Identification of CKAP4/p63 as a Major Substrate of the Palmitoyl Acyltransferase DHHC2, a Putative Tumor Suppressor, Using a Novel Proteomics Method

Abstract

The recent discovery of at least 23 enzymes that mediate palmitoylation (palmitoyl acyltransferases or PATs 1 ) suggests that regulation of palmitoylation is far more complex than previously assumed. Defining PAT-substrate specificities in living vertebrate systems is a critical next step in understanding the complexity of regulated palmitoylation and the potential consequences of malfunctioning PATs on downstream signaling events. To date, five PAT genes have been implicated in human diseases ranging from schizophrenia to cancer and Huntington disease, so defining the substrates of individual PATs will provide important clues linking these known mutations to an associated disease. One reason why our understanding of palmitoylation has not kept pace with that of other post-translational protein modifications has been the lack of rapid and sensitive methods to study this phenomenon. Recent advances in the field have provided tools that can identify palmitoylated proteins and match known palmitoyl proteins to PATs already suspected of mediating their palmitoylation (1-3). But until now, there has been no method to identify substrates of specific PATs in a living vertebrate system without prior knowledge of a possible relationship between PAT and substrate. Palmitoyl-cysteine isolation capture and analysis (PICA) 2 solves this problem. In this study, we used PICA and HeLa (human cervical carcinoma cells) to identify CKAP4/p63 as a physiologically important substrate of DHHC2, a PAT that has been linked to the metastatic potential of liver and colon cancer (4).The ability of PICA to identify PAT substrates is based on the principle that it quantifies the differential frequency of palmitoylation of individual proteins in control conditions versus conditions in which the function of a single PAT is reduced by siRNA-mediated gene knockdown (Fig. 1). The process to identify substrates of DHHC2 consisted of four basic steps. In the first part we generated two distinct pools of palmitoylated proteins in HeLa cells: one from untreated, control HeLa cells and the other from HeLa cells in which the activity of one PAT, DHHC2, was reduced. These two distinct pools of palmitoylated proteins were then captured, and the differences in the degree of palmitoylation of individual proteins among the two pools were identified. To do this, first we reduced the expression of ZDHHC2 mRNA (and consequently the function of the encoded enzyme, DHHC2) in HeLa cells using siRNA-mediated gene knockdown, resulting in a reduced level of palmitoylation of DHHC2 substrates. Total protein from knockdown and control cells was prepared by first blocking all free (including non-palmitoylated) thiols with methyl methanethiosulfonate (MMTS). This was followed by selective exposure of all

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

“…Similar to the results seen for phosphorylation, the blocking of palmitoylation decreases CKAP4 binding to microtubules [22]. A group of at least 23 enzymes called palmitoyl acyltransferases (PATs), which are characterized by an Asp-His-His-Cys (DHHC) motif, have been determined to mediate palmitoylation [23].…”

Prognostic significance of cytoskeleton‐associated membrane protein 4 and its palmitoyl acyltransferase DHHC2 in hepatocellular carcinoma

“…10 Previous reports suggested that CKAP4 palmitoylation by DHHC2 is required for its trafficking from the endoplasmic reticulum to the plasma membrane and for its nuclear localization, which is critical to the antiproliferation effect of CKAP4. 19 It is therefore of interest to investigate the expression and clinical significance of DHHC2 in patients with HCC.…”

“…Recent studies have shown that palmitoylation by DHHC2, a putative tumor suppressor, is required for CKAP4 trafficking from the endoplasmic reticulum to the plasma membrane and for its nuclear localization. 10 Additional studies have reported that CKAP4 contributes to gentamicin-induced apoptosis in proximal tubule cells, likely through dimerization of CKAP4, which is dependent on its palmitoylation. 11 In the current study, we examined the pattern of expression of CKAP4 and DHHC2 in HCC tumor tissues and normal-like specimens.…”

Origins of cancer symposium 2015: posttranslational modifications and cancer

“…Using a technique called palmitoyl-cysteine isolation capture and analysis, she identified substrates of ZDHHC2 in HeLa cells, including CKAP4/p63, a receptor for antiproliferative factor (APF). APF is increased in patients suffering from interstitial cystitis [5]. Following up on this finding, she demonstrated that knock-down of ZDHHC2 inhibited the anti-proliferative activity of APF in cancerous and normal epithelium.…”