Interactomes of Glycogen Synthase Kinase‑3 Isoforms

Functional differentiation
of the two isoforms of the protein-serine/threonine
kinase, glycogen synthase kinase-3 (GSK-3), is an unsettled area of
research. The isoforms are highly similar in structure and are largely
redundant, though there is also evidence for specific roles. Identification
of isoform-specific protein interactors may elucidate the differences
in function and provide insight into isoform-selective regulation.
We therefore sought to identify novel GSK-3 interaction partners and
to examine differences in the interactomes of the two isoforms using
both affinity purification and proximity-dependent biotinylation (BioID)
mass spectrometry methods. While the interactomes of the two isomers
are highly similar in HEK293 cells, BioID in HeLa cells yielded a
variety of preys that are preferentially associated with one of the
two isoforms. DCP1B, which favored GSK-3α, and MISP, which favored
GSK-3β, were evaluated for reciprocal interactions. The differences
in interactions between isoforms may help in understanding the distinct
functions and regulation of the two isoforms as well as offer avenues
for the development of isoform-specific strategies.

丝氨酸/苏氨酸蛋白激酶糖原合成酶激酶3（glycogen synthase kinase-3, GSK-3）的两种同工型的功能分化，一直是尚未定论的研究领域。这两种同工型在结构上高度相似，且在很大程度上功能冗余，但亦有证据表明二者各自存在特异性功能。鉴定同工型特异性的蛋白质相互作用因子，或可阐明二者在功能上的差异，并为理解同工型选择性调控机制提供线索。因此，本研究旨在鉴定新型的GSK-3相互作用伴侣，并通过亲和纯化与邻近依赖生物素标记（BioID）质谱法两种方法，探究两种同工型的相互作用组差异。尽管在HEK293细胞中两种同工型的相互作用组高度相似，但在HeLa细胞中通过BioID鉴定到了多种优先结合两种同工型之一的靶蛋白。其中，偏好结合GSK-3α的DCP1B，以及偏好结合GSK-3β的MISP，被用于开展双向互作验证。两种同工型之间的互作差异，或有助于理解二者独特的功能与调控机制，同时也为开发同工型特异性的干预策略提供了新的方向。