p48 Ebp1 acts as a downstream mediator of Trk signaling in neurons, contributing neuronal differentiation

Two Ebp1 isoproteins, p48 and p42, regulate cell survival and differentiation distinctively. Here we show that p48 is the major isoform in hippocampal neurons and is localized throughout the entire neuron. Notably, reduction of p48 Ebp1 expression inhibited BDNF-mediated neurite outgrowth in hippocampal neurons. The p48 protein acts as a downstream effector of the Trk receptor, which mediates the functions of nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) in hippocampal cells. Trk receptor activation by both NGF and BDNF induced phosphorylation of Ebp1 at the S360 upon the activation of protein kinase Cδ (PKCδ) and triggered dissociation of p48 from retinoblastoma (Rb). Although both NGF and BDNF activate mitogen-activated protein kinase (MAPK; extracellular signal-related kinase (ERK)) as well as phosphatidylinositide 3-kinase (PI3K)/Akt, their activation is regulated in different time-frame upon growth factor specificity, especially, eliciting PKCδ mediated p48 S360 phosphorylation. Thus, p48 Ebp1 contributes to neuronal cell differentiation and growth factor specificity through the activation of PKCδ, acting as a crucial downstream effector of neurotrophin signaling.     

Nuclear Akt associates with PKC-phosphorylated Ebp1, preventing DNA fragmentation by inhibition of caspase-activated DNase

Akt promotes cell survival through phosphorylation. The physiological functions of cytoplasmic Akt have been well defined, but little is known about the nuclear counterpart. Employing a cell-free apoptotic assay and NGF-treated PC12 nuclear extracts, we purified Ebp1 as a factor, which contributes to inhibition of DNA fragmentation by CAD. Depletion of Ebp1 from nuclear extracts or knockdown of Ebp1 in PC12 cells abolishes the protective effects of nerve growth factor, whereas overexpression of Ebp1 prevents apoptosis. Ebp1 (S360A), which cannot be phosphorylated by PKC, barely binds Akt or inhibits DNA fragmentation, whereas Ebp1 S360D, which mimics phosphorylation, strongly binds Akt and suppresses apoptosis. Further, phosphorylated nuclear but not cytoplasmic Akt interacts with Ebp1 and enhances its antiapoptotic action independent of Akt kinase activity. Moreover, knocking down of Akt diminishes the antiapoptotic effect of Ebp1 in the nucleus. Thus, nuclear Akt might contribute to suppressing apoptosis through interaction with Ebp1.     

P42 Ebp1 functions as a tumor suppressor in non-small cell lung cancer

Although the short isoform of ErbB3-binding protein 1 (Ebp1), p42 has been considered to be a potent tumor suppressor in a number of human cancers, whether p42 suppresses tumorigenesis of lung cancer cells has never been clarified. In the current study we investigated the tumor suppressor role of p42 in non-small cell lung cancer cells. Our data suggest that the expression level of p42 is inversely correlated with the cancerous properties of NSCLC cells and that ectopic expression of p42 is sufficient to inhibit cell proliferation, anchorage-independent growth, and invasion as well as tumor growth in vivo. Interestingly, p42 suppresses Akt activation and overexpression of a constitutively active form of Akt restores the tumorigenic activity of A549 cells that is ablated by exogenous p42 expression. Thus, we propose that p42 Ebp1 functions as a potent tumor suppressor of NSCLC through interruption of Akt signaling. [BMB Reports 2015; 48(3): 159-165]     

PHLPP and a second isoform, PHLPP2, differentially attenuate the amplitude of Akt signaling by regulating distinct Akt isoforms

Akt/protein kinase B controls cell growth, proliferation, and survival. We recently discovered a novel phosphatase PHLPP, for PH domain leucine-rich repeat protein phosphatase, which terminates Akt signaling by directly dephosphorylating and inactivating Akt. Here we describe a second family member, PHLPP2, which also inactivates Akt, inhibits cell-cycle progression, and promotes apoptosis. These phosphatases control the amplitude of Akt signaling: depletion of either isoform increases the magnitude of agonist-evoked Akt phosphorylation by almost two orders of magnitude. Although PHLPP1 and PHLPP2 both dephosphorylate the same residue (hydrophobic phosphorylation motif) on Akt, they differentially terminate Akt signaling by regulating distinct Akt isoforms. Knockdown studies reveal that PHLPP1 specifically modulates the phosphorylation of HDM2 and GSK-3alpha through Akt2, whereas PHLPP2 specifically modulates the phosphorylation of p27 through Akt3. Our data unveil a mechanism to selectively terminate Akt-signaling pathways through the differential inactivation of specific Akt isoforms by specific PHLPP isoforms.     

Lysine 263 residue of NPM/B23 is essential for regulating ATP binding and B23 stability

Here, we show that Nucleophsomin/B23 provides lysine 263 as a critical binding site for ATP. Mutagenesis of lysine 263 to asparagine (K263N) disrupts B23 from ATP binding. While B23 WT exclusively localizes to the nucleolus, the B23-K263N is redistributed from the nucleolus to the nucleoplam. Notably, the K263N mutant is unstable, and displayed rapid degradation. Alteration of K263 induced B23 instability through increased ubiquitination and proteaosomal degradation. Moreover, mutation of K263 impedes the mitogenic effect of B23 in PC12 cells. Thus, K263 is a critical site for ATP binding and required for B23 stability, confining B23 in the nucleolus.     

Role of protein kinase C in arginine vasopressin-stimulated ERK and p70S6 kinase phosphorylation

We previously showed in rat renal glomerular mesangial cells, that arginine vasopressin (AVP)-stimulated cell proliferation was mediated by epidermal growth factor receptor (EGF-R) transactivation, and activation (phosphorylation) of ERK1/2 and p70S6 kinase (Ghosh et al. [2001]: Am J Physiol Renal Physiol 280:F972-F979]. In this paper, we extend these observations and show that different protein kinase C (PKC) isoforms play different roles in mediating AVP-stimulated ERK1/2 and p70S6 kinase phosphorylation and cell proliferation. AVP treatment for 0-60 min stimulated the serine/threonine phosphorylation of PKC isoforms alpha, delta, epsilon, and zeta. The activation of PKC was dependent on EGF-R and phosphatidylinositol 3-kinase (PI3K) activation. In addition, inhibition of conventional and novel PKC isoforms by chronic (24 h) exposure to phorbol 12-myristate 13-acetate (PMA) inhibited AVP-induced activation of ERK and p70S6 kinase as well as EGF-R phosphorylation. Rottlerin, a specific inhibitor of PKCdelta, inhibited both ERK and p70S6 kinase phosphorylation and cell proliferation. In contrast, a PKCepsilon translocation inhibitor decreased ERK1/2 activation without affecting p70S6 kinase or cell proliferation, while a dominant negative PKCzeta (K281W) cDNA delayed p70S6 kinase activation without affecting ERK1/2. On the other hand, G?6976, an inhibitor of conventional PKC isoforms, did not affect p70S6 kinase, but stimulated ERK1/2 phosphorylation without affecting cell proliferation. Our results indicate that PKCdelta plays an important role in AVP-stimulated ERK and p70S6 kinase activation and cell proliferation.     

Ebp1, an ErbB-3 binding protein, interacts with Rb and affects Rb transcriptional regulation

Ebp1, an ErbB-3 binding protein, inhibits the proliferation and induces the differentiation of human breast cancer cells. The mechanisms of these effects are unknown. Rb, the product of the retinoblastoma gene, is an important modulator of cell cycle progression and cellular differentiation. We report that Rb is a binding target for Ebp1. Ebp1 was localized to both the nucleus and the cytoplasm of logarithmically growing AU565 breast cancer cells and HeLa cells as determined by confocal immunofluorescent microscopy. Ebp1 was present in Rb immunoprecipitates derived from AU565 breast cancer cells. GST-Rb also bound endogenous Ebp1. Using GST-Ebp1 constructs, we determined that the 72 C-terminal amino acids of Ebp1 were sufficient to bind Rb. Dephosphorylation of Ebp1 enhanced the interaction of Ebp1 with Rb. The overexpression of Ebp1 in MCF-7 and AU565 (Rb(+)) cells inhibited the activity of the E2F1 regulated cyclin-E promoter. Ebp1 bound E2F1 indirectly via Rb in lysates of MCF-7 cells. The interaction of Ebp1 with Rb may prove to be an important mechanism of Ebp1 induced changes in cell proliferation and differentiation.     

Protein kinase C-delta phosphorylates Ebp1 and prevents its proteolytic degradation, enhancing cell survival

ErbB3-binding protein (Ebp1) promotes cell survival by preventing apoptotic DNA fragmentation through a complex with active nuclear Akt. Ebp1 phosphorylation by protein kinase C (PKC)-delta mediates its binding to nuclear Akt. In this study, we show that Ebp1 itself acts as a substrate of active caspase 3 during the programmed cell death. PKC-delta phosphorylation on Ebp1 protects it from apoptotic degradation initiated in cell-free apoptotic solution. Moreover, Ebp1 is evidently cleaved in PKC-delta-deficient cells but not in wild-type cells. Ebp1 translated from first ATG is resistant to apoptotic cleavage; by contrast, Ebp1 from second and third ATG demonstrates robust degradation, and PKC phosphorylation on S360 suppresses its cleavage by active caspase 3. Ebp1 can be digested at both D53 and D196 sites, but cleavage at D196 appears to be a prerequisite for its further degradation at D53 site. Compared with wild-type Ebp1, D196A mutant markedly protects cells from apoptosis. Thus, PKC-delta antagonizes apoptosis through phosphorylating Ebp1 and protects it from apoptotic degradation.     

Ebp1 is a dsRNA-binding protein associated with ribosomes that modulates eIF2alpha phosphorylation

dsRNA-binding domains (dsRBDs) characterize an expanding family of proteins involved in different cellular processes, ranging from RNA editing and processing to translational control. Here we present evidence that Ebp1, a cell growth regulating protein that is part of ribonucleoprotein (RNP) complexes, contains a dsRBD and that this domain mediates its interaction with dsRNA. Deletion of Ebp1's dsRBD impairs its localization to the nucleolus and its ability to form RNP complexes. We show that in the cytoplasm, Ebp1 is associated with mature ribosomes and that it is able to inhibit the phosphorylation of serine 51 in the eukaryotic initiation factor 2 alpha (eIF2alpha). In response to various cellular stress, eIF2alpha is phosphorylated by distinct protein kinases (PKR, PERK, GCN2, and HRI), and this event results in protein translation shut-down. Ebp1 overexpression in HeLa cells is able to protect eIF2alpha from phosphorylation at steady state and also in response to various treatments. We demonstrate that Ebp1 interacts with and is phosphorylated by the PKR protein kinase. Our results demonstrate that Ebp1 is a new dsRNA-binding protein that acts as a cellular inhibitor of eIF2alpha phosphorylation suggesting that it could be involved in protein translation control.     

Angiogenin activates Erk1/2 in human umbilical vein endothelial cells

Angiogenin is a potent angiogenic factor that binds to endothelial cells and is endocytosed and rapidly translocated to the nucleus where it is concentrated in the nucleolus and binds to DNA. Angiogenin also activates cell-associated proteases, induces cell invasion and migration, stimulates cell proliferation, and organizes cultured cells to form tubular structures. The intracellular signaling pathways that mediate these various cellular responses are not well understood. Here we report that angiogenin induces transient phosphorylation of extracellular signal-related kinase1/2 (Erk1/2) in cultured human umbilical vein endothelial cells. Angiogenin does not affect the phosphorylation status of stress-associated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) and p38 mitogen-activated protein (MAP) kinases. PD98059--a specific inhibitor of MAP or Erk kinase 1 (MEK 1), the upstream kinase that phosphorylates Erk1/2--abolishes angiogenin-induced Erk phosphorylation and cell proliferation without affecting nuclear translocation of angiogenin. In contrast, neomycin, a known inhibitor of nuclear translocation and cell proliferation, does not interfere with angiogenin-induced Erk1/2 phosphorylation. These data indicate that both intracellular signaling pathways and direct nuclear functions of angiogenin are required for angiogenin-induced cell proliferation and angiogenesis.     

Bradykinin-mediated cell proliferation depends on transactivation of EGF receptor in corneal fibroblasts

In previous studies, bradykinin (BK) has been shown to induce cell proliferation through BK B2 receptor (B2R) via p42/p44 MAPK in Statens Seruminstitut Rabbit Corneal Cells (SIRCs). In addition to this pathway, EGFR transactivation pathway has been implicated in linking a variety of G-protein coupled receptors to MAPK cascades. Here, we further investigate whether these transactivation mechanisms participating in BK-induced cell proliferation in SIRCs. Using an immunofluorescence staining and RT-PCR, we initially characterize that SIRCs were corneal fibroblasts and predominantly expressed B2R by BK. Inhibition of p42/p44 MAPK by the inhibitors of Src, EGFR, and Akt or transfection with respective siRNAs prevents BK-induced DNA synthesis in SIRCs. The mechanisms underlying these responses were mediated through phosphorylation of Src and EGFR via the formation of Src/EGFR complex which was attenuated by PP1 and AG1478. Moreover, BK-induced p42/p44 MAPK and Akt activation was mediated through EGFR transactivation, which was diminished by the inhibitors of MMP-2/9 and heparin-binding EGF-like factor (HB-EGF). Finally, increased nuclear translocation of Akt and p42/p44 MAPK turns on early gene expression leading to cell proliferation. These results suggest that BK-induced cell proliferation is mediated through c-Src-dependent transactivation of EGFR via MMP2/9-dependent pro-HB-EGF shedding linking to activation of Akt and p42/p44 MAPK in corneal fibroblasts.     

Peanut lectin stimulates proliferation of colon cancer cells by interaction with glycosylated CD44v6 isoforms and consequential activation of c-Met and MAPK: functional implications for disease-associated glycosylation changes

Peanut agglutinin lectin (PNA) binds the Thomsen-Friedenreich (TF) oncofetal carbohydrate antigen (galactose beta1-3N-acetylgalactosamine alpha) that shows increased expression in colon cancer, adenomas, and inflammatory bowel disease. PNA is mitogenic, both in vitro and in vivo, for colon epithelial cells. In these cells, PNA binds predominantly to cell-surface TF antigen expressed by high molecular weight isoforms of the transmembrane glycoprotein CD44 that are generated in inflamed and neoplastic colonic epithelia by altered RNA splicing. Our aim was to identify the signaling mechanism underlying the proliferative response to PNA. This was investigated in HT29, T84, and Caco2 colon cancer cells. Parallel lectin and immunoblotting of PNA affinity-purified HT29 cell membrane extracts showed PNA binding to high molecular weight CD44v6 isoforms. Within 5 min, PNA (25 microg/mL) caused a 6-fold increase in phosphorylation of hepatocyte growth factor receptor c-Met, known to co-associate with CD44v6. This was followed by the downstream activation of p44/p42 mitogen-activated protein kinase (MAPK) over 15-20 min. The presence of 100 microg/mL asialofetuin, a TF antigen-expressing glycoprotein, blocked both PNA-induced c-Met and MAPK activation. A similar PNA-induced c-Met and MAPK phosphorylation was also seen in T84 cells that express CD44v6 but not in Caco2 cells that lack CD44v6. PNA-induced cell proliferation was completely blocked by 1 microM PD98059, an inhibitor of MAPK activation (p < 0.0001). The expression of TF antigen by CD44 isoforms in colonic epithelial cells allows lectin-induced mitogenesis that is mediated by phosphorylation of c-Met and MAPK. It provides a mechanism by which dietary, microbial, or endogenous galactose-binding lectins could affect epithelial proliferation in the cancerous and precancerous colon.     

14-3-3sigma is a p37 AUF1-binding protein that facilitates AUF1 transport and AU-rich mRNA decay

Short-lived cytokine mRNAs contain an AU-rich destabilizing element (ARE). AUF1 proteins bind the ARE, undergo shuttling, and promote cytoplasmic ARE-mRNA decay through a poorly understood mechanism. We therefore identified AUF1-interacting proteins that may play a role in ARE-mRNA decay. We used mass-spectrometry to identify 14-3-3sigma protein as an AUF1-interacting protein. 14-3-3sigma binds selectively and strongly to p37 AUF1 and to a lesser extent to the p40 isoform, the two isoforms most strongly associated with ARE-mRNA decay, but not to the two larger isoforms, p42 and p45. The 14-3-3sigma interaction site on p37 was mapped to a region found only in the two smaller AUF1 isoforms and which overlaps a putative nuclear localization signal (NLS). Stable overexpression of 14-3-3sigma significantly increased cytoplasmic accumulation of p37 AUF1 and reduced the steady-state level and half-life of a reporter ARE-mRNA. siRNA silencing of AUF1 eliminated the effect of 14-3-3sigma overexpression. 14-3-3sigma therefore binds to p37 AUF1, retains it in the cytoplasm probably by masking its NLS, and enhances rapid turnover of ARE-mRNAs.     

Tumor suppressor ARF degrades B23, a nucleolar protein involved in ribosome biogenesis and cell proliferation

The tumor suppressor ARF induces a p53-dependent and -independent cell cycle arrest. Unlike the nucleoplasmic MDM2 and p53, ARF localizes in the nucleolus. The role of ARF in the nucleolus, the molecular target, and the mechanism of its p53-independent function remains unclear. Here we show that ARF interacts with B23, a multifunctional nucleolar protein involved in ribosome biogenesis, and promotes its polyubiquitination and degradation. Overexpression of B23 induces a cell cycle arrest in normal fibroblasts, whereas in cells lacking p53 it promotes S phase entry. Conversely, knocking down B23 inhibits the processing of preribosomal RNA and induces cell death. Further, oncogenic Ras induces B23 only in ARF null cells, but not in cells that retain wild-type ARF. Together, our results reveal a molecular mechanism of ARF in regulating ribosome biogenesis and cell proliferation via inhibiting B23, and suggest a nucleolar role of ARF in surveillance of oncogenic insults.     

Epidermal growth factor regulates astrocyte expression of the interleukin-4 receptor via a MAPK-independent pathway

Human astrocytes express the interleukin (IL)-4 receptor alpha chain (IL-4R alpha) in vitro and in vivo but mechanisms governing astrocyte IL-4R alpha expression have not been established. We hypothesized that epidermal growth factor (EGF) and IL-4, agents that profoundly affect astrocyte proliferation, might also alter IL-4R alpha expression. Exposure to EGF for 24 h enhanced IL-4R alpha mRNA levels; in contrast, IL-4 yielded no increase. Immunoblotting demonstrated that EGF but not IL-4 increased astrocyte IL-4R alpha protein after 2--4 days of exposure. Similarly, EGF but not IL-4 strongly activated phosphorylation of p42/p44 extracellular regulated kinase isoforms, a reaction blocked by the mitogen-activated protein kinase (MAPK) inhibitor, PD98059. PD98059 also blocked EGF-stimulated DNA synthesis but not IL-4R alpha mRNA levels, while antibody to the EGF receptor (erbB1) blocked both EGF effects. Data suggest that astrocyte IL-4R alpha expression is upregulated by EGF but not by IL-4 in an EGF-receptor-dependent manner and that mechanisms are independent of MAPK activation.