Phosphorylation of RalB is important for bladder cancer cell growth and metastasis.

Abstract

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.

• Authors: Hong Wang, Charles Owens, Nidhi Chandra, Mark R Conaway, David L Brautigan, Dan Theodorescu
• Journal: Cancer research (Cancer Res) Vol. 70 Issue 21 Pg. 8760-9 (Nov 01 2010) ISSN: 1538-7445 [Electronic] United States
• PMID: 20940393 (Publication Type: Journal Article, Research Support, N.I.H., Extramural)
• Copyright: ©2010 AACR.

Chemical References

• Actins
• RNA, Messenger
• RNA, Small Interfering
• Ralb protein, human
• Cyclic AMP-Dependent Protein Kinases
• Protein Kinase C
• RALA protein, human
• ral GTP-Binding Proteins
• Tetradecanoylphorbol Acetate

Topics

• Actins (metabolism)
• Animals
• Apoptosis
• Blotting, Western
• Cell Adhesion
• Cell Movement
• Cell Proliferation
• Cyclic AMP-Dependent Protein Kinases (metabolism)
• Cytoskeleton (metabolism)
• Female
• Fluorescent Antibody Technique
• Humans
• Immunoprecipitation
• Lung Neoplasms (metabolism, prevention & control, secondary)
• Mice
• Mice, Nude
• Mutagenesis, Site-Directed
• Mutation (genetics)
• Phosphorylation
• Protein Kinase C (metabolism)
• RNA, Messenger (genetics)
• RNA, Small Interfering (pharmacology)
• Rabbits
• Reverse Transcriptase Polymerase Chain Reaction
• Tetradecanoylphorbol Acetate (pharmacology)
• Urinary Bladder Neoplasms (metabolism, pathology, prevention & control)
• Wound Healing
• ral GTP-Binding Proteins (antagonists & inhibitors, genetics, metabolism)