Abstract
Glycogen 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 ∼2.4% 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-3β phosphorylated these proteins in vitro RNA-Seq of WT and Gsk3 DKO ESCs identified ∼190 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 ∼47 proteins (1.4%) whose levels increased and ∼78 (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.
Highlights
Glycogen synthase kinase-3 (GSK-3) is a constitutively active, ubiquitously expressed protein kinase that regulates multiple signaling pathways
Wild-type embryonic stem cells (ESCs) were cultured in parallel in light amino acid medium
Deletion of GSK-3␣ and GSK-3 and activation of Wnt signaling by -catenin accumulation were confirmed by Western blotting (Fig. 1C)
Summary
To characterize the GSK-3 phosphoproteome, we performed SILAC and quantitative MS using wild-type and Gsk DKO mouse ESCs (Fig. 1A). Filtering the data set to include only phosphopeptides with an absolute -fold change of Ն1.5-fold, we found 89 phosphopeptides from 65 unique proteins (3.4% of all phosphoproteins identified) that had significantly reduced phosphorylation in DKO ESCs compared with wild type (Fig. 2 (A and B) and supplemental Table 1) Of these phosphorylation events, 88% were on a serine residue and 12% were on threonine. We did not observe statistically significant enrichment of any residues, the most common residues at the ϩ4-position in the set of GSK-3– dependent phosphopeptides were serine and threonine (supplemental Fig. 3B) Taken together, these analyses suggest a bias for serine or threonine at the ϩ4-position and indicate that the majority of GSK-3– dependent phosphorylation sites do not contain the (S/T)XXX(S/T) consensus. The SILAC/MS data identified GSK-3– dependent phosphorylation of RBM8A (without a change in overall protein level, supplemental Fig. 4) at serine 166 and/or serine 168, which lie within adjacent RS motifs. For a complete list of phosphopeptides, see supplementary Table 1A)
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