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. Author manuscript; available in PMC: 2018 Oct 3.
Published in final edited form as: Nature. 2018 Apr 3;556(7700):249–254. doi: 10.1038/s41586-018-0018-1

Extended Data Figure 3. PFKFB4 functions as a protein kinase by phosphorylating SRC-3 at the S857 residue.

Extended Data Figure 3

a, In vitro PFKFB4 kinase assay in presence of purified SRC-3 protein, fructose-6-phosphate (F6P), ATP and increasing concentration of recombinant PFKFB4 enzyme followed by SDS-PAGE. Immunoblotting with p-Ser/Thr antibody shows the level of phosphorylated Ser/Thr-SRC-3 protein. b, In vitro PFKFB4 kinase assay in presence of purified SRC-3 protein, PFKFB4 enzyme and varying concentrations of F6P and ATP followed by SDS-PAGE. Immunoblotting with p-Ser/Thr antibody shows the level of p-SRC-3 protein. c, Coomassie blue stain showing the levels of GST-fused SRC-3 fragments used in in vitro kinase reactions performed in Fig. 2b. d, Proteomics analysis of in vitro kinase assay using the GST-SRC-3-CID fragment in the presence of PFKFB4 enzyme and ATP followed by mass spectrometric analyses. Mass spectrum shows the green phosphorylation peak. e, Proteomics analysis of an in vitro kinase assay using a S857A-mutated GST-SRC-3-CID protein in the presence of PFKFB4 enzyme and ATP, followed by mass spectrometric analyses. Mass spectrum failed to detect phosphorylation peaks in the S857A mutated SRC-3-CID protein. f, Expression of PFKFB1, PFKFB2, PFKFB3 and PFKFB4 in MDA-MB-231 cells expressing shRNAs targeting PFKFB4 (sh#09 and sh#20). mRNA levels were normalized to internal housekeeping gene actin. [Mean ± s.d., n=3, biological replicates, two-way ANOVA with Tukey’s Multiple comparisons test, *P<0.05]. g, Protein levels of pSRC-3-S857, total-SRC-3 and actin in MDA-MB-231 cells stably expressing NTshRNA, SRC-3shRNA, or expression of shRNA-resistant S857A (shSRC-3+S857A) mutant or wild-type SRC-3 in SRC-3 depleted cells (shSRC-3+WT-SRC-3) cultured in 25mM glucose. Protein bands were quantified by Image J after normalization to β-actin. h, MDA-MB-231 cells stably expressing NT-shRNA or shRNA targeting PFKFB4 were grown in presence of 25mM glucose or were glucose starved for 4 hours followed by incubation with Streptolysin O (SLO) for 5 min. FBP (10µM) was added to glucose starved cells for additional 1 hour, followed by cell lysis and immunoblotting. Protein bands were quantified by Image J after normalization to β-actin and the NT shRNA lane was set to 1. i, Relative luciferase activity (RLU) showing the transcriptional activity of SRC-3 in MDA-MB-231 cells transduced with Adv. GFP or Adv. PFKFB4 cultured in presence of 5mM, 15mM or 25mM glucose. [Mean ± s.d., n=6 (pBIND) and n=3 (pBIND-SRC-3) biological cell samples, two-way ANOVA with Tukey’s Multiple comparisons test, *P<0.000001]. Data shown in 3a-c and 3f-h are representative of 3 biologically independent experiments with similar results, and 3d-e are representative of 2 biologically independent experiments each run with three different reactions all showing similar results and peptide coverage.