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. 1. Introduction Cytoskeleton-associated protein 4 (CKAP4; also known as CLIMP-63, ERGIC-63, and p63) is a 63？kDa, reversibly palmitoylated and phosphorylated, type II transmembrane (TM) protein, originally identified as a resident of the endoplasmic reticulum/Golgi intermediate complex (ERGIC) [1–5]. The first report describing CKAP4 [1] (referring to it as “p62”) demonstrated that its palmitoylation peaked during mitosis and suggested that palmitoylation may be an important regulator of vesicular transport between various membranous compartments. Soon thereafter, Schweizer and colleagues cloned CKAP4 and identified membrane-proximal cysteine 100 (C100) as the site for palmitoylation [4]. More recently, DHHC2 was identified as the palmitoyl acyltransferase (PAT) that palmitoylates CKAP4 at C100 [6]. CKAP4 is localized prominently to the endoplasmic reticulum (ER). One major function of CKAP4 is to anchor rough ER to microtubules, organizing the overall structure of ER with respect to the microtubule network [3–5, 7, 8]. The binding of CKAP4 to microtubules is regulated by phosphorylation of three critical serine residues (S3, S17, and S19) located in its cytosolic, N-terminal domain (Figure 1) [2]. CKAP4 is unique among microtubule binding proteins in at least one respect: it is a TM protein. However, it is similar to many other microtubule-binding proteins in that phosphorylation blocks its ability to bind microtubules [9]. Overexpression of a mutant version of CKAP4 that mimics phosphorylation of three