Our results indicate that prenylation and subsequent membrane trafficking of small GTPases is a dynamically regulated process defined by changes in the nucleotide-bound state of the GTPase and by interactions with SmgGDS splice variants. The assumption that newly synthesized small GTPases immediately enter the prenylation pathway, without any regulatory controls, is no longer tenable. This newly defined ability to regulate prenylation might provide many advantages to cells, including the ability to store nonprenylated small GTPases that could be rapidly released into the prenylation pathway when they are needed, rather than depending on energy-dependent transcription and translation.
The most striking and consistent pattern we observed is that SmgGDS-607 interacts with the nonprenylated forms of all of the small GTPases tested, whereas SmgGDS-558 is restricted to recognizing the prenylated forms of these small GTPases. The finding that SmgGDS-607 co-expression slows the prenylation of the DN forms of Rap1A, RhoA, and Rac1 indicates that SmgGDS-607 regulates the entrance of these GTPases into the prenylation pathway. It might seem perplexing that SmgGDS-607 regulates the prenylation of DN GTPases more than WT GTPases, especially because DN GTPases are not endogenously expressed in cells. However, the biological significance of this observation is explained by reports that the DN mutation prolongs the time that small GTPases remain in the nucleotide-free state or the GDP-bound state (36
), which are states attained by endogenous small GTPases in cells. Thus, the preferential regulation of DN GTPases by SmgGDS-607 indicates that SmgGDS-607 preferentially regulates small GTPases in the nucleotide-free or GDP-bound forms. Consistent with this interpretation, it was previously found that WT-Rac1 remains in the nucleotide-free form when it is bound to SmgGDS (40
). Based on these observations, it is intriguing to speculate that SmgGDS-607 intercepts newly synthesized, nucleotide-free, or GDP-bound GTPases to regulate their entrance into the prenylation pathway.
The demonstration that insertion of the DN mutation alone alters prenylation indicates that changes in the nucleotide-bound state control the entrance of small GTPases into the prenylation pathway. We found that the DN mutation slows the prenylation of Rap1A and RhoA but enhances the prenylation of Rac1. Because it is unlikely that the actual mechanism of prenylation differs between Rap1A, RhoA, and Rac1, all of which are geranylgeranylated (20
), these dissimilarities likely represent differences in how these GTPases enter the prenylation pathway in response to unique signals and differing interactions with SmgGDS-607 or other proteins. Interestingly, we found that neither the DN mutation nor interactions with SmgGDS-607 detectably alter the prenylation of K-Ras, which is farnesylated (23
). It is possible that K-Ras is less responsive than the other GTPases because farnesylated GTPases are regulated differently than geranylgeranylated GTPases.
Our observation that co-expression of SmgGDS-607 diminishes, rather than enhances, the prenylation of RhoA, Rac1, and Rap1A suggests that SmgGDS-607 functions as a gatekeeper that prohibits these GTPases from entering the prenylation pathway until the correct signals are received. Due to this regulatory function, it might be expected that silencing SmgGDS-607 expression would allow the unimpeded entrance of small GTPases into the prenylation pathway. Consistent with the possibility that small GTPases can continue to be prenylated when SmgGDS-607 expression is diminished, we found that silencing SmgGDS-607 does not significantly alter events that depend on prenylated GTPases, including NSCLC soft agar colony formation (, A and B) and the membrane localization of GFP-tagged Rap1A in NSCLC cells ().
Multiple signals might release small GTPases from SmgGDS-607 and allow their entrance into the prenylation pathway. One of the most likely signals to release a GTPase from SmgGDS-607 is GDP/GTP exchange, because SmgGDS has been reported to act as a weak GEF for all of the small GTPases tested in this study (9
). However, it is possible that SmgGDS splice variants do not have intrinsic GEF activity but instead act as scaffolds that bind GEFs for small GTPases. A scaffold function for SmgGDS is supported by the discovery that SmgGDS forms complexes with Rac1 and β-Pix, which is a GEF for Rac1 (41
). The presence of ARM repeats in SmgGDS also supports its role as a scaffold, because ARM repeats often occur in proteins with scaffold functions (42
). In their role as scaffold proteins, SmgGDS splice variants may form complexes with small GTPases and different regulatory proteins at specific steps in the prenylation pathway. Release into the prenylation pathway, or subsequent transport to or from the ER, may depend on GDP/GTP exchange induced by a GEF in the SmgGDS-GTPase complex. Alternatively, signaling events such as phosphorylation of proteins in the SmgGDS-GTPase complex may trigger release of GTPases to allow their processing or transport.
To incorporate our findings into a model of signal-dependent prenylation (), we propose that SmgGDS-607 retains nonprenylated GTPases in a state that slows their entry into the prenylation pathway. When the appropriate signal is delivered, SmgGDS-607 facilitates GDP/GTP exchange by the GTPase either directly or in complex with another protein, promoting the entry of the GTPase into the prenylation pathway (A). Because WT GTPases can undergo GDP/GTP exchange, they can be released from SmgGDS-607 and enter the prenylation pathway (A). In contrast, because DN GTPases do not bind GTP, DN GTPases are relatively nonresponsive to the signals that release the GTPase from the SmgGDS-607 complex (B). Overexpression of SmgGDS-607 would increase the formation of these nonresponsive DN-GTPase-SmgGDS-607 complexes. This possibility explains why SmgGDS-607 overexpression reduces the prenylation of DN-Rap1A, DN-RhoA, and DN-Rac1 (C). Overexpressed SmgGDS-607 would also slow the prenylation of WT GTPases, if the required signal is not delivered to release the WT GTPase from the overexpressed SmgGDS-607. This possibility explains why SmgGDS-607 overexpression reduces the prenylation of WT-Rap1A, as indicated by the results of the Triton X-114 fractionation assay (D).
FIGURE 6. Models of the regulation of prenylation and trafficking of PBR-containing small GTPases by SmgGDS splice variants. A, SmgGDS-607 may act directly or in cooperation with another protein (X) to stimulate GDP/GTP exchange and promote entry of nonprenylated (more ...)
In this model, SmgGDS-607 might sequester newly synthesized small GTPases in the cytoplasm as reserves for rapid prenylation (C
), consistent with reports of regulated activation of PTases (43
) and rapid prenylation of small GTPases in response to different signals (47
). SmgGDS-607 might also act as a scaffold or GEF to stimulate nonconventional cytoplasmic signaling by nonprenylated small GTPases (D
). Our results also support the possibility that SmgGDS-607 transfers nonprenylated small GTPases to the PTase or facilitates a guanine nucleotide exchange event necessary for prenylation (E
), likely in response to a signal.
The binding of prenylated PBR-containing small GTPases by SmgGDS-558 suggests several possible mechanisms by which SmgGDS-558 might promote signaling and membrane localization of small GTPases. SmgGDS-558 might promote the release of newly prenylated small GTPases from PTases (F
), consistent with a recently proposed model that unidentified cellular proteins assist in the release of farnesylated proteins from farnesyltransferase (48
). SmgGDS-558 might also escort newly prenylated small GTPases to the ER for CAAX
). The possibility that SmgGDS-558 transfers PBR-containing GTPases from the ER to the PM (H
) is consistent with SmgGDS-558 preferentially interacting with PBR-containing small GTPases, because PBR-containing GTPases follow a unique ER to PM route that differs from the Golgi to PM route followed by small GTPases lacking a PBR (6
). Finally, SmgGDS-558 might also escort prenylated GTPases from the PM to endomembranes (I
), thereby promoting signaling from endomembranes (J
). These multiple mechanisms provide novel ways to regulate small GTPase prenylation and membrane trafficking that merit further investigation.
Our discovery that SmgGDS splice variants participate in the prenylation of small GTPases has potential therapeutic implications for NSCLC and prostate cancer, which are cancers that overexpress SmgGDS (12
). There is much interest in developing chemotherapeutic drugs that block the passage of small GTPases through the prenylation pathway or disrupt the functioning of the isoprenoid moiety. Drug targets include PTases (49
), Rce1, and isoprenylcysteine carboxylmethyltransferase (3
), enzymes that synthesize the isoprenoid moiety (51
) and the attached isoprenoid moiety itself (52
). Unfortunately, some of the developed drugs can disrupt other metabolic pathways and block the prenylation of proteins unrelated to small GTPases, which may result in unwanted side effects. In contrast, SmgGDS splice variants are potentially more specific therapeutic targets, because they interact only with PBR-containing small GTPases (11
). Further studies are needed to validate SmgGDS splice variants as potential drug targets in cancer.
Our study supports the view that, in addition to the widely accepted model that prenylation of small GTPases occurs constitutively, cells possess the capability of regulating the prenylation of small GTPases. Expression of SmgGDS-607 and SmgGDS-558 provides cells with a mechanism to potentially coordinate the entry, transit, and exit of small GTPases moving through the prenylation pathway to their final sites of activity at membranes. The participation of SmgGDS splice variants in these events raises the possibility that SmgGDS splice variants, as well as other uncharacterized regulatory proteins, could be exploited to therapeutically control the prenylation and localization of small GTPases.