In previous studies, we have shown ezrin to be an important mediator of breast cancer cell migration (Ng et al., 2001
) and a number of recent articles have highlighted the significant contribution of ezrin to the clinical development of metastasis in both osteosarcoma and rhabdomyosarcoma (Khanna et al., 2004
; Yu et al., 2004
; Elliott et al., 2005
). Mechanistically, ERM proteins have been implicated in the regulation of the Rho family of small GTPases either through inhibition of the Rho-specific GDP dissociation inhibitor RhoGDI (Takahashi et al., 1997
) or through interaction with the Cdc42/Rho-specific guanine exchange factor Dbl (Vanni et al., 2004
). The functional consequence of the ERM-Dbl complex on Rho GTPase activity has to date however only been demonstrated for RhoA. For instance, in the context of uropod formation in lymphocytes, the C-terminal threonine-phosphorylated form of full-length ezrin was shown to associate with Dbl through its NH2
-terminal domain and causes Rho activation (Lee et al., 2004
). Here we define a novel mechanism by which ezrin is important and necessary for spatial activation of Cdc42 in MDA-MB-231 cells. We observe that Dbl is recruited to GM1-positive microdomains in the plasma membrane in an ERM-dependent manner, and that onco-Dbl induced morphological changes are dependent on Cdc42 and functional ERM proteins. This suggests a mechanism whereby Dbl interacts with ezrin and/or other ERM proteins and is thereby recruited to the plasma membrane and to lipid raft microdomains, enabling a localized activation of Cdc42. Furthermore, these breast cancer cells do not have a detectable amount of GTP-bound RhoA and do not ordinarily form stress fibers or focal adhesions. The effect of N-ERMAD(E244K), through an abrogation of endogenous ERM-Dbl association, is therefore likely to be specific via a down-regulation of Cdc42 activity.
Previous experiments detected ERM proteins in lipid rafts using quantitative proteomic mass spectrometry analyses (Foster et al., 2003
; Li et al., 2003
). Using density gradient fractionation techniques and caveolin as a lipid raft marker, we found only a small fraction of endogenous ezrin to localize to the fractions that contain caveolin (#6-#8; Supplementary Figure S2). The low percentage of total ezrin residing in lipid rafts at any one time makes relatively insensitive biochemical techniques such as sucrose gradient fractionation unsuitable for a quantitative analysis of changes in distribution.
On the basis of structure-function analyses and BIAcore assays, we have modified the dominant inhibitory strategy, which is based on ezrin N-ERMAD, and created a point mutant (E244K) that has an impaired ability to bind to the C-ERMAD. This mutated N-ERMAD domain retains the ability to inhibit PKC-mediated cell migration in a dominant negative manner. Notably, the C-terminal threonine phosphorylation of endogenous ERM induced by phorbol ester was inhibited by both N-ERMAD and N-ERMAD(E244K) despite the 400-fold reduction of N-ERMAD:C-ERMAD affinity caused by the point mutation. Hence, for the first time, we have demonstrated that the functional inhibition by the N-ERMAD domain is enhanced despite a decreased association with endogenous ERM proteins. As both N-ERMAD and N-ERMAD(E244K) locate at the plasma membrane, we postulate that the decrease in N-ERMAD:C-ERMAD association increases the availability of N-ERMAD(E244K) and that the dominant inhibition is instead achieved by abrogating the interaction between endogenous ERM and various N- ERMAD–interacting partners that are involved in the conformational activation of ERM proteins (Fievet et al., 2004
; reviewed in Bretscher et al., 2002
). The T567D form rescues because it does not require the activation machinery, i.e., it is already in the open conformation to bind to Dbl at the right location, where the membrane is locally tethered to the cytoskeleton. Potential N-ERMAD–interacting partners that contribute to the activation process include the various protein kinases that phosphorylate the conserved C-terminal threonine (myotonic dystrophy kinase-related Cdc42-binding kinase [Nakamura et al., 2000
], protein kinase Cα [Ng et al., 2001
], and Nck-interacting kinase [Baumgartner et al., 2006
]) as well as the phosphoinositide PIP2
(Fievet et al., 2004
head) binding was shown to require several lysine residues from subdomains A and C of N-ERMAD, which form the positively charged molecular surface to interact with the negatively charged membrane inositol polyphosphates and, in the absence of PIP2
, moesin does not bind CD44 in vitro (Hamada et al., 2000
). Although the effect of the E244K on PIP2
binding was not formally tested, the non-PIP2
-binding mutated (K253/254N, K262/263N) form of ezrin N-ERMAD was shown to be restricted to the cytoplasm, an effect that we did not observe with the E244K mutation. The identification of the precise binding protein that is “titrated” away by the N-ERMAD(E244K) will be the subject of further investigation.
Expression of N-ERMAD(E244K) resulted in a decrease of Cdc42 recruitment to lipid rafts. This was in addition to a decrease in the localized association of Cdc42 with its downstream effector PAK, and a global reduction in active, GTP-bound Cdc42. No changes were observed in the closely related GTPase Rac1, which led us to further investigate specific GEFs involved in regulation of GTPase activity. We identified Dbl as an upstream activator and exchange factor specific to Cdc42 in MDA-MB-231 cells, which was partially inhibited by the presence of N-ERMAD(E244K). Coexpression of N-ERMAD(E244K) could not reverse the phenotype after expression of onco-Dbl or mimic the phenotype of N-ERMAD(E244K) expression alone, possibly due to the much enhanced activity state and the decrease in protein turnover of onco-Dbl when compared with the endogenous proto-Dbl (Ron et al., 1989
; Kamynina et al., 2007
). Conversely, no changes were detected when analyzing the cell morphology induced by Cdc42-specific FGD1, Rac-specific Tiam1, or Rho-specific p190RhoGEF, in the absence or presence of N-ERMAD(E244K). Here we report that the morphological changes induced by onco-Dbl are dependent on the downstream effector Cdc42, and notably, the function of ERM proteins.
N-ERMAD(E244K) expression inhibits both the correct spatial recruitment of Dbl to the plasma membrane as well as the lipid raft recruitment and activation of Cdc42. Furthermore, in support of our hypothesis that the N-ERMAD(E244K)-mediated inhibition of endogenous Dbl recruitment to the plasma membrane was caused by a dominant inhibition of C-terminal phosphorylation of endogenous ERM proteins, this effect was shown to be reversed by overexpression of the phosphomimetic (T567D), rather than the nonphosphorylatable (T567A) form of ezrin. Together, these findings suggest that the active (i.e., C-terminally phosphorylated) form of ERM proteins, through forming a protein complex with Dbl, contributes toward Cdc42 activation in the correct subcellular localization, in response to promigratory signals. The activation of lipid raft localized Cdc42 (and its downstream effector PAK1; Krautkramer et al., 2004
) in turn can stimulate rapid lipid raft patch accumulation (Golub and Caroni, 2005
) and hence promote actin cytoskeleton accumulation and sustained protrusive activity at the leading edge.
Correct subcellular localization of activated species of GTPases is emerging as a common mechanism in the regulation of different dynamic cellular processes. Rap1 promotes cell spreading by localizing the Rac-specific GEFs VAV2 and Tiam1 to the plasma membrane and concurrent Rac1 activation (Arthur et al., 2004
). Moreover, inactivation of Rap1 can inhibit cell spreading promoted by constitutive active forms of VAV2 and Tiam1. This indicates that even the VAV2/Tiam1-promoted increase in Rac1 activity is not sufficient to promote cell spreading highlighting the significance of proper subcellular localization (Arthur et al., 2004
). In Saccharomyces cerevisiae
, targeting of the Cdc42p-specific GEF Cdc24p to the incipient bud site is essential for the recruitment and activation of Cdc42p, and deletion of the Dbl-homology domain responsible for targeting Cdc24p to the budding site is lethal and unable to complement the growth defects of Cdc24
Δ cells (Ziman and Johnson, 1994
; Toenjes et al., 1999
; Shimada et al., 2004
). This indicates an evolutionarily conserved mechanism for proper localized GTPase activation. In the work presented here, we suggest a similar mechanism whereby ERM proteins are involved in the activation of Cdc42 in lipid raft microdomains at the leading edge of migrating cancer cells, via recruitment of the Cdc42/Rho-specific GEF Dbl.