Ebp1 isoforms distinctively regulate cell survival and differentiation

Abstract

Ebp1, an ErbB3 receptor-binding protein, inhibits the proliferation and induces the differentiation of human cancer cells. Ebp1 binds nuclear Akt and prevents DNA fragmentation by inhibiting caspase-activated DNase. Here, we show that Ebp1 possesses two different isoforms, p48 and p42, which differentially mediate PC12 cell survival and differentiation. The longer-form p48 localizes in both the cytoplasm and the nucleus and suppresses apoptosis, whereas the shorter-form p42 predominantly resides in the cytoplasm and promotes cell differentiation. EGF strongly stimulates p42 to bind ErbB3, and the association depends on PKC-mediated phosphorylation of Ebp1. By contrast, p48 does not bind to ErbB3 regardless of EGF treatment. Overexpression of p48 provokes cell proliferation, which is inhibited by p42. Moreover, nerve growth factor elicits extensive sprouting in p42 stably transfected PC12 cells, whereas p48 cells reveal modest neurite outgrowth. Although mitogen-activated protein kinase cascade remains similar in both cells, Akt is more active in p48 cells than in p42 cells. Thus, Ebp1 might regulate cell survival and differentiation through two distinctive p48 and p42 isoforms.

Fig. 1.

Ebp1 encodes two isoforms: p48 and p42. (A) Subcellular fractionation of PC12 cells. (Top) In whole-cell lysate, Ebp1 from rat PC12 and human HEK293 cells appeared as one band on SDS/PAGE. PC12 cells were treated with NGF for various time points. The cytosolic and nuclear fractions were prepared. p48 distributed in both the cytoplasmic and nuclear fractions, where p42 exclusively resided in the cytoplasmic fraction. (Middle) The identity of each fraction was verified with its specific marker. (Bottom) Immunofluorescent staining revealed that endogenous Ebp1 distributed in both the cytoplasm and the nucleus. (B) Ebp1 subcellular distribution. GFP-tagged Ebp1 with first (p48), second (p44), and third (p42) ATG constructs were transfected into PC12 cells and examined under a fluorescent microscope. p48 distributed in both the cytoplasm and the nucleolus, whereas both p44 and p42 occurred exclusively in the cytoplasm. (C) RT-PCR analysis of Ebp1. Total RNA was extracted from various cells and tissues. The first-strand cDNA was prepared with oligo(dT) primer. The two PCR products were ligated into pCR2.1 vector (Invitrogen) and sequenced. (D) ClustalW-formatted sequence alignment. The small band skipped 29 nucleotides containing the second ATG. The primers used in the assay are underlined.

Fig. 2.

PKC phosphorylation of p48 regulates its nucleolar residency. (A) EGF provokes p48 relocation from the nucleolus to the nucleoplasm. HEK293 cells were transfected with GFP–Ebp1 p48 and stimulated with EGF for 30 min. (Left) EGF treatment elicited p48 translocation from the nucleolus to the nucleoplasm. (Right) Quantitative analysis revealed that EGF stimulation substantially decreased p48 Ebp1’s nucleolar residency. (B) GFP–Ebp1 S360A (Upper) but not S360D (Lower) resided in the nucleolus.

Fig. 3.

p42 suppresses cell growth and p48 enhances cell growth. (A) Growth curve for Ebp1 isoforms’ stably transfected PC12 cell lines. The stably transfected PC12 cells were cultured in 10% FBS medium in the absence of tetracycline. The cell number was counted at different time points. The data shown are mean ± SD of triplicate experiments. (B) Immunoblotting analysis of cell-cycle proteins. p42 and p48 cell lines were induced and incubated with 50 ng/ml NGF for different times. At each time point, cell lysate was prepared and analyzed by immunoblotting with anti-cyclin A, PCNA, and cyclin D1 antibodies.

Fig. 4.

p42 but not p48 promotes PC12 cell differentiation. Quantitative analysis of PC12 cell differentiation. A total of 100–200 cells were counted and scored for the expression of neurites longer than two cell bodies. Data are mean ± SD values of triplicate determinations taken from a single experiment. The experiments were replicated three times.

Fig. 5.

p48 but not p42 inhibits apoptosis. (A) Ebp1 p48 but not p42 isoforms inhibit apoptosis. p48 and p42 stable cell lines were induced and treated with 250 nM staurosporine for 24 h. Induction of p48 but not p42 prevented DNA fragmentation even in the absence of NGF. (Left) In the presence of NGF, DNA fragmentation was completely blocked in both cells. (Right) PARP cleavage coupled to the DNA fragmentation pattern. (B) Caspase-3 activity assay. p42 and p48 cell lines were cultured in the presence or absence of tetracycline for 24 h, followed by treatment with 250 nM staurosporine for various times. The data shown are mean ± SD of triplicate samples. (C) p48 enhances cell survival. Induced p48 cells, p42 cells, and control PC12 cells were cultured in the medium containing NGF for 3 and 5 days. The differentiated cells were then cultured in NGF-free medium for 36 h. The soluble DNA was extracted and analyzed on a 2% agarose gel. (D) p48 but not p42 prevents DNA fragmentation. The NGF-differentiated cells (5 days) and undifferentiated cells were treated with 250 nM staurosporine for 24 h, and the degraded DNA was analyzed.

Fig. 6.

p42 but not p48 associates with ErbB3 receptor. (A) p42 but not p48 binds ErbB3 receptor. HEK293 cells were cotransfected with ErbB3 and GST-p42 or p48, and then stimulated with EGF. Ebp1 was pulled down with glutathione beads, and the precipitated proteins were analyzed with anti-ErbB3 antibody. EGF stimulation increased the interaction between ErbB3 and p42 but not between ErbB3 and p48. (B) p42 S360D but not S360A binds ErbB3. HEK293 cells were cotransfected with ErbB3 and GST-WT p42, S360A, and S360D, then stimulated with EGF. Ebp1 was pulled down with glutathione beads, and the precipitated proteins were analyzed with anti-ErbB3 antibody.

Fig. 7.

p48 selectively binds nuclear Akt and sustains its kinase activity. (A) p48 but not p42 strongly interacts with nuclear Akt. p48 and p42 stable cell lines were induced in the medium without tetracycline for 24 h, infected with adenovirus expressing myristolated-Akt or nuclear Akt, and stimulated with NGF for 30 min. Ebp1 was immunoprecipitated and its associated proteins were analyzed with anti-Akt antibody. (Left) NGF stimulated nuclear but not cytoplasmic Akt to bind p48. (Right) By contrast, NGF enhanced cytoplasmic but not nuclear Akt to interact with p42. (B) Akt activation is stronger in p48 but not in p42 cells. p48 and p42 stable cell lines were induced and treated with NGF for 30 min. Cell lysate was monitored by immunoblotting with anti-phospho-Akt-473, anti-Akt, anti-phospho-Erk, and anti-Erk1/2 antibodies. Akt was immunoprecipitated from Ebp1 cells and incubated with purified active PP2A (1 unit, 0.5 μg) at 30°C for 1 h in the presence or absence of calyculin A. PP2A dephosphorylated Akt immunoprecipitated from p42 but not p48 cells, which can be inhibited by phosphatase inhibitor calyculin A (10 μM) pretreatment. (C) Akt activation was sustained and stronger in p48 cells compared with control and p42 cells.