Cytoskeleton‐Associated Protein 4 (CKAP4) Knockdown Disrupts the Actin Filament Network in Bladder Carcinoma Cells

Abstract

Cytoskeleton‐associated protein 4 (CKAP4) is a 63kDa, non‐glycosylated type II integral membrane protein that plays an essential role in the maintenance of endoplasmic reticulum structure by anchoring the rough ER to microtubules in epithelial cells. More recently, CKAP4 has been identified as a biologically relevant receptor for four unique ligands—tissue plasminogen activator, surfactant protein A, Dickkopf1, and antiproliferative factor. While research shows that CKAP4 interacts directly with the microtubule network, its potential role within the actin filament network has not been previously studied. To explore this putative role, we used RNA interference to reduce CKAP4 expression in T24 bladder carcinoma cells and then fixed and stained the actin cytoskeleton using phalloidin‐488. We observed a marked difference in actin filament morphology and a disruption of the actin filament network in cells treated with CKAP4 siRNA versus cells treated with control siRNA or vehicle alone; moreover, CKAP4 knock down generated a significant reduction in actin polymerization. To understand the observed phenotypic changes, we performed differential gene expression analysis using quantitative PCR. Our data showed that CKAP4 knock down led to an increase in the expression of genes known to promote cell motility and dynamic changes in the cytoskeleton with a concomitant reduction in the expression of genes involved in stress fiber formation. Western blot analysis of selected, differentially expressed proteins supported the gene expression data. Collectively, these results suggest that CKAP4 may play an important role in regulating the actin cytoskeleton.