Cytoskeleton-associated Protein 4 Research Articles

CKAP4 is a Dickkopf1 receptor and is involved in tumor progression.

Dickkopf1 (DKK1) is a secretory protein that antagonizes oncogenic Wnt signaling by binding to the Wnt coreceptor low-density lipoprotein receptor-related protein 6 (LRP6). DKK1 may also regulate its own signaling to promote cancer cell proliferation, but the mechanism is not understood. Here, we identified cytoskeleton-associated protein 4 (CKAP4) as a DKK1 receptor and evaluated CKAP4-mediated DKK1 signaling in cancer cell proliferation. We determined that DKK1 binds CKAP4 and LRP6 with similar affinity but interacts with these 2 receptors with different cysteine-rich domains. DKK1 induced internalization of CKAP4 in a clathrin-dependent manner, further supporting CKAP4 as a receptor for DKK1. DKK1/CKAP4 signaling activated AKT by forming a complex between the proline-rich domain of CKAP4 and the Src homology 3 domain of PI3K, resulting in proliferation of normal cells and cancer cells. Expression of DKK1 and CKAP4 was frequent in tumor lesions of human pancreatic and lung cancers, and simultaneous expression of both proteins in patient tumors was negatively correlated with prognosis and relapse-free survival. An anti-CKAP4 antibody blocked the binding of DKK1 to CKAP4, suppressed AKT activity in a human cancer cell line, and attenuated xenograft tumor formation in immunodeficient mice. Together, our results suggest that CKAP4 is a potential therapeutic target for cancers that express both DKK1 and CKAP4.

CKAP4 is identified as a receptor for Dickkopf in cancer cells.

The secretory protein Dickkopf-1 (DKK-1) is a known Wnt antagonist and has been shown to suppress tumorigenesis in some cancer cells; however, it is also upregulated in many types of cancer and associated with poor prognosis. Wnt-independent mechanisms by which DKK-1 promotes cancer cell proliferation are not well understood. In this issue of the JCI, Kimura and colleagues demonstrate that DKK-1 interacts with cytoskeleton-associated protein 4 (CKAP4) to promote activation of AKT. They show that both DKK-1 and CKAP4 are frequently upregulated in pancreatic and lung cancers. Importantly, targeting this interaction with an anti-CKAP4 antibody prevented tumor formation in murine xenograft models. These results identify a previously unrecognized DKK-1-mediated pathway and suggest CKAP4 as a potential therapeutic target for certain cancers.

Proteomic analysis of balding and non-balding mesenchyme-derived dermal papilla cells from androgenetic alopecia patients using on-line two-dimensional reversed phase-reversed phase LC–MS/MS

Androgenetic alopecia (AGA) is the most common and progressive disorder of hair loss with psychological effects. However, the exact mechanisms of baldness have not yet been elucidated. Until now, there has been no report using current proteomic approaches to examine balding and non-balding mesenchyme-derived DPCs simultaneously. To achieve the goal of identifying differentially expressed proteins in balding DPCs compared to non-balding DPCs, we present a strategy combining gel-assisted digestion and automatic on-line two-dimensional reversed phase-reversed phase (2D RP-RP) LC-MS/MS with informatics-assisted label-free protein quantitation. This strategy efficiently quantified the proteins of balding and non-balding DPCs from patients, and 128 up-regulated and 12 down-regulated proteins among 690 distinct proteins were identified in balding DPCs compared to non-balding DPCs. Up-regulated proteins belonging to these pathways in the balding DPCs, including argininosuccinate synthase 1 (ASS1), phosphoribosylaminoimidazole carboxylase (PAICS; ADE2), cytoskeleton-associated protein 4 (CKAP4), gelsolin (GSN), Ras GTPase-activating-like protein (IQGAP1), and S-phase kinase-associated protein 1 (SKP1), were confirmed by Western blot analysis. Gelsolin (GSN), Ras GTPase-activating-like protein (IQGAP1), plectin-1 (PLEC1), and nonerythroid α-spectrin (SPTAN1) were confirmed by immunofluorescence analysis. This proteomic approach to DPCs should help in understanding the pathogenesis of AGA. The results demonstrated the first deep proteomic approach to mesenchyme-derived dermal papilla cells (DPCs), which are a useful model system to investigate the mechanisms of baldness, from human hair and also identified differentially expressed proteins in balding DPCs compared to non-balding DPCs. This study should help in understanding the pathogenesis of androgenetic alopecia (AGA) and furthermore some proteins differentially expressed could be studied as therapeutic targets for AGA.

Antiproliferative Factor Regulates Connective Tissue Growth Factor (CTGF/CCN2) Expression in T24 Bladder Carcinoma Cells

Antiproliferative factor (APF) is a sialoglycopeptide elevated in the urine of patients with interstitial cystitis—a chronic, painful bladder disease. APF inhibits the proliferation of normal bladder epithelial and T24 bladder carcinoma cells in vitro by binding to cytoskeleton associated protein 4 (CKAP4) and altering the transcription of genes involved in proliferation, adhesion, and tumorigenesis; however, specific molecular mechanisms and effector genes that control APF's antiproliferative effects are unknown. In this study, we found that there was a 7.5‐fold upregulation of connective tissue growth factor (CTGF/CCN2) expression in T24 bladder carcinoma cells treated with APF, and Western blot revealed a dose‐dependent increase in CCN2 protein levels with secretion into the culture medium following APF treatment. CCN2 overexpression enhanced APF's antiproliferative activity while CCN2 knockdown by siRNA diminished it. Using a luciferase reporter construct, we found that APF treatment resulted in a 5‐fold activation of the CCN2 proximal promoter and that siRNA‐mediated knockdown of CKAP4 inhibited CCN2 upregulation. Additionally, we show that CKAP4 translocates to the nucleus and binds to the CCN2 proximal promoter in an APF‐dependent manner, providing evidence that CCN2 regulation by APF involves CKAP4 nuclear translocation and binding to the CCN2 promoter. NIH 1R03AR057193‐01; KIZ grant

Antiproliferative factor regulates connective tissue growth factor (CTGF/CCN2) expression in T24 bladder carcinoma cells

Antiproliferative factor (APF) is a sialoglycopeptide elevated in the urine of patients with interstitial cystitis (IC)-a chronic, painful bladder disease of unknown etiology. APF inhibits the proliferation of normal bladder epithelial and T24 bladder carcinoma cells in vitro by binding to cytoskeleton-associated protein 4 (CKAP4) and altering the transcription of genes involved in proliferation, cellular adhesion, and tumorigenesis; however, specific molecular mechanisms and effector genes that control APF's antiproliferative effects are unknown. In this study, we found that there was a 7.5-fold up-regulation of connective tissue growth factor (CTGF/CCN2) expression in T24 bladder carcinoma cells treated with APF. Western blot revealed a dose-dependent increase in CCN2 protein levels, with secretion into the culture medium after APF treatment. CCN2 overexpression enhanced APF's antiproliferative activity, whereas CCN2 knockdown diminished APF-induced p53 expression. Using a luciferase reporter construct, we found that APF treatment resulted in fivefold activation of the CCN2 proximal promoter and, of importance, that small interfering RNA-mediated knockdown of CKAP4 inhibited CCN2 upregulation. In addition, we demonstrate that CKAP4 translocates to the nucleus and binds to the CCN2 proximal promoter in an APF-dependent manner, providing evidence that CCN2 regulation by APF involves CKAP4 nuclear translocation and binding to the CCN2 promoter.

Antiproliferative Factor-Induced Changes in Phosphorylation and Palmitoylation of Cytoskeleton-Associated Protein-4 Regulate Its Nuclear Translocation and DNA Binding

Cytoskeleton-associated protein 4 (CKAP4) is a reversibly palmitoylated and phosphorylated transmembrane protein that functions as a high-affinity receptor for antiproliferative factor (APF)—a sialoglycopeptide secreted from bladder epithelial cells of patients with interstitial cystitis (IC). Palmitoylation of CKAP4 by the palmitoyl acyltransferase, DHHC2, is required for its cell surface localization and subsequent APF signal transduction; however, the mechanism for APF signal transduction by CKAP4 is unknown. In this paper, we demonstrate that APF treatment induces serine phosphorylation of residues S3, S17, and S19 of CKAP4 and nuclear translocation of CKAP4. Additionally, we demonstrate that CKAP4 binds gDNA in a phosphorylation-dependent manner in response to APF treatment, and that a phosphomimicking, constitutively nonpalmitoylated form of CKAP4 localizes to the nucleus, binds DNA, and mimics the inhibitory effects of APF on cellular proliferation. These results reveal a novel role for CKAP4 as a downstream effecter for APF signal transduction.

Palmitoylation of Cytoskeleton Associated Protein 4 by DHHC2 Regulates Antiproliferative Factor-mediated Signaling

Previously, we identified cytoskeleton-associated protein 4 (CKAP4) as a major substrate of the palmitoyl acyltransferase, DHHC2, using a novel proteomic method called palmitoyl-cysteine identification, capture and analysis (PICA). CKAP4 is a reversibly palmitoylated and phosphorylated protein that links the ER to the cytoskeleton. It is also a high-affinity receptor for antiproliferative factor (APF), a small sialoglycopeptide secreted from bladder epithelial cells of patients with interstitial cystitis (IC). The role of DHHC2-mediated palmitoylation of CKAP4 in the antiproliferative response of HeLa and normal bladder epithelial cells to APF was investigated. Our data show that siRNA-mediated knockdown of DHHC2 and consequent suppression of CKAP4 palmitoylation inhibited the ability of APF to regulate cellular proliferation and blocked APF-induced changes in the expression of E-cadherin, vimentin, and ZO-1 (genes known to play a role in cellular proliferation and tumorigenesis). Immunocytochemistry revealed that CKAP4 palmitoylation by DHHC2 is required for its trafficking from the ER to the plasma membrane and for its nuclear localization. These data suggest an important role for DHHC2-mediated palmitoylation of CKAP4 in IC and in opposing cancer-related cellular behaviors and support the idea that DHHC2 is a tumor suppressor.