We have shown here that the atypical Rho family small GTPase Wrch-1 undergoes serum-stimulated Src-mediated tyrosine phosphorylation at residue Y254 in its C-terminal membrane targeting domain, a modification that dynamically alters its subcellular localization by promoting relocalization from the plasma membrane. We also showed that Y254 is the major site of phosphorylation and that mutation of Y254 to a nonphosphorylatable phenylalanine residue (Y254F) prevents relocalization of the plasma membrane pool, remains GTP bound, enhances recruitment of the GTPase-binding domain of the Wrch-1 effector PAK1 (PAK-PBD), and enhances Wrch-1-mediated effects on growth transformation and on epithelial morphogenesis in 3D culture. In contrast, both serum-stimulated WT Wrch-1 and phospho-mimetic Y254E are restricted from the plasma membrane and are inactive, failing to recruit PAK-PBD or to activate the effector kinases PAK or Pyk2 unless mutationally activated to be GAP insensitive (Q107L) and therefore constitutively active. These results indicate that C-terminal tyrosine phosphorylation of Wrch-1 may be important for downregulation of its biological activities, and they provide evidence supporting a potential mechanism whereby loss of interaction with an unidentified plasma membrane-associated GEF or increased association with a novel endosomally localized Wrch-1 GAP turns off previously active Wrch-1-GTP.
Our results demonstrate that Wrch-1 is GTP bound and active at the plasma membrane, where it is capable of recruiting and activating downstream effectors such as PAK1 and Pyk2. In contrast, serum-stimulated, Src-dependent phosphorylation at Y254 results in loss of Wrch-1 from the plasma membrane and loss of activity, and the endosomal pool of Wrch-1 is inactive. That the constutively active, GAP-insensitive mutant Q107L remains capable of effector recruitment at endosomes even when targeted there by the C-terminal phospho-mimetic mutation Y254E indicates that phosphorylation-mediated relocalization does not decrease Wrch-1 effector activation simply by removing Wrch-1 from its relevant effectors, at least some of which are still present at endosomes. For example, the well-validated Wrch-1 effector PAK is not only present at endosomes but is recruited and activated preferentially there by Chp/Wrch-2 in response to tumor necrosis factor alpha (12
). Instead, our results support a model in which loss of Wrch-1 from the plasma membrane pool upon C-terminal phosphorylation at Y254 actively promotes its deactivation, since the major location is then the endosomal pool, which is not active.
Relocalization from the plasma membrane may decrease interaction of Wrch-1 with a plasma membrane-localized GEF or enhance its interaction with an endosomally localized GAP. No GEFs or GAPs for Wrch-1 have been identified, although both are presumed to exist. It is possible that known Cdc42-activating GEFs such as the FGD family, Tuba, Asef, or Intersectin may promote Wrch-1 activity at the plasma membrane, although these tend to be activated by growth factors or serum, whereas Wrch-1 is inactivated upon serum stimulation. On the other hand, endosomal GAPs, such as p50RhoGAP (40
) and ARAP1 (14
), have been identified for other Rho family GTPases, but it is not known whether they promote GTP hydrolysis on Wrch-1. Other mechanisms of endosomal translocation and subsequent deactivation are well-documented for receptor tyrosine kinases (RTKs). Upon autophosphorylation and activation following growth factor stimulation, many RTKs become ubiquitinated and undergo relocalization to the endosomal compartment, which attenuates their signaling (15
). Additionally, ubiquitination of the small GTPase H-Ras has been shown to promote its endosomal trafficking and thereby to attenuate its signaling through the Raf-MEK-extracellular signal-regulated kinase pathway (21
). We have observed that Wrch-1 is also ubiquitinated but that this modification does not appear to grossly alter its localization (data not shown). Thus, our present data best support the model of C-terminal phosphorylation of Wrch-1 leading to its downregulation by decreasing proximity to one or more GEFs and/or enhancing proximity to one or more GAPs.
Accumulating evidence suggests that phosphorylation of the C-terminal membrane targeting domains on small GTPases combines with other sequences and posttranslational modifications to dynamically regulate the localization and function of these proteins. To date, all the phosphorylation sites so identified, whether in Ras or Rho GTPases, have been Ser/Thr residues just upstream of C-terminal farnesyl or geranylgeranyl isoprenoid modifications (6
). However, the atypical Rho family GTPase Wrch-1 is neither modified by isoprenylation nor possesses cognate serine or threonine residues. Instead, our study provides the first report of direct regulation of Rho family GTPase subcellular localization and function by tyrosine phosphorylation of its membrane targeting domain. Although Cdc42 has been reported to be tyrosine phosphorylated by Src upon epidermal growth factor stimulation (45
), this phosphorylation occurs at residue Y64 within the switch II region rather than in the Cdc42 C terminus, which lacks any tyrosine residue. Whether tyrosine phosphorylation of Cdc42 alters its subcellular localization was not explored, but it was reported to promote binding of Cdc42 to RhoGDI. However, Wrch-1 is not modified by an isoprenoid, a feature required for binding of RhoGDI (42
), and does not interact with RhoGDI (4
). Thus, tyrosine phosphorylation of Wrch-1 and Cdc42 occurs on distinct domains and has distinct consequences. Chp/Wrch-2, the closest relative of Wrch-1, is also palmitoylated and unprenylated (12
), but, like Cdc42, Chp lacks a tyrosine residue near its C terminus. This is perhaps not entirely surprising, as many closely related isoforms of small GTPases differ from each other mostly in their membrane targeting domains, possibly to provide signaling diversity.
Wrch-1 tyrosine phosphorylation at Y254 occurs only two residues away from the critical palmitoylation site C256, raising the question of whether one modification sterically hinders the other. It is clear that tyrosine phosphorylation of Wrch-1 does not require prior palmitoylation, as even a nonpalmitoylatable Cys→Ser mutant becomes tyrosine phosphorylated upon serum stimulation (data not shown). Indirect evidence suggests that this phosphorylation can occur simultaneously with palmitoylation: phosphorylation results in an increased association with internal membranes, whereas an inability to be palmitoylated results in a complete lack of Wrch-1 membrane association (5
). Taken together, these results suggest that tyrosine phosphorylation normally occurs on palmitoylated Wrch-1.
We have shown here that Wrch-1 tyrosine phosphorylation requires Src and that Src can directly phosphorylate Wrch-1 in vitro. Although Y254, the major residue for tyrosine phosphorylation, does not occur in the context of a known Src kinase consensus site, and algorithms such as NetPhos or ScanSite do not predict its phosphorylation by Src, there is currently no reliable predictor of whether a given protein is in fact a substrate for Src in vivo. Even enolase, a commonly used positive control for Src phosphorylation, does not contain a known Src consensus site. However, many tyrosine kinases require phosphorylation themselves in order to be active, and this activating or priming phosphorylation step may be accomplished in cis or in trans. Therefore, other than direct phosphorylation of Wrch-1 by Src, another possibility is that Src activity promotes binding to Wrch-1 and/or activation of another tyrosine kinase that can phosphorylate it. Whether Wrch-1 is a direct or an indirect downstream target of Src-mediated phosphorylation in cells remains to be determined.
Recent studies have suggested several context-dependent functional connections between Wrch-1 and Src that are likely to be pertinent regardless of whether the connection is direct or indirect. In osteoclasts, Wrch-1 colocalizes with Src in podosomes, and increased Wrch-1 activity perturbs the podosome belt (31
). Thus, Src-mediated tyrosine phosphorylation of Wrch-1 could contribute to the dynamic regulation of podosome formation and assembly. In addition, Wrch-1 negatively regulates macrophage colony-stimulating factor (M-CSF)-stimulated osteoclast migration (9
), and Src has recently been shown to be activated in osteoclasts downstream of M-CSF stimulation (48
). Therefore, M-CSF may promote osteoclast migration by activating Src, to then downregulate Wrch-1 through tyrosine phosphorylation on Y254. In contrast to osteoclasts, where it decreases migration, Wrch-1 increases the migration of fibroblasts (13
), where it is localized to focal adhesions (31
) and regulates their assembly. Src and several of its substrates are major components of focal adhesions. In PAE cells, Wrch-1 but not Cdc42 requires Src to induce formation of filopodia (34
), but it is unknown whether Src is required for an effect on Wrch-1 itself or on a downstream target not shared with Cdc42.
We have observed that Y254, the major site of Src-mediated tyrosine phosphorylation, negatively regulates Wrch-1-mediated anchorage-independent growth and epithelial cell morphogenesis, because mutation to the nonphosphorylatable Y254F conferred greater activity on Wrch-1 than the wild-type tyrosine residue. Thus, Src-mediated tyrosine phosphorylation at Y254 may normally act as a brake for Wrch-1 function. Although Src is often thought of simply as an oncogene that leads inexorably to cellular dedifferentiation, it is clear that it can exert dual functions. For example, Src serves dual functions during epithelial cell morphogenesis in Drosophila melanogaster
, where it both antagonizes E-cadherin-mediated cell adhesion and simultaneously stimulates E-cadherin transcription (37
). Similarly, if Src modulates Wrch-1 through opposing functions, then proper cycling between the phosphorylated and the unphosphorylated state of Wrch-1 is likely to be required for the correct final outcome, regardless of whether Wrch-1 is a direct or indirect target of Src kinase activity. It will certainly also be of interest to determine which phosphatase(s) contributes to restoration of the unphosphorylated state of Wrch-1.
Our observations identify important contributors to Wrch-1 regulation and lend further credence to the emerging paradigm that C-terminal phosphorylation of small GTPases may serve as a key mechanism to dynamically regulate their localization, activation, and function. Thus, further investigations into such phosphorylation events will be critical for a better understanding of the regulation of Rho GTPases.