The Effects And Mechanism Of The H3T3A Mutant On The H3T3 Phosphorylation

Histone phosphorylation is one of the most important histone modificationmanners.HistoneH3T3 phosphorylation is a landmark event of mitosis,and the phosphorylated H3T3 canrecruit the CPC complex to the centromere,then the CPC complex can phosphorylate a series of protein substrate s,thus regulating the chromosome separation,spindle assembly and maintenance,cytokinesis and so on.Haspin is an enzyme that catalyzes the phosphorylation of H3T3.As a constitutiveenzyme,Haspin is inactive during interphase.However,duringmitotic phase,CDK1,PLK1 and Aurora B kinase can phosphorylate multiple sites atHaspinN-terminus,thus activatating Haspin.Repo-Man/PP1?complexdephosphorylatesH3T3.Repo-Man,a chromosome scaffolding protein,can dephosphorylate H3T3 by forming a complex with PP1?.Thus in prometaphase,the H3T3phosphorylation is charged by Haspin and Repo-Man/PP1?with antistatic function oneach other,making phosphorylation of H3T3occuring only on the centromere.Previous study showed that the phosphorylation of H3T3 was significantly reducedin the H3T3A transgenic fruit flies,compared with wild-type H3 transgenic fruit flies,but the mechanism is unclear.O ur previous results showedthat the reaction rate was relatively slow at the initial stage when the catalytic domain of Haspin was incubated with the polynucleosome,and once the reaction began,the reaction rate did not change with the increase of the enzyme concentration.The reaction rate was only related to the speed of Haspinmovement on po lynucleosome.However,When the catalytic domain of Haspin was incubated with H3 monomer,the kinetics of this reaction was fit to Michaelis-Menten equation.With the increase of the enzyme concentration or the substrate concentration,the reaction rate was increased.Thus,we speculated that Haspin maycatalyze the phosphorylation of H3T3 in a processive manner.In a processive mechanism,when the enzyme binds to the substrate which has multiple catalytic sites,the enzymes is always bound to the subtstrate until all possible sites in substrate have been catalyzed.We used Time course method and Start-Trap strategy to verify the catalytic mechanism of Haspin.Time course experimentsshowed that when two forms of Haspin(MBP-Haspin-HBIS-Deletion,His₆-Haspin_492-798)were incatated with three forms of H3 substrates?H3 monomer,chicken polynucleosome,reconstituted ploynucleosome?,with the increase of the enzyme concentration,the catalytic reaction rate was increased.This result does not support the processive cytalytic model.The results of the Start-Trap experiment showed that when the inhibitor H3?2-25?peptide was added in the middle of the reaction,the addition of inhibitors inhibitedthe rate and extent of the reaction,indicating that the inhibitor would bind to the free Haspin kinase and terminate the reaction.So we think that Haspin may not be a processive kinase.Basedonthese data,we further discussedthe effect and mechanism on the H3T3 phosphorylation when the H3T3A mutants are incorparated into the normal chromosome.Due to big differences between the in vitro kinase reaction and the intracellular environment,the kinetic data in vitro and the catalytic mechanism of Haspin in cellular environment may have much different.Therefore,we constructed H3 and H3T3A-induced stable cell lines of HEK293T and He La cells using Tet-on system.By using Western Blot analysis,we found thatwhen the H3T3A mutatntwasincorporated into the normalchromosomes,the endogenous H3T3ph,H3S10ph wasdramatilcally decreased,the phosphorylation of Haspin was also decreased.The phosphorylation of H3T3ph and Haspin was restored after treatment with PP1 and PP2A phosphorylase inhibitors M-LR and Okadaic acid.However PP2A inhibitor LB-100 couldnot restorethe phosphorylation of H3T3 and Haspin.Based on these data,we hypothesize that H3T3A may promotethe activity of PP1 by anunknown mechanism,subsequently causing the dephosphorylation of Haspinand Aurora B,inhibiting the H3T3 phosphorylation pathway and promoting the H3T3 dephosphorylation pathway,fianllyleading to a dramatic reduction of H3T3 phosphorylation level.