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Vol. 20, Issue 5, 1454-1463, March 1, 2009
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*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
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) 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, DHHC9, DHHC17, and DHHC11 with cancer (Oyama et al., 2000; Ducker et al., 2004; Mansilla et al., 2007; Yamamoto et al., 2007). In several of these examples, the absence of PAT expression and subsequent failure to palmitoylate target substrates is the underlying problem.
Although now recognized as a PAT, DHHC2 was previously known as ream, an acronym for reduced expression associated with metastasis. As the name suggests, this gene was first identified because its expression level was consistently and significantly reduced in clonal murine colorectal adenocarcinoma cell lines with high metastatic potential, but not in clonal lines derived from the same tumor that did not metastasize (Tsuruo et al., 1983; Oyama et al., 2000). It was concluded that ream expression is inversely related to the metastatic potential of a cell, leading to speculation that this gene normally suppresses one or more of the processes by which cancer cells escape from blood vessels, invade into and proliferate in a target organ, and induce angiogenesis and form metastatic foci.
Human ZDHHC2 maps to a region of chromosome 8 (p21.3-22) that is frequently deleted in many types of cancer, including colorectal (Fujiwara et al., 1993, 1994; Ichii et al., 1993) hepatocellular carcinoma (Emi et al., 1993; Fujiwara et al., 1994), nonsmall cell lung (Fujiwara et al., 1993; Ohata et al., 1993), and cancers of the breast (Yaremko et al., 1996; Anbazhagan et al., 1998), urinary bladder (Knowles et al., 1993), and prostate (Bova et al., 1993). Loss of heterozygosity on chromosomal band 8p22 has been shown to be a common event in some epithelial tumors, pointing toward the likelihood that the region harbors potential tumor suppressor genes (Emi et al., 1993; Fujiwara et al., 1993; Ichii et al., 1993; Ohata et al., 1993).
Because DHHC2 has no other known signaling properties beyond palmitoylation, knowledge of its target substrates in a cancer cell line could yield significant clues about the role of DHHC2-mediated palmitoylation in metastasis and tumor suppression. In previous work, we used a novel, proteomic method called palmitoyl-cysteine identification, capture and analysis (PICA) to identify the target substrates of DHHC2 in HeLa cells, a cervical adenocarcinoma cell line. We determined that cytoskeleton-associated protein 4 (CKAP4, also known as p63, ERGIC-63, and CLIMP-63) is a principle, physiologically important substrate of DHHC2 (Zhang et al., 2008).
CKAP4 is a reversibly palmitoylated, type II transmembrane protein that has been shown to anchor rough endoplasmic reticulum (ER) to microtubules in epithelial cells (i.e., COS and HeLa; Schweizer et al., 1993a,b, 1994, 1995a; Vedrenne and Hauri, 2006). This function requires a direct interaction between the cytoplasmic N-terminal tail of the protein to microtubules and is regulated by phosphorylation of three critical serine residues (Klopfenstein et al., 1998). More recently, CKAP4 has been identified as a functional cell surface receptor for antiproliferative factor (APF; Conrads et al., 2006), a low-molecular-weight, Frizzled-8 protein–related sialoglycopeptide secreted from bladder epithelial cells in patients suffering from the chronic, painful bladder disorder, interstitial cystitis (IC; Keay et al., 2000, 2004a). APF profoundly inhibits normal bladder epithelial cell growth (Keay et al., 1996, 2000, 2004a). APF also inhibits the proliferation of bladder carcinoma cells and HeLa cells in vitro with an IC50 of 
1 nM (Keay et al., 2004a, 2006; Conrads et al., 2006). CKAP4 has been shown to mediate the antiproliferative effects of APF, which is also known to alter transcription of at least 13 genes known to be involved in the regulation of proliferation and tumorigenesis (including E-cadherin, vimentin, cyclin D1, p53, and ZO-1; Keay et al., 2003; Conrads et al., 2006; Kim et al., 2007).
In this study, we examined the effects of reduced CKAP4 palmitoylation on APF-mediated signaling by silencing the expression of DHHC2 with targeted siRNA. Our data show that DHHC2-mediated palmitoylation of CKAP4 is a critical event regulating CKAP4 subcellular distribution and APF-stimulated changes in cellular proliferation and gene expression.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell Culture and Transfections
HeLa (ATCC CCL-2; American Type Culture Collection, Manassas, VA) cells were maintained in DMEM containing 10% fetal bovine serum (FBS), 100 U/ml penicillin, 100 µg/ml streptomycin, and 1 µg/ml fungizone (all from Invitrogen). Cells were transfected using FuGENE6 reagent (Roche, Basel, Switzerland) according to the manufacturer's instructions. To obtain stable clones, cells were diluted into 96-well plates (100 cells/well) 24 h after transfection and selected in the presence of 0.4 mg/ml Geneticin (G418; Invitrogen).
Normal primary bladder (NB) epithelial cells were isolated from patients as previously described (Keay et al., 1996, 2000, 2004a; Conrads et al., 2006) Cells were propagated in DMEM-F12 (Mediatech, Manassas, VA) with 10% heat-inactivated FBS, 1% antibiotic/antimycotic solution, 1% L-glutamine, 0.25 U/ml insulin (Sigma, St. Louis, MO), and 5 ng/ml human epidermal growth factor (R&D Systems, Minneapolis, MN) at 37°C in a 5% CO2 atmosphere and characterized by binding of AE-1/AE-3 pancytokeratin antibodies (Signet, Emeryville, CA) as previously described (Keay et al., 1996, 2004b).
Small Interfering RNA
Double-stranded small interfering RNA (siRNA; ON-TARGETplus) targeting ZDHHC2 and nonsense siRNA (ON-TARGETplus Control siRNA) were purchased from Dharmacon (Lafayette, CO). HeLa cells were trypsinized for 5 min at 37°C and centrifuged in DMEM growth medium, and the cell pellet was resuspended in serum-free medium at a density of 1 x 106 cells/ml. Two hundred microliters of the cell suspension was then transferred to a sterile 2-mm cuvette with 14 µg siRNA and electroporated at 160 V/500 µF capacitance using a Bio-Rad Gene Pulser Xcell (Hercules, CA). The cells were immediately transferred to 96-well plates for thymidine incorporation assay or to LabTek multiwell glass slides (Nalge Nunc, Rochester, NY) for immunocytochemistry. To determine the effectiveness of siRNA-mediated knockdown, we used quantitative real-time PCR (qRT-PCR), to measure the abundance of ZDHHC2 mRNA at times 0, 12, 24, 48, 72, and 96 h after transfection of the siRNA. These experiments were run in triplicate.
[3H]Thymidine Incorporation
Cell proliferation was measured by [3H]thymidine incorporation into the DNA of HeLa or NB epithelial cells. Briefly, synthetic APF or inactive control peptide (NeoMPS, San Diego, CA) was resuspended in acetonitrile/distilled water (1:1), diluted in serum-free DMEM, and applied to HeLa or NB cells; cell controls received acetonitrile/distilled water diluted in serum-free DMEM alone. Cells were then incubated at 37°C in a 5% CO2 atmosphere for 48 h. The cells were then labeled with 1 µCi/well [3H]thymidine for 4 h, trypsinized, insoluble cell contents harvested and methanol-fixed onto glass fiber filter paper, and the amount of radioactivity incorporated determined. Significant inhibition of [3H]thymidine incorporation was defined as a mean decrease in cpm of greater than 2 SDs from the mean of control cells for each plate.
Immunocytochemistry
HeLa cells stably transfected with WT CKAP4 or CKAP4 C100S were seeded at a density of 2 x 104 cells/well in eight-well LabTek chamber slides (Nalge Nunc) and grown to semiconfluence in DMEM medium containing 10% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin, 1 µg/ml fungizone, and 0.4 mg/ml G418 (all from Invitrogen). Cells were fixed for 20 min with 3% paraformaldehyde in phosphate-buffered saline (PBS), permeabilized with 0.1% Triton X-100 in PBS, and blocked in PBS/5% NGS (normal goat serum). Cells transfected with DHHC2 siRNA and treated with synthetic APF (Peptides International, Louisville, KY) were fixed using ethanol/acetone (1:1) for 15 min at room temperature and washed three times with 1x PBS before blocking in PBS/5% normal goat serum (NGS). The following primary antibodies were used: mouse mAb G1/296 against CKAP4 ("anti-CLIMP-63", diluted 1:100, Alexis Biochemicals, San Diego, CA), rabbit pAb against calreticulin (diluted 1:1000, Abcam, Cambridge, MA), and fluorescein isothiocyanate (FITC)-conjugated mouse mAb against the V5 epitope (diluted 1:500, Invitrogen). Secondary antibodies were FITC-labeled goat anti-rabbit or goat anti-mouse (diluted 1:1000, Invitrogen) and tetramethyl rhodamine isothiocyanate (TRITC)-labeled goat anti-mouse (diluted 1:1000, Jackson ImmunoResearch Laboratories, West Grove, PA). Slides were mounted in SlowFade Antifade reagent (Invitrogen) and imaged using a Nikon TE2000 epifluorescence microscope (Melville, NY).
qRT-PCR
Total RNA was extracted from synthetic APF-treated, inactive control peptide-treated, or control untreated NB epithelial cells using the RNeasy Plus Mini Kit (Qiagen, Valencia, CA) according to the manufacturer's protocol. RNA was assessed by visualization of the 28S/18S rRNA ratio on a 1% agarose gel, and total RNA concentration was determined by measuring the absorbance of each sample at 260 nm and 280 nm using a Gene Quant RNA/DNA Calculator (GE Healthcare, Piscataway, NJ). qRT-PCR for gene expression was performed using Quantitect Primers (Qiagen), SYBR Green RT-PCR kit reagents (Qiagen), and a Roche System II Light-Cycler (software version 3.5). Samples were tested in triplicate runs, and specific mRNA levels were quantified and compared with mRNA levels for β-actin using real-time PCR analysis software from Applied Biosystems (Foster City, CA).
Western Blot Analysis
Cells were lysed in ice-cold RIPA buffer containing protease inhibitors (Pierce, Rockford, IL), sonicated, and centrifuged for 15 min at 4°C. The supernatant protein concentration was measured using a Folin reagent-based protein assay kit (Bio-Rad). Proteins were separated by electrophoresis using 4–12% NuPAGE Novex Bis-Tris polyacrylamide gels in MOPS running buffer (Invitrogen) and then transferred to nitrocellulose. Membranes were blocked for 2 h at room temperature in TBST buffer (Tris-buffered saline, pH 7.4, with 0.1% Tween 20) containing 5% nonfat milk and incubated with specific antibodies against vimentin (diluted 1:2000; BD PharMingen, San Jose, CA) or ZO-1 (diluted 1:125; Zymed Laboratories, San Francisco, CA) overnight at 4°C. The membranes were subsequently washed with TBST, incubated for 1 h at room temperature in HRP-conjugated goat anti-mouse (diluted 1:40000; Santa Cruz Biotechnology, Santa Cruz, CA) or goat anti-rabbit (diluted 1:10000; Pierce) secondary antibodies, and developed by enhanced chemiluminescence (Pierce). To assess equal loading of protein, the membranes were stripped and reprobed for β-actin (diluted 1:5000; Sigma). The membranes were exposed to film (BioMax AR, Eastman Kodak, Rochester, NY), and the resulting images were scanned at 300 dpi. The protein bands of interest were quantified using ImageJ (http://rsb.info.nih.gov/ij/), and the integrated signal densities were normalized first to β-actin (the loading control) and subsequently expressed in terms of the fractional abundance relative to untreated control cells.
Immunoprecipitation
HeLa cells were transfected with mCFP:DHHC2 or mock-transfected using FuGENE6 reagent (Roche) according to the manufacturer's instructions. Forty-eight hours later, cells were washed in PBS, lysed in 500 µl ice-cold lysis buffer (25 mM HEPES, 150 mM NaCl, 5 mM MgCl2, 1% NP-40, and protease inhibitor cocktail), and incubated on ice for 90 min with vortexing. After normalizing for equal protein concentration, lysates were immunoprecipitated with a mAb against GFP (JL-8 BD Biosciences, San Jose, CA; also recognizes the GFP spectral mutant, CFP) overnight at 4°C with rocking. Protein G Sepharose 4B (Invitrogen) was added the following day, and the samples were incubated for an additional 18 h at 4°C with rocking. Proteins in the immunoprecipitation complex were washed four times in ice-cold lysis buffer and then heated in SDS sample buffer before separation by SDS-PAGE and transfer to nitrocellulose. Western blot analysis was performed as described above using a mAb against CKAP4 (G1/296, diluted 1:1000; Alexis Biochemicals).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Supplemental Figures S2 and S3).
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80% of the cells were expressing CFP-DHHC2, as determined by epifluorescence microscopy. As seen in Figure 1C, Western blot analysis detected a 63-kDa band characteristic of CKAP4 in lysates from cells expressing CFP-DHHC2 but not in lysates from mock-transfected cells, indicating that under these conditions CKAP4 and CFP-DHHC2 coassociate. This result supports our previous findings that CKAP4 is a substrate of DHHC2.
Palmitoylation by DHHC2 Is Required for CKAP4 Trafficking from the ER to the Plasma Membrane
CKAP4 has been extensively characterized as an ER-resident protein (Schweizer et al., 1993a,b, 1994, 1995b; Klopfenstein et al., 2001). In a previous study, we showed that siRNA-mediated knockdown of DHHC2 expression blocks CKAP4 trafficking from the ER to the plasma membrane (PM; Zhang et al., 2008). To further examine the effect of palmitoylation on CKAP4 localization, we first labeled nonpermeabilized mock- or DHHC2 siRNA-transfected HeLa cells with an mAb against CKAP4 (Figure 2). In agreement with published reports, CKAP4 was localized on the cell surface of mock-transfected HeLa cells (Figure 2A) (Bates et al., 2008). In contrast, CKAP4 was not detected on the cell surface of HeLa cells transfected with DHHC2 siRNA (Figure 2B). Subsequently, we colabeled mock- or DHHC2 siRNA-transfected HeLa cells with antibodies against CKAP4 and calreticulin, an ER marker. As shown in Figure 3A, CKAP4 colocalizes with calreticulin in the ER but is also present on the PM and in the nucleus. However, when DHHC2 expression is knocked down, CKAP4 is no longer detected on the PM, which is consistent with our previous findings (see Figure 5; Zhang et al., 2008), and it is also no longer present in the nucleus (Figure 3A). We then generated HeLa cell lines stably expressing epitope-tagged WT CKAP4 or the palmitoylation-incompetent mutant, CKAP4 C100S, to examine the effect of complete depalmitoylation on CKAP4 subcellular distribution. Cells were fixed and incubated with an anti-V5-FITC–conjugated mAb, and the stably expressed proteins were visualized by epifluorescence microscopy. As shown in Figure 3B, WT CKAP4 is present on the plasma and perinuclear membranes and in the nucleus, similar to endogenous CKAP4 localization, whereas CKAP4 C100S is confined to the ER. Importantly, in cells stably expressing CKAP4 C100S, the ER retains its reticulated structure, radiating throughout the cell. However, when palmitoylation of endogenous CKAP4 is inhibited after DHHC2 knockdown, the ER contracts around the nucleus to a greater degree (Figure 4, D). These data corroborate our earlier findings indicating that palmitoylation is required for trafficking of CKAP4 from the ER to the PM (Zhang et al., 2008), and they suggest that palmitoylation may also be required for CKAP4 nuclear translocation.
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). To examine the subcellular distribution of CKAP4 in HeLa cells treated with APF, we performed immunocytochemistry. As shown in Figure 4, CKAP4 is clearly localized to the PM and perinuclear membrane of HeLa cells (Figure 4A) and may also be present in the nucleus. However, APF treatment results in an obvious increase in nuclear translocation (Figure 4B). To determine if palmitoylation of CKAP4 by DHHC2 is required for its nuclear localization, we transfected HeLa cells with DHHC2 siRNA and then exposed the cells to APF for 48 h. In cells with reduced DHHC2 expression, CKAP4 is not detected in the nucleus after APF treatment (Figure 4C). These data indicate that CKAP4 must be palmitoylated by DHHC2 in order to translocate to the nucleus in response to APF-treatment as well as in response to other factors in untreated cells. These findings are consistent with the hypothesis that palmitoylation is required for translocation of CKAP4 to both the nucleus and the PM (where it can bind to APF).
CKAP4 Must Be Palmitoylated by DHHC2 for APF to Block HeLa and NB Cell Proliferation
Both HeLa and NB epithelial cells express endogenous CKAP4 and have been shown to be sensitive to the antiproliferative effects of APF (Conrads et al., 2006). To determine the effect of reduced CKAP4 palmitoylation on the proliferative response of HeLa and NB cells to APF, we knocked down the expression of DHHC2 using siRNA. Forty-eight hours after siRNA transfection, cells were incubated with varying concentrations of APF. As shown in Figure 5, A and B, DHHC2 knockdown profoundly inhibited the ability of both cell types to respond to APF. These results suggest that DHHC2-mediated palmitoylation of CKAP4 is necessary for APF-induced antiproliferative effects in HeLa and NB cells.
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; Zhang et al., 2005; Kim et al., 2007). To determine if palmitoylation of CKAP4 by DHHC2 is required for APF-induced changes in gene and protein expression, confluent NB epithelial cells were treated with APF or inactive peptide control for 48 h, and the mRNA and protein levels of vimentin, ZO-1, and E-cadherin were determined by qRT-PCR and Western blot analyses, respectively. As shown in Figure 6, A–C, APF-mediated decreased expression of ZO-1 and vimentin and increased expression of E-cadherin were inhibited by knocking down the expression of DHHC2 with siRNA. Western blot analyses confirmed that ZO-1 and vimentin protein levels were reduced after APF treatment of NB cells and that the ability of APF to induce these changes was blocked in cells transfected with DHHC2 siRNA (Figure 7A; although an increase in E-cadherin mRNA levels could be measured in NB cells after APF treatment, E-cadherin protein expression, in all conditions, remained below the threshold required for Western blot detection). These data demonstrate that palmitoylation of CKAP4 by DHHC2 is required for APF-induced changes in specific mRNA and protein expression in NB cells.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). In this report, we furthered those findings by demonstrating the physical interaction of endogenous CKAP4 with CFP-DHHC2 and by characterizing the functional significance of this interaction in HeLa and NB cells. We have shown that palmitoylation of CKAP4 by DHHC2 is a critical event required for APF-initiated signaling events, including 1) increased nuclear translocation of CKAP4, 2) inhibition of cellular proliferation, and 3) changes in gene and protein expression (i.e., vimentin, ZO-1, and E-cadherin; Keay et al., 2003; Zhang et al., 2005; see Figure 8 for a summary).
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; Zhang et al., 2005). These changes in cellular behavior are mediated through high-affinity binding of APF to CKAP4, as CKAP4 gene knockdown and immunodepletion abrogate APF signaling (Conrads et al., 2006). In addition, no other functional high-affinity receptor for APF other than CKAP4 was found. Our data show that palmitoylation of CKAP4 by DHHC2 is necessary for APF-mediated changes in proliferation, gene expression, and nuclear translocation of CKAP4. These results support the idea that the downstream effects of APF are mediated through CKAP4 and that palmitoylation of CKAP4 by DHHC2 is necessary, as loss of DHHC2 expression by siRNA-mediated knockdown is sufficient to inhibit these APF- and CKAP4-dependent signaling events. Importantly, the effects of reduced DHHC2 expression on APF-dependent signaling cannot be attributed to a reduction in CKAP4 abundance (as was the case with other DHHC2 substrates, CD9 and CD151; Sharma et al., 2008), because we have shown previously that CKAP4 expression is unaffected by DHHC2 knockdown in HeLa cells (Zhang et al., 2008).
Palmitoylation plays an important role in regulating the subcellular distribution and function of many proteins with key roles in diverse signaling networks (Resh, 2006; Greaves and Chamberlain, 2007; Linder and Deschenes, 2007; Nadolski and Linder, 2007). Taken together, our findings suggest that palmitoylation of CKAP4 by DHHC2 may facilitate trafficking of CKAP4 from the ER to the PM (where it can physically associate with APF). It is important to note that the general appearance of depalmitoylated CKAP4 was different when DHHC2 was knocked down versus when CKAP4 C100S was stably expressed: CKAP4 C100S was localized to an extended, tubular ER network held in place by palmitoylated, endogenous CKAP4, an organization that was lost after DHHC2 knockdown and depalmitoylation of endogenous CKAP4. Vedrenne and et al. (2005) reported a similar reorganization of the ER in cells expressing mutant versions of CKAP4 that were unable to bind to or bundle microtubules. One of these mutants replaced the three critical, phosphorylatable serines (residues 3, 17, and 19) with glutamic acids to mimic a state of phosphorylation. In cells expressing these phosphomimicking mutants, the ER retracted around the nucleus leaving the microtubular network intact. This suggests that stable anchoring of the ER to microtubules by CKAP4 is required to maintain its spatial distribution.
The markedly increased nuclear abundance of CKAP4 in HeLa cells in response to APF suggests that CKAP4 and/or APF could have a direct role in mediating gene and protein expression. We have not observed increased nuclear localization of CKAP4 in untreated HeLa cells, and nuclear translocation after binding of tPA or SPA was also not apparent in previous studies (Razzaq et al., 2003; Gupta et al., 2006). The C-terminus of CKAP4 is predicted to fold into an extensive coiled-coil domain containing a heptad repeat (residues 468-503) that is predicted to be a leucine zipper (http://www.predictprotein.org/). This portion of the protein is either outside of the cell if CKAP4 is on the PM or in the lumen of the ER if CKAP4 is a resident of the ER. The coiled-coil domain has been shown to be required for the formation of CKAP4 oligomers, as oligomerization is lost when this region is deleted (Klopfenstein et al., 2001). Leucine zipper-containing transcription factors bind to DNA as dimers (Lee, 1992; Sauve et al., 2004), a state likely to be adopted by CKAP4 in the cell. The sequence of residues that follows (503-602) is predicted by InterProScan (www.ebi.ac.uk/Tools/InterProScan/) to be helical as well. Interestingly, it is also relatively rich in the positively charged, basic amino acids histidine, arginine, and lysine. Examination of this sequence in helical wheel programs (e.g., http://cti.itc.virginia.edu/cmg/Demo/wheel/wheelApp.html) shows that the helix is amphipathic, suggesting that this region may also be involved in intermolecular interactions. The fact that CKAP4—an ER- and PM-localized, palmitoylated, transmembrane protein—translocates to the nucleus is remarkable in and of itself. To our knowledge, there is no other example of such a protein entering the nucleus. The mechanism by which this occurs is not obvious and requires further investigation. Furthermore, there have been no reported examples of palmitoylated proteins in the nucleus, suggesting that CKAP4 may be depalmitoylated and/or truncated before nuclear translocation.
Recently, DHHC2 has also been shown to palmitoylate the tetraspanins CD9 and CD151, promoting physical associations between them and protecting them from lysosomal degradation (Sharma et al., 2008). This discovery provides one plausible mechanism by which DHHC2 may function as a tumor suppressor. In this study by Sharma et al. (2008), a 70% reduction in ZDHHC2 mRNA expression (via siRNA-mediated knockdown) resulted in lysosomal targeting and rapid degradation of CD9 unless the lysosomal pH was increased by applying bafilomycin A1 (an inhibitor or vacuolar protein-translocating APTases) to the cells. This treatment spared CD9 from degradation and allowed Sharma and colleagues to determine that DHHC2 knockdown reduced palmitoylation of CD9 by 35–55%—a value similar to that observed for CKAP4 after DHHC2 knockdown (Zhang et al., 2008). However, an important question that remains for CD9 is which of its palmitoyl cysteines are palmitoylated by DHHC2; there is only one palmitoyl cysteine in CKAP4–Cysteine 100. It is possible that hypopalmitoylation of both CKAP4 and CD9 may increase tumor or metastatic behavior. In either case, the importance of maintaining DHHC2 expression to suppress metastatic cellular behavior is becoming clearer.
Although there is relatively little known about the 23 members of the mammalian PAT family in terms of their regulation and specificity for substrates, the remarkable number of known associations between disease and the genes that encode PATs demonstrates the importance of palmitoylation for human health. The significance of DHHC2-mediated palmitoylation of CKAP4 is likely to extend beyond IC and cancer. CKAP4 has also been identified as a functional, cell-surface receptor for tissue plasminogen activator (tPA) in vascular smooth muscle cells and for surfactant protein A (SP-A) in rat type II pneumocytes (Razzaq et al., 2003; Gupta et al., 2006). Like APF, tPA regulates cellular proliferation, migration, and invasion in the vasculature: behaviors that are also critically relevant to IC and cancer. tPA binding to CKAP4 on the plasma membrane regulates the response of vascular smooth muscle cells (VSMCs) to a variety of blood vessel injuries (Razzaq et al., 2003). After vascular injury, tPA stimulates VSMC migration and remodeling of the surrounding extracellular matrix, key features that promote vascular repair. SP-A levels are decreased in the lungs of patients with cystic fibrosis and respiratory distress syndrome, as well as chronic lung diseases (Heinrich et al., 2006), and it is thought that CKAP4 may play a role in SP-A recycling and SP-A signaling by mediating transport of SP-A from the ER to the plasma membrane and/or in SP-A binding at the PM and subsequent internalization (Gupta et al., 2006). Although it is not known whether CKAP4 palmitoylation is required for mediating the effects of tPA in smooth muscle cells or SP-A in type II pneumocytes, the functional significance of CKAP4 palmitoylation in cell membrane localization of CKAP4 and in APF-mediated signaling in IC illustrate the wide-ranging significance of palmitoylation.
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ACKNOWLEDGMENTS |
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Footnotes |
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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.
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) or with nonsense siRNA (
) as a control for nonsequence-specific effects on day 1 and serum-starved on day 2, and varying concentrations of APF or control peptide (0.25-250 nM) were added to the medium on day 3; cells were then cultured for an additional 48 h under conditions of serum starvation. (B) NB epithelial cells were electroporated with DHHC2 double-stranded siRNA (
) as a control for nonsequence-specific effects on day 1 and serum-starved on day 2, and varying concentrations of APF or control peptide (0.25–250 nM) were added to the medium on day 3; cells were then cultured for an additional 48 h under conditions of serum starvation. For both cell types, cellular proliferation was assessed by inhibition of [3H]thymidine incorporation. Each data point represents the mean and SD of three independent experiments. Data are presented as percent inhibition of [3H]thymidine incorporation compared with controls. The ability of APF to block proliferation was inhibited in the presence of DHHC2 siRNA for all APF concentrations except 250 nM for HeLa cells or 25 and 250 nM for NB cells.
