Rho Kinase Phosphorylation Promotes Ezrin-Mediated Metastasis in Hepatocellular Carcinoma

Abstract

During progression of hepatocellular carcinoma, multiple genetic and epigenetic alterations act to posttranslationally modulate the function of proteins that promote cancer invasion and metastasis. To define such abnormalities that contribute to liver cancer metastasis, we carried out a proteomic comparison of primary hepatocellular carcinoma and samples of intravascular thrombi from the same patient. Mass spectrometric analyses of the liver cancer samples revealed a series of acidic phospho-isotypes associated with the intravascular thrombi samples. In particular, we found that Thr567 hyperphosphorylation of the cytoskeletal protein ezrin was tightly correlated to an invasive phenotype of clinical hepatocellular carcinomas and to poor outcomes in tumor xenograft assays. Using phospho-mimicking mutants, we showed that ezrin phosphorylation at Thr567 promoted in vitro invasion by hepatocarcinoma cells. Phospho-mimicking mutant ezrinT567D, but not the nonphosphorylatable mutant ezrinT567A, stimulated formation of membrane ruffles, suggesting that Thr567 phosphorylation promotes cytoskeletal-membrane remodeling. Importantly, inhibition of Rho kinase, either by Y27632 or RNA interference, resulted in inhibition of Thr567 phosphorylation and a blockade to cell invasion, implicating Rho kinase-ezrin signaling in hepatocellular carcinoma cell invasion. Our findings suggest a strategy to reduce liver tumor metastasis by blocking Rho kinase-mediated phosphorylation of ezrin.

Spatial Control of Proton Pump H,K-ATPase Docking at the Apical Membrane by Phosphorylation-coupled Ezrin-Syntaxin 3 Interaction

“…Our previous study demonstrated that phosphorylation of the conserved Thr-567 residue of ezrin alters the physiology of gastric parietal cells (26) and participates in hepatocarcinoma metastasis (30). We recently established a protocol in which phosphorylation-mediated protein conformational change can be studied at the single molecule level using AFM (18).…”

Cell Polarity Kinase MST4 Cooperates with cAMP-dependent Kinase to Orchestrate Histamine-stimulated Acid Secretion in Gastric Parietal Cells

“…Our early study has demonstrated that persistent phosphorylation of the conserved Thr-567 residue of ezrin alone alters the physiology of gastric parietal cell polarity (26), and this persistent phosphorylation also participates in hepatocarcinoma metastasis (27). We have recently established a protocol in which phosphorylationmediated protein conformational change can be studied at the single-molecule level using atomic force microscopy (17).…”

TGFβ and IGF1R signaling activates protein kinase A through differential regulation of ezrin phosphorylation in colon cancer cells

“…Phosphorylation at threonine 567 (T567) transitions ezrin from inactive oligomers into active monomers by unmasking the active sites through dissociation of FERM and Cterminal domains (11)(12)(13)(14)(15). Ezrin plays an important role in cell motility and invasion and has been implicated in metastasis of several types of cancer including CRC (16)(17)(18)(19)(20)(21). In addition, ezrin plays a key role in cell survival of colon cancer cells and regulates expression of Inhibitor of Apoptosis Proteins (IAP), XIAP and survivin, which have been shown to be involved in cell survival and metastasis (5,22).…”