RESUMEN
The cohesin complex holds sister chromatids together until all chromosomes are properly attached to the mitotic spindle. Cleavage of the cohesin subunit Rad21 at the metaphase to anaphase transition allows separation of sister chromatids and is fundamental for the creation of identical daughter cells. Sororin blocks removal of cohesin from chromosomes from S phase until mitosis. In mitosis, Sororin is phosphorylated by Cdk1 releasing it from the cohesin complex. Aurora B phosphorylation of Sororin may play a similar role as Cdk1. Using PhosTag electrophoresis, we detect multiple Sororin species suggesting that phosphorylation of Sororin in mitosis is heterogeneous. Mutating the Cdk1 consensus site S21 to alanine eliminates many of the phosphorylated species suggesting that S21 is a key site of phosphorylation in vivo. Inhibiting Aurora B reduces phosphorylation of Sororin in cells, but only if Cdk1 sites are intact suggesting that some phosphorylation events on Sororin may be sequential. Surprisingly, mutating Aurora B consensus sites in Sororin causes it to relocalize to the nucleolus during interphase and blocks its interaction with chromosomes in Aurora B-inhibited cells. These observations indicate that phosphorylation plays unexpected roles in regulating the subcellular localization of Sororin.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Aurora Quinasa B/fisiología , Proteínas de Ciclo Celular/metabolismo , Quinasas Ciclina-Dependientes/fisiología , Procesamiento Proteico-Postraduccional , Secuencia de Aminoácidos , Sitios de Unión , Proteína Quinasa CDC2 , Nucléolo Celular/metabolismo , Secuencia de Consenso , Células HeLa , Humanos , Datos de Secuencia Molecular , Fosforilación , Transporte de Proteínas , Especificidad por SustratoRESUMEN
The chromosomal passenger complex (CPC) senses tension defects at the kinetochore to activate the spindle assembly checkpoint, and helps to position the cleavage furrow. The CPC, consisting of INCENP, Survivin, Borealin and Aurora B localizes to the inner centromere at metaphase and re-localizes to the spindle midzone at anaphase; several CPC functions are regulated by post-translational modification. Borealin is phosphorylated at multiple sites and phosphorylation at S219 causes Borealin to migrate more slowly upon electrophoresis. Here we find that Cdk1 can induce a mobility shift of Borealin, suggesting that S219 phosphorylation is under Cdk1 control. However, Cdk1 is inefficient at phosphorylating purified Borealin in vitro. A yeast orthologue of Borealin, Npl1, is dephosphorylated by the phosphatase Cdc14. We find no difference in the mobility shift of Borealin in human cells lacking either Cdc14A or Cdc14B. In contrast, the phosphatase inhibitor okadaic acid does delay the dephosphorylation of Borealin as cells exit mitosis. The proteasome inhibitor MG132 reduces Borealin phosphorylation in mitosis and increases the steady-state level of Borealin, especially in mutants lacking the C-terminus. However, a second, structurally unrelated proteasome inhibitor, lactacystin did not up-regulate Borealin. These results suggest that the effect of MG132 on Borealin is due to the inhibition of an intracellular protease other than the proteasome.