| The cell cycle occurs during cell proliferation and is a very common and important life process.Eukaryotic cells,have a distinct cell cycle,which is divided into the G1 phase for the first cell growth phase,the S phase for DNA and protein synthesis,the G2 phase for cell regrowth,and the M phase for cell division.In eukaryotes,cell cycle regulation relies on cell cycle checkpoints,the core components of which are cyclins and cyclin dependent kinases（CDK）.Organisms with a distinct cell cycle are found not only in eukaryotes but also in archaea.Archaea belong to the third domain of life and have a closer relationship with eukaryotes.The cell cycle of archaea is best studied in Sulfolobus,but the details of its regulation are still unclear.The genome of Sulfolobus also encdes a variety of protein kinases,which are involved in the regulation of various intracellular activities,but whether these protein kinases are involved in cell cycle regulation has not been reported.In this research,the S.islandicus REY15A was chosen to investigate the role of protein kinases in cell cycle regulation.The main experimental results in this study are as follows.1.There are nine protein kinase in S.islandicus.Based on the analysis of transcriptomic data during a cell cycle of synchronized S.islandicus E233S cells,it was determined that SisePK2 was the only protein kinase with apparent periodic changes with transcription of sisePK2 reaching the highest transcript level in the G2 phase.Subsequently,the transcriptomic results were confirmed by RT-qPCR..In addition,the protein levels at different time points in the cell cycle was examined by Western blotting.The amount of SisePK2 protein was quantified and its protein level reached its highest after 5 h after release of the synchronized cells.The above results suggest that SisePK2 may be involved in the regulation of cell cycle.2.To further investigate the role of SisePK2 in cell cycle regulation,ΔePK2 strain was constructed..There was no difference between ΔePK2 and the control E233S strain in terms of growth curve and cell morphology,and the deletion did not cause changes in cell division protein expression.However,cell cycle synchronization experiments by acetic acid treatment revealed that ΔePK2 could not be exhibits as a distinct peak in monochromosome-containing cells after synchronization,suggesting that SisePK2 affected the cell cycle progression.3.SisePK2 overexpression strains Sis/pSeSD-ePK2 and SisePK2 kinase active site Sis/pSeSD-ePK2-K393A,Sis/pSeSD-ePK2D498A,Sis/pSeSD-ePK2D532A,Sis/pSeSD-ePK2K393AD498A and Sis/pSeSD-ePK2K393AD498AD532A were constructed,growth and microscopy analysis revealed that when SisePK2 was overexpressed,the cellsappeared to grow slower and larger,and the cell diameter in Sis/pSeSD-ePK2 was close to twice as the normal cell diameter,which indicated that SisePK2 overexpression would inhibit the normal cell division process proceeded.Flow cytometry analysis showed an increase in intracellular DNA content in Sis/pSeSD-ePK2,indicating that SisePK2 overexpression did not affect intracellular DNA synthesis,and that the cell cycle was stalled at G2 phase after cell cycle synchronization in Sis/pSeSD-ePK2.This suggests that SisePK2 overexpression affects the G2 phase after the S phase.Overexpression of SisePK2 with mutated kinase active site,cell size was largely restored,but cell growth remained slow.Based on the structural domain composition of SisePK2,six truncated mutants were constructed for the TPR motifs and the kinase structural domain on SisePK2,but none of these truncated mutants had a distinct phenotype,and it is assumed that the in vivo function of SisePK2 is dependent on the intact structure.4.SisePK2 with a C-terminal His tag was purified from S.islandicus,and massspectrometry was performed on SisePK2 and the co-purified proteins.SisPrmA,which it the subunit of proteasome,was indentified.SisPrmA has been documented to affect the cell cycle by degrading cell division proteins in S.acidocaldarius.Furthermore,when the mimic-phosphorylated SisPrmA protein was overexpressed in S.islandicus,cell growth was slowed down,suggesting that SisPrmA may be one of the phosphorylated substrates for the regulation of cell cycle by SisePK2.5.To further investigate the phosphorylation substrates of SisePK2 affecting the cell cycle,the phosphorylation levels of E233S,ΔePK2,and Sis/pSeSD-ePK2 were examined with samples taken at 0.5 h,1.5 h,and 2.5 h after synchronization,and there were significant differences in protein phosphorylation levels.The proteins with phosphorylation differences were identified by phosphorylation mass spectrometry,and two transcription factors,SiRe0263 and SiRe2067,showed obvious differences in phosphorylation.It was speculated to be potential phosphorylation substrates of SisePK2,but their specific regulatory roles still need to be further explored.In conclusion,this study identified a kinase,SisePK2,which is involved in cell cycle regulation in archaea,and determined the phase of its function in the cell cycle.The study also identified its potential substrate,SisPrmA,and two transcription factors,SiRe0263 and SiRe2076,as its possible substrates which may be involved in cell cycle regulation.This study has laid a foundation for further characterization of the regulation of protein kinases in archaea. |
