14-3-3 Binding to Pim-phosphorylated Ser166 and Ser186 of human Mdm2 - Potential interplay with the PKB/Akt pathway and p14

Here we show that 14-3-3 proteins bind to Pim kinase-phosphorylated Ser166 and Ser186 on the human E3 ubiquitin ligase mouse double minute 2 (Mdm2), but not protein kinase B (PKB)/Akt-phosphorylated Ser166 and Ser188. Pim-mediated phosphorylation of Ser186 blocks phosphorylation of Ser188 by PKB, indicating potential interplay between the Pim and PKB signaling pathways in regulating Mdm2. In cells, expression of Pim kinases promoted phosphorylation of Ser166 and Ser186, interaction of Mdm2 with endogenous 14-3-3s and p14^ARF, and also increased the amount of Mdm2 protein by a mechanism that does not require Pim kinase activities. The implications of these findings for regulation of the p53 pathway, oncogenesis and drug discovery are discussed.

Structured summary

MINT-6823587:PIM3 (uniprotkb:Q86V86) phosphorylates (MI:0217) MDM2 (uniprotkb:Q00987) by protein kinase assay (MI:0424)MINT-6823623:MDM2 (uniprotkb:Q00987) physically interacts (MI:0218) with p14ARF (uniprotkb:Q8N7268N726) by coimmunoprecipitation (MI:0019)MINT-6823537:PKB (uniprotkb:P31749) phosphorylates (MI:0217) MDM2 (uniprotkb:Q00987) by protein kinase assay (MI:0424)MINT-6823574:PIM2 (uniprotkb:QP1W9) phosphorylates (MI:0217) MDM2 (uniprotkb:Q00987) by protein kinase assay (MI:0424)MINT-6823555:PIM1 (uniprotkb:P11309)P phosphorylates (MI:0217) MDM2 (uniprotkb:Q00987) by protein kinase assay (MI:0424)     

Control of p53 nuclear accumulation in stressed cells

Wild-type p53 accumulates in the nucleus following stress. Current models suggest this nuclear accumulation involves phosphorylation at p53 N-terminal sites, and inhibition of murine double minute (MDM)2-dependent nuclear export. We monitored the effects of stress on MDM2-dependent nuclear export of wild-type p53 and a mutant lacking N-terminal phosphorylation sites. Etoposide and ionizing radiation inhibited nuclear export of wild-type p53 and the phosphor-mutant to comparable extents, indicating nuclear export inhibition does not require N-terminal phosphorylation. Cytoplasmic p53 accumulated in the nucleus of transfected cells treated with the nuclear export-inhibitor leptomycin B (LMB). Interestingly, LMB caused less p53 nuclear accumulation than stress treatment, suggesting stress-induced nuclear accumulation of p53 does not result solely from inhibited nuclear export.     

FKBP25, a novel regulator of the p53 pathway, induces the degradation of MDM2 and activation of p53

The p53 tumour suppressor protein is tightly controlled by the E3 ubiquitin ligase, mouse double minute 2 (MDM2), but maintains MDM2 expression as part of a negative feedback loop. We have identified the immunophilin, 25 kDa FK506-binding protein (FKBP25), previously shown to be regulated by p53-mediated repression, as an MDM2-interacting partner. We show that FKBP25 stimulates auto-ubiquitylation and proteasomal degradation of MDM2, leading to the induction of p53. Depletion of FKBP25 by siRNA leads to increased levels of MDM2 and a corresponding reduction in p53 and p21 levels. These data are consistent with the idea that FKBP25 contributes to regulation of the p53-MDM2 negative feedback loop.

Structured summary

MINT-6823686:MDM2 (uniprotkb:Q00987) physically interacts (MI:0218) with FKBP25 (uniprotkb:Q00688) by anti bait coimmunoprecipitation (MI:0006)MINT-6823707, MINT-6823722:MDM2 (uniprotkb:Q00987) physically interacts (MI:0218) with FKBP25 (uniprotkb:Q62446) by pull down (MI:0096)MINT-6823775:P53 (uniprotkb:Q04637) physically interacts (MI:0218) with MDM2 (uniprotkb:Q00987) by anti bait coimmunoprecipitation (MI:0006)MINT-6823735, MINT-6823749:FKBP25 (uniprotkb:Q62446) binds (MI:0407) to MDM2 (uniprotkb:Q00987) by pull down (MI:0096)MINT-6823761:Ubiquitin (UNIPROTKB:62988)P physically interacts (MI:0218) with MDM2 (uniprotkb:Q00987) by pull down (MI:0096)MINT-6823669:MDM2 (uniprotkb:Q00987) physically interacts (MI:0218) with FKBP25 (uniprotkb:Q00688) by two hybrid (MI:0018)     

TRIAD1 inhibits MDM2-mediated p53 ubiquitination and degradation

Murine double minute (MDM2) is an E3 ligase that promotes ubiquitination and degradation of tumor suppressor protein 53 (p53). MDM2-mediated regulation of p53 has been investigated as a classical tumorigenesis pathway. Here, we describe TRIAD1 as a novel modulator of the p53-MDM2 axis that induces p53 activation by inhibiting its regulation by MDM2. Ablation of TRIAD1 attenuates p53 levels activity upon DNA damage, whereas ectopic expression of TRIAD1 promotes p53 stability by inhibiting MDM2-mediated ubiquitination/degradation. Moreover, TRIAD1 binds to the C-terminus of p53 to promote its dissociation from MDM2. These results implicate TRIAD1 as a novel regulatory factor of p53-MDM2.Structured summary of protein interactions:p53 physically interacts with Mdm2 and Triad1 by anti tag coimmunoprecipitation (View Interaction: 1, 2, 3)Mdm2physically interacts with Triad1 by anti tag coimmunoprecipitation (View interaction)p53physically interacts with Mdm2 by anti tag coimmunoprecipitation (View interaction)Triad1binds to p53 by pull down (View interaction)Mdm2physically interacts with p53 by anti tag coimmunoprecipitation (View interaction)p53physically interacts with Triad1 by anti tag coimmunoprecipitation (View interaction)     

Over-expression of the human MDM2 p53 binding domain by fusion to a p53 transactivation peptide

MDM2 binds to the tumor suppressor protein p53 and regulates the level of p53 in cells. Although it is possible to prepare a small amount of the region of MDM2 that binds to p53, the expression level of this fragment of MDM2 is relatively low, limiting the studies involving this protein. Here, we describe a construct for the optimized bacterial expression and purification of the MDM2 p53 binding domain. We found that the expression level of the soluble MDM2 p53 binding domain in bacteria was increased dramatically by fusing it to its interaction partner, the p53 transactivation peptide. Attachment of the p53 transactivation peptide (residues 17-29) to the N-terminus of MDM2 resulted in a more than 200-fold increase of soluble protein expression of the p53 binding domain in bacteria. To obtain the final MDM2 p53 binding domain (residues 5-109) we inserted a tobacco etch virus protease recognition site between the P53 peptide and the MDM2 p53 binding domain. To weaken the protein/peptide interaction and facilitate the separation of the protein from the complex, we introduced a point mutation of one of the key interaction residues (F19A or W23A) in the p53 peptide. The advantages of our new construct are high yield and easy purification of the MDM2 protein.     

Transportin-1 and Transportin-2: Protein nuclear import and beyond

Nearly 20 years after its identification as a new β-karyopherin mediating the nuclear import of the RNA-binding protein hnRNP A1, Transportin-1 is still commonly overlooked in comparison with its best known cousin, Importin-β. Transportin-1 is nonetheless a considerable player in nucleo-cytoplasmic transport. Over the past few years, significant progress has been made in the characterization of the nuclear localization signals (NLSs) that Transportin-1 recognizes, thereby providing the molecular basis of its diversified repertoire of cargoes. The recent discovery that mutations in the Transportin-dependent NLS of FUS cause mislocalization of this protein and result in amyotrophic lateral sclerosis illustrates the importance of Transportin-dependent import for human health. Besides, new functions of Transportin-1 are emerging in processes other than nuclear import. Here, we summarize what is known about Transportin-1 and the related β-karyopherin Transportin-2.     

Subcellular localization of rice histone deacetylases in organelles

Histone deacetylases (HDACs) are known to function in the nucleus. Here, we report on the organellar localization of three rice HDACs, OsSIR2b, OsHDAC6, and OsHDAC10. The 35S:OsSIR2b-GFP and 35S:OsHDAC10-GFP constructs were introduced into tobacco BY2 cells. Co-localization analysis of the green fluorescent protein and MitoTracker fluorescent signals in the transformed BY2 cells indicated that OsSIR2b and OsHDAC10 are localized in the mitochondria. Transgenic Arabidopsis lines harboring 35S:OsHDAC6-GFP and 35S:OsHDAC10-GFP constructs were similarly analyzed, revealing that OsHDAC6-GFP is localized exclusively in chloroplasts, whereas OsHDAC10-GFP is localized in both mitochondria and chloroplasts. The presence of OsHDAC6-GFP and OsHDAC10-GFP in chloroplasts was verified by immunodetection.     

The maximal size of protein to diffuse through the nuclear pore is larger than 60kDa

It has generally been believed that the diffusion limit set by the nuclear pore for protein is 60kDa. We here studied the cellular localization of several artificial proteins and found that the diffusion limit set by the nuclear pore is not as small as previously thought. The results indicate that the maximal size of protein to diffuse through the nuclear pore complex could be quite larger than 60kDa, thus greatly extending the diffusion limit that the nuclear pore can accommodate.     

A novel lysine-rich domain and GTP binding motifs regulate the nucleolar retention of human guanine nucleotide binding protein, GNL3L

A variety of G-proteins and GTPases are known to be involved in nucleolar function. We describe here a new evolutionarily conserved putative human GTPase, guanine nucleotide binding protein-like 3-like (GNL3L). Genes encoding proteins related to GNL3L are present in bacteria and yeast to metazoa and suggests its critical role in development. Conserved domain search analysis revealed that the GNL3L contains a circularly permuted G-motif described by a G5-G4-G1-G2-G3 pattern similar to the HSR1/MMR1 GTP-binding protein subfamily. Highly conserved and critical residues were identified from a three-dimensional structural model obtained for GNL3L using the crystal structure of an Ylqf GTPase from Bacillus subtilis. We demonstrate here that GNL3L is transported into the nucleolus by a novel lysine-rich nucleolar localization signal (NoLS) residing within 1-50 amino acid residues. NoLS identified here is necessary and sufficient to target the heterologous proteins to the nucleolus. We show for the first time that the lysine-rich targeting signal interacts with the nuclear transport receptor, importin-beta and transports GNL3L into the nucleolus. Interestingly, depletion of intracellular GTP blocks GNL3L accumulation into the nucleolar compartment. Furthermore, mutations within the G-domains alter the GTP binding ability of GNL3L and abrogate wild-type nucleolar retention even in the presence of functional NoLS, suggesting that the efficient nucleolar retention of GNL3L involves activities of both basic NoLS and GTP-binding domains. Collectively, these data suggest that GNL3L is composed of distinct modules, each of which plays a specific role in molecular interactions for its nucleolar retention and subsequent function(s) within the nucleolus.     

Both the p33 and p55 Subunits of the Helicobacter pylori VacA Toxin Are Targeted to Mammalian Mitochondria

Helicobacter pylori infection causes peptic ulcers and gastric cancer. A major toxin secreted by H. pylori is the bipartite vacuolating cytotoxin A, VacA. The toxin is believed to enter host cells as two subunits: the p55 subunit (55 kDa) and the p33 subunit (33 kDa). At the biochemical level, it has been shown that VacA forms through the assembly of large multimeric pores composed of both the p33 subunit and the p55 subunit in biological membranes. One of the major target organelles of VacA is the mitochondria. Since only the p33 subunit has been reported to be translocated into mitochondria and the p55 subunit is not imported, it has been contentious as to whether VacA assembles into pores in a mitochondrial membrane. Here we show the p55 protein is imported into the mitochondria along with the p33 protein subunit. The p33 subunit integrally associates with the mitochondrial inner membrane, and both the p33 subunit and the p55 subunit are exposed to the mitochondrial intermembrane space. Their colocalization suggests that they could reassemble and form a pore in the inner mitochondrial membrane.     

Hispolon promotes MDM2 downregulation through chaperone-mediated autophagy

Amplification and overexpression of murine double minute (MDM2) has been observed in several human cancers. Some chemotherapeutic agents cause MDM2 ubiquitination and degradation in a proteasome-dependent system. In addition to the proteasome system, chaperone-mediated autophagy (CMA) is a lysosomal pathway for selective misfolded protein degradation. Molecular chaperone heat shock cognate 70 protein (Hsc70) recognizes the misfolded proteins, which are then delivered to lysosome-associated membrane protein type 2A (LAMP2A) for lysosomal degradation. Our previous study reported that hispolon was able to induce cell apoptosis and downregulate MDM2 expression. In this study, our results showed that the proteasome inhibitor, MG132, could not inhibit hispolon-induced MDM2 downregulation. In contrast, both inhibition of lysosomes with NH₄Cl and inhibition of LAMP2A using siRNA partially attenuated hispolon-induced MDM2 downregulation. To determine whether Hsc70 recognizes MDM2 on amino acids 135-141, SMP14 antibody was used to compete with Hsc70 for interaction with MDM2. After Hsc70 knockdown, SMP14 antibody immunoprecipitated increased MDM2. We also found that hispolon induced increased association of Hsp70, Hsc70, Hsp90 and LAMP2A with MDM2. This association was inhibited in cells pretreated with geldanamycin (GA), an Hsp90 inhibitor. GA also attenuated hispolon-induced MDM2 downregulation. Meanwhile, inhibition of Hsc70 using siRNA attenuated hispolon-induced MDM2 downregulation. Our study provides the first example of the ability of hispolon to mediate MDM2 downregulation in lysosomes through the CMA pathway.     

The tRNA aminoacylation co-factor Arc1p is excluded from the nucleus by an Xpo1p-dependent mechanism

Arc1p, a yeast tRNA-binding protein, forms a complex with the aminoacyl-tRNA synthetases, methionyl tRNA synthetase (MetRS) and glutamyl tRNA synthetase (GluRS). Although this complex localizes normally in the cytoplasm, in the absence of Arc1p the two free synthetases are also found inside the nucleus. In this work, in order to localize free Arc1 we abolished complex assembly by deleting the appended domains from both MetRS and GluRS. Surprisingly, free Arc1p remained cytoplasmic even when fitted with a strong nuclear localization signal (NLS). However, NLS-Arc1p accumulated in the nucleus when Xpo1/Crm1, the export receptor for NES-containing cargo proteins, was mutated. Thus, the cytoplasmic location of Arc1p is maintained by Xpo1p-dependent nuclear export and Arc1p could act as an adapter in the nucleocytoplasmic trafficking of tRNA and/or the tRNA-aminoacylation machinery.     

p73 and MDM2 confer the resistance of epidermoid carcinoma to cisplatin by blocking p53

p73 responds to DNA damage and exerts its pro-apoptotic function. However, p73 might contribute to the development of drug-resistance in certain tumor cells. In this study, we found that p73 and MDM2 correlate with cisplatin-resistant phenotype of human epidermoid carcinoma-derived cells. p73 and MDM2 were kept at low levels in the cisplatin-sensitive KB-3-1 cells, whereas p53 was induced to be phosphorylated at Ser-15 in response to cisplatin. In contrast, p73 and MDM2 were expressed at higher levels, and cisplatin-mediated p53 phosphorylation was undetectable in the cisplatin-resistant KCP-4 cells. Enforced expression of p73 in KB-3-1 cells caused an accumulation of unphosphorylated form of p53 and MDM2, and conferred the cisplatin resistance. Collectively, our results suggest that a loss of the cisplatin sensitivity is at least in part due to a lack of cisplatin-induced p53 phosphorylation, and p73 might cooperate with MDM2 to be involved in this process.