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

Abstract

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. INTRODUCTIONPalmitoylation is the posttranslational addition of the 16-carbon palmitate group to specific cysteine residues of proteins (Smotrys and Linder, 2004) via a labile thioester bond. Unlike other forms of lipidation, such as myristoylation and prenylation, palmitoylation is reversible which allows for dynamic regulation of protein-membrane interactions, trafficking between membrane compartments (Wedegaertner and Bourne, 1994;Jones et al., 1997;Moran et al., 2001;Zacharias et al., 2002), and synaptic plasticity (el-Husseini Ael and Bredt, 2002). For many years it was believed that palmitoylation occurred primarily by autocatalytic mechanisms (Bizzozero et al., 1987;Bano et al., 1998); however, the recent discovery of a family of palmitoyl acyltransferase (PAT) enzymes that catalyze protein palmitoylation has reversed this notion, expanding the complexity of the mechanisms by which palmitoylation is regulated (Lobo et al., 2002;Roth et al., 2002;Fukata et al., 2004;Linder and Deschenes, 2007).PATs are encoded by the ZDHHC gene family and are characterized by an Asp-His-His-Cys motif (DHHC) within a cysteine-rich domain (CRD). The DHHC and CRD domains are essential for palmitoyl acyltransferase activity (Roth et al., 2002;Fukata et al., 2004;Sharma et al., 2008). Twenty-three genes encoding proteins with DHHC-CRD domains have been identified in mouse and human databases (Fukata et al., 2004). Of these, seven have already been shown to be associated with human disease: DHHC8 with schizophrenia (Mukai et al., 2004); DHHC17/HIP14 with Huntington's disease (Yanai et al., 2006); DHHC15 and DHHC9 with X-linked mental retardation (Mansouri et al., 2005;Raymond et al., 2007); and DHHC2, D...

Quantitative Proteomics Identifies a β-Catenin Network as an Element of the Signaling Response to Frizzled-8 Protein-Related Antiproliferative Factor

“…Thus, it is unlikely that APF can interact with Wnt and consequently lead to the down-regulation of ␤-catenin. Instead, APF might down-modulate ␤-catenin by inhibiting the production of HB-EGF (8) and/or antagonizing the binding of tissue plasminogen activator (tPA) with CKAP4, a high-affinity receptor for APF (6,7). This speculation is based on previous findings that HB-EGF may activate the phosphatidylinositol 3-kinase/Akt pathway and in turn inhibit GSK3␤ and stabilize ␤-catenin (37), and that CKAP4 is also a receptor for tPA (38), which may activate ␤-catenin signaling by transactivating EGFR (39).…”

Cytoskeleton-Associated Protein 4: Functions Beyond the Endoplasmic Reticulum in Physiology and Disease

“…It has also been shown that knockdown of DHHC2 by siRNA inhibited plasma membrane localization and the ability of APF to inhibit proliferation [14]. CKAP4 has also been predicted to undergo posttranslational modification by tyrosine sulfation, the addition of a negatively charged sulfate group to a tyrosine residue, at tyrosine 323 [25,26].…”

“…For example, CKAP4 has been shown to be required for APF-mediated signaling [11,[13][14][15][16], for gentamycin-induced cytotoxicity [17], for regulation of tPA function [10], and for SP-A-induced surfactant uptake by pneumocytes [18]. This evidence for CKAP4 mediating a wide array of cellular responses important in physiological and pathological processes, such as interstitial cystitis, druginduced cytotoxicity, pericullar proteolytic activity, and lung lipid homeostasis, provides new insights into this classically ER-resident protein.…”

IMPAIRED REGULATORY MECHANISMS OF THE mTOR SIGNALING PATHWAY IN OSTEOARTHROSIS

“…О р и г и н а л ь н ы е и с с л е д о в а н и я Науч-практич ревматол 2012; 50(6): [33][34][35][36][37] Экспрессия генов ключевых метаболических путей, регулирующих сигнальный путь mTOR, в крови больных ОА на разных стадиях забо-левания (а -и). С -здоровые лица, LOW -подгруппа больных ОА с низкой экспрессией гена mTOR; HIGH -подгруппа больных ОА с вы-сокой экспрессией гена mTOR; ES -больные ОА на поздней стадии заболевания Обсуждение У больных ОА в подгруппе LOW метаболические на-рушения, приводящие к ингибированию гена mTOR, могут быть связаны с недостаточностью энергетического метабо-лизма, смещением баланса в сторону АМФ, что приводит к активации сигнального пути АМПК -ингибитора mTOR [18].…”

“…Пальмитат в клетках регулирует 162 гена, индуцирует апоптоз и старение многих типов кле-ток [32]. Кроме того, пальмитоирование мембранных бел-ков блокирует пролиферативную активность клеток [33]. Возможно, именно это нарушение метаболизма является критическим для активного разрушения хряща, а также для развития болевого синдрома у больных ОА [16,17] на поздней стадии заболевания.…”