Biochem. J. (2001) 354
(73–78) (Printed in Great Britain)
Membrane recruitment of DOCK180 by binding to PtdIns(3,4,5)P3
Shin KOBAYASHI*, Toshiyuki SHIRAI†, Etsuko KIYOKAWA‡, Naoki MOCHIZUKI*, Michiyuki MATSUDA*1 and Yasuhisa FUKUI†
*Department of Pathology, Research Institute, International Medical Center of Japan, 1-21-1 Toyama, Shinjuku-ku, Tokyo 162-8655, Japan, †Department of Applied Biological Chemistry, The Graduate School of Agriculture and Life Sciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan, and ‡Department of Pathology, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan
DOCK180 was originally identified as one of two major proteins bound to the Crk oncogene product and became an archetype of the CDM family of proteins, including Ced-5 of Caenorhabditis elegans and Mbc of Drosophila melanogaster. Further study has suggested that DOCK180 is involved in the activation of Rac by the CrkII–p130Cas complex. With the use of deletion mutants of DOCK180, we found that the C-terminal region containing a cluster of basic amino acids was required for binding to and activation of Rac. This region showed high amino-acid sequence similarity to the consensus sequence of the phosphoinositide-binding site; this led us to examine whether this basic region binds to phosphoinositides. For this purpose we used PtdIns(3,4,5)P3-APB beads, as reported previously [Shirai, Tanaka, Terada, Sawada, Shirai, Hashimoto, Nagata, Iwamatsu, Okawa, Li et al. (1998) Biochim. Biophys. Acta 1402, 292–302]. By using various competitors, we demonstrated the specific binding of DOCK180 to PtdIns(3,4,5)P3. The expression of active phosphoinositide 3-kinase (PI-3K) did not enhance a DOCK180-induced increase in GTP-Rac; however, the expression of PI-3K translocated DOCK180 to the plasma membrane. Thus DOCK180 contained a phosphoinositide-binding domain, as did the other guanine nucleotide exchange factors with a Dbl homology domain, and was translocated to the plasma membrane on the activation of PI-3K.
Key words: CDM family proteins, pleckstrin homology domain, Rac.
Abbreviations used: DH domain, Dbl homology domain; EGFP, enhanced green fluorescent protein; GAP, GTPase-activating protein; GEF, guanine nucleotide exchange factor; GST, glutathione S-transferase; HA, haemagglutinin; PH domain, pleckstrin homology domain; PI-3K, phosphoinositide 3-kinase, PtdSer, phosphatidylserine.
1To whom correspondence should be addressed (e-mail mmatsuda@ri.imcj.go.jp).
Received 9 May 2000/27 September 2000; accepted 20 November 2000
The Biochemical Society, London ©
2001
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