights
Defects in Bruton’s tyrosine kinase (Btk) are responsible for X-chromosome linked agammaglobulinemia (XLA) in humans [4, 5] and X-chromosome linked immunodeficiency in mice [6, 7]
The pleckstrin homology and Tec homology (PHTH) module has been shown to be important for interaction with Vav, protein kinase C isoforms, G proteins G␥ and G␣12, F-actin, tyrosine kinase FAK, phosphotyrosine phosphatase PTPD1, transcriptional factors Stat 3 and BP-135/ TFII-I, and substrate BCR downstream signaling 1 (BRDG1) [1,2,3]
It had been proposed that the PHTH module could form intramolecular interactions with other parts of Btk family kinases, leading to suppression of its kinase activity [15, 29]
Summary
Protein Purification—Full-length Btk, PHTH-deleted Btk, and the catalytic domain of Btk were purified from Sf9 cells. Purified Btk or Btk⌬PHTH kinase (10 nM) in Btk kinase buffer (30 mM Hepes, pH 7.4, 10 mM MgCl2, and 10 M ATP) was combined with 70 M Btk substrate peptide. Vav (amino acid residues from 170 –375 of mouse Vav) or GST-CDB3 was used as substrate. Purification of these recombinant proteins has been described previously [11, 12]. When Km(ATP) was determined, the concentration of the peptide substrate was fixed at 100 M. Km and Vmax were determined by fitting data to the Michaelis-Menten equation. kcat was calculated as Vmax/[E] [14]
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