Phosphorylation of the N‐terminal domain of p48 Ebp1 by CDK2 is required for tumorigenic function of p48

Abstract

The long isoform of ErbB3 binding protein 1 (Ebp1), p48, strongly promotes tumorigenesis of glioblastoma, accelerating cell proliferation and transformation, while the short isoform, p42, which lacks the N-terminal 54 amino acids, inhibits tumor growth. However, it is unclear if the N-terminal domain of p48 regulates the oncogenic function of p48. Here, we show that p48, but not p42, interacts with cyclin-dependent kinase 2 (CDK2) through its N-terminal domain, resulting in the specific phosphorylation of serine 34 of p48. Overexpression of wild-type p48 greatly enhanced tumor cell growth, whereas phospho-ablated mutant S34A of p48, which is mutated at the CDK2 phosphorylation site, antagonizes cell proliferation and transformation. Moreover, phospho-ablated mutant S34A abrogated the ability of p48 to accelerate tumor cell growth in a mouse engraft model. Thus, our findings indicate that p48Ebp1 acts as an oncoprotein through selective interaction and/or modification of the N-terminal domain that does not exist in its short isoform p42.

Expression and Role of the ErbB3-Binding Protein 1 in Acute Myelogenous Leukemic Cells

“…High expression of Ebp1 in breast cancers predicts a poor clinical outcome, whereas tamoxifen treatment of MCF-7 breast cancer cells markedly decreases both the transcription and protein expression of p48 Ebp1 (26). It has also been shown recently that the phosphorylation of p48 Ebp1 by CDK2 is required for its tumorigenic function (6). Although further studies are needed to prove that p48 Ebp1 is an oncogenic protein, the findings to date strongly suggest it has tumor-promoting activities.…”

“…p48 Ebp1 is the predominant form of Ebp1 expressed in many cell types (4)(5)(6)8). Western blot analyses of AML cells using an antibody that also detects the p42 isoform show only a single band of 48 kDa.…”

“…Ebp1 encodes two alternatively spliced isoforms, p48 and p42, that have different functions (4). The p48 isoform is located predominantly in the nucleolus and cytoplasm and promotes cell proliferation and cell survival (4)(5)(6), whereas the p42 isoform is predominantly cytoplasmic and inhibits cell growth (4,7). Ebp1 also interacts with a variety of other important regulatory proteins that include Akt and Nucleophosmin 1 (NPM1; 8,9).…”

“…Purified cells were transduced with recombinant retroviruses made from pMSCV-neo constructs encoding MLL-AF9 and MLL-AF10, and plated in methylcellulose media (M3231, Stemcell Technologies) containing SCF, IL3, IL6, and GM-CSF. Transformed cells (1Â10 6 ) from methylcellulose cultures were transplanted intravenously into lethally irradiated C57BL/6 mice (900 rads) with 2Â10 5 syngeneic bone marrow cells (19). Mice were subsequently sacrificed and bone marrow cells from control animals and from MLL-AF9 and MLL-AF10 mice were collected for analysis by Western blot analysis.…”

Regulation of ribosomal RNA synthesis in T cells: requirement for GTP and Ebp1

“…39 The two isoforms of Ebp1, p48 and p42, 22 have distinct subcellular localizations and functions. The p48 isoform contains an additional 54 amino acids at the amino terminus and promotes cell proliferation, 23,40 whereas the shorter p42 form is frequently ubiquitinated and degraded and is rarely detected on immunoblots. 23,41 In both primary T cells and Jurkat T cells, only the 48 kDa protein was detectable on western blots.…”