A Novel Post-translational Modification of Nucleolin,SUMOylation at Lys-294, Mediates Arsenite-induced Cell Death by Regulating gadd45α mRNA Stability

Nucleolin is a ubiquitously expressed protein and participates in many important biological processes, such as cell cycle regulation and ribosomal biogenesis. The activity of nucleolin is regulated by intracellular localization and post-translational modifications, including phosphorylation, methylation, and ADP-ribosylation. Small ubiquitin-like modifier (SUMO) is a category of recently verified forms of post-translational modifications and exerts various effects on the target proteins. In the studies reported here, we discovered SUMOylational modification of human nucleolin protein at Lys-294, which facilitated the mRNA binding property of nucleolin by maintaining its nuclear localization. In response to arsenic exposure, nucleolin-SUMO was induced and promoted its binding with gadd45α mRNA, which increased gadd45α mRNA stability and protein expression, subsequently causing GADD45α-mediated cell death. On the other hand, ectopic expression of Mn-SOD attenuated the arsenite-generated superoxide radical level, abrogated nucleolin-SUMO, and in turn inhibited arsenite-induced apoptosis by reducing GADD45α expression. Collectively, our results for the first time demonstrate that nucleolin-SUMO at K294R plays a critical role in its nucleus sequestration and gadd45α mRNA binding activity. This novel biological function of nucleolin is distinct from its conventional role as a proto-oncogene. Therefore, our findings here not only reveal a new modification of nucleolin protein and its novel functional paradigm in mRNA metabolism but also expand our understanding of the dichotomous roles of nucleolin in terms of cancer development, which are dependent on multiple intracellular conditions and consequently the appropriate regulations of its modifications, including SUMOylation.     

gadd45 is not required for activation of c-Jun N-terminal kinase or p38 during acute stress.

Cells respond to environmental stress with activation of c-Jun N-terminal kinase (JNK) and p38. Recent studies have implicated Gadd45 and two related proteins, MyD118/Gadd45beta and CR6/Gadd45gamma, as initiators of JNK/p38 signaling via their interaction with an upstream kinase MTK1. It was proposed that stress-induced expression of the Gadd45-related proteins leads to MTK1 activation and subsequent JNK/p38 activation. Using embryo fibroblasts from gadd45-null mice, we have addressed the requirement for Gadd45 in mediating JNK/p38 activation during acute stress. Comparison of JNK/p38 activities in response to methyl methanesulfonate, hydrogen peroxide, UVC irradiation, sorbitol, and anisomycin treatment of gadd45(+/+) and gadd45(-/-) fibroblasts revealed no deficiency in JNK/p38 activation in gadd45(-/-) fibroblasts. In addition, in wild type cells, JNK and p38 activation significantly preceded gadd45 induction with all stresses. Examination of myd118/gadd45beta and cr6/gadd45gamma expression in gadd45(+/+) and gadd45(-/-) fibroblasts revealed similar induction patterns in the two cell types, which, like gadd45 expression, was delayed relative to JNK/p38 activation. We conclude that gadd45 expression is not required for activation of JNK/p38 by environmental stresses, nor are stress-induced increases in myd118/gadd45beta and cr6/gadd45gamma expression necessary for kinase activation in response to such insults.     

Identification of nucleolin as an AU-rich element binding protein involved in bcl-2 mRNA stabilization

bcl-2 mRNA contains an AU-rich element (ARE) that functions in regulating bcl-2 stability. Our earlier studies indicated that taxol- or okadaic acid-induced bcl-2 mRNA destabilization in HL-60 cells is associated with decreased binding of trans-acting factors to the ARE. To identify factors that play a role in the regulation of bcl-2 mRNA stability, bcl-2 ARE-binding proteins were purified from HL-60 cells. Three polypeptides of 100, 70, and 32 kDa were isolated from a bcl-2 ARE affinity matrix. Matrix-assisted laser desorption ionization mass spectroscopy analysis identified these proteins as full-length nucleolin and proteolytic fragments of nucleolin. RNA gel shifts assays indicated that recombinant nucleolin (residues 284-707) binds specifically to bcl-2 ARE RNA. In addition, recombinant nucleolin decreases the rate of decay of mRNA in HL-60 cell extracts in an ARE-dependent manner. Taxol or okadaic acid treatment of HL-60 cells results in proteolysis of nucleolin in a similar time frame as drug-induced bcl-2 mRNA down-regulation. These findings suggest that nucleolin functions as a bcl-2-stabilizing factor and that taxol and okadaic acid treatment induces apoptosis in HL-60 cells through a process that involves down-regulation of nucleolin and destabilization of bcl-2 mRNA.     

Regulation of Gadd45a mRNA expression in vascular smooth muscle under growth and stress conditions

In order to identify differentially expressed genes under growth conditions, quiescent vascular smooth muscle cells (VSMCs) were stimulated with foetal calf serum (FCS) or platelet-derived growth factor-BB (PDGF-BB) for different time periods. Analysing the gene expression by the differential display (DD) method, we identified the cDNA of the growth arrest and DNA damage inducible gene 45a (Gadd45a, also known as gadd45 and gadd45a). Treatment with FCS or PDGF-BB led to a transient down-regulating of Gadd45a expression during the G0/G1 phase and maximal expression when cells had completed division. We found that expression of p53 and BRCA1 mRNA precedes Gadd45a mRNA expression with a maximal induction in the S phase. As in smooth muscle cells, a similar pattern of the Gadd45a mRNA expression was observed in knockout Gadd45a(-/-) cultured mouse embryonic fibroblasts (MEFs). However, no differences between Gadd45a(+/+) and Gadd45a(-/-) cell lines were observed regarding their kinetics of cell division. These experiments suggest a function of Gadd45a when cells exit the cell cycle rather than when regulating the entry into the S phase.     

The nascent polypeptide-associated complex (NAC) controls translation initiation in cis by recruiting nucleolin to the encoding mRNA

Protein aggregates and abnormal proteins are toxic and associated with neurodegenerative diseases. There are several mechanisms to help cells get rid of aggregates but little is known on how cells prevent aggregate-prone proteins from being synthesised. The EBNA1 of the Epstein-Barr virus (EBV) evades the immune system by suppressing its own mRNA translation initiation in order to minimize the production of antigenic peptides for the major histocompatibility (MHC) class I pathway. Here we show that the emerging peptide of the disordered glycine–alanine repeat (GAr) within EBNA1 dislodges the nascent polypeptide-associated complex (NAC) from the ribosome. This results in the recruitment of nucleolin to the GAr-encoding mRNA and suppression of mRNA translation initiation in cis. Suppressing NAC alpha (NACA) expression prevents nucleolin from binding to the GAr mRNA and overcomes GAr-mediated translation inhibition. Taken together, these observations suggest that EBNA1 exploits a nascent protein quality control pathway to regulate its own rate of synthesis that is based on sensing the nascent GAr peptide by NAC followed by the recruitment of nucleolin to the GAr-encoding RNA sequence.     

Alpha-melanocyte-stimulating hormone reduces impact of proinflammatory cytokine and peroxide-generated oxidative stress on keratinocyte and melanoma cell lines

We have previously shown that alpha-melanocyte-stimulating hormone (alpha-MSH) can oppose tumor necrosis factor alpha activation of NF-kappaB (1-2 h) and intercellular adhesion molecule 1 up-regulation (mRNA by 3 h and protein by 24 h) in melanocytes and melanoma cells. The present study reports on the ability of four MSH peptides to control intracellular peroxide levels and glutathione peroxidase (GPx) activity in pigmentary and nonpigmentary cells. In human HBL melanoma and HaCaT keratinocytes tumor necrosis factor alpha and H(2)O(2) both activated GPx in a time- and concentration-dependent manner (by 30-45 min). alpha-MSH peptides were found to inhibit the stimulated GPx activity and had biphasic dose-response curves. MSH 1-13 and MSH [Nle(4)-d-Phe(7)] achieved maximum inhibition at 10(-10) and 10(-12) m, respectively. Higher concentrations (10-100 fold) of MSH 4-10 and MSH 11-13 were required to produce equivalent levels of inhibition. alpha-MSH was also capable of reducing peroxide accumulation within 15 min, and again this inhibition was biphasic. The data support a role of alpha-MSH in acute protection of cells to oxidative/cytokine action that precedes NF-kappaB and GPx activation. The rapidity and potency of the response to alpha-MSH in pigmentary and nonpigmentary cells suggest this to be a central role of this peptide in cutaneous cells.     

hnRNP C increases amyloid precursor protein (APP) production by stabilizing APP mRNA.

We have previously shown that heterogeneous nuclear ribonucleoprotein C (hnRNP C) and nucleolin bound specifically to a 29 nt sequence in the 3'-untranslated region of amyloid precursor protein (APP) mRNA. Upon activation of peripheral blood mononuclear cells, hnRNP C and nucleolin acquired APP mRNA binding activity, concurrent with APP mRNA stabilization. These data suggested that the regulated interaction of hnRNP C and nucleolin with APP mRNA controlled its stability. Here we have directly examined the role of the cis element and trans factors in the turnover and translation of APP mRNA in vitro . In a rabbit reticulocyte lysate (RRL) translation system, a mutant APP mRNA lacking the 29 nt element was 3-4-fold more stable and synthesized 2-4-fold more APP as wild-type APP mRNA. Therefore, the 29 nt element functioned as an APP mRNA destabilizer. RNA gel mobility shift assays with the RRL suggested the presence of endogenous nucleolin, but failed to show hnRNP C binding activity. However, wild-type APP mRNA was stabilized and coded for 6-fold more APP when translated in an RRL system supplemented with exogenous active hnRNP C. Control mRNAs lacking the 29 nt element were unaffected by hnRNP C supplementation. Therefore, occupancy of the 29 nt element by hnRNP C stabilized APP mRNA and enhanced its translation.     

G alpha 12/13 basally regulates p53 through Mdm4 expression

G alpha(12/13), which belongs to the G alpha(12) family, participates in the regulation of diverse physiologic processes. In view of the control of G alpha(12/13) in cell proliferation, this study investigated the role of G alpha(12/13) in the regulation of p53 and mdm4. Immunoblotting and immunocytochemistry revealed that p53 was expressed in control embryonic fibroblasts and was largely localized in the nuclei. G alpha(12) deficiency decreased p53 levels and its DNA binding activity, accompanying p21 repression with Bcl(2) induction, whereas G alpha(13) deficiency exerted weak effects. G alpha(12) or G alpha(13) deficiency did not change p53 mRNA expression. ERK1/2 or Akt was not responsible for p53 repression due to G alpha(12) deficiency. Mdm4, a p53-stabilizing protein, was repressed by G alpha(12) deficiency and to a lesser extent by G alpha(13) deficiency, whereas mdm2, PTEN, beta-catenin, ATM, and Chk2 were unaffected. p53 accumulation by proteasomal inhibition during G alpha(12) deficiency suggested the role of G alpha(12) in p53 stabilization. Constitutively active G alpha(12) (G alpha(12)QL) or G alpha(13) (G alpha(13)QL) promoted p53 accumulation with mdm4 induction in MCF10A cells. p53 accumulation by mdm4 overexpression, but no mdm4 induction by p53 overexpression, and small interfering RNA knockdown verified the regulatory role of mdm4 for p53 downstream of G alpha(12/13). In control or G alpha(12)/G alpha(13)-deficient cells, genotoxic stress led to p53 accumulation. At concentrations increasing the flow cytometric pre-G(1) phase, doxorubicin or etoposide treatment caused serine phosphorylations in G alpha(12)-/- or G alpha(12/13)-/- cells, but did not induce mdm4. G alpha(12/13)QL transfection failed to phosphorylate p53 at serines. Our results indicate that G alpha(12/13) regulate basal p53 levels via mdm4, which constitutes a cell signaling pathway distinct from p53 phosphorylations elicited by genotoxic stress.