Makorin Ring Finger Protein 1 (MKRN1) is a transcriptional co-regulator and an E3 ligase. Here, we show that MKRN1 simultaneously functions as a differentially negative regulator of p53 and p21. In normal conditions, MKRN1 could destabilize both p53 and p21 through ubiquitination and proteasome-dependent degradation. As a result, depletion of MKRN1 induced growth arrest through activation of p53 and p21. Interestingly, MKRN1 used earlier unknown sites, K291 and K292, for p53 ubiquitination and subsequent degradation. Under severe stress conditions, however, MKRN1 primarily induced the efficient degradation of p21. This regulatory process contributed to the acceleration of DNA damage-induced apoptosis by eliminating p21. MKRN1 depletion diminished adriamycin or ultraviolet-induced cell death, whereas ectopic expression of MKRN1 facilitated apoptosis. Furthermore, MKRN1 stable cell lines that constantly produced low levels of p53 and p21 exhibited stabilization of p53, but not p21, with increased cell death on DNA damage. Our results indicate that MKRN1 exhibits dual functions of keeping cells alive by suppressing p53 under normal conditions and stimulating cell death by repressing p21 under stress conditions.

SummaryThe capsid protein of the West Nile virus (WNV) functions as an apoptotic agonist via the induction of mitochondrial dysfunction and the activation of caspases-9 and -3. Here, we have determined that the WNV capsid (WNVCp) is capable of binding to and sequestering HDM2 into the nucleolus. WNVCp was shown to interfere with the formation of the HDM2 and p53 complex, thereby causing the stabilization of p53 and the subsequent induction of its target apoptotic protein, Bax. Whereas WNVCp was capable of inducing the p53-dependent apoptotic process in wild-type mouse embryonic fibroblasts (MEF) or SH-SY5Y cells, it exerted no significant effects on p53-null MEF or on p53-knockdown SH-SY5Y cells. This suggests that WNVCp-mediated apoptosis requires p53. Furthermore, when WNV was transfected into cells, endogenous Hdm2 and WNVCp were able to interact physically. WNVCp expressed in wild-type MEF proved able to induce the translocation of the endogenous Hdm2 into the nucleolus. Consistently, WNV was highly pathogenic in the presence of p53, and was less so in the absence of p53. The results of these studies suggest that the apoptotic mechanism mediated by WNV might occur in accordance in a fashion similar to that of the tumour-suppressing mechanism mediated by ARF.

The biological mechanisms for maintaining the basal level of p53 in normal cells require nuclear exclusion and cytoplasmic degradation. Here, we showed that Jab1 facilitates p53 nuclear exclusion and its subsequent degradation in coordination with Hdm2. p53 was excluded from the nucleus in the presence of Jab1; this exclusion was prevented by leptomycin B treatment. Nuclear export of p53 was accompanied by a decrease in the levels of p53, as well as of its target proteins, which include p21 and Bax. Domain analyses of Jab1 showed that the N-terminal domain, 1-110, was capable of inducing cytoplasmic translocation of p53. Furthermore, 110 -191 was required to facilitate the degradation of p53. Neither of these mutants incorporated into the CSN complex, indicating that Jab1 could affect the levels of p53 independent of intact CSN complex. Conversely, Jab1 was incapable of translocating and degrading two p53 mutants, W23S and 6KR, neither of which could be modified by Hdm2. Moreover, Jab1 did not affect the cellular localization or protein levels of p53 in p53 and Hdm2 double-null mouse embryo fibroblasts. We further observed that the ablation of endogenous Jab1 by small interfering RNA prevented Hdm2-mediated p53 nuclear exclusion. Under stressed conditions, which could sequester Hdm2 in its inactive state, Jab1 did not affect p53. Our studies implicate that Jab1 is required to remove post-translationally modified p53 and provide a novel target for p53-related cancer therapies.

The clinical manifestations of West Nile virus (WNV), a member of the Flavivirus family, include febrile illness, sporadic encephalitis, and paralysis. The capsid (Cp) of WNV is thought to participate in these processes by inducing apoptosis through mitochondrial dysfunction and activation of caspase-9 and caspase-3. To further identify the molecular mechanism of the WNV capsid protein (WNVCp), yeast two-hybrid assays were employed using WNV-Cp as bait. Jab1, the fifth subunit of the COP9 signalosome, was subsequently identified as a molecule that interacts with WNVCp. Immunoprecipitation and glutathione S-transferase pulldown assays confirmed that direct interaction could occur between WNVCp and Jab1. Immunofluorescence microscopy demonstrated that the overexpressed WNVCp, which localized to the nucleolus, was translocated to the cytoplasm upon its co-expression with Jab1. When treated with leptomycin B, Jab1-facilitated nuclear exclusion of WNVCp was prevented, which indicated that the CRM1 complex is required for Jab1-facilitated nuclear export of WNVCp. Moreover, Jab1 promoted the degradation of WNVCp in a proteasome-dependent way. Consistent with this, WNVCpmediated cell cycle arrest at the G 2 phase in H1299 was prevented by exogenous Jab1. Finally, an analysis of WNVCp deletion mutants indicated that the first 15 amino acids were required for interaction with Jab1. Furthermore, the double-point mutant of the WNVCp, P5A/P8A, was incapable of binding to Jab1. These results indicate that Jab1 has a potential protective effect against pathogenic WNVCp and might provide a novel target site for the treatment of disease caused by WNV.

Net1 is a RhoA guanine nucleotide exchange factor (GEF) that is overexpressed in a subset of human cancers and contributes to cancer cell motility and invasion in vitro. However, the molecular mechanism accounting for its role in cell motility and invasion has not been described. In the present work, we show that expression of both Net1 isoforms in breast cancer cells is required for efficient cell motility. Although loss of Net1 isoform expression only partially blocks RhoA activation, it inhibits lysophosphatidic acid (LPA)-stimulated migration as efficiently as knockdown of RhoA itself. However, we demonstrate that the Net1A isoform predominantly controls myosin light-chain phosphorylation and is required for trailing edge retraction during migration. Net1A interacts with focal adhesion kinase (FAK), localizes to focal adhesions, and is necessary for FAK activation and focal adhesion maturation during cell spreading. Net1A expression is also required for efficient invasion through a Matrigel matrix. Analysis of invading cells demonstrates that Net1A is required for amoeboid invasion, and loss of Net1A expression causes cells to shift to a mesenchymal phenotype characterized by high ␤ 1 -integrin activity and membrane type 1 matrix metalloproteinase (MT1-MMP) expression. These results demonstrate a previously unrecognized role for the Net1A isoform in controlling FAK activation during planar cell movement and amoeboid motility during extracellular matrix (ECM) invasion.

West Nile virus capsid protein (WNVCp) displays pathogenic toxicity via the apoptotic pathway. However, a cellular mechanism protective against this toxic effect has not been observed so far. Here, we identified Makorin ring finger protein 1 (MKRN1) as a novel E3 ubiquitin ligase for WNVCp. The cytotoxic effects of WNVCp as well as its expression levels were inhibited in U2OS cells that stably expressed MKRN1. Immunoprecipitation analyses revealed an interaction between MKRN1 and WNVCp. Domain analysis indicated that the C terminus of MKRN1 and the N terminus of WNVCp were required for the interaction. MKRN1 could induce WNVCp ubiquitination and degradation in a proteasome-dependent manner. Interestingly, the WNVCp mutant with amino acids 1 to 105 deleted WNVCp was degraded by MKRN1, whereas the mutant with amino acids 1 to 90 deleted was not. When three lysine sites at positions 101, 103, and 104 of WNVCp were replaced with alanine, MKRN1-mediated ubiquitination and degradation of the mutant were significantly inhibited, suggesting that these sites are required for the ubiquitination. Finally, U2OS cell lines stably expressing MKRN1 were resistant to cytotoxic effects of WNV. In contrast, cells depleted of MKRN1 were more susceptible to WNVCp cytotoxicity. Confirming this, overexpression of MKRN1 significantly reduced, but depletion of MKRN1 increased, WNV proliferation in 293T cells. Taken together, our results suggest that MKRN1 can protect cells from WNV by inducing WNVCp degradation.

Net1 isoform A (Net1A) is a RhoA GEF that is required for cell motility and invasion in multiple cancers. Nuclear localization of Net1A negatively regulates its activity, and we have recently shown that Rac1 stimulates Net1A relocalization to the plasma membrane to promote RhoA activation and cytoskeletal reorganization. However, mechanisms controlling the subcellular localization of Net1A are not well understood. Here, we show that Net1A contains two nuclear localization signal (NLS) sequences within its Nterminus and that residues surrounding the second NLS sequence are acetylated. Treatment of cells with deacetylase inhibitors or expression of active Rac1 promotes Net1A acetylation. Deacetylase inhibition is sufficient for Net1A relocalization outside the nucleus, and replacement of the N-terminal acetylation sites with arginine residues prevents cytoplasmic accumulation of Net1A caused by deacetylase inhibition or EGF stimulation. By contrast, replacement of these sites with glutamine residues is sufficient for Net1A relocalization, RhoA activation and downstream signaling. Moreover, the N-terminal acetylation sites are required for rescue of F-actin accumulation and focal adhesion maturation in Net1 knockout MEFs. These data indicate that Net1A acetylation regulates its subcellular localization to impact on RhoA activity and actin cytoskeletal organization.

Maintaining proper telomere length requires the presence of the telomerase enzyme. Here we show that telomerase reverse transcriptase (TERT), a catalytic component of telomerase, is recruited to promyelocytic leukemia (PML) nuclear bodies through its interaction with PML-IV. Treatment of interferon-α (IFNα) in H1299 cells resulted in the increase of PML proteins with a concurrent decrease of telomerase activity, as previously reported. PML depletion, however, stimulated telomerase activity that had been inhibited by IFNα with no changes in TERT mRNA levels. Upon treatment with IFNα, exogenous TERT localized to PML nuclear bodies and binding between TERT and PML increased. Immunoprecipitation and immunofluorescence analyses showed that TERT specifically bound to PML-IV. Residues 553-633 of the C-terminal region of PML-IV were required for its interaction with the TERT region spanning residues 1-350 and 595-946. The expression of PML-IV and its deletion mutant, 553-633, suppressed intrinsic telomerase activity in H1299. TERT-mediated immunoprecipitation of PML or the 553-633 fragment demonstrated that these interactions inhibited telomerase activity. H1299 cell lines stably expressing PML-IV displayed decreased telomerase activity with no change of TERT mRNA levels. Accordingly, telomere length of PML-IV stable cell lines was shortened. These results indicate that PML-IV is a negative regulator of telomerase in the post-translational state.