Alpha(1) adrenergic agonist induction of p21(waf1/cip1) mRNA stability in transfected HepG2 cells correlates with the increased binding of an AU-rich element binding factor

Stimulation of transfected HepG2 cells (TFG2) with the alpha(1)-adrenergic agonist phenylephrine (PE) significantly activated p21(waf1/cip1) gene expression without affecting p53 gene expression. Northern blotting and reporter assay demonstrated that this induction was due to PE stimulation of p21(waf1/cip1) mRNA stability. To further define the underlying mechanism, we prepared a chloramphenicol acetyltransferase (CAT)-p21(waf1/cip1) 3'-untranslated region (3'-UTR) hybrid construct by inserting the 3'-UTR of p21(waf1/cip1) mRNA just downstream from the CAT coding sequence and transfected it into TFG2 cells. PE treatment enhanced the activity of this construct by 6-fold. Deletion analyses indicated that an AU-rich element (AURE) located between 553 to 625 within the p21(waf1/cip1) 3'-UTR was required for this induction. RNA gel shift assays demonstrated that this AURE bound an RNA-binding protein. This protein has been purified 5000-fold from PE-treated TFG2 cells by heparin-Sepharose and RNA affinity chromatography. SDS-polyacrylamide gel electrophoresis, UV cross-linking, and Northwestern analyses indicated the molecular mass of this protein as 24 and 52 kDa. Finally, PE treatment markedly enhanced this RNA-protein binding by a p42/44 mitogen-activated protein kinase-dependent mechanism. These data suggest that the AURE located between 553 and 625 within the p21(waf1/cip1) mRNA 3'-UTR, which binds an RNA-binding protein, is responsible for PE-induced p21(waf1/cip1) mRNA stability.     

RNA-binding protein Csx1 is phosphorylated by LAMMER kinase, Lkh1, in response to oxidative stress in Schizosaccharomyces pombe

Recent studies have shown that global gene expression during oxidative stress in Schizosaccharomyces pombe is regulated by stress-induced activation and binding of Csx1 to atf1(+) mRNA. However, the kinase responsible for the activation of Csx1 has not been identified. Here, we describe, for the first time, that Csx1 is phosphorylated by S. pombe LAMMER kinase, Lkh1, under oxidative conditions and that the stress-activated binding of the Csx1 to the atf1(+) mRNA was also affected by Lkh1 and Spc1. These data indicate that concerted actions of Spc1 and Lkh1 are required for the activation of Csx1 during oxidative condition in the fission yeast S. pombe.     

Roles for p21waf1/cip1 and p27kip1 during the adaptation response to massive intestinal resection

The magnitude of gut adaptation is a decisive factor in determining whether patients are able to live independent of parenteral nutrition after massive small bowel loss. We previously established that the cyclin-dependent kinase inhibitor (CDKI) p21(waf1/cip1) is necessary for enterocyte proliferation and a normal adaptation response. In the present study, we have further elucidated the role of this CDKI in the context of p27(kip1), another member of the Cip/Kip CDKI family. Small bowel resections (SBRs) or sham operations were performed in control (C57/BL6), p21(waf1/cip1)-null, p27(kip1)-null, and p21(waf1/cip1)/p27(kip1) double-null mice. Morphological (villus height/crypt depth) alterations in the mucosa, the kinetics of enterocyte turnover (rates of enterocyte proliferation and apoptosis), and the protein expression of various cell cycle-regulatory proteins were recorded at various postoperative times. Enterocyte compartment-specific mRNA expression was investigated using laser capture microdissection. Resection-induced adaptation in control mice coincided with increased protein expression of p21(waf1/cip1) and decreased p27(kip1) within 3 days postoperatively. Identical changes in mRNA expression were detected in crypt but not in villus enterocytes. Adaptation occurred normally in control and p27(kip1)-null mice; however, mice deficient in both p21(waf1/cip1) and p27(kip1) failed to increase baseline rates of enterocyte proliferation and adaptation. The expression of p21(waf1/cip1) protein and mRNA in the proliferative crypt compartment is necessary for resection-induced enterocyte proliferation and adaptation. The finding that deficient expression of p27(kip1) does not affect adaptation suggests that these similar CDKI family members display distinctive cellular functions during the complex process of intestinal adaptation.     

Phosphorylation of the cyclin-dependent kinase inhibitor p21Cip1 on serine 130 is essential for viral cyclin-mediated bypass of a p21Cip1-imposed G1 arrest

K cyclin encoded by Kaposi's sarcoma-associated herpesvirus confers resistance to the cyclin-dependent kinase (cdk) inhibitors p16Ink4A, p21Cip1, and p27Kip1 on the associated cdk6. We have previously shown that K cyclin expression enforces S-phase entry on cells overexpressing p27Kip1 by promoting phosphorylation of p27Kip1 on threonine 187, triggering p27Kip1 down-regulation. Since p21Cip1 acts in a manner similar to that of p27Kip1, we have investigated the subversion of a p21Cip1-induced G1 arrest by K cyclin. Here, we show that p21Cip1 is associated with K cyclin both in overexpression models and in primary effusion lymphoma cells and is a substrate of the K cyclin/cdk6 complex, resulting in phosphorylation of p21Cip1 on serine 130. This phosphoform of p21Cip1 appeared unable to associate with cdk2 in vivo. We further demonstrate that phosphorylation on serine 130 is essential for K cyclin-mediated release of a p21Cip1-imposed G1 arrest. Moreover, we show that under physiological conditions of cell cycle arrest due to elevated levels of p21Cip1 resulting from oxidative stress, K cyclin expression enabled S-phase entry and was associated with p21Cip1 phosphorylation and partial restoration of cdk2 kinase activity. Thus, expression of the viral cyclin enables cells to subvert the cell cycle inhibitory function of p21Cip1 by promoting cdk6-dependent phosphorylation of this antiproliferative protein.     

Cooperative regulation of DOG2, encoding 2-deoxyglucose-6-phosphate phosphatase, by Snf1 kinase and the high-osmolarity glycerol-mitogen-activated protein kinase cascade in stress responses of Saccharomyces cerevisiae

We screened the genome of Saccharomyces cerevisiae for the genes responsive to oxidative stress by using the lacZ transposon-insertion library. As a result, we found that expression of the DOG2 gene coding for 2-deoxyglucose-6-phosphate phosphatase was induced by oxidative stress. The expression of DOG2 was also induced by osmotic stress. We found a putative cis element (STRE, a stress response element) in the DOG2 promoter adjacent to a consensus sequence to which the Mig1p repressor is known to bind. The basal levels of DOG2 gene expression were increased in a mig1Delta mutant, while the derepression of DOG2 was not observed in a snf1Delta mutant under glucose-deprived conditions. Induction of the DOG2 gene expression by osmotic stress was observed in any of the three disruptants pbs2Delta, hog1Delta, and snf1Delta. However, the osmotic induction was completely abolished in both the snf1Delta pbs2Delta mutant and the snf1Delta hog1Delta mutant. Additionally, these single mutants as well as double mutants failed to induce DOG2 expression by oxidative stress. These results suggest that Snf1p kinase and the high-osmolarity glycerol-mitogen-activated protein kinase cascade are likely to be involved in the signaling pathway of oxidative stress and osmotic stress in regulation of DOG2.     

The enhancer of decapping proteins,Edc1p and Edc2p,bind RNA and stimulate the activity of the decapping enzyme

A major pathway of eukaryotic mRNA turnover initiates with deadenylation, which allows a decapping reaction leading to 5'-3' exonucleolytic degradation. A key control point in this pathway is the decapping of the mRNA. Two proteins, Edc1 and Edc2, were genetically identified previously as enhancers of the decapping reaction. In this work, we demonstrate that Edc1p and Edc2p are RNA-binding proteins. In addition, recombinant Edc1p or Edc2p stimulates mRNA decapping in cell-free extracts or with purified decapping enzyme. These results suggest that Edc1p and Edc2p activate decapping directly by binding to the mRNA substrate and enhancing the activity of the decapping enzyme. Interestingly, edc1Delta strains show defects in utilization of glycerol as a carbon source and misregulation of several mRNAs in response to carbon-source changes. This identifies a critical role for decapping and Edc1p in alterations of gene expression in response to carbon-source changes.     

CARM1 regulates proliferation of PC12 cells by methylating HuD

HuD is an RNA-binding protein that has been shown to induce neuronal differentiation by stabilizing labile mRNAs carrying AU-rich instability elements. Here, we show a novel mechanism of arginine methylation of HuD by coactivator-associated arginine methyltransferase 1 (CARM1) that affected mRNA turnover of p21^cip1/waf1 mRNA in PC12 cells. CARM1 specifically methylated HuD in vitro and in vivo and colocalized with HuD in the cytoplasm. Inhibition of HuD methylation by CARM1 knockdown elongated the p21^cip1/waf1 mRNA half-life and resulted in a slow growth rate and robust neuritogenesis in response to nerve growth factor (NGF). Methylation-resistant HuD bound more p21^cip1/waf1 mRNA than did the wild type, and its overexpression upregulated p21^cip1/waf1 protein expression. These results suggested that CARM1-methylated HuD maintains PC12 cells in the proliferative state by committing p21^cip1/waf1 mRNA to its decay system. Since the methylated population of HuD was reduced in NGF-treated PC12 cells, downregulation of HuD methylation is a possible pathway through which NGF induces differentiation of PC12 cells.