Cdc42-interacting Protein 4 Mediates Binding of the Wiskott-Aldrich Syndrome Protein to Microtubules

doi:10.1074/jbc.275.11.7854

Cdc42-interacting Protein 4 Mediates Binding of the Wiskott-Aldrich Syndrome Protein to Microtubules*

From the Metabolism Branch, NCI, National Institutes of Health, Bethesda, Maryland 20892

Abstract

The Wiskott-Aldrich syndrome is an inherited X-linked immunodeficiency characterized by thrombocytopenia, eczema, and a tendency toward lymphoid malignancy. Lymphocytes from affected individuals have cytoskeletal abnormalities, and monocytes show impaired motility. The Wiskott-Aldrich syndrome protein (WASP) is a multi-domain protein involved in cytoskeletal organization. In a two-hybrid screen, we identified the protein Cdc42-interacting protein 4 (CIP4) as a WASP interactor. CIP4, like WASP, is a Cdc42 effector protein involved in cytoskeletal organization. We found that the WASP-CIP4 interaction is mediated by the binding of the Src homology 3 domain of CIP4 to the proline-rich segment of WASP. Cdc42 was not required for this interaction. Co-expression of CIP4 and green fluorescent protein-WASP in COS-7 cells led to the association of WASP with microtubules. In vitro experiments showed that CIP4 binds to microtubules via its NH₂ terminus. The region of CIP4 responsible for binding to active Cdc42 was localized to amino acids 383–417, and the mutation I398S abrogated binding. Deletion of the Cdc42-binding domain of CIP4 did not affect the colocalization of WASP with microtubules in vivo. We conclude that CIP4 can mediate the association of WASP with microtubules. This may facilitate transport of WASP to sites of substrate adhesion in hematopoietic cells.

Footnotes

↵* 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.
↵‡ To whom correspondence should be addressed: Metabolism Branch, NCI, National Institutes of Health, Rm. 4N-115, Bldg. 10, 9000 Rockville Pike, Bethesda, MD 20892. Tel.: 301-496-5387; Fax: 301-496-9956; E-mail: dstew@helix.nih.gov.
Abbreviations:

WASP

Wiskott-Aldrich syndrome protein

CIP4

Cdc42-interacting protein 4

GST

glutathione S-transferase

PCR

polymerase chain reaction

PBS

phosphate-buffered saline

SH

Src homology

PH

pleckstrin homology

GFP

green fluorescent protein

WIP

WASP-interacting protein

PIPES

1,4-piperazinediethanesulfonic acid

PAGE

polyacrylamide gel electrophoresis

TRITC

tetramethylrhodamine B isothiocyanate

GBD

GTPase binding domain

CRIB

Cdc42/Rac interacting and binding

GDPβS

guanyl-5′-yl thiophosphate

GTPγS

guanosine 5′-O-(thiotriphosphate)

PACSIN

protein kinase C and casein kinase 2 substrate in neurons

PSTPIP

proline, serine, threonine phosphatase-interacting protein

IGF1-Rβ

insulin-like growth factor 1-receptor β
- Received October 9, 1999.
- Revision received December 6, 1999.
The American Society for Biochemistry and Molecular Biology, Inc.