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Vol. 20, Issue 5, 1454-1463, March 1, 2009

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

Sonia L. Planey^*,, Susan K. Keay^,, Chen-Ou Zhang, and David A. Zacharias^*

*Whitney Laboratory, Department of Neuroscience, University of Florida, St. Augustine, FL 32080; and University of Maryland School of Medicine and VA Medical Center, Baltimore, MD 21201

Submitted August 18, 2008; Revised December 22, 2008; Accepted January 5, 2009
Monitoring Editor: Benjamin Margolis

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.

Present address: The Commonwealth Medical College, Department of Basic Sciences, Tobin Hall, 501 Madison Ave, Scranton, Pennsylvania, 18510.

Address correspondence to: Sonia L. Planey (splaney{at}tcmedc.org)

Abbreviations used: APF, antiproliferative factor; CKAP4, cytoskeleton-associated protein 4/p63; CRD, cysteine-rich domain; DHHC, Asp-His-His-Cys; ER, endoplasmic reticulum; FITC, fluorescein isothiocyanate; IC, interstitial cystitis; LZ, leucine zipper; MT, microtubule; NB, normal bladder; NGS, normal goat serum; PAT, palmitoyl acyl transferase; PICA, palmitoyl-cysteine isolation, capture and analysis; PM, plasma membrane; qRT-PCR, quantitative real-time PCR; TRITC, tetramethyl rhodamine isothiocyanate; VSMC, vascular smooth muscle cells.