AKT levels were increased following ATO treatment

we showed that, as well as Csn5, CK2 also associated with topoII in a reaction to AR42. Hence, we hypothesized that phosphorylation of topoII by CK2 caused the organization of topoII with the Csn5 Fbw7 complex in AR42 treated cells. in support of this theory are shown in Fig. 6C, where in fact the CK2 inhibitor DMAT abrogated the interaction of topoII with Fbw7 and Csn5. Exposure AG-1478 of PLC5 cells to AR42 induced a concentration dependent increase in topoII phosphorylation, followed by parallel increases in its relationship with Fbw7 and Csn5, culminating in topoII proteolysis. However, pharmacological inhibition of CK2 by DMAT avoided increases above basal levels of its consequent association and AR42 induced topoII phosphorylation with Fbw7 and Csn5, thereby protecting topoII from drug induced deterioration. Glycogen synthase kinase Mitochondrion 3B dependent binding of topoII to Fbw7 through a recognition motif at the C terminus Fbw7 recognizes the Cdc4 phosphodegron motif of PXX in several of its target proteins, including cyclin E, Myc, Jun, SV40 large T antigen, and the sterol regulatory element binding protein. In this CPD motif, phosphorylation at the Thr residue by GSK3B together with that at the Ser residue by a priming kinase is required for binding. Investigation of the topoII sequence unveiled two possible Fbw7 recognition motifs, 1361SPKLS1365 and 1393SPPAT1397 in the C terminal domain. It's especially significant that the former motif encompasses a well-characterized GSK3B phosphorylation motif and overlaps with a putative CK2 recognition website 1365SNKE1368, suggesting that CK2 may be the kinase for GSK3B mediated phosphorylation of topoII. The involvement of GSK3B in AR42 mediated degradation was corroborated by many lines of evidence. First, pharmacological inhibition of GSK3B by SB 216763 protected cells against the suppressive effect of AR42 on phrase. 2nd, co immunoprecipitation suggests that AR42 generated a concentration dependent increase in the organization of topoII with GSK3B. Third, ectopic GSK3B expression canagliflozin resembled serving dependently the results of AR42 to the levels of topoII expression and phosphorylation, and its relationship with Fbw7. The involvement of the 1361SPKLSNKE1368 pattern in controlling topoII protein stability through relationships with Fbw7, GSK3B and CK2 was supported by mutational analyses. Banner labeled topoII mutants were created by changing the Ser1361, Ser1365, Glu1368, Ser1393, or Thr1397 residue with Ala via site directed mutagenesis, and then expressed in PLC5 cells in the presence or absence of ectopically expressed CK2. Ectopic CK2 expression was used to copy HDAC inhibitor induced CK2 up-regulation and accompanying topoII degradation since treatment with AR42 and other HDAC inhibitors induced the expression of the transfected Flag topoII, possibly through the epigenetic activation of transcription.