Mutation of the Pleckstrin Homology Domain of Bruton's Tyrosine Kinase in Immunodeficiency Impaired Inositol 1,3,4,5-Tetrakisphosphate Binding Capacity

doi:10.1074/jbc.271.48.30303

Mutation of the Pleckstrin Homology Domain of Bruton's Tyrosine Kinase in Immunodeficiency Impaired Inositol 1,3,4,5-Tetrakisphosphate Binding Capacity*

From the ^‡ Molecular Neurobiology Laboratory, Tsukuba Life Science Center, The Institute of Physical and Chemical Research (RIKEN), 3-1-1 Koyadai, Tsukuba, Ibaraki 305, Japan,
^§ Department of Molecular Neurobiology, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108, Japan, and
^∥ Calciosignal Net Project, Exploratory Research for Advanced Technology (ERATO), 2-9-3 Shimo-meguro, Meguro-ku, Tokyo 153, Japan

^∥ To whom correspondence should be addressed:
Molecular Neurobiology Laboratory, Tsukuba Life Science Center, The Institute of Physical and Chemical Research (RIKEN), 3-1-1 Koyadai, Tsukuba, Ibaraki 305, Japan.
Tel.: 81-298-36-9170; Fax: 81-298-36-9040; E-mail: fukuda{at}rtc.riken.go.jp.

Abstract

Bruton's tyrosine kinase (Btk), a cytoplasmic protein-tyrosine kinase, plays a pivotal role in B cell activation and development. Mutations in the pleckstrin homology (PH) domain of the Btk gene cause human X-linked agammaglobulinemia (XLA) and murine X-linked immunodeficiency (Xid). In this paper, we report that the PH domain of Btk functions as an inositol 1,3,4,5-tetrakisphosphate (IP₄), inositol 1,3,4,5,6-pentakisphosphate, and inositol 1,2,3,4,5,6-hexakisphosphate (IP₆) binding domain (K_d of approximately 40 nM for IP₄), and that all of the XLA (Phe replaced by Ser at position 25 (F25S), R28H, T33P, V64F, and V113D) and Xid mutations (R28C) found in the PH domain result in a dramatic reduction of IP₄ binding activity. Furthermore, the rare alternative splicing variant, with 33 amino acids deleted in the PH domain, corresponding to exon 3 of the Btk gene, also impaired IP₄ binding capacity. In contrast, a gain-of-function mutant called Btk*, which carries a E41K mutation in the PH domain, binds IP₆ with two times higher affinity than the wild type. Our data suggest that B cell differentiation is closely correlated with the IP₄ binding capacity of the PH domain of Btk.

Footnotes

↵* This work was supported in part by grants from the Japanese Ministry of Education, Science, Sports, and Culture (to K. M.) and the Japan Society for the Promotion of Science (to M. F.). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

Btk

Bruton's tyrosine kinase

PH

pleckstrin homology

TH

Tec homology

SH

Src homology

XLA

X-linked agammaglobulinemia

Xid

X-linked immunodeficiency

PKC

protein kinase C

IP₄

inositol 1,3,4,5-tetrakisphosphate

IP₅

inositol 1,3,4,5,6-pentakisphosphate

IP₆

inositol 1,2,3,4,5,6-hexakisphosphate

IP₃

inositol 1,4,5-trisphosphate

PCR

polymerase chain reaction

GST

glutathione S-transferase.
↵² T. Kojima, M. Fukuda, and K. Mikoshiba, manuscript in preparation.
- Received September 3, 1996.
- Revision received October 4, 1996.