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...
Adeno-associated viral (AAV) vectors are promising vehicles for hemophilia gene therapy, with favorable clinical trial data seen in the treatment of hemophilia B. In an effort to optimize the expression of human coagulation factor VIII (hFVIII) for the treatment of hemophilia A, an extensive study was performed with numerous combinations of liver-specific promoter and enhancer elements with a codon-optimized hFVIII transgene. After generating 42 variants of three reduced-size promoters and three small enhancers, transgene cassettes were packaged within a single AAV capsid, AAVrh10, to eliminate performance differences due to the capsid type. Each hFVIII vector was administered to FVIII knockout (KO) mice at a dose of 10 genome copies (GC) per mouse. Criteria for distinguishing the performance of the different enhancer/promoter combinations were established prior to the initiation of the studies. These criteria included prominently the level of hFVIII activity (0.12-2.12 IU/mL) and the pattern of development of anti-hFVIII antibodies. In order to evaluate the impact of capsid on hFVIII expression and antibody formation, one of the enhancer and promoter combinations that exhibited high hFVIII immunogenicity was evaluated using AAV8, AAV9, AAVrh10, AAVhu37, and AAVrh64R1 capsids. The capsids subdivided into two groups: those that generated anti-hFVIII antibodies in ≤20% of mice (AAV8 and AAV9), and those that generated anti-hFVIII antibodies in >20% of mice (AAVrh10, AAVhu37, and AAVrh64R1). The results of this study, which entailed extensive vector optimization and in vivo testing, demonstrate the significant impact that transcriptional control elements and capsid can have on vector performance.
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