Glycogen synthase kinase 3β interacts with and phosphorylates the spindle-associated protein astrin

Emerging evidence shows that glycogen synthase kinase 3 beta (GSK3 beta) is involved in mitotic division and that inhibiting of GSK3 beta kinase activity causes defects in spindle microtubule length and chromosome alignment. However, the purpose of GSK3 beta involvement in spindle microtubule assembly and accurate chromosome segregation remains obscure. Here, we report that GSK3 beta interacts with the spindle- associated protein Astrin both in vitro and in vivo. Additionally, Astrin acts as a substrate for GSK3 beta and is phosphorylated at Thr- 111, Thr- 937 ((S/ T) P motif) and Ser- 974/ Thr-978 ((S/ T) XXX(S/T)- p motif; p is a phosphorylatable residue). Inhibition of GSK3 beta impairs spindle and kinetochore accumulation of Astrin and spindle formation at mitosis, suggesting that Astrin association with the spindle microtubule and kinetochore may be dependent on phosphorylation by GSK3 beta. Conversely, depletion of Astrin by small interfering RNA has no detectable influence on the localization of GSK3 beta. Interestingly, in vitro assays demonstrated that Astrin enhances GSK3 beta- mediated phosphorylation of other substrates. Moreover, we showed that coexpression of Astrin and GSK3 beta differentially increases GSK3 beta- mediated Tau phosphorylation on an unprimed site. Collectively, these data indicate that GSK3 beta interacts with and phosphorylates the spindle- associated protein Astrin, resulting in targeting Astrin to the spindle microtubules and kinetochores. In turn, the GSK3 beta-Astrin complex may facilitate further physiological and pathological phosphorylation.