This study reports phosphorylation of RalA and RalB by different protein kinases. RalB is phosphorylated at Ser198 by PKC, while RalA is phosphorylated at Ser194 by Aurora A kinase (19
) and PKA. The S198 site is phosphorylated in stably expressed RalB and more importantly, in endogenous RalB in living cells activated by phorbol ester. The Ser198 site (KKS198
FK) in RalB conforms to a motif recognized by PKC [(R/K)X(S)(Hyd)(R/K)] and mutation of this Ser to Ala reduced PKC phosphorylation about 70%, with the other 30% of phosphorylation presumably occurring at another site(s). Mutation of Ser182 and Ser192 in addition to Ser198 did not eliminate phosphorylation of RalB and mass spectrometry did not locate other sites of phosphorylation up to residue 175 of RalB. While the identity of secondary PKC phosphorylation site(s) in RalB remains unknown, Ser198 clearly is the primary PKC phosphorylation site, with functional consequences. Similarly, RalA was shown to be phosphorylated at multiple sites, besides major Ser194 sites, Ser183 was phosphorylated in response to suppression of phosphatase PP2A Aβ subunit (20
The phosphorylation of S198 is important for anchorage-independent cell growth, cell motility and actin cytoskeletal organization, in vivo bladder cancer tumorigenicity driven by active RalB. We also evaluated the role of RalB phosphorylation in regulating experimental lung metastasis in bladder cancer by using loss of function experiments. Similar to what was noted in prostate cancer for bone metastasis for the DU145 cell line (11
), RalB depletion by RNAi reduced lung colonization and increased survival in UMUC3 cells. Importantly, when we restored Ral expression to endogenous levels by expressing wild type RalB, and phosphosite mutant RalB(S198A) only the former restored the metastatic competence of the cells. This indicates phosphorylation of RalB is important for the development of lung metastasis in human bladder cancer cells.
Our finding that RalA and RalB are phosphorylated by different kinases may explain in part the different cellular actions of these paralogs, despite nearly identical overall protein sequence. The situation with RalA and RalB may be similar to the Ras family member Rap1 that is phosphorylated by PKA. Phosphorylation alters the ability of Rap1 to associate and regulate its downstream targets (27
). Previous reports claimed that phosphorylation of RalA at Ser194 by Aurora-A kinase regulates RalA activity as measured by effector RalBP1 binding and function in cells (19
). We compared the binding of WT and S198A RalB with downstream effectors RalBP1, Sec5 and Exo84. We found co-precipitation with these effectors was not different. The phosphorylation of RalB may affect binding to another yet undetermined protein in bladder cancer cells. Phosphorylation of RhoA by PKA induces relocalization of RhoA (31
). Recently, it was reported that RalA phosphorylation at S194 by Aurora-A kinase translocated RalA from the plasma membrane (21
). Here in human bladder cells we also observed phosphorylation of RalB by PKC in inducing RalB translocation from plasma membrane to perinuclear regions, and it depends on S198 phosphorylation. This might be the functional consequence of this RalB phosphorylation.
RalB phosphorylation by PKC has interesting implications for human bladder cancer. RalB has been implicated in bladder cancer migration and metastasis (10
) and PKC is known to play an important role in cellular differentiation and in malignancy and metastasis (33
). PKC activity is elevated in invasive human bladder carcinoma cell line EJ and inhibition of kinases by staurosporine inhibited invasion of this cell line (36
). PMA also upregulated vascular endothelial growth factor (VEGF) expression via PKC signaling in human bladder transitional carcinoma cell line RT4 and this was inhibited by staurosporine. VEGF is known as a key factor in human tumorigenesis and metastatic potential (37
) and has been shown to be regulated by Ral GTPases (38
). In colon carcinoma cells PKCα contributes to high migratory activity (39
) and a PKC inhibitor effectively inhibits migration and invasion of bladder carcinoma cells (40
). Taken together, there appears to be substantial evidence for involvement of RalB and PKC in human bladder progression. Importantly, by linking PKC and RalB our results may expose one of the underlying mechanisms whereby PMA and other PKC activators act as tumor promoters in cancer since RalB is involved in multiple cancer types (9
). Based on this data, work to determine which PKC isoforms affect S198 in RalB appears warranted.
In summary, RalB phosphorylation translocates RalB from membrane to perinuclear in an S198-dependent fashion, is necessary for RalB effects on cell migration and anchorage-independent growth in vitro and tumor growth and metastasis in vivo, irrespective of RalB activation by GTP loading. Importantly, these results suggest that RalB functions as an “AND” gate in signaling, where both GTP loading and S198 phosphorylation are required for full biological activity. We should look for circumstances where RalGEFs and PKCs are coincidently activated, as conditions for RalB action. Since PKCs are implicated in bladder cancer progression and react with S198, inhibition of PKC, activation of the S198 phosphatase or mimicking the phosphosite to interfere with RalB targeting could prove useful for treating metastatic bladder cancer.