A Novel Human Phosphatidylethanolamine-binding Protein Resists Tumor Necrosis Factor α-induced Apoptosis by Inhibiting Mitogen-activated Protein Kinase Pathway Activation and Phosphatidylethanolamine Externalization

Abstract

The phosphatidylethanolamine (PE)-binding proteins (PEBPs) are an evolutionarily conserved family of proteins with pivotal biological functions. Here we describe the cloning and functional characterization of a novel family member, human phosphatidylethanolaminebinding protein 4 (hPEBP4). hPEBP4 is expressed in most human tissues and highly expressed in tumor cells. Its expression in tumor cells is further enhanced upon tumor necrosis factor (TNF) ␣ treatment, whereas hPEBP4 normally co-localizes with lysosomes, TNF␣ stimulation triggers its transfer to the cell membrane, where it binds to Raf-1 and MEK1. L929 cells overexpressing hPEBP4 are resistant to both TNF␣-induced ERK1/2, MEK1, and JNK activation and TNF␣-mediated apoptosis. Co-precipitation and in vitro protein binding assay demonstrated that hPEBP4 interacts with Raf-1 and MEK1. A truncated form of hPEBP4, lacking the PE-binding domain, maintains lysosomal co-localization but has no effect on cellular responses to TNF␣. Given that MCF-7 breast cancer cells expressed hPEBP4 at a high level, small interfering RNA was used to silence the expression of hPEBP4. We demonstrated that down-regulation of hPEBP4 expression sensitizes MCF-7 breast cancer cells to TNF␣-induced apoptosis. hPEBP4 appears to promote cellular resistance to TNF-induced apoptosis by inhibiting activation of the Raf-1/MEK/ERK pathway, JNK, and PE externalization, and the conserved region of PE-binding domain appears to play a vital role in this biological activity of hPEBP4.The phosphatidylethanolamine-binding protein (PEBP) 1 family consists of a number of 21-23-kDa basic proteins, first identified in bovine brain, with preferential in vitro affinity for phosphatidylethanolamine, a component of the cell membrane. This family is an evolutionarily conserved group found in species of flowering plants (Antirrhinum (1)), parasites (Plasmodium falciparium (2)), nematodes (Toxocara canis (3)), insects (Drosophila melanogaster (4)), and mammals, including cattle, monkeys, and humans (5). A number of functions have been suggested for the mammalian PEBP proteins, including lipid binding and inhibition of serine proteases (6). These proteins can also act as precursors for a bioactive peptide HCNP (hippocampal cholinergic neurostimulating peptide), important in hippocampus development (5). Plant PEBP homologues are involved in the control of a morphogenic switch between shoot growth and flower structures (7). Yeast two-hybrid screen analysis has shown that human PEBP1 (hPEBP1, also called Raf kinase inhibitory protein or RKIP) acts as a suppressor of Raf-1 kinase activity and mitogen-activated protein kinase signaling in fibroblasts via its ability to sequester and inactivate Raf-1 and MEK1 (8, 9). Both Raf-1 and MEK bind to the highly conserved phosphatidylethanolamine-binding domain of hPEBP; hPEBP induces dissociation of Raf-1⅐MEK complexes and behaves as a competitive inhibitor of MEK phosphorylation. Mapping of the binding domains has shown that MEK and Raf-1 bind to overlapping sites in hPEBP...

hPEBP4 Resists TRAIL-induced Apoptosis of Human Prostate Cancer Cells by Activating Akt and Deactivating ERK1/2 Pathways

“…hPEBP4 is preferentially expressed in breast cancer and ovarian cancer cells (18). We demonstrate that silencing of hPEBP4 can potentiate TNF␣-induced apoptosis in MCF-7 breast cancer cells via ERK1/2 and JNK activation, indicating hPEBP4 is a candidate target molecule for breast cancer treatment (19).…”

“…Cell Transfection-The expression vectors hPEBP4-B (the hPEBP4 expression vector) and p75PEBP4-B (the PE-binding domain-truncated vector) were constructed as described previously (18), and transfected into DU145 cells using Lipofectamine reagent with pcDNA3.1/Myc-His (Ϫ) B as a mock control. 48 h after transfection, the cells were screened under 0.8 mg/ml G418 (Merck, Darmstadt, Germany) for 20 days.…”

“…The cell lysates were mixed with Ni-nitrilotriacetic acid beads (Qiagen, Hilden, Germany), gently agitated overnight at 4°C, and washed four times in lysis buffer containing 10 mM imidazole and 0.1% Triton X-100, and Western blot was performed as previously described (19). For immunoprecipitation (IP) of endogenous hPEBP4 from TRAIL-stimulated LNCaP cell lysates with anti-hPEBP4 polyclonal antibody (18), LNCaP cell lysates were precleared with protein A-Sepharose beads (Sigma), and IP performed using anti-hPEBP4 polyclonal antibody cross-linked to protein A-Sepharose beads as previously described (19).…”

“…Prostate Cancer-Our previous study showed that hPEBP4 was highly expressed in human breast cancer tissues, and hPEBP4 was also highly expressed in human breast cancer, ovarian cancer, and prostate cancer cell lines (18). To address the relationship between hPEBP4 expression and the progression of prostate cancer, here we examined the expression pattern of hPEBP4 in human prostate cancer tissues by tissue microarrays.…”

“…Human phosphatidylethanolamine-binding protein 4 (hPEBP4) is a novel member of the PEBP family identified by us as an anti-apoptotic molecule (18). hPEBP4 is preferentially expressed in breast cancer and ovarian cancer cells (18).…”