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. 2022 Jul 27;14(9):e15904. doi: 10.15252/emmm.202215904

Figure 5. MAP4K3/GLK phosphorylates and stabilizes ACE2 proteins.

Figure 5

  • A
    Immunoblotting analyses of ACE2 and Flag‐tagged GLK in HCC827 cells transfected with either Flag‐GLK or Flag‐GLK (K45E) kinase‐dead mutant. For immunoblotting of phosphorylated ACE2, Phos‐tagged SDS–PAGE gel (Phos‐tag™) was used, followed by immunoblotting with anti‐ACE2 antibody. Asterisk denotes unglycosylated ACE2 proteins.
  • B
    In vitro kinase assays using purified proteins. ACE2 serine phosphorylation, Flag‐tagged GLK (anti‐GLK), Flag‐tagged GLK (K45E) kinase‐dead mutant (anti‐GLK), and Flag‐tagged ACE2 proteins (anti‐ACE2) were detected by immunoblotting.
  • C
    Mass spectrometry analysis of GLK‐phosphorylated ACE2 proteins after in vitro kinase assays. ACE2 Ser776 and Ser783 residues were phosphorylated by GLK wild‐type but not GLK (K45E) kinase‐dead mutant.
  • D
    Mass spectrometry analysis of the ACE2 peptides from the serum exosomes of COVID‐19 patients. The ACE2 protein peptide sequences containing phospho‐Ser776 or phospho‐Ser783 residue of ACE2 proteins detected in the serum exosomes of COVID‐19 patients (Cohort #3) are shown. Exosomes were isolated using ExoQuick kits, and soluble proteins were removed by ExoQuick ULTRA columns. Exosomes were further purified by immunoprecipitation using a combination of anti‐CD9, anti‐CD63, and CD81 magnetic beads.
  • E
    In vitro kinase assays using immunoprecipitated Flag‐tagged ACE2 or Flag‐tagged GLK immunocomplexes. ACE2 serine phosphorylation, ACE2, and GLK were detected by immunoblotting using anti‐phospho‐serine, anti‐ACE2, and anti‐GLK antibodies, respectively.
  • F
    Immunoblotting analyses of Myc‐tagged GLK, Flag‐tagged ACE2, and tubulin proteins in HEK293T cells co‐transfected with Flag‐ACE2 wild‐type or individual ACE2 (S776/783A, S776A, or S783A) mutants plus either empty vector or Myc‐GLK.
  • G
    Immunoblotting analyses of Flag‐tagged ACE2 and tubulin proteins in HEK293T cells transfected with Flag‐ACE2 wild‐type or individual phosphomimetic ACE2 (S776/783E, S776E, or S783E) mutants.
  • H
    Flag‐tagged ACE2 proteins were immunoprecipitated from lysates of HEK293T cells transfected with Flag‐ACE2 wild‐type or a phosphomimetic ACE2 (S776/783E) mutant, followed by immunoblotting with anti‐Lys48‐linked ubiquitination or anti‐Flag antibody. Cells were treated with 25 μM MG132 for 2 h before being harvested.

Data information: Data shown are representative results of three (A, B, E, F, G, H) independent experiments.

Source data are available online for this figure.