bioRxiv preprint doi: https://doi.org/10.1101/2020.12.07.415257; this version posted December 7, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. Identifying a GSK3-TRARG1 signaling axis reveals BCL9L as a novel regulator of GLUT4 trafficking Xiaowen Duan1, Dougall M. Norris2, Sean J. Humphrey1, Pengyi Yang3,6, Kristen C. Cooke1, Will P. Bultitude4, Benjamin L. Parker1,a, Olivia J. Conway2, James G. Burchfield1, James R. Krycer1, Frances M. Brodsky4, David E. James1,5♯, Daniel J. Fazakerley1,2♯ 1Charles Perkins Centre, School of Life and Environmental Sciences, The University of Sydney, Sydney, NSW 2006, Australia 2Metabolic Research Laboratories, Wellcome-Medical Research Council Institute of Metabolic Science, University of Cambridge, Cambridge, CB2 0QQ, UK 3Charles Perkins Centre, School of Mathematics and Statistics, The University of Sydney, Sydney, NSW 2006, Australia 4Research Department of Structural and Molecular Biology, Division of Biosciences, University College London, Gower Street, London WC1E 6BT, UK 5School of Medicine, The University of Sydney, Sydney, NSW 2006, Australia 6Computational Systems Biology Group, Children’s Medical Research Institute, The University of Sydney, Westmead, NSW 2006, Australia aPresent Address is Department of Physiology, School of Biomedical Sciences, The University of Melbourne, Parkville, VIC, Australia. Corresponding authors: Daniel J. Fazakerley E-mail: [email protected] or David E. James E-mail: [email protected] Running title: TRARG1 is a novel substrate of GSK3 Keywords: glycogen synthase kinase 3 (GSK3), glucose transporter type 4 (GLUT4), trafficking regulator of GLUT4-1 (TRARG1), protein phosphorylation, phosphoproteomics, proteomics, insulin signaling, protein-protein interaction, BCL9L 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.07.415257; this version posted December 7, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. Abstract required for the insulin-sensitizing action of the PPARγ agonist rosiglitazone [5, 6]. However, the Trafficking regulator of GLUT4-1, TRARG1, mechanisms by which insulin signals to TRARG1 positively regulates insulin-stimulated GLUT4 or how TRARG1 promotes insulin sensitivity trafficking and insulin sensitivity. However, the remains unknown. mechanism(s) by which this occurs remain(s) unclear. Using biochemical and mass The most characterized signaling pathway spectrometry analyses we found that TRARG1 is relevant to GLUT4 trafficking is the PI3K/Akt dephosphorylated in response to insulin in a signaling axis, which is activated by a series of PI3K/Akt-dependent manner and is a novel upstream signaling events triggered by the substrate for GSK3. Priming phosphorylation of binding of insulin to its receptor at the cell surface. mouse TRARG1 at serine 84 allows for GSK3- Akt is necessary and sufficient for insulin- directed phosphorylation at serines 72, 76 and 80. stimulated GLUT4 trafficking [7], and is thought A similar pattern of phosphorylation was to act predominantly via phosphorylation of the observed in human TRARG1, suggesting that our Rab GTPase-activating protein (GAP), TBC1D4. findings are translatable to human TRARG1. Phosphorylation of TBC1D4 inhibits its GAP Pharmacological inhibition of GSK3 increased activity, and permits its target Rab GTPases to cell surface GLUT4 in cells stimulated with a mediate the translocation of GSVs to the PM [8]. submaximal insulin dose, and this was impaired However, loss of TBC1D4 or its cognate Rab following Trarg1 knockdown, suggesting that proteins did not completely mimic or inhibit TRARG1 acts as a GSK3-mediated regulator in insulin-stimulated GLUT4 translocation [9-11], GLUT4 trafficking. TRARG1 dephosphorylation suggesting that there may be additional sites of in response to insulin or GSK3 inhibition insulin action within the GLUT4 trafficking regulated its protein-protein interactions, pathway [12]. decreasing the interaction between TRARG1 and BCL9L, JAGN1, PYGO2 and HSD17B12. In addition to phosphorylating substrates such as Knock-down of Bcl9l promoted GLUT4 TBC1D4, Akt also modulates cellular translocation to the plasma membrane. These data metabolism through increased or diminished place TRARG1 within the insulin signaling activity of other kinases. For example, Akt network and provide insights into how TRARG1 directly phosphorylates glycogen synthase kinase regulates GLUT4 trafficking in adipocytes. 3 (GSK3) at the regulatory Ser21 or 9 residue (α and β isoform, respectively), which inhibits its kinase activity [13] and leads to Introduction dephosphorylation and activation of glycogen synthase (GS) and thereby glycogen synthesis. Insulin maintains whole-body glucose GSK3 is an unique kinase in that 1) it is homeostasis by regulating glucose transport into constitutively active and inhibited in response to muscle and adipose tissue and by inhibiting extracellular stimulation and 2) its substrates glucose output from liver. Defects in these usually need priming by another kinase [14]. processes cause insulin resistance, which is a Given the role of GSK3 in glycogen synthesis, it precursor to metabolic disorders including Type has generally been thought that GSK3 mainly 2 diabetes. Insulin signaling augments glucose participates in glucose metabolism via regulation transport into adipose tissue and muscle by of glycogen synthesis [15-19]. However, promoting the translocation of the glucose although there are conflicting reports [20-22], transporter GLUT4 from a specialized evidence from studies using small molecules to intracellular storage compartment (GLUT4 acutely inactivate GSK3 shows that GSK3 may storage vesicles; GSVs) to the plasma membrane also regulate glucose transport and GLUT4 (PM) [1]. Previous proteome studies of translocation [21, 22]. intracellular membranes highly enriched in GLUT4 have revealed major GSV resident In the present study, we identified a range of post- proteins including TBC1D4 (AS160), Sortilin, translational modifications (PTMs) on TRARG1 IRAP, LRP1, VAMP2 [2-4] and Trafficking and integrated TRARG1 into the insulin signaling regulator of GLUT4-1 (TRARG1). TRARG1 pathway by showing that insulin promotes positively regulated insulin-stimulated glucose TRARG1 dephosphorylation through the transport and GLUT4 trafficking, and was PI3K/Akt axis. Specifically, we report that 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.07.415257; this version posted December 7, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. TRARG1 is a novel direct downstream substrate di-gly modifications) and acetylation (Fig. 1B, of the protein kinase GSK3, phosphorylating Supplemental table S1). This analysis revealed murine TRARG1 at three sites within a highly TRARG1 to be extensively post-translationally phosphorylated patch between residues 70 and 90 modified, with a particularly high number of in the cytosolic portion of TRARG1. Further, phosphorylated sites within a short cytosolic potentiation of submaximal insulin-stimulated region between residues 70 and 90. To test if GLUT4 trafficking induced by GSK3 inhibition these specific PTMs affected TRARG1 mobility was impaired by Trarg1 knockdown. GSK3- in SDS-PAGE, we generated a series of HA- mediated TRARG1 phosphorylation did not alter TRARG1 mutants, including Ser/Thr to Ala/Glu TRARG1 subcellular localization but regulated mutants (7A and 7E mutants where S74, S76, S79, TRARG1 protein-protein interactions (PPIs). S84, S85, T88, S90 were mutated to A or E; or TRARG1 interactors, for example BCL9L, a 12A and 11E mutants where all S/T sites between transcriptional co-activator of β-catenin, played a S70 and S90 were mutated to A or E, with the negative role in GLUT4 translocation to the PM. 12A mutant containing an extra Y91 mutated to These findings have revealed new information on A) where A prevents and E mimics how TRARG1 is regulated by insulin and phosphorylation [23], a Lys-null mutant (KàR) provided insights into how it may regulate to prevent ubiquitination or acetylation of Lys GLUT4 trafficking in adipocytes. and a Cys-null mutant (CàS) to prevent palmitoylation of Cys. Of these mutants, only the Results Ser/Thr to Ala mutant expressed in HEK-293E cells or 3T3-L1 adipocytes exhibited a similar TRARG1 phosphorylation altered its electrophoretic mobility as the lower band of HA- migration in SDS-PAGE TRARG1, while Ser/Thr to Glu mutant migrated to a similar position as the higher bands of HA- We previously reported that a proportion of TRARG1 (Fig. 1C and S1C). These data suggest TRARG1 exhibited reduced electrophoretic that the apparent higher molecular weight mobility following separation by SDS-PAGE. TRARG1 bands are dependent on PTMs on These apparent higher molecular weight species Ser/Thr, but not Lys or Cys. of TRARG1 were enriched in the PM but not low or high density microsomal (LDM, HDM) Consistent with these data, treatment of lysates fractions (Fig. S1A), and only present in from 3T3-L1 adipocytes (Fig.