phosphothreonine and Osteosarcoma

Gambogic acid (GA) has been wildly studied to show potent anti-tumor effects in vivo and in vitro. We have confirmed that GA stabilized and activated p53 through down-regulating the expression of MDM2 in variety of cancer cell lines. However, GA-induced p53 activation could be partially reversed by caffeine, a PI3k inhibitor. Therefore, questions of whether GA induces post-translational modifications of p53 and subsequent activation of p53; and if that is the case, which upstream signaling pathway(s) is (are) responsible for that are proposed. Here, the relationship between p53 activation and its post-translational modifications was investigated in the human cancer cell lines HepG2 and A549 in response to GA or adriamycin treatment. GA induces p53 phosphorylation at sites Ser15 and Ser20 in a concentration- or time-dependent way, which was a direct result of DNA damage, as gamma-HA2X foci and 'comet' DNA fragments were detected. GA induces p53 phosphorylation through activation of an ATM- and Rad3-related pathway, and GA-induced phosphorylation of Chk1 is also involved. Upon treatment with GA, ATR activation is clearly associated with p53 phosphorylation, as well as activation of its target gene p21(Waf/CIP1). Furthermore, we found the dephosphorylation of Cdk1 at Thr161 induced by GA was abrogated, followed by a remarkable disruption of G2/M arrest when the cells were pre-incubated with caffeine. Interestingly, the sensitivity to caffeine enhanced the cytotoxicity of GA as well. Taken together, these data showed an important role of the DNA damage response mediated by ATR-Chk1 in p53/p21(Waf/CIP1) activation and downstream G2/M arrest during GA treatment.

Topics: Androstadienes; Antineoplastic Agents; Bone Neoplasms; Caffeine; CDC2 Protein Kinase; Cell Cycle; Cell Line, Tumor; Comet Assay; Cyclin-Dependent Kinase Inhibitor p21; DNA Damage; Down-Regulation; Doxorubicin; Hep G2 Cells; Humans; Osteosarcoma; Phosphorylation; Phosphothreonine; Signal Transduction; Threonine; Tumor Suppressor Protein p53; Wortmannin; Xanthones

Polo-like kinases (Plks) contain a conserved Polo-box domain, shown to bind to phosphorylated Ser-pSer/pThr-Pro motifs. The Polo-box domain of Plk-1 mediates substrate interaction and plays an important role in subcellular localization. Intriguingly, the major interactions between the PBD and the optimal recognition peptide are mediated by highly conserved residues in the PBD, suggesting there is little target specificity conveyed by the various PBDs. However, here we show that the affinity of the purified Plk1-3 PBDs to both a physiological Cdc25C derived phospho-peptide and an optimal recognition phospho-peptide differs significantly among family members. To decipher the role of the PBDs and kinase domains in inferring Plk specificity, we exchanged the PBD of Plk1 (PBD1) with the PBD of Plk2, 3, or 4 (PBD2-4). The resulting hybrid proteins can restore bipolar spindle formation and centrosome maturation in Plk1-depleted U2OS cells to various degrees. In these experiments PBD2 was most efficient in complementing PBD-function. Using the MPM2 antibody that recognizes a large set of mitotic phospho-proteins, we could show that PBD1 and PBD2 display some limited overlap in target recognition. Thus, PBDs convey a significant deal of target specificity, indicating that there is only a limited amount of functional redundancy possible within the Plk family.

Topics: Bone Neoplasms; cdc25 Phosphatases; Cell Cycle Proteins; Centrosome; Humans; Mitosis; Osteosarcoma; Phosphopeptides; Phosphorylation; Phosphoserine; Phosphothreonine; Polo-Like Kinase 1; Protein Serine-Threonine Kinases; Protein Structure, Tertiary; Proto-Oncogene Proteins; Recombinant Proteins; Spindle Apparatus; Substrate Specificity; Surface Plasmon Resonance; Tumor Cells, Cultured; Tumor Suppressor Proteins

Expression of the B-Myb transcription factor is upregulated during late G1 phase of the cell cycle by an E2F-dependent transcriptional mechanism. B-Myb is specifically phosphorylated during S phase, suggesting that a cyclin-dependent kinase (Cdk) regulates its activity. Consistent with this notion, the S phase-specific cyclin A/Cdk2 was found previously to enhance B-Myb transactivation activity in cotransfected cells. In this study we provide evidence that B-Myb is a direct physiological target for cyclin A/Cdk2. We demonstrate that B-Myb is an in vitro substrate for cyclin A/Cdk2, but not for cyclin D1/Cdk4 or cyclin E/Cdk2. By mutating candidate Cdk2 phosphorylation sites, we show that B-Myb is phosphorylated at Thr447, Thr490, Thr497 and Ser581 by cyclin A/Cdk2 in vitro and that these sites are also phosphorylated in cycling U-2 OS cells. Inhibition of endogenous Cdk2 by dominant negative Cdk2 attenuated phosphorylation of Thr447, Thr490 and Thr497, but had no effect upon Ser581 modification. B-Myb transactivation activity was significantly reduced in a mutant containing amino acid substitutions at all four identified cyclin A/Cdk2 sites and was constitutively low in Saos-2 cells where endogenous cyclin A/Cdk2 activity was unable to phosphorylate ectopically expressed B-Myb. These data indicate that phosphorylation by cyclin A/Cdk2 is directly involved in enhancing B-Myb transactivation activity and that levels of endogenous cyclin A/Cdk2 activity may contribute to cell line-specific B-Myb function.

Topics: Bone Neoplasms; CDC2-CDC28 Kinases; Cell Cycle Proteins; Cyclin A; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinases; DNA-Binding Proteins; G1 Phase; Gene Expression Regulation; Humans; Osteosarcoma; Phosphorylation; Phosphoserine; Phosphothreonine; Protein Processing, Post-Translational; Protein Serine-Threonine Kinases; Recombinant Fusion Proteins; S Phase; Structure-Activity Relationship; Substrate Specificity; Trans-Activators; Transcriptional Activation; Transfection; Tumor Cells, Cultured