Targeting of Tiam1 to the Plasma Membrane Requires the Cooperative Function of the N-terminal Pleckstrin Homology Domain and an Adjacent Protein Interaction Domain

doi:10.1074/jbc.272.45.28447

Targeting of Tiam1 to the Plasma Membrane Requires the Cooperative Function of the N-terminal Pleckstrin Homology Domain and an Adjacent Protein Interaction Domain*

From The Netherlands Cancer Institute, Division of Cell Biology, 121 Plesmanlaan, 1066 CX Amsterdam, The Netherlands

Abstract

The Rho-like GTPases Cdc42, Rac, and Rho play key roles in the regulation of the actin cytoskeleton and are implicated in transcriptional activation and cell transformation. We have previously identified the invasion-inducing Tiam1 gene, which encodes an activator of Rac. In fibroblasts, Tiam1 induces Rac-mediated membrane ruffling, which requires the N-terminal pleckstrin homology (PHn) domain. Here we show that this PHn domain is part of a protein interaction domain, which mediates membrane localization of Tiam1. After subcellular fractionation, up to 50% of Tiam1 is recovered in the Triton X-100-insoluble high speed pellet that contains small protein complexes. The regions in Tiam1 that are responsible for these protein interactions comprise the PHn domain, an adjacent putative coiled coil region (CC), and an additional flanking region (Ex). Deletions in each of these regions abolish membrane localization of Tiam1 and membrane ruffling, suggesting that they function cooperatively. Indeed, only polypeptides encompassing the PHn-CC-Ex region, and not the PHn-CC or the Ex region, localize at the membrane. These results indicate that the N-terminal PH domain is part of a larger functional Tiam1 domain that mediates protein complex formation and membrane localization of Tiam1.

Footnotes

↵* This work was supported by grants from the Dutch Cancer Society and the Netherlands Organization for Scientific Research (to J. G. C.).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: The Netherlands Cancer Institute, Division of Cell Biology (H1), 121 Plesmanlaan, 1066 CX Amsterdam, The Netherlands. Tel.: 31-20-5121932; Fax: 31-20-5121944; E-mail: JColl{at}NKI.NL.
↵1 The abbreviations used are: PH, pleckstrin homology; PHn, N-terminal pleckstrin homology; DH, Dbl homology; DHR, discs large homology region; GEF, guanine nucleotide-exchange factor; MAPK, mitogen-activated protein kinase; FITC, fluorescein isothiocyanate; CSK, cytoskeleton; aa, amino acids; PAGE, polyacrylamide gel electrophoresis; TRITC, tetramethylrhodamine isothiocyanate.
↵2 F. N. van Leeuwen and E. E. Sander, unpublished results.
- Received April 30, 1997.
- Revision received July 8, 1997.