Nuclear Localization Signal and p53 Binding Site in MAP/ERK Kinase Kinase 1 (MEKK1).

Previously, we showed that Mekk1 translocates to the nucleus, interacts with tumor suppressor protein p53, and co-represses PKD1 transcription via an atypical p53 binding site on the minimal PKD1 promoter (JBC 285:38,818-38,831, 2010). In this study, we report the mechanisms of Mekk1 nuclear transport and p53 binding. Using GFP-linked constitutively active-Mekk1 (CA-Mekk1) and a deletion strategy, we identified a nuclear localization signal (HRDVK) located at amino acid (aa) residues 1,349-1,353 in the C-terminal Mekk1 catalytic domain. Deletion of this sequence in CA-Mekk1 and full-length Mekk1 significantly reduced their nuclear translocation in both HEK293T and COS-1 cells. Using co-immunoprecipitation, we identified an adjacent sequence (GANLID, aa 1,354-1,360) in Mekk1 responsible for p53 binding. Deletion of this sequence markedly reduced the interaction of Mekk1 with p53. Mekk1 does not appear to affect phosphorylation of Ser15, located in the Mdm2 interaction site, or other Ser residues in p53. However, Mekk1 mediates p53 protein stability in the presence of Mdm2 and reduces p53 ubiquitination, suggesting an interference with Mdm2-mediated degradation of p53 by the ubiquitin-proteasome pathway.

Regulation of HFE expression by poly(ADP-ribose) polymerase-1 (PARP1) through an inverted repeat DNA sequence in the distal promoter.

Hereditary hemochromatosis (HH) is a common autosomal recessive disorder of iron overload among Caucasians of northern European descent. Over 85% of all cases with HH are due to mutations in the hemochromatosis protein (HFE) involved in iron metabolism. Although the importance in iron homeostasis is well recognized, the mechanism of sensing and regulating iron absorption by HFE, especially in the absence of iron response element in its gene, is not fully understood. In this report, we have identified an inverted repeat sequence (ATGGTcttACCTA) within 1700bp (-1675/+35) of the HFE promoter capable to form cruciform structure that binds PARP1 and strongly represses HFE promoter. Knockdown of PARP1 increases HFE mRNA and protein. Similarly, hemin or FeCl3 treatments resulted in increase in HFE expression by reducing nuclear PARP1 pool via its apoptosis induced cleavage, leading to upregulation of the iron regulatory hormone hepcidin mRNA. Thus, PARP1 binding to the inverted repeat sequence on the HFE promoter may serve as a novel iron sensing mechanism as increased iron level can trigger PARP1 cleavage and relief of HFE transcriptional repression.

CMV promoter is repressed by p53 and activated by JNK pathway.

Viral promoters are widely utilized in commercial and customized vectors to drive expression of genes of interest including reporter, effector and transfection control, because of their high transcription efficiency in a variety of primary and transformed cell lines. However, we observed altered rate of transcription for these promoters under conditions such as presence of an effector protein. These variations in viral promoter driven expressions can potentially lead to incorrect conclusion, especially in comparative and quantitative experiments. We found significantly reduced viral promoter activity in cells overexpressing tumor suppressor protein p53, whereas markedly induced transcription in cells overexpressing MAP/ERK kinase kinase 1 (Mekk 1). Using deletion constructs generated from the CMV promoter, we found the transcription reduction by p53 is possibly mediated through the TATA motif present in proximal CMV promoter. The activation of the CMV promoter by Mekk 1, on the other hand, is attributed to the proximal CRE binding site in the promoter. These findings may be of interest to investigators who use CMV (or other viral) promoter driven vectors for either comparative or quantitative gene expression, or effect on promoter activity.

Cytotoxicity analysis of active components in bitter melon (Momordica charantia) seed extracts using human embryonic kidney and colon tumor cells.

Bitter melon (Momordica charantia) seed extracts (BMSE) have been used as traditional medicine for treating various ailments, although in many cases, the active component(s) are unidentified. In this study, bitter melon seeds were extracted in water, ethanol, or ethanol: water (1:1). The aqueous seed extracts (BMSE-W) exhibited marked cytotoxicity towards human embryonic kidney 293T (HEK293T) and human colon tumor 116 (HCT1116) cells. The activity in BMSE-W was unaffected by heat and proteinases treatments, and eluted in the total volume of size-exclusion HPLC, suggesting the small, organic nature of the active component(s). Gas chromatographic-mass spectrometic (GC-MS) analysis of the HPLC fractions identified methoxy-phenyl oxime (MPO) as a major active component. Acetophenone oxime, a commercially available structural homolog of MPO, demonstrated cytotoxicity comparable with that of the BMSE-W. The oxime functional group was found to be critical for activity. Increased poly-(ADP-ribose)-polymerase and beta-actin cleavage, and chromatin condensation observed in treated cells suggested apoptosis as a plausible cause for the cytotoxicity. This study, for the first time, identified a cytotoxic oxime in BMSE-W.