RalA and RalB are monomeric G proteins that are 83% identical in amino acid sequence but have paraloguespecific effects on cell proliferation, metastasis, and apoptosis. Using in vitro kinase assays and phosphositespecific antibodies, here we show phosphorylation of RalB by protein kinase C (PKC) and RalA by protein kinase A. We used mass spectrometry and site-directed mutagenesis to identify S198 as the primary PKC phosphorylation site in RalB. Phorbol ester [phorbol 12-myristate 13-acetate (PMA)] treatment of human bladder carcinoma cells induced S198 phosphorylation of stably expressed FLAG-RalB as well as endogenous RalB. PMA treatment caused RalB translocation from the plasma membrane to perinuclear regions in a S198 phosphorylation-dependent manner. Using RNA interference depletion of RalB followed by rescue with wild-type RalB or RalB(S198A) as well as overexpression of wild-type RalB or RalB(S198A) with and without PMA stimulation, we show that phosphorylation of RalB at S198 is necessary for actin cytoskeletal organization, anchorage-independent growth, cell migration, and experimental lung metastasis of T24 or UMUC3 human bladder cancer cells. In addition, UMUC3 cells transfected with a constitutively active RalB(G23V) exhibited enhanced subcutaneous tumor growth, whereas those transfected with phospho-deficient RalB(G23V-S198A) were indistinguishable from control cells. Our data show that RalA and RalB are phosphorylated by different kinases, and RalB phosphorylation is necessary for in vitro cellular functions and in vivo tumor growth and metastasis. Cancer Res; 70(21); 8760-9. ©2010 AACR.

RalA expression in human prostate cancer is associated with cell migration and is necessary for bone metastasis. However, the downstream effectors of RalA that mediate these functions remain unclear. Here we examined cell migration after small interfering RNA-mediated depletion of Ral effectors Ral binding protein 1 (RalBP1/RLIP), exocyst complex component 2 (Sec5), and phospholipase D1 (PLD1) and found that RalBP1 and RalA depletion inhibited cell migration to a similar extent. Stable lentivirus short hairpin interfering RNA-mediated depletion of RalA and RalBP1 in PC3 human prostate cancer cells inhibited bone metastasis after intracardiac inoculation. Depletion of RalBP1 diminished orthotopic tumor growth of PC3 cells and inhibited spontaneous metastasis from this site. Interestingly, the expression of wild-type or RalA mutants deficient in RalBP1 binding was effective at rescuing the reduced metastatic capacity of RalA-depleted PC3 cells, suggesting that RalA depletion does not reduce this solely by diminished interaction with RalBP1. To determine whether the role of RalBP1 in metastasis is relevant beyond prostate cancer, we studied the requirement of RalBP1 expression in an experimental metastasis model of human bladder cancer, a tumor type with high RalBP1 expression. Depletion of RalBP1 in UMUC3 cells resulted in decreased lung colonization while having a minimal effect on subcutaneous tumor growth. Our studies are the first to suggest that the expression of RalBP1 is necessary for human cancer cell metastasis. Furthermore, we show that the requirement for RalA expression for manifestation of this phenotype is not entirely dependent on a RalA-RalBP1 interaction.

RalA and RalB are members of the Ras family of monomeric G proteins that are 83% identical in amino acid sequence, but have paralog-specific effects on cancer cell proliferation, metastasis and apoptosis. Using in vitro kinase assays and phosphosite specific antibodies, here we show paralog-specific phosphorylation of RalA by PKA and RalB by PKC. Mutagenesis identified Ser194 as the single PKA phosphorylation site in RalA and S198 as the primary PKC phosphorylation site in RalB. Phorbol ester treatment of human bladder carcinoma cells induced phosphorylation of S198 in both stably expressed FLAG-RalB and endogenous RalB. Compared to activated RalB(G23V) that localized at plasma membrane, phosphosite mutant RalB(G23V-S198A) colocalized with GFP-Rab11, a recycling endosome marker. Phorbol ester treatment enhanced anchorage independent growth of UMUC3 human bladder carcinoma cells stably transfected with wild type RalB or activated RalB(G23V), but not with phosphosite mutants RalB(S198A) or RalB(G23V-S198A). Furthermore, UMUC3 cells transfected with RalB(G23V) exhibited enhanced subcutaneous tumor growth compared to vector transfected cells, but cells expressing RalB(G23V-S198A) had growth indistinguishable from the controls. Our data demonstrate that RalA and RalB are specifically phosphorylated by different kinases, and RalB phosphorylation regulates its intracellular localization and in vivo tumor growth promotion. Phosphorylation of RalA and RalB by different kinases may contribute to the biological specificity of these closely related proteins. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 311.

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