RasGRF is a Ras family GEF cloned by virtue of its homology with the Saccharomyces cerevisiae
CDC25 gene product that stimulates nucleotide exchange on S. cerevisiae
The RasGRF GEFs family includes RasGRF1 and RasGRF2, that exhibit an 80% of overall homology (revised in ref. 27
). Both contain a number of functional motifs involved in diverse signaling control mechanisms and protein-protein interactions. The carboxyl-terminal Cdc25 domain is sufficient to catalyze nucleotide exchange on Ras and to induce cellular transformation in fibroblasts.26
In its amino terminus, RasGRF GEFs contain a Dbl homology domain (DH) which is generally present in GEFs for the Rho family of small G proteins. The DH domain is flanked by two Pleckstrin homology domains (PH) also present in Rho family GEFs and other unrelated proteins. The presence of regulatory domains for Rho and Ras GTPases make of RasGRF a confluence point in the control of the signals flowing through both pathways. Even though the rasgrf
genes are preferentially expressed in the central nervous system, both RasGRF proteins can also be found in several other tissues, whilst their functional roles at those locations remain less defined and require further studies (reviewed in ref. 27
Most interestingly, RasGRF functions can be inhibited by its interaction with Cdc42 in its inactive GDP bound form. As such, Cdc42-GDP negatively regulates the activation of the Ras/ERK cascade and of TC21 as induced by RasGRF.28,29
Reciprocally, we have recently demonstrated that the effects of Cdc42 on cytoskeletal dynamics are inhibited by RasGRF1/2, independently of their functions as Ras activators, by outcompeting bona fide Cdc42 exchange factors. In this respect, RasGRF GEFs are unique because they can behave as Rho GTPase inhibitors. Remarkably, an important consequence of Cdc42 inhibition by RasGRF overexpression is decreased actomyosin contractility.11
In Ras GRF1/2, the DH domain is responsible for binding to Cdc42, thereby regulating the switch between rounded and elongated invasion strategies. On the other hand, the DH domain is also required for RasGRF translocation to the membrane.29
As such, it is possible that the cross-talk between Cdc42 and RasGRF could be spatially restricted. As a precedent, RasGRF has been described to activate Ras and TC21 in particular sub-cellular locations.28,30
Thus, it is possible that the control of actomyosin contractility may take place at a particular sub-cellular location. This level of complexity in the interplay between RasGRF and Cdc42 remains to be investigated.
By controlling the balance between Ras and Rho signal output the interaction between RasGRF1/2 and Cdc42 could ultimately determine cell fate. For example, it could be hypothesized that, under normal circumstances, cells with moderate to low levels of both RasGRF1/2 and Cdc42 will exhibit low levels of Ras/TC21 and Cdc42 signals, evoking normal/physiological responses that depending on the cell context would result in normal proliferative and migratory states ().
Figure 1 Schematic representation of the balance between Cdc42 and RasGRF pathways. (A) The balance between both signaling pathways is in equilibrium and both are active to a similar extent. (B) RasGRF expression is higher and there is more signaling through the (more ...)
On the other hand, high levels of RasGRF1/2 would result in high flux through TC21 and Ras pathways and, at the same time, in the inhibition of Cdc42 and its associated actomyosin contractility (). The latter would allow cell elongation and protrusive activity. In line with this notion, it has been shown that RasGRF favors neuronal differentiation under physiological conditions.27
In agreement, neurite outgrowth requires downregulation of actomyosin contractility.31
Thus, it is tempting to speculate that in neurons, RasGRF could contribute to neurite outgrowth by downregulating Cdc42-mediated acto-myosin contractility. A similar mechanism could take place in certain cancer contexts and at the onset of tumor progression. Before local invasion and metastatic dissemination process takes place, RasGRF could promote Ras signals () while inhibiting Cdc42 mediated processes. This would result in an initial promotion of cancer cell proliferation via Ras-ERK signaling while preventing invasion.
All the melanoma cell lines used in our study11
harbor either BRAF or N-Ras mutations, which render constitutive activation of the ERK pathway.32
In such a scenario, one would imagine that a Ras activator—RasGRF—would have less of a role in activating the ERK pathway. Surprisingly, all the lines displayed detectable levels of RasGRF2.11
Metastatic melanoma cells express genes associated with a diverse range of cell lineages. Something that may partly explain the diverse modes of motility that melanoma cells can exhibit.33
So, the question that arises is why would a melanoma cell retain RasGRF expression?
- Our work11 supports a prominent role for RasGRF2 in suppressing the invasive “highly contractile” rounded phenotype11 by inhibiting the activation of Cdc42. An attractive possibility is that RasGRF would serve a key role in the switch between rapid, highly contractile and proteolytic/low contractile phenotypes (). Indeed, a system for providing agile inter-convertibility between these forms of migration would facilitate tumor cells to metastasize. When measured in vivo, rounded movement can be 10–100 times faster than protrusive movement. But, while rounded movement can be much faster, elongated movement would still be required whenever progress through a rigid extra-cellular matrix requiring proteolysis is necessary. So the ability to convert to this form of movement is essential wherever tumor cells meet such barriers (reviewed in ref. 9). Taking this into consideration, melanoma cells would exploit RasGRF1/2 functions in order to finely tune the transition from rounded to elongated types of movement—whenever dictated by environmental conditions. Melanoma cells could maintain RasGRF expression in order to regulate this phenotypic switch, independently of its role as a regulator of the Ras/ERK pathway. The main metastatic sites in melanoma are lung, liver, brain and bone and noticeably we have shown how depletion of RasGRF2 favors initial stages of melanoma lung colonization. It would be very interesting to assess the effects of re-expressing RasGRF2, once melanoma cells have invaded the lung, in order to promote proliferation in this newly colonized site via the activation of the Ras-ERK pathway.
- RasGRF could still contribute to ERK mediated functions at later stages of melanoma. In support of this notion, constitutive activation of the RAS/ERK pathway through a mechanism involving RasGRF1 has been described to promote secretion of the protease MMP934 that could help in metastasis.
In contrast to melanoma, a number of reports have described reduced levels of RasGrf2 expression in human and rodent tumors of pancreatic, mammary, colon and lung origin, frequently in association with aberrant methylation of the RasGrf2 genomic locus (reviewed in ref. 27
). In these cases, RasGRF function has been lost and the situation would be most likely as shown in . As such, RasGRF2 deficiency favors the development and dissemination of lymphomas in mouse models.35
Lymphocytes have been described to rely on amoeboid shape to traffic through interstitial tissues.36
In this particular scenario of tumor cells relying mainly on amoeboid strategies, depletion of RasGRF would be favorable as a mechanism to sustain efficient amoeboid highly contractile phenotype. Alternatively, other negative regulators of acto-myosin contractility could have a role once RasGRF2 is lost. Regarding this possibility and quite intriguingly, RasGRF1 has been identified in a signature for B-chronic lymphocytic leukemia (B-CLL).37
Even though both RasGRF1 and RasGRF2 can activate Ras27
and inhibit Cdc42,11
a thorough comparative analysis of their capabilities in these processes, in physiologically relevant contexts, has not been performed. Thus, the possibility exists that the presence of one or the other isoform could tilt the threshold either towards Ras activation or Cdc42 inhibition.