Here we identify Ser 31 as a phosphorylation site in RhoGDI2 that is induced by PMA treatment of cells, and we show that the conventional type PKCα is the predominant kinase involved in this phosphorylation. Comparison of the S31E-RhoGDI2 phospho-mimetic mutant to wild type RhoGDI2 revealed reduced Rac1 binding, an increase in Rac1 activation, and an inability to extract Rac1 from membranes. The results suggest PKC phosphorylation is a potential mechanism to inactivate the RhoGDI2 metastasis suppressor protein.
Structures of RhoGDI1 or RhoGDI2 bound to Rac identified two highly similar regions of RhoGDIs: a structurally stable C-terminal immunoglobulin like domain that binds the isoprenyl tail of GTPases, and a flexible N-terminal regulatory region consisting of a helical hairpin that binds to the switch I and II regions of Rac2 and inhibits GDP dissociation through stabilization of Mg+2
binding of switch I (24
). The N-terminal region of the GDIs is essential for GTPase binding, as truncation of the first 41 residues disrupts protein structure and results in a loss of function (26
). Ser 31 of RhoGDI2 is found in the N-terminal region of the protein and caps the first helix of the helical hairpin structure (24
). The Ser 31 residue is conserved among all members of the RhoGDI family. Interestingly, the corresponding site in RhoGDI1 (Ser 34) has been identified as a substrate for conventional PKCs; however, Ser 34 RhoGDI1 phosphorylation led to a selective decrease in binding to RhoA, but not to Rac or Cdc42 (28
). While these results suggest that both RhoGDI1 and RhoGDI2 are regulated by PKCs, phosphorylation does not produce identical effects on how different GDIs interact with Rho family GTPases. The mechanism through which this occurs is unclear, but may be related to the different binding affinities RhoGDI1 and RhoGDI2 have for GTPases.
Our analysis of the RhoGDI2-Rac2 complex indicates that the Ser 31 hydroxyl group of RhoGDI2 is involved in two hydrogen bonds with Glu 34 that form a cap at the N-terminus of helix 1 (). Because Ser 31 interacts with an acidic side chain, phosphorylation or substitution of Ser 31 with an acidic residue would cause electrostatic repulsion in addition to the loss of the stabilizing hydrogen bonds, and would be expected to disrupt RhoGDI2 structure and decrease its interactions with Rac. Furthermore, the residues surrounding Ser 31 make important contacts with Rac (24
), and it is foreseeable that phosphorylation of Ser31 could also disrupt these contacts and further interfere with GTPase binding. NMR studies of RhoGDI1 or RhoGDI2 showed that the N-terminal 60 residues of the proteins were flexible and disordered when free in solution, but became ordered and formed a helical hairpin structure upon addition of Rac (26
). This model suggests that the N-terminal region of RhoGDI2 exists in an equilibrium between a disordered state and an ordered state, and that binding of RhoGDIs to a GTPase stabilizes the ordered state and the helical hairpin structure that is essential for GTPase binding (26
). From this model and our data presented here, we propose that phosphorylation of Ser 31 drives RhoGDI2 towards an unstructured form and prevents binding of RhoGDI2 to a GTPase.
While results obtained with the S31A-RhoGDI2 mutant vary, they are generally consistent with our structural analysis, and our proposed model of RhoGDI2 regulation by Ser 31 phosphorylation predicts the relative severity of the deficiencies caused by the amino acid substitutions or Ser 31 phosphorylation. Substitution of Ser 31 to Ala would eliminate the Ser 31 hydroxyl group hydrogen bonding with Glu 34 and would destabilize the protein and its interactions with Rac, although not to as great of an extent as phosphorylation of Ser 31 or mutation to an acidic residue. Thus, when viewed in terms of the equilibrium between an ordered and disordered state, mutation of Ser 31 to Ala would slightly shift the equilibrium towards a more disordered state. This could explain the intermediate effect of S31A-RhoGDI2 on Rac1 binding (). The effect of S31A-RhoGDI2 expression on Rac1 membrane distribution () also trends toward an intermediate effect between that of WT and S31E-RhoGDI2, although the effect of S31A-RhoGDI2 is not statistically significant from either WT or S31E-RhoGDI2 in this assay. On the other hand, Rac1 activation levels in the presence of S31A-RhoGDI2 are decreased to a similar extent as in samples expressing WT RhoGDI2 (). While binding to Rac1 is decreased in the S31A-RhoGDI2 mutant, it does still bind to Rac1, and this seems to be enough to depress Rac1 activation levels. It stands to reason that the S31A-RhoGDI2 mutant would behave similarly to WT RhoGDI2 in Rac1 activation assays given that WT RhoGDI2 is largely unphosphorylated in unstimulated cells (See , non-stimulated cells).
While several phosphorylation sites have been identified on RhoGDI1, only one other major phosphorylation site in RhoGDI2, Tyr 153, was identified prior to this work. Similar to Ser 31 phosphorylation, Src mediated phosphorylation of Tyr 153 results in decreased Rac1 binding and inability of RhoGDI2 to extract Rac1 from membranes. Because Tyr153 and Ser31 are in different structural domains of RhoGDI2, we imagine that even though phosphorylation reduces Rac1 binding in each case, it probably does so via different mechanisms. Interestingly, the phospho-mimetic Y153E-RhoGDI2 mutant has enhanced metastasis suppressor function in experimental lung metastasis assays of bladder cancer cells (18
), indicating that Src phosphorylation at Tyr 153 is a means of positively regulating RhoGDI2 metastasis suppressor function in bladder. The mechanisms through which this occurs are unclear, but thought to be related to the redistribution of RhoGDI2 to membranes, occuring upon Tyr 153 phosphorylation. This is in contrast to Ser 31 phosphorylation, which does not appear to redistribute the protein as the majority of the S31E-RhoGDI2 phospho-mimetic mutant remains in the cytosol ().
Given the effect of Tyr 153 on RhoGDI2 function, it is clear that phosphorylation of RhoGDI2 is an important mechanism to modulate its metastasis suppressor function. It is intriguing to speculate that PKCα mediated Ser 31 phosphorylation may also play a role in modulating RhoGDI2 metastasis suppression. Interestingly, PKCα protein levels have been shown to increase in bladder cancer with increasing tumor grade (29
), and PKCα was found to be more active in transitional cell carcinomas of bladder when compared to adjacent normal tissue (30
). Further, studies using pharmacological inihibition of conventional PKCs have shown that PKC α or β can enhance migration, invasion and cell growth of bladder cancer cells (31
), altogether suggesting that PKCα may be pro-tumorigenic, although extensive work remains to verify this. Thus, PKCα phosphorylation at Ser31 of RhoGDI2 may be an alternate means of modulating RhoGDI2 metastasis suppressor activity in tumor cells. Experiments to test this hypothesis will be the subject of future work.