5899

RALB

v-ral simian leukemia viral oncogene homolog B;RALB;v-ral simian leukemia viral oncogene homolog B

RAS-like protein B|ras related GTP binding protein B|ras-related protein Ral-B|v-ral simian leukemia viral oncogene homolog B (ras related; GTP binding protein)

2q14.2

protein-coding

RalA and RalB are monomeric G proteins that are 83% identical in amino acid sequence but have paralogue-specific effects on cell proliferation, metastasis, and apoptosis. Using in vitro kinase assays and phosphosite-specific 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.

BACKGROUND: The Ral guanine nucleotide-exchange factors (RalGEFs) serve as key effectors for Ras oncogene transformation of immortalized human cells. RalGEFs are activators of the highly related RalA and RalB small GTPases, although only the former has been found to promote Ras-mediated growth transformation of human cells. In the present study, we determined whether RalA and RalB also had divergent roles in promoting the aberrant growth of pancreatic cancers, which are characterized by the highest occurrence of Ras mutations. RESULTS: We now show that inhibition of RalA but not RalB expression universally reduced the transformed and tumorigenic growth in a panel of ten genetically diverse human pancreatic cancer cell lines. Despite the apparent unimportant role of RalB in tumorigenic growth, it was nevertheless critical for invasion in seven of nine pancreatic cancer cell lines and for metastasis as assessed by tail-vein injection of three different tumorigenic cell lines tested. Moreover, both RalA and RalB were more commonly activated in pancreatic tumor tissue than other Ras effector pathways. CONCLUSIONS: RalA function is critical to tumor initiation, whereas RalB function is more important for tumor metastasis in the tested cell lines and thus argues for critical, but distinct, roles of Ral proteins during the dynamic progression of Ras-driven pancreatic cancers.

A hallmark of metastasis is organ specificity; however, little is known about the underlying signaling pathways responsible for the colonization and growth of tumor cells in target organs. Since tyrosine kinase receptor activation is frequently associated with prostate cancer progression, we have investigated the role of a common signaling intermediary, activated Ras, in prostate cancer metastasis. Three effector pathways downstream of Ras, Raf/extracellular signal-regulated kinase (ERK), phosphatidylinositol 3-kinase, and Ral guanine nucleotide exchange factors (RalGEFs), were assayed for their ability to promote the metastasis of a tumorigenic, nonmetastatic human prostate cancer cell line, DU145. Oncogenic Ras promoted the metastasis of DU145 to multiple organs, including bone and brain. Activation of the Raf/ERK pathway stimulated metastatic colonization of the brain, while activation of the RalGEF pathway led to bone metastases, the most common organ site for prostate cancer metastasis. In addition, loss of RalA in the metastatic PC3 cell line inhibited bone metastasis but did not affect subcutaneous tumor growth. Loss of Ral appeared to suppress expansive growth of prostate cancer cells in bone, whereas homing and initial colonization were less affected. These data extend our understanding of the functional roles of the Ral pathway and begin to identify signaling pathways relevant for organ-specific metastasis.