gsk525762 mg Since the localization of LO depends on phospho
Since the localization of 5-LO depends on phosphorylation, we also analyzed the phosphorylation profile of 5-LO-WT and all isoforms by Western blot using specific gsk525762 mg against the phosphorylation sites S271 and S523. Whereas phosphorylation at S271 activates the 5-LO, inhibits nuclear export and leads to nuclear localization, phosphorylation of S523 prevents nuclear import and has an inhibitory effect on 5-LO activity [35–37]. It has been shown for NIH3T3 cells that 5-LO is strongly phosphorylated at S271 and that pharmacological inhibition of 5-LO phosphorylation at S271 or the S271 to Ala mutation leads to the cytosolic localization . In this context it should be mentioned that, to our knowledge, the cellular 5-LO localization has never been correlated with the phosphorylation status of 5-LO at S271 in primary leukocytes. For HEK293 cells, it has been reported that the phosphomimetic mutant GFP-5LO-S271E shows a cytosolic localization whereas GFP-5-LO-WT and the S271A mutant were in the nucleus . The authors concluded that cell stress and p38 MAPK activation induces nuclear export (not import) of 5-LO in HEK293 cells. This could explain why the heavily phosphorylated isoforms p12 and Δ13 are localized in the cytosol in HEK293 cells. However, in our hands, mutation of S271 to A in 5-LO-WT converts the nuclear to a mainly cytosolic localization. Furthermore, the Δ4 isoform is not phosphorylated at S271 but it is also located in the cytosol in HEK293T cells. Therefore, it is likely that structural alterations within the catalytic domains of the alternative isoforms play a significant role for their cytosolic localization.
Evaluation of the results showed that 5-LO-WT was only slightly phosphorylated at S271 and not phosphorylated at S523. This is in agreement with a previous publication and corresponds with our observation that 5-LO-WT is localized in the nucleus . The isoforms 5-LOp12 and 5-LO∆13 were prominently phosphorylated at S271 while 5-LO∆4 showed only a low phosphorylation status. 5-LOp12 is hyper-phosphorylated at S523 which explains its cytosolic localization, whereas 5-LO∆13 and 5-LO∆4 are virtually not phosphorylated at S523. For 5-LO∆4, the missing NLS around K158 might be the reason for its cytosolic localization. For 5-LO∆13, Allain et al. already published that the isoform is hyper-phosphorylated at S523 which is in contrast to our results . One possible explanation would be the influence of geneticin which was used for the generation of stable transfectants in , whereas the transfectants in this study were generated by stable integration into the genome by transposase. The continuous geneticin-mediated cell stress might lead to an upregulation of 5-LO kinases in the cells. Another explanation could be that the previous study was performed with untagged 5-LOΔ13 whereas we used mCherry tagged 5-LOΔ13. Furthermore, it should be considered that Allain et al. used cells which were cotransfected with FLAP whereas this was not the case here. Interestingly, 5-LOp12 is strongly phosphorylated at both phosphorylation sites. The amino acid S523 is in very close proximity to the amino acids of 5-LOp12 which are removed by alternative splicing (530–553). As the phosphorylation site S523 is still accessible for PKA, the deletion of these amino acids in the isoform does not seem to cause misfolding of the protein in this area.
Introduction The incidence of cerebrovascular diseases has been increased rapidly nowadays and such diseases are becoming a larger burden for patients, families or even the entire society. Numerous diseases, such as cerebral arteriosclerosis, Alzheimer's disease and Parkinson's syndrome, are closely related to ischemic injury, free radical metabolism disorders, cell damage and apoptosis (Lam et al., 2016, Rani et al., 2016). At present, research of various types of oxidation and free radical resistance drugs has become a popular topic (Raina and Sen, 2017).