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

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 invasioninducing 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. Pleckstrin homology (PH)1 domains are protein motifs that have been identified in numerous signaling molecules (Refs. 1 and 2 and for reviews see Refs. 3 and 4). Proteins containing a PH domain include serine/threonine and tyrosine kinases, phospholipases, cytoskeletal proteins, and regulators of small GTPases of the Ras superfamily. PH domains have been implicated in the (transient) localization of proteins to the plasma membrane (3, 5-7). The precise binding properties of PH domains in vivo are largely unknown, but in vitro studies indicate that PH domains can bind to specific phosphoinositol lipids as well as to ␤␥ subunits of heterotrimeric G proteins (3, 4), both providing a link with membrane localization. Many PH domains show in vitro a rather promiscuous interaction with phospholipids and ␤␥ subunits, which raises the question how specificity is achieved in vivo.

Ankyrin–Tiam1 Interaction Promotes Rac1 Signaling and Metastatic Breast Tumor Cell Invasion and Migration

“…In breast tumor cells, such as SP-1 cells, Tiam1 is detected as a 200-kD protein (Fig. 1), which is similar to the Tiam1 described in other cell types (Woods and Bryant 1991; Michiels et al 1995; Nobes and Hall 1995; Van Leeuwen et al 1995, Van Leeuwen et al 1997; Hordijk et al 1997; Stam et al 1997; Bourguignon et al 2000). Tiam1, isolated from SP-1 cells, is also capable of carrying out GDP/GTP exchange for Rac1 in vitro (Fig.…”

“…6 A, a–c) is required for the activation of Rac1 signaling pathways leading to membrane ruffling and c-Jun NH 2 -terminal kinase activation (Michiels et al 1997; Stam et al 1997). Using a 49-kD E. coli –derived CBP-tagged Tiam1 fragment (i.e., amino acids 393–738 of Tiam1; Fig.…”

“…6 C) contains not only the putative ankyrin-binding domain (amino acids 717–727), but also the NH 2 -terminal pleckstrin homology (PHn), the coiled-coil region (CC) and an additional adjacent region (Ex) (also designated as PHn-CC-Ex domain; Michiels et al 1997). This Tiam1 fragment has been shown to be responsible for Tiam1's membrane localization, Rac1-dependent membrane ruffling, and C-Jun NH 2 -terminal kinase activation in fibroblasts and COS cells (Michiels et al 1997; Stam et al 1997). In this study, we have found that cotransfection of SP1 cells with Tiam1 fragment cDNA and C1199 Tiam1 cDNA effectively blocks tumor cell–specific behaviors (e.g., C1199 Tiam1 association with ankyrin in the cell membrane [Fig.…”

Tiam1 mediates neurite outgrowth induced by ephrin-B1 and EphA2

“…On the other hand, Tiam1 has been reported to activate by the lipid products of PI3-kinase in vitro (Fleming et al, 2000). Because PHn (amino-terminal PH domain) is required for the membrane localization and the binding of Tiam1 to the lipid products of PI3-kinase (Stam et al, 1997), the overexpression of PHnTSS is considered to block PI3-kinasedependent Tiam1 activation. Therefore, the inhibitory effect by N392-PHnTSS (Tiam1 1À669 ) on the neurite outgrowth of NB1 cells may include a mechanism that may not depend on the association of Tiam1 with EphA2 (Figure 8).…”

“…Although Rac1 was also slightly activated after stimulation with EphB2-Fc in ephrin-B1-expressing cells ( Figure 5A, lane 3), the amount of GTP-bound Rac1 was markedly increased in response to the stimulation by EphB2-Fc when both Tiam1 and ephrin-B1 are expressed ( Figure 5A, compare lanes 4 and 5). Because the PHnTSS region of Tiam1 works as a dominant-negative mutant of Tiam1 (Stam et al, 1997), we next examined whether PHnTSS of Tiam1 blocks the Rac1 activity induced by the activation of ephrin-B1. The activation of Rac1 induced by the stimulation of EphB2-Fc was completely abolished by the coexpression of PHnTSS of Tiam1, but not by the coexpression of Tiam1 (N392) ( Figure 5A, lanes 8 and 9).…”