Rac promotes normal cell proliferation and cancer invasion (Sahai and Marshall, 2002
). To exert its functions, Rac needs to dissociate from Rho-GDI and attach to the inner aspect of the plasma membrane. Our study provides evidence that the tumor suppressor Merlin mediates contact inhibition of growth by blocking recruitment of Rac to matrix adhesions. Upon activation, PAK reverses this inhibition by phosphorylating, and thus inactivating, Merlin. These results place Merlin at the center of a novel tumor suppressor–oncogene circuit and provide a framework to interpret its function in tumorigenesis.
We have observed that PAK signaling is sufficient to release endothelial cells from contact inhibition. By contrast, activation of other mitogenic signaling pathways, including those involving β-catenin, phosphatidylinositol-3 kinase, and ERK, does not exert this effect. Upon introduction in sparse cells, dominant-negative PAK promotes growth arrest, suggesting that PAK kinase activity is required for normal cell cycle progression. Activated PAK promotes recruitment of Rac to matrix adhesions in confluent cells, whereas dominant-negative Rac blocks PAK's ability to release cells from contact inhibition, indicating that PAK promotes mitogenesis by inducing recruitment of Rac to matrix adhesions. Two distinct mutant forms of Merlin, Merlin-ΔBB and Merlin-S518D, which are presumed to exert a dominant-negative effect, promote recruitment of Rac to matrix adhesions and release cells from contact inhibition. By contrast, an unphosphorylatable form of Merlin, Merlin-S518A, suppresses PAK's ability to release HUVEC from contact inhibition. These observations indicate that PAK promotes recruitment of Rac to matrix adhesions and mitogenesis by phosphorylating and thereby inactivating Merlin.
Several lines of evidence indicate that Merlin's ability to suppress Rac function is necessary and sufficient for contact inhibition of primary endothelial cells. Constitutively active PAK, dominant-negative Merlin, and membrane-targeted Rac are each sufficient to release HUVEC from contact inhibition. Conversely, dominant-negative PAK promotes growth arrest when introduced in cells that are not contact inhibited. In addition, siRNA-mediated knockdown of Merlin is sufficient to promote recruitment of Rac to matrix adhesions and mitogenesis of confluent HUVEC, excluding the possibility that the open form of Merlin directly promotes recruitment of Rac to the membrane and mitogenesis. Thus, our results support the hypothesis that the closed, dephosphorylated form of Merlin inhibits proliferation and mediates contact inhibition by suppressing activation of Rac, whereas the open, phosphorylated form does not exert this effect. In addition, our results suggest that the open, phosphorylated form may inhibit the growth-suppressive function of the closed, dephosphorylated form. Future studies will be required to elucidate the molecular basis of this effect. Finally, we cannot exclude the possibility that the open form of Merlin has additional functions, as it has been reported that Merlin-S518D enhances filopodia formation in rat Schwannoma cells (Surace et al., 2004
Under growth-permissive conditions, joint integrin-RTK signaling causes recruitment and activation of Rac and, thus, of PAK. We propose that, upon phosphorylation by PAK, Merlin loses its ability to inhibit recruitment of Rac to the membrane, thus facilitating Rac–PAK signaling. This positive feed-forward mechanism of signal amplification would cause robust activation of Rac. Finally, activated Rac would promote cell proliferation through target effectors that were distinct from PAK. We speculate that, upon cell–cell contact cadherin signaling suppresses activation of PAK, leading to the accumulation of unphosphorylated Merlin. This form of Merlin would finally block proliferation by suppressing recruitment of Rac to the membrane (). Several aspects of this model require further experimental examination. Specifically, it will be important to address the mechanism by which cadherin-dependent adhesion suppresses PAK and the mechanism by which Merlin inhibits recruitment of Rac to the membrane. In addition, future studies will have to examine whether the growth-suppressive pathway identified here also operates in other normal cell types.
Merlin's function as a tumor suppressor has been investigated intensively. Merlin−/−
fibroblasts and keratinocytes display defective cadherin-dependent junctions and do not undergo growth arrest upon becoming confluent (Lallemand et al., 2003
), suggesting that Merlin exerts its tumor suppressor function by stabilizing cell–cell junctions. In this model, Merlin functions upstream of cadherins, either by facilitating their assembly or by preventing their disassembly, and it thus inhibits proliferative signaling indirectly. In contrast, we have observed that dominant-negative or siRNA-mediated inhibition of Merlin rescues HUVEC from contact inhibition of growth without disrupting cell–cell junctions. We attribute this apparent discrepancy to the use of different methods of inhibiting Merlin function and/or different cell types. The effect of genetic ablation is permanent and complete, whereas the effect of dominant-negative inhibition or siRNA-mediated knockdown is transient and often incomplete. In addition, it is known that Rac signaling participates in both the assembly and the disassembly of cell–cell junctions, and the extent to which it promotes these contrasting functions may vary with cell type (Braga, 2002
). It is thus possible, and indeed likely, that genetic ablation of Merlin causes a more robust up-regulation of Rac signaling than dominant-negative or siRNA-mediated inhibition. In addition, fibroblasts and keratinocytes may be more sensitive to the effect of loss of Merlin function on cell–cell junctions than endothelial cells. Regardless of what specific mechanism explains this apparent discrepancy, our results indicate that inhibition of Merlin can rescue cells from contact inhibition without disrupting their junctions, supporting the hypothesis that Merlin regulates proliferation by a direct signaling mechanism. Similarly, studies in Drosophila Melanogaster
support the notion that the ERM protein Moesin promotes epithelial organization by inhibiting Rho signaling rather than by functioning as a cytoskeletal linker (Speck et al., 2003
The biochemical function of Merlin is poorly understood. Merlin interacts with the integrin signaling component paxillin (Fernandez-Valle et al., 2002
) that functions, together with PKL, as a scaffold to recruit PAK and PIX, a GEF for Rac, to focal complexes (Turner, 2000
). Hence, Merlin may inhibit recruitment of Rac by the paxillin–PKL–PIX complex until it becomes phosphorylated and thus inactivated by PAK. In addition, PAK phosphorylates Rho-GDI at Ser 101/174, promoting its dissociation from Rac (DerMardirossian et al., 2004
). Although this mechanism can contribute to the recruitment of Rac to matrix adhesions, our results clearly show that PAK functions in this process also by phosphorylating Merlin. Merlin has been shown to bind to Rho-GDI in vitro (Maeda et al., 1999
), raising the possibility that Merlin stabilizes the association of Rac with Rho-GDI. However, we have failed to detect association of endogenous Merlin and Rho-GDI in endothelial cells by coimmunoprecipitation (unpublished data). Finally, recent studies suggest that integrins control recruitment of Rac to the membrane by organizing cholesterol-rich membrane microdomains (del Pozo et al., 2004
), and dephosphorylated Merlin tends to accumulate in these microdomains (Stickney et al., 2004
), suggesting the possibility that Merlin inhibits recruitment of Rac to these membrane domains.
Rho family GTPases promote cell proliferation and migration, and they mediate Ras transformation in vitro. However, unlike Ras proteins Rho GTPases do not acquire mutations that impair their GTPase activity and cause constitutive activation in human cancer (Sahai and Marshall, 2002
). It is possible that these mutations are not sufficient to cause transformation because they do not overcome the barrier imposed by cytoplasmic segregation. PAK1 is amplified in some ovarian and breast cancers (Bekri et al., 1997
; Schram et al., 2003
). In addition, elevated levels of PAK1 have been detected in breast and colorectal cancer (Balasenthil et al., 2004
; Carter et al., 2004
). Finally, PAK4 is overexpressed in various carcinoma lines (Callow et al., 2002
). Based on our results, we speculate that PAK may promote tumorigenesis by functionally inactivating Merlin and elevating Rac signaling.