Terminal prostanoid synthases show distinct functional coupling with upstream COX isoxymes

ucleotides. As shown in of endogenous survivin with a notable decrease in cells with 2N DNA content, and a concomittant increase in cells with 4N content. In addition, the abundance of cells with a DNA content >4N increased dramatically from 2 to 17%. By contrast, cells exhibiting restored proliferation by expression of the siRNA resistant form of wildtype survivin, showed little difference in FACS profile, whether transfected with control or survivin-specific siRNA. Similarly to the siRNA sensitive control cells, in the absence of endogenous survivin, cells expressing T48A or T48E exhibited a decreased G1 population, an increased G2 & M population, and an elevation in the number of cells with >4N DNA content, as compared with their profiles in the PF-562271 site presence of control siRNA. Collectively these data demonstrate that neither T48A nor T48E can substitute for wildtype survivin to support cell proliferation. Localization of survivin-GFP in the absence of CK2 phosphorylation. Next we looked at the interphase localization of T48A and T48E mutants in our stable HeLa cell lines. In the presence of endogenous survivin both forms were cytoplasmic,; however, upon siRNA depletion they relocated to the nucleus where they formed discrete subnuclear foci. Given this relocation we wondered whether the distribution of survivin-GFP in the absence of CK2 activity would phenocopy this behavior. To this end HeLa cells expressing GFP or survivin-GFP were treated with the CK2 inhibitor 4,5,6,7-tetrabromobenzotriazole for 6 h. As with the T48 mutants TBB treatment did not affect the mitotic localization of survivin-GFP in HeLa cells, but did induce nuclear accumulation and focus formation in interphase cells. By contrast the distribution of the GFP control was unaffected by TBB. As inhibition of CK2 activity has been shown to induce G2 arrest, we were concerned that the nuclear foci were simply a manifestation of G2 arrest, thus we examined the cell cycle distribution of untreated control and 100 M TBBtreated cells by DNA-FACS-profiling. However, as indicated in 540 Cell Cycle Volume 10 Issue 3 Survivin T48 mutants display altered affinities for survivin and borealin in vitro. As survivin can self-associate and is an integral component of the chromosomal passenger complex during mitosis we next asked whether mutation of T48 compromised its interaction with itself or any other CPP. To this end recombinant GST, GST-survivin and GST-T48 survivin variants www.landesbioscience.com Cell Cycle 541 were purified and incubated on glutathione beads in the presence of in vitro translated 35S-methionine labelled CPPs, PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19822663 in a series of in vitro pull down assays. Using this method both T48 mutants interacted with all CPCs examined. Most noteably, after normalizing band intensities of IVT-CPC abundance with GST-protein loadings in each sample, borealin was found to have a three-fold increase in affinity for the mutant forms, compared with the wild-type protein. These data suggest that mutation of T48 enhances the affinity of survivin for borealin. Mutation of T97, restores the proliferative potential of T48 mutants. Prior to determining that T48 was the sole phosphorylation target of CK2 in survivin we analyzed the activity of T97 mutants, see reference 45, and double mutants, T48AT97A and T48ET97E. Intriguingly, this second mutation restored proliferation of T48 mutants in our siRNA assay. By microscopy, we observed that both double mutants localized to the centromeres, midzon