Fig. 4: PRPS1 O-GlcNAcylation inhibits its phosphorylation by AMPK.
From: Direct stimulation of de novo nucleotide synthesis by O-GlcNAcylation
a,b, The indicated siRNAs were introduced into H1299 cells (a); the Myc-vector or Myc-OGT plasmids were introduced into H1299 cells (b). Immunoblot analyses were conducted with indicated antibodies (n = 4). c,d, HEK293T cells were transfected by indicated plasmids. Subsequent immunoprecipitation (IP) and immunoblotting procedures were carried out to examine interaction with AMPK (c) (n = 3) and S180 phosphorylation levels (d) (n = 4) of PRPS1 WT, S83A, T166A, 2A or S180A. e,f, The recombinant O-GlcNAcylated and non-O-GlcNAcylated GST-PRPS1 proteins obtained from the in vitro O-GlcNAcylation assays were subjected to in vitro pulldown of AMPK (e) or AMPK in vitro kinase assays (f) (n = 3). g, The stoichiometry analysis of S180 phosphorylation on O-GlcNAcylated or non-O-GlcNAcylated peptides (aa 160–185) by AMPK through MS (n = 3). h, The FLAG-PRPS1 plasmids were introduced into Ampkα1/α2 WT or depleted MEFs. Indicated cells were treated with or without 2 mM AICAR for 1 h. IP and immunoblot analyses were performed with anti-FLAG beads and indicated antibodies (n = 2). i, HEK293T cells were transfected with indicated plasmids. IP and immunoblot analyses were performed with anti-FLAG beads and indicated antibodies. Quantification of PRPS1 O-GlcNAcylation levels is shown on the right (n = 3). j,k, HEK293T cells were transfected with indicated plasmids and treated with or without 2 mM AICAR for 1 h (j); HEK293T cells were transfected with indicated plasmids, including PRPS1 S180 phosphorylation mimic mutant (S180D) (k). IP and immunoblot analyses were conducted with anti-FLAG beads and indicated antibodies (n = 3). l, Immunoblot analyses were performed with the indicated antibodies in Ampkα1/α2 WT or depleted MEFs (left); the Ampkα1/α2-depleted MEFs were treated with or without 50 µM OSMI-1 for 24 h (right) (n = 3). m,n, The FLAG-PRPS1 plasmids were introduced into Ampkα1/α2-depleted MEFs. Cells were treated with or without 50 µM OSMI-1 for 24 h; FLAG-PRPS1 was eluted in fractions on the size-exclusion column in the same settings. Indicated samples were subjected to immunoblot analyses (m). FLAG-PRPS1 was purified and subjected to PRPS1 enzymatic activity assays (n). FLAG-PRPS1 protein levels in reactions were examined by western blot (lower panel) (n = 3). Every error bar signifies mean ± s.e.m. A two-tailed Student’s t-test was employed for statistical evaluation.
