Phosphoproteomics reveals that glycogen synthase kinase-3 phosphorylates multiple splicing factors and is associated with alternative splicing

Abstract

Edited by Eric R. FearonGlycogen synthase kinase-3 (GSK-3) is a constitutively active, ubiquitously expressed protein kinase that regulates multiple signaling pathways. In vitro kinase assays and genetic and pharmacological manipulations of GSK-3 have identified more than 100 putative GSK-3 substrates in diverse cell types. Many more have been predicted on the basis of a recurrent GSK-3 consensus motif ((pS/pT)XXX(S/T)), but this prediction has not been tested by analyzing the GSK-3 phosphoproteome. Using stable isotope labeling of amino acids in culture (SILAC) and MS techniques to analyze the repertoire of GSK-3-dependent phosphorylation in mouse embryonic stem cells (ESCs), we found that ϳ2.4% of (pS/pT)XXX(S/T) sites are phosphorylated in a GSK-3-dependent manner. A comparison of WT and Gsk3a;Gsk3b knock-out (Gsk3 DKO) ESCs revealed prominent GSK-3-dependent phosphorylation of multiple splicing factors and regulators of RNA biosynthesis as well as proteins that regulate transcription, translation, and cell division. Gsk3 DKO reduced phosphorylation of the splicing factors RBM8A, SRSF9, and PSF as well as the nucleolar proteins NPM1 and PHF6, and recombinant GSK-3␤ phosphorylated these proteins in vitro. RNASeq of WT and Gsk3 DKO ESCs identified ϳ190 genes that are alternatively spliced in a GSK-3-dependent manner, supporting a broad role for GSK-3 in regulating alternative splicing. The MS data also identified posttranscriptional regulation of protein abundance by GSK-3, with ϳ47 proteins (1.4%) whose levels increased and ϳ78 (2.4%) whose levels decreased in the absence of GSK-3. This study provides the first unbiased analysis of the GSK-3 phosphoproteome and strong evidence that GSK-3 broadly regulates alternative splicing. Glycogen synthase kinase-3 (GSK-3)2 is a ubiquitously expressed, highly conserved serine/threonine kinase that plays a central role in multiple signaling pathways, most prominently as an antagonist of insulin/AKT and Wnt/␤-catenin signaling pathways (1). Unlike most protein kinases, GSK-3 is constitutively active and in general is inhibited by upstream signaling. GSK-3 was first identified as a protein kinase that phosphorylates and inhibits glycogen synthase, the rate-limiting enzyme in glycogen synthesis (2) and was later shown to antagonize canonical Wnt signaling (1). However, GSK-3 is now known to regulate a broad range of cellular processes, including cytoskeletal organization, circadian rhythm, cell growth and survival, immune responses, and developmental processes (1).In metazoans, GSK-3 is encoded by two similar genes, Gsk3a and Gsk3b, with 98% amino acid sequence identity in the catalytic domains of mammalian GSK-3␣ and GSK-3␤. The two genes are partially redundant, and mice with complete loss of Gsk3a are viable due to compensation by Gsk3b. However, Gsk3b loss-of-function mutations in mice are embryonic or neonatal lethal (3,4), and the Gsk3a;Gsk3b DKO is lethal in early embryogenesis (5, 6). Furthermore, DKO mouse ESCs maintain expression of pluripotency markers and are unab...

Phosphoproteomics links glycogen synthase kinase-3 to RNA splicing

“…Specifically, it will be interesting to discover whether GSK-3-mediated regulation of alternative splicing is a prerequisite for ESC differentiation toward the mesendodermal lineage and how the two functionally redundant isoforms, GSK-3␣ and GSK-3␤, cooperatively act in this context. In summary, the study by Shinde et al (2) not only reinforces the previously established GSK-3 substrates but also significantly expands the list of GSK-3 substrates to functional pathways such as RNA splicing. The use of WT and DKO mESCs for identification of GSK-3 substrates eliminates potential nonspecific effects of small molecule inhibitors, which had been frequently utilized in previous studies.…”

“…However, the identification of genuine substrates for a given kinase in specific cellular context is incredibly challenging: Not only is protein phosphorylation in biological processes highly context-dependent, but the extent to which a particular substrate is phosphorylated is influenced by substrate specificity, availability, and competition in kinase-substrate interaction networks. In this Editors' Pick, Shinde et al (2) shed new light on the substrates of one important kinase in their systematic analysis of GSK-3-dependent 2 protein phosphorylation in mESCs using quantitative mass spectrometry. Their data provide a global picture of GSK-3 function that prompts new thinking about substrate motifs, identifies new substrates, and points to unexpected functional space ripe for further investigation.…”

“…To address this issue, Shinde et al (2) employed mESCs as a model cell type, as the double knock-out (DKO) of both isoforms can be tolerated (i.e. does not cause any defect in mESC self-renewal).…”

“…2 The abbreviations used are: GSK-3, glycogen synthase kinase-3; DKO, double knock-out; mESC, mouse embryonic stem cell.…”

“…To further understand GSK-3's activity in phosphorylating splicing factors in mESCs, Shinde et al (2) first performed an in vitro phosphorylation assay, followed by MS analysis to determine phosphosites of proteins involved in RNA splicing. Notably, the authors found that GSK-3␤ directly phos- The authors declare that they have no conflicts of interest with the contents of this article.…”