Basic aspects of RhoGAPs
As discussed earlier, Rho GTPase GDP/GTP cycling is controlled by two classes of regulatory proteins that accelerate the otherwise very slow intrinsic guanine nucleotide exchange and GTP hydrolysis activity [36
]. There are two major RhoGEF families, one comprised of the Dbl family (69 members) and the other the DOCK family (11 members) [37
]. Similarly, the number of RhoGAPs (>70) also greatly outnumber the number of Rho GTPases [38
]. That there exist multiple GEFs and GAPs, as well as effectors, for a single Rho GTPase reflects the critical role of these proteins as nodes in complex signaling networks.
Much recent evidence has uncovered a striking role for the mutation or aberrant expression of many RhoGEFs and RhoGAPs in a multitude of cellular processes and disease states, especially cancer and the related processes of cytoskeletal organization and cell migration [40
]. For this review, we will focus on RhoGAPs and particularly the DLC family of RhoGAPs. All RhoGAPs share a conserved RhoGAP catalytic domain [38
]. Otherwise, they show little sequence similarity in their remaining sequences, which typically contain multiple additional protein-protein and protein-lipid interacting domains, as well as many putative phosphorylation sites. These domains dictate the specific subcellular localization and regulation of each RhoGAP [39
]. Such mechanisms of control allow high spatial and temporal control of the termination of GTPase activation, preventing inappropriate or prolonged signals. The spatial regulation of Rho activation in turn influences effector utilization, thus providing a critical mechanism of regulation of Rho GTPase function. Thus, the GAPs serve a critical signaling nodes, incorporating diverse cellular stimuli in order determine specific Rho GTPase outcomes.
In contrast to RhoGEFs, much less is known regarding RhoGAP regulation. A few RhoGAPs have been studied in molecular detail, including p190RhoGAP and Deleted in liver cancer-1 (DLC-1). For example, p190RhoGAP is tyrosine phosphorylated and binds to the RasGAP, p120RasGAP, both regulating its function [41
]. However, very little is known about the full cellular regulation mechanisms of most of the RhoGAPs. This will be a great but important challenge in the Rho GTPase field, as different RhoGAPs likely play different and specific roles in different aspects of Rho GTPase function. Uncovering the signaling mechanisms that regulate RhoGAPs in specific cellular contexts will be necessary for a full understanding of Rho GTPase function. The future challenges will especially include an understanding of 1) which Rho GTPases are the targets of each RhoGAP in vivo
, 2) which downstream signaling targets of each Rho GTPase do the GAPs affect, 3) which RhoGAPs are redundant and which are specific to certain Rho functions and 4) which Rho GTPases are most involved in disease processes and could serve as drug targets to specifically affect certain aspects of GTPase function without altering others. Below we discuss what is known regarding DLC-1 and related RhoGAPs.
DLC-1 in Cell Migration and Invasion
First identified as a gene deleted in liver cancer, subsequent studies found loss of DLC-1 gene expression in a wide variety of human cancers, including lung and breast [8
]. Interestingly, the rate of heterozygous loss of DLC-1 in some cancer types approaches that of p53, underscoring its potential role as a tumor suppressor [42
]. Additionally, recent genome-wide sequencing analyses of human tumors have identified missense mutations in DLC-1 [43
]. Likewise, several studies have demonstrated that reintroduction of DLC-1 into liver, lung or breast cancer cell lines results in decrease tumorigenic growth [45
], and in a recent mouse model of liver cancer, loss of DLC-1 together with Myc oncogene activation resulted in increased tumor growth [42
]. Together, these results demonstrate an important role for DLC-1 in cancer and support a tumor suppressor role for DLC-1. DLC-2 and DLC-3 are highly related isoforms of DLC-1 and similar less extensive observations also support their role as tumor suppressors [8
]. Whether loss of DLC-2 and DLC-3 as well as that of DLC-1 must occur in cancer will be a subject of future investigation.
All RhoGAPs contain an ~150 amino acid RhoGAP catalytic domain [38
]. DLC-1 and related isoforms contain two additional domains, an N-terminal SAM domain and a C-terminal START domain (). In vitro
, we determined that the isolated RhoGAP domain acts as a potent GAP for RhoA, RhoB, RhoC, and to a lesser degree for Cdc42, but not Rac1 [48
]. SAM domains (~70 amino acids) are putative protein interaction modules found in a diverse spectrum of signaling and nuclear proteins, typically as components of multi-domain proteins (e.g., Eph-related tyrosine kinases, Ets transcription factors). SAM domains have been shown to homo- and hetero-oligomerize, and can additionally bind both non-SAM domain-containing proteins. Structural studies of the SAM domain of DLC-2 suggest that it may bind lipids (although the specificity and in vivo
relevance is unknown [49
]). START domains (~120 amino acids) are lipid-binding domains found in proteins that transfer lipids between organelles. START domains are found in StAR, HD-ZIP and other signaling proteins. Representatives of the START domain family have been shown to bind different ligands such as sterols (StAR protein) and phosphatidylcholine.
Much less is known about the function of the SAM and START domains in DLC function. Our studies have found that the SAM domain appears to be involved in autoinhibition of the RhoGAP activity [50
]. Between the SAM and RhoGAP domains is a long unstructured region that includes a phosphorylation-independent binding site (Y442 in human DLC-1) for the Src homology 2 domains of tensin family of adaptor proteins [51
]. This binding region is necessary for focal adhesion localization as well as tumor suppression, suggesting that RhoGAP activity at or near focal adhesions and not total cell RhoGAP activity is necessary for tumor suppression. A phosphorylation site for rat DLC-1 has also been identified in this N-terminal region (S329), by activated AKT and RSK1 kinases, although the functional consequence of this modification has not been determined (). Future work will involve understanding how DLC-1 is regulated by its additional domains and phosphorylation, as well as the precise the downstream mechanisms through which DLC-1 acts as a tumor suppressor.
Since aberrant activation of RhoA and RhoC has been implicated in oncogenesis, a logical hypothesis is that loss of DLC-1 function will lead to hyperactivation of these Rho GTPases, resulting in their stimulation of cell proliferation. Consistent with this possibility, two studies suggest that tumor suppression by DLC-1 works through RhoA [53
], although our study demonstrated partial RhoGAP independent tumor suppression by DLC-1 [48
]. Our studies also found that ectopic expression of DLC-1 in DLC-1 deficient lung tumor cells reduced the level of activated RhoA. Additionally, although FA association does not appear to alter DLC-1 RhoGAP activity in vivo
or in vitro (unpublished results), FA association appears to be required for proper spatial regulation of Rho GTPases for tumor suppression.
As indicated above the Rho and Cdc42 GTPases play multiple critical roles in the regulation of actin organization, cell polarity, and cell migration. Thus the aberrant function of DLC-1 seen in many types of cancers would be expected to influence the activation state of these critical GTPases and to have significant effects on cytoskeletal organization and cell motility. We have recently explored this aspect in some detail [50
]. Thus, overexpression of various activated forms of DLC-1 leads to profound changes in cytoskeletal organization with disruption of focal adhesions and the rapid, continuing and apparently random extension of long protrusions, as well as inability to retract the tail of the cell. These effects are likely mediated via reduction in Rho activity. Using single cell tracking assays, we have also found that overexpression of a form of DLC-1 (DLC-1ΔSAM) that lacks the SAM domain (and is thus activated), but which retains the remainder of the N-terminal region, has a profound effect on cell motility. DLC-1ΔSAM increases the velocity of migration but reduces directionality; the overall result in bulk migration assays is a reduction in migration, presumably due to loss of directionality, an effect that may be mediated via DLC-1's inhibition of CDC42 and consequent effects on polarity. These effects on migration are not seen with active forms of DLC-1 that lack the N-terminal region. DLC-1ΔSAM is capable of associating with focal adhesions, but when it is expressed extensive disruption of these structures occurs. Thus it is not clear whether the profound effects of DLC-1ΔSAM on migration are due to its ability to localize to residual focal adhesions, or due to other interactions mediated through the N-termus.
Further evidence for a key role for focal adhesion localized DLC-1 comes from our studies of a truncated version of the protein (DLC-1N) that contains the N-terminal domain but not the GAP domain; we also constructed a second version with a mutation at the Y442 site (DLC-1N Y442A). DLC-1N retains the ability to localize to focal adhesions, but the version with the mutation at 442 does not. Overexpression of DLC-1N leads to almost complete paralysis of cell movement, whereas the 442 mutant had no effect. Using fluorescence microscopy and immunochemistry we have demonstrated that DLC-1N displaces endogenous DLC-1 from focal adhesions. This strongly argues that, not only GAP activity, but also proper focal adhesion localization of DLC-1, play important roles in the control cell migration. Presumably focal adhesion associated DLC-1 can orchestrate localized effects on Rho and CDC42 that are critical for control of cell velocity and directionality.
In addition to the work described above, a number of other studies have demonstrated that DLC-1 inhibits cell motility or invasiveness in cultures of liver, breast, ovarian and lung cancer cell lines [48
]. Further, the observed effects of DLC-1
on cell motility are consistent with its proposed role as a metastasis suppressor gene [8
]. For example, using gene chip array analysis several groups have found that DLC-1 is unde-rexpressed in highly metastatic cells [55
In summary, the RhoGAP DLC-1 and related family members play important roles both in the regulation of tumorigenesis and in associated cytoskeletal activities that impact on the motile, invasive and metastatic characteristics of cancer cells. Whether these two aspects of DLC-1 function occur through the same or different GTPase pathways will be an important goal of future research. The functions of DLC-1 seem to depend not only on its enzymatic activity as a GAP but also on its focal contact localization and its interactions with other proteins that regulate and localize its function.