Gel lanes were sliced into different bands and in-gel digested overnight at 37C with trypsin. be a strong stimulator of SRC-3 that co-activates estrogen receptor (ER). PFKFB4 phosphorylates SRC-3 at serine 857 (S857) enhancing its transcriptional activity, whereas either suppression of PFKFB4 or ectopic expression of a phosphorylation-deficient SRC-3 mutant S857A (SRC-3S857A) significantly abolishes SRC-3-mediated transcriptional output. Functionally, PFKFB4-driven SRC-3 activation drives glucose flux towards pentose phosphate pathway enabling purine synthesis by transcriptionally upregulating the expression of enzyme transketolase (TKT). In addition, two enzymes adenosine monophosphate deaminase-1 (AMPD1) and xanthine dehydrogenase (XDH) involved in purine metabolism were identified as SRC-3 targets which may or may not be directly involved in purine synthesis. Mechanistically, phosphorylation at S857 increases coactivator interaction with the transcription factor ATF4 stabilizing SRC-3/ATF4 recruitment to target gene promoters. Ablation of SRC-3 or PFKFB4 suppresses breast tumor growth and prevents metastasis to the lung from an orthotopic setting as does an SRC-3S857A mutant. PFKFB4 and pSRC-3-S857 levels are elevated and significantly correlate in ER positive tumors whereas, in patients with basal subtype, PFKFB4-SRC-3 drives a common protein signature that positively correlates with the poor survival of breast malignancy patients. These findings suggest that the Warburg-pathway enzyme PFKFB4 functions as a molecular fulcrum Rabbit Polyclonal to GTF3A coupling sugar metabolism to transcriptional activation by stimulating SRC-3 crucial to promote aggressive metastatic tumors. Among the scenery of genetic alterations that drive aggressive metastatic tumors, transcriptional coregulator SRC-3 is one of the abundantly deregulated oncogenes3C5. Importantly, dynamic interactions between SRC-3 and its subsequent recruitment to target genes are delicately regulated by post-translational modifications on SRC-36. Phosphorylation of SRC-3 can alter its transcriptional activity, protein stability and subcellular localization7C9, and deregulated kinase signaling hyper-activating SRC-3 is usually a sodium 4-pentynoate hallmark of many tumors10, 11. As a starting point for identifying kinases that modulate SRC-3 transcriptional activity, we performed an unbiased RNAi screening assay using a kinome library containing siRNAs targeting 636 human kinases (median 3 sodium 4-pentynoate siRNAs per kinase) in the presence of a GAL4-DNA binding domain-fused-SRC-3 (pBIND-SRC-3)12 and GAL4 DNA binding sites made up of luciferase reporter gene (pG5-luc) (Fig. 1a). The concentration of pBIND-SRC-3 construct needed to obtain luciferase readings in a linear range was standardized along with the dose of kinase siRNAs to observe significant alterations in SRC-3 intrinsic activity (Extended Data Fig. 1a, b). As a positive control we used siRNAs targeting protein kinase C zeta (PRKCZ1), a kinase known to activate SRC-313, and compared the repression of the coregulator activity upon kinase knockdown with the non-targeting control GFP-siRNAs (Extended Data sodium 4-pentynoate Fig. 1c). Kinome-wide screening identified several kinases as modulators of SRC-3 activity (Fig. 1b, Extended Data Fig. 1d, Supplementary Table 1), either as stimulators or repressors compared to sodium 4-pentynoate the controls (Extended Data Fig. 1e). Open in a separate window Physique 1 PFKFB4 is an essential activator of transcriptional coregulator SRC-3a, Schematics showing the RNAi kinome library screening with SRC-3 transcriptional activity assay using GAL4 DNA binding site-luciferase reporter (pG5-luc) along with GAL4-DNA binding domain name (DBD)-full-length SRC-3 fusion (pBIND-SRC-3) or control pBIND as readout. b, Log2 fold switch in SRC-3 activity with three siRNAs/kinase represented as Set A, Set B and Set C in the 3D plot (represents biologically impartial samples. Ten kinases were designated as reproducible and significant hits sodium 4-pentynoate in the screen (Fig. 1c and Extended Data Fig. 1f), among which metabolic kinase PFKFB4 was identified as the most strong positive regulator of SRC-3 activity. A secondary screen coupled with growth assays to identify the top-hit kinases driving malignancy cell proliferation also recognized PFKFB4 to be the most dominant kinase regulating cellular proliferation (Extended Data Fig. 1g). Silencing of PFKFB4 with different shRNAs and siRNAs decreased SRC-3 activity (Extended Data Fig. 2a, b) in multiple malignancy lines with reduced PFKFB4 levels (Extended Data Fig. 2c, d), whereas ectopic overexpression of PFKFB4 using an adenoviral contamination (Adv. PFKFB4) enhanced SRC-3 activity (Fig. 1d). Interestingly, SRC-3 protein levels were increased upon ectopic PFKFB4 expression (Fig. 1e), but not mRNA levels (Extended Data Fig. 2e), and proximity ligation assays (PLA) support a direct SRC-3-PFKFB4 interaction, consistent with PFKFB4-dependent regulation of SRC-3.