Pyruvate kinase M2 isoform (PKM2) catalyzes the last step of glycolysis and plays an important role in tumor cell proliferation. Recent studies have reported that PKM2 also regulates apoptosis. However, the mechanisms underlying such a role of PKM2 remain elusive. Here we show that PKM2 translocates to mitochondria under oxidative stress. In the mitochondria, PKM2 interacts with and phosphorylates Bcl2 at threonine (T) 69. This phosphorylation prevents the binding of Cul3-based E3 ligase to Bcl2 and subsequent degradation of Bcl2. A chaperone protein, HSP90α1, is required for this function of PKM2. HSP90α1's ATPase activity launches a conformational change of PKM2 and facilitates interaction between PKM2 and Bcl2. Replacement of wild-type Bcl2 with phosphorylation-deficient Bcl2 T69A mutant sensitizes glioma cells to oxidative stress-induced apoptosis and impairs brain tumor formation in an orthotopic xenograft model. Notably, a peptide that is composed of the amino acid residues from 389 to 405 of PKM2, through which PKM2 binds to Bcl2, disrupts PKM2-Bcl2 interaction, promotes Bcl2 degradation and impairs brain tumor growth. In addition, levels of Bcl2 T69 phosphorylation, conformation-altered PKM2 and Bcl2 protein correlate with one another in specimens of human glioblastoma patients. Moreover, levels of Bcl2 T69 phosphorylation and conformation-altered PKM2 correlate with both grades and prognosis of glioma malignancy. Our findings uncover a novel mechanism through which mitochondrial PKM2 phosphorylates Bcl2 and inhibits apoptosis directly, highlight the essential role of PKM2 in ROS adaptation of cancer cells, and implicate HSP90-PKM2-Bcl2 axis as a potential target for therapeutic intervention in glioblastoma.

Indirect evidence has implicated an interaction between the cytoskeleton and the protein synthetic machinery. Two recent reports have linked the elongation factor 1a (EF-1a) which is involved in protein synthesis, with the microtubular cytoskeleton. In situ hybridization has, however, revealed that the messages for certain cytoskeletal proteins are preferentially associated with actin filaments. ABP-50 is an abundant actin filament bundling protein of native relative molecular mass 50,000 (50K) isolated from Dictyostelium discoideum. Immunofluorescence studies show that ABP-50 is present in filopodia and other cortical regions that contain actin filament bundles. In addition, ABP-50 binds to monomeric actin in the cytosol of unstimulated cells and the association of ABP-50 with the actin cytoskeleton is regulated during chemotaxis. Through complementary DNA sequencing and subsequent functional analysis, we have identified ABP-50 as D. discoideum EF-1a. The ability of EF-1a to bind reversibly to the actin cytoskeleton upon stimulation could provide a mechanism for spatially and temporally regulated protein synthesis in eukaryotic cells.

ABP-50 is the elongation factor-] alpha (EF-1 alpha) of Dictyostelium discoideum (Yang et al.: Nature 347:494-496, 1990). ABP-50 is also an actin filament binding and bundling protein (Demma et al.: J . Biol. Chem. 265:2286-2291. In the present study we have investigated the compartmentalization of ABP-50 in both resting and stimulated cells. Immunofluorescence microscopy shows that in addition to being colocalized with F-actin in surface extensions in unstimulated cells, ABP-50 exhibits a diffuse distribution throughout the cytosol. Upon addition of CAMP, a chemoattractant, ABP-50 becomes localized in the filopodia that are extended as a response to stimulation. Quantification of ABP-50 in Triton-insoluble and -soluble fractions of resting cells indicates that 10% of the total ABP-50 is recovered in the Triton cytoskeleton, while the remainder is in the soluble cytosolic fraction. Stimulation with cAMP increases the incorporation of ABPJO into the Triton cytoskeleton. The peak of incorporation of ABP-50 at 90 sec is concomitant with filopod extension. Immunoprecipitation of the cytosolic ABP-50 from unstimulated cells using affinity-purified polyclonal anti ABP-50 results in the coprecipitation of non-filamentous actin with ABP-50. Purified ABP-50 binds to G-actin with a Kd of approximately 0.09 p M . The interaction between ABP-50 and G-actin is inhibited by GTP but not by GDP, while the bundling of F-actin by ABP-50 is unaffected by guanine nucleotides. We conclude that a significant amount of ABP-50 is bound to either G-or F-actin in vivo and that the interaction between ABP-50 and F-actin in the cytoskeleton is regulated by chemotactic stimulation.

O‐GlcNAcylation catalysed by O‐GlcNAc transferase (OGT) is a reversible post‐translational modification. O‐GlcNAcylation participates in transcription, epigenetic regulation, and intracellular signalling. Dysregulation of O‐GlcNAcylation in response to high glucose or OGT expression has been implicated in metabolic diseases and cancer. However, the underlying mechanisms by which OGT regulates hepatoma development remain largely unknown. Here, we employed the lentiviral shRNA‐based system to knockdown OGT to analyse the contribution of OGT in hepatoma cell proliferation and stem‐like cell potential. The sphere‐forming assay and western blot analysis of stem‐related gene expression were used to evaluate stem‐like cell potential of hepatoma cell. We found that the level of total O‐GlcNAcylation or OGT protein was increased in hepatocellular carcinoma. OGT activated stem‐like cell potential in hepatoma through eukaryotic initiation factor 4E (eIF4E) which bound to stem‐related gene Sox2 5'‐untranslated region. O‐GlcNAcylation of eIF4E at threonine 168 and threonine 177 protected it from degradation through proteasome pathway. Expression of eIF4E in hepatoma was determined by immunostaining in 232 HCC patients, and Kaplan‐Meier survival analysis was used to determine the correlation of eIF4E expression with prognosis. High glucose promoted stem‐like cell potential of hepatoma cell through OGT‐eIF4E axis. Collectively, our findings indicate that OGT promotes the stem‐like cell potential of hepatoma cell through O‐GlcNAcylation of eIF4E. These results provide a mechanism of HCC development and a cue between the pathogenesis of HCC and high glucose condition.

dSelK (G-rich), a homolog of human and mouse SelK, is one of three selenoproteins in Drosophila melanogaster. It is the only trans-membrane selenoprotein in D. melanogaster integrated into both the endoplasmic reticulum (ER) membrane and the Golgi apparatus. The gene expression profile of Drosophila Schneider 2 (S2) cells after the dsRNA interference (dsRNAi) targeting of dSelK was examined with the GeneChip Drosophila Genome 2.0 Array (Affymetrix), a high-density oligonucleotide microarray encompassing nearly the full Drosophila genome. The results showed that the transcriptional expression of eight genes whose proteins are located on (or related to) the ER or the Golgi apparatus was highly induced or repressed by the dsRNAi treatment. The mRNA levels of the inositol 1,4,5-tris-phosphate receptor (IP3 receptor), whose gene product is integrated into the ER membrane and regulates the release of Ca2+ from the ER to the cytosol, were significantly downregulated. In contrast, the expression of inositol 1,4,5-tris-phosphate kinase 1, which is a cytosolic protein with opposing functions to the IP3 receptor, was significantly upregulated. Quantitative real-time PCR verified these results. The concentration of intracellular free Ca2+ of the Drosophila S2 cells was significantly decreased after the knockdown of dSelK, whereas overexpression of dSelK significantly increased the intracellular free Ca2+ concentration. These results indicate that dSelK in D. melanogaster is involved in regulating the release of Ca2+ from the ER to the cytosol and may play important roles in the signal transduction pathways involving Ca2+ mobilization.