Pleckstrin Homology Domains Interact with Filamentous Actin

doi:10.1074/jbc.274.28.19752

Pleckstrin Homology Domains Interact with Filamentous Actin*

From the Division of Allergy, La Jolla Institute for Allergy and Immunology, San Diego, California 92121, the ^§Division of Experimental Medicine, Brigham and Women’s Hospital, Boston, Massachusetts 02115, the ^¶Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California 92037, and the ^‖Department of Clinical Molecular Biology, Faculty of Medicine, Kyoto University, Kawahara-cho Shogoin, Sakyo-ku, Kyoto 606, Japan

Abstract

A fraction of Bruton’s tyrosine kinase (Btk) co-localizes with actin fibers upon stimulation of mast cells via the high affinity IgE receptor (FcεRI). In this study, a molecular basis of the Btk co-localization with actin fibers is presented. Btk and other Tec family tyrosine kinases have a pleckstrin homology (PH) domain at their N termini. The PH domain is a short peptide module frequently found in signal-transducing proteins and cytoskeletal proteins. Filamentous actin (F-actin) is shown to be a novel ligand for a subset of PH domains, including that of Btk. The actin-binding site was mapped to a 10-residue region of the N-terminal region of Btk. Basic residues in this short stretch are demonstrated to be involved in actin binding. Isolated PH domains induced actin filament bundle formation. Consistent with these observations, Btk binds F-actinin vitro and in vivo. Wild-type Btk protein is in part translocated to the cytoskeleton upon FcεRI cross-linking, whereas Btk containing a mutated PH domain is not. Phosphatidylinositol 3,4,5-trisphosphate-mediated membrane translocation of Btk was enhanced in cytochalasind-pretreated, FcεRI-stimulated mast cells. These data indicate that PH domain-mediated F-actin binding plays a role in Btk co-localization with actin filaments.

Footnotes

↵* This work was supported in part by National Institutes of Health Grants AI33617, AI38348 (to T. K.), AR38910 (to P. J.), GM42388 (to J. R. A.), and U19 AI42244 (to T. K. and J. R. A.). This is Publication Number 161 from the La Jolla Institute for Allergy and Immunology.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.
↵‡ Present address: The Fourth Military Medical University, Xi’an, 710032, People’s Republic of China.
↵** To whom correspondence should be addressed: La Jolla Institute for Allergy and Immunology, 10355 Science Center Dr., San Diego, CA 92121. Tel.: 619-558-3500; Fax: 619-558-3526, E-mail: toshi_kawakami@liai.org.
Abbreviations:

Btk

Bruton’s tyrosine kinase

BMMC

bone marrow-derived mouse mast cells

DNP

dinitrophenyl

F-actin

filamentous actin

FcεRI

the high affinity IgE receptor

G-actin

globular actin

GST

glutathioneS-transferase

OSBP

oxysterol-binding protein

PH

pleckstrin homology

PIP₂

phosphatidylinositol 4,5-bisphosphate

PIP₃

phosphatidylinositol 3,4,5-trisphosphate

PKC

protein kinase C

PLC

phospholipase C

PTK

protein-tyrosine kinase

SH

Src homology

TH

Tec homology

mAb

monoclonal antibody

PAGE

polyacrylamide gel electrophoresis

PCR

polymerase chain reaction

MARCKS

myristoylated alanine-rich C kinase substrate
- Received January 4, 1999.
- Revision received April 6, 1999.
The American Society for Biochemistry and Molecular Biology, Inc.