Induction of Id1 and Id3 by Latent Membrane Protein 1 of Epstein-Barr Virus and Regulation of p27/Kip and Cyclin-Dependent Kinase 2 in Rodent Fibroblast Transformation

Abstract

Latent membrane protein 1 (LMP1), the Epstein-Barr virus oncoprotein, activates NF-B, phosphatidylinositol 3-kinase, mitogen-activated protein kinase, and c-Jun N-terminal kinase signaling. To determine global transcriptional changes induced by LMP1 in epithelial cells, genomic analysis of C33A cells stably expressing LMP1 was performed. Relatively few genes were induced by LMP1. Expression of two members of the Id (inhibitor of differentiation) family of proteins, Id1 and Id3, was induced in the presence of LMP1 and confirmed by mRNA and protein in C33A and Rat-1 cells. In Rat-1 foci transformed by LMP1, Id1 protein was also increased. Id proteins are known negative regulators of E-box proteins that positively regulate p16 and potentially other cyclin-dependent kinase inhibitors (cdki's). In LMP1-expressing Rat-1 cells, cdki p27 was specifically downregulated. Decreased p27 was correlated with increased levels of Cdk2 and increased levels of phosphorylated retinoblastoma protein. This study describes new properties of LMP1 that likely contribute to transformation and oncogenesis.Epstein-Barr virus (EBV) is a ubiquitous human pathogen that is associated with several malignancies (16, 28). Latent membrane protein 1 (LMP1) is considered the EBV oncoprotein and is expressed in many of the cancers associated with EBV. LMP1 transforms rodent fibroblasts by conferring anchorage-independent growth and loss of contact inhibition (32). Fibroblasts expressing LMP1 form tumors in nude mice and can grow under reduced-serum conditions. LMP1 is also essential for EBV-mediated transformation of B lymphocytes (13).LMP1 is an integral membrane protein with a short aminoterminal cytoplasmic tail, six membrane-spanning domains, and a cytoplasmic carboxy-terminal domain. LMP1 functions as a constitutively active tumor necrosis factor (TNF) receptor, as oligomerization of LMP1 molecules via interactions of the transmembrane domains brings the carboxyl-terminal domains in close proximity to induce ligand-independent signaling (6, 8, 9). The carboxyl-terminal domain contains two signaling domains, C-terminal activation regions (CTARs). CTAR1 binds TNF receptor-associated factors (TRAFs), and CTAR2 binds the TNF receptor-associated death domain protein (designated TRADD) that recruits other signaling molecules. Signaling from the C-terminal domain activates NF-B and leads to activation of several important signaling pathways, including the mitogen-activated protein kinase (MAPK), c-Jun N-terminal kinase, and phosphatidylinositol 3-kinase (PI3K) pathways (6).LMP1 alters the cellular environment by inducing the expression of a number of genes. Genomic analyses of EBVinfected and LMP1-expressing lymphocytes indicate that most of the genes that are induced during EBV infection are a result of LMP1 and NF-B signaling (3, 4). LMP1 induces the expression of cell surface receptors, epidermal growth factor receptor (EGFR) (22), CD40, CD54, and CD95. LMP1 also induces antiapoptotic proteins such as A20 (8), Bcl-2, cIAP, and Bfl-1, as well as inv...

Id3 is a novel regulator of p27kip1 mRNA in early G1 phase and is required for cell-cycle progression

“…Id2/Id3 have been reported to regulate p27 in very specific contexts (Everly et al, 2004;Kee and BronnerFraser, 2005); however, their effect on p27 transcriptional control during cell proliferation had never been …”

“…In addition, it has also been shown that E2A and Ids proteins participate to the transcriptional regulation of the cyclin-dependent kinase inhibitors (CKIs) p21 ink4a (Id1) (Zheng et al, 2004) in different biological models including senescence. Recent studies have shown that besides p21 and p16, Id1 and Id3 are also involved in the regulation of p27 protein, notably in Xenopus neural crest progenitors (Kee and Bronner-Fraser, 2005) and in Epstein-Barr virus infected cells (Everly et al, 2004); however, the molecular basis of this inhibition has never been elucidated.…”

ID1 Facilitates the Growth and Metastasis of Non-Small Cell Lung Cancer in Response to Nicotinic Acetylcholine Receptor and Epidermal Growth Factor Receptor Signaling

“…ID1 has been implicated in regulating a variety of cellular processes, including growth, senescence, differentiation, apoptosis, angiogenesis, and neoplastic transformation (3). A number of studies have suggested several mechanisms for cell cycle progression by ID1, including inhibition of cyclin-dependent kinase (CDK) inhibitors such as p16, p21, and p27, leading to Rb phosphorylation and subsequent cell cycle progression (12,42,45,46). In spite of the similar structures and functional bases for the four ID proteins, they play different regulatory roles throughout embryonic development and tumorigenesis.…”

“…A549 cells that stably express dominant negative Src (CKD104, kinase dead) were generated by transfecting A549 cells with a plasmid construct that expresses mutant kinase-dead Src and selecting for puromycin resistance. A549 cells transfected with empty vector (called A549-EV [12]) were used as the control.…”

Id proteins expression in prostate cancer: high-level expression of Id-4 in primary prostate cancer is associated with development of metastases

“…Overexpression of Id-1 inhibits expression of p16, 36,37 p21, 38 and p27, 39 which leads to an increased activity of cyclin dependent kinase 2 (CDK2) and increased Retinoblastoma protein phosphorylation. Id-2, on the other hand, inhibits Retinoblastoma protein through direct binding and Id-2 itself is restrained by Retinoblastoma protein.…”