phosphothreonine and Glioma

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.

Topics: Amino Acid Sequence; Apoptosis; Carcinogenesis; Cell Line, Tumor; Cullin Proteins; Glioma; HSP90 Heat-Shock Proteins; Humans; Hydrogen Peroxide; Mitochondria; Oxidative Stress; Phosphorylation; Phosphothreonine; Prognosis; Protein Binding; Protein Stability; Protein Transport; Proteolysis; Proto-Oncogene Proteins c-bcl-2; Pyruvate Kinase; Ubiquitin-Protein Ligases

Apoptin, a protein of the chicken anemia virus (CAV), consists of 121 amino acids (aa) and represents a novel, potentially tumor-specific therapeutic and diagnostic agent. The C-terminal part of Apoptin (aa 81-121) is believed to contain a bipartite nuclear localization signal (NLS) (NLS1: aa 82-88 and NLS2: aa 111-121), which is only active in tumor cells after phosphorylation of threonine(108) by tumor-specific cytoplasmic phosphokinases. Furthermore, a nuclear export signal (NES) (aa 97-105) seems to enable nuclear export of Apoptin only in healthy cells. The specificity for tumor cell nuclei also applies to the truncated C-terminal part of Apoptin (aa 81-121), which therefore represents a highly attractive peptide sequence for peptide synthesis. Here we describe for the first time the synthesis of fluorescein isothiocyanate (FITC)- and Dansyl-labelled conjugates containing this C-terminal part of Apoptin, with either phosphorylated or nonphosphorylated threonine(108). The phosphorylated conjugates were synthesized in an attempt to achieve nuclear accumulation in healthy cells, which lack cytoplasmic tumor-specific phosphokinases. Surprisingly, all the conjugates accumulated rapidly within the cell nuclei of both tumor and non-tumor cells from the bladder, brain and prostate and led to cell death. By coupling Apoptin(81-121) to FITC and DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) at either the C- or N-terminus we could exlude that the coupling site is decisive for tumor cell-specific nuclear localization. The labels FITC, DOTA and Dansyl were not responsible for cell death in healthy cells because cell death was not prevented by using an unlabelled Apoptin(81-121) peptide. Cellular and nuclear uptake of the FITC-labelled Apoptin(81-121) peptide was almost completely abolished after altering the NLS2 (replacement of five arginines with serines).

Topics: Amino Acid Sequence; Apoptosis; Astrocytes; Brain; Capsid Proteins; Cell Line, Tumor; Cell Nucleus; Cells, Cultured; Dansyl Compounds; Flow Cytometry; Fluorescein-5-isothiocyanate; Glioma; Heterocyclic Compounds, 1-Ring; Humans; Male; Microscopy, Confocal; Microscopy, Fluorescence; Nuclear Export Signals; Nuclear Localization Signals; Peptide Fragments; Phosphothreonine; Prostate; Prostatic Neoplasms; Urinary Bladder; Urinary Bladder Neoplasms; Urothelium

PTEN is a tumor suppressor protein that dephosphorylates phosphatidylinositol 3,4,5 trisphosphate and antagonizes the phosphatidylinositol-3 kinase signaling pathway. We show here that PTEN can also inhibit cell migration through its C2 domain, independent of its lipid phosphatase activity. This activity depends on the protein phosphatase activity of PTEN and on dephosphorylation at a single residue, threonine(383). The ability of PTEN to control cell migration through its C2 domain is likely to be an important feature of its tumor suppressor activity.

Topics: Animals; Catalysis; Catalytic Domain; Cell Line, Tumor; Cell Movement; Chlorocebus aethiops; COS Cells; Glioma; Humans; Mutation; Phosphoprotein Phosphatases; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphothreonine; Precipitin Tests; Protein Structure, Tertiary; PTEN Phosphohydrolase; Recombinant Proteins; Sequence Deletion; Transfection; Tumor Suppressor Proteins