Glycogen Synthase Kinase 3β Interacts with and Phosphorylates the Spindle-associated Protein Astrin

Abstract

Emerging evidence shows that glycogen synthase kinase 3␤ (GSK3␤) is involved in mitotic division and that inhibiting of GSK3␤ kinase activity causes defects in spindle microtubule length and chromosome alignment. However, the purpose of GSK3␤ involvement in spindle microtubule assembly and accurate chromosome segregation remains obscure. Here, we report that GSK3␤ interacts with the spindle-associated protein Astrin both in vitro and in vivo. Additionally, Astrin acts as a substrate for GSK3␤ 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␤ 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␤. Conversely, depletion of Astrin by small interfering RNA has no detectable influence on the localization of GSK3␤. Interestingly, in vitro assays demonstrated that Astrin enhances GSK3␤-mediated phosphorylation of other substrates. Moreover, we showed that coexpression of Astrin and GSK3␤ differentially increases GSK3␤-mediated Tau phosphorylation on an unprimed site. Collectively, these data indicate that GSK3␤ interacts with and phosphorylates the spindle-associated protein Astrin, resulting in targeting Astrin to the spindle microtubules and kinetochores. In turn, the GSK3␤-Astrin complex may also facilitate further physiological and pathological phosphorylation.Glycogen synthase kinase 3 (GSK3), 2 a serine/threonine kinase active in several signaling pathways, is involved in the regulation of cell fate, including Wnt and Hedgehog signal transduction, protein synthesis, glycogen metabolism, mitosis and apoptosis (1-4). GSK3 has two structurally similar isoforms in mammals, GSK3␣ and GSK3␤, that are ubiquitously expressed and differ in their N-and C-terminal regions (2, 5). Earlier reports indicated that not only are the developmental profiles of GSK3␣ and GSK3␤ expression different but also the regulation and functions of these two proteins are not always identical (6 -9). Factors known to influence the functions of GSK3␤ include the phosphorylation of GSK3␤ itself, the subcellular localization of GSK3␤, the protein-protein interaction of GSK3␤, and the phosphorylation state of GSK3␤ substrates (1, 3, 4, 10). Insulin-mediated inhibition of GSK3 was mediated through a phosphorylation-dependent mechanism, with the phosphorylation at position Ser-21 and Ser-9 in GSK3␣ and GSK3␤, respectively (11).GSK3␤ also has a preference for pre-phosphorylated substrates, recognizing the consensus sequence (S/T)XXX(S/T)-p. In this sequence the first S/T residue is the target for GSK3␤ phosphorylation, X is any amino acid, P denotes the phosphorylatable residue, and the S/T located at the C terminus is the phosphorylation priming site (12). On the other hand, a number of proteins, including Axin and Tau, are phosphorylated by GSK3␤ without pre-phosphorylation. Recombinant Tau...

Astrin regulates meiotic spindle organization, spindle pole tethering and cell cycle progression in mouse oocytes

“…33 GSK3β interacts with and phosphorylates Astrin by the coiled-coil domain to target Astrin to the spindle microtubules and kinetochores. 11 Therefore, the C-terminal coiled-coil domain is able to compete effectively with endogenous Astrin protein for binding to docking proteins, and has a dominant effect on spindle organization; such a mechanism is similar to Astrin depletion.…”

“…9,10 Phosphorylation of Astrin by phosphorylated by p34cdc2 kinase has been determined in vitro, 7 to serve as a substrate for GSK3β and it is specifically phosphorylated at Thr-111, Thr-937 (S/TP motif ) and Ser-974/Thr-978 (S/T-XXX-S/T motif ), suggesting that its association with spindle microtubules may be regulated by phosphorylation. 11 Astrin also interacts with Aurora-A and regulates its localization in mitotic spindles. 10 All of the previous studies on Astrin had mainly been focused on human mitotic cells, but its role in mammalian meiosis is not known.…”

Phosphorylation of Astrin Regulates Its Kinetochore Function

“…Astrin is known to be phosphorylated at multiple sites (33,34), it is unclear whether Astrin phosphorylation by mitotic kinases regulates its KT targeting. This uncertainty prompted us to research other phosphorylation sites, the mitotic kinase(s) responsible for Astrin targeting to KTs, and the resulting KT structural ensemble.…”

“…Several kinases also regulate the function of Astrin. Glycogen synthase kinase 3␤ (Gsk3␤) phosphorylates Astrin at Thr-111, Thr-937, Ser-974, and Thr-978 to regulate its spindle-forming ability but has no effect on localization (33). Aurora A regulates separase activity and the interaction of Astrin with Kinastrin/Skap and Clasp1␣ by phosphorylating Astrin at Ser-115 to promote mitotic progression (34).…”

The mitosis-regulating and protein-protein interaction activities of Astrin are controlled by Aurora-A-induced phosphorylation

“…Astrin is diffused in the cytoplasm, with some concentrated spots near centrosomes in the interphase (23), and it is then translocated to kinetochores, spindle poles, and subsequently midbodies in mitosis (8,20,23), revealing Astrin as a chromosome passenger (1). The spindle pole or kinetochore targeting of Astrin is regulated by glycogen synthase kinase-3␤ (GSK-3␤) (11), hNinein (10), and Aurora-B (29) and through the interaction with Src kinase-associated phosphoprotein (SKAP) (14). Knockdown of Astrin by siRNA results in the malformation of the spindle, leading to mitosis arrest and subsequent apoptosis (20).…”