In vitro and In vivo evidence demonstrating chronic absence of Ref-1 Cysteine 65 impacts Ref-1 folding configuration, redox signaling, proliferation and metastasis in pancreatic cancer.

Ref-1/APE1 (Redox Effector/Apurinic Endonuclease 1) is a multifunctional enzyme that serves as a redox factor for several transcription factors (TFs), e.g., NF-kB, HIF-1α, which in an oxidized state fail to bind DNA. Conversion of these TFs to a reduced state serves to regulate various biological responses such as cell growth, inflammation, and cellular metabolism. The redox activity involves a thiol exchange reaction for which Cys65 (C65) serves as the nucleophile. Using CRISPR editing in human pancreatic ductal adenocarcinoma (PDAC) cells, we changed C65 to Ala (C65A) in Ref-1 to evaluate alteration of Ref-1 redox dynamics as well as chronic loss of Ref-1 redox activity on cell signaling pathways, specifically those regulated by NF-kB and HIF-1α. The redox activity of Ref-1 requires partial unfolding to expose C65, which is buried in the folded structure. Labeling of Ref-1 with polyethylene glycol-maleimide (PEGm) provides a readout of reduced Cys residues in Ref-1 and thereby an assessment of partial unfolding in Ref-1. In comparing Ref-1WT vs Ref-1C65A cell lines, we found an altered distribution of oxidized versus reduced states of Ref-1. Accordingly, activation of NF-kB and HIF-1α in Ref-1C65A lines was significantly lower compared to Ref-1WT lines. The bioinformatic data revealed significant downregulation of metabolic pathways including OXPHOS in Ref-1C65A expressing clones compared to Ref-1WT line. Ref-1C65A also demonstrated reduced cell proliferation and use of tricarboxylic acid (TCA) substrates compared to Ref-1WT lines. A subcutaneous as well as PDAC orthotopic in vivo model demonstrated a significant reduction in tumor size, weight, and growth in the Ref-1C65A lines compared to the Ref-1WT lines. Moreover, mice implanted with Ref-1C65A redox deficient cells demonstrate significantly reduced metastatic burden to liver and lung compared to mice implanted with Ref-1 redox proficient cells. These results from the current study provide direct evidence that the chronic absence of Cys65 in Ref-1 results in redox inactivity of the protein in human PDAC cells, and subsequent biological results confirm a critical involvement of Ref-1 redox signaling and tumorigenic phenotype.

Phosphorylation site specificity of the Pak-mediated regulation of Raf-1 and cooperativity with Src.

The p21-activated kinase, Pak, has recently been shown to phosphorylate Raf-1 on serine 338 (S338), a critical regulatory residue. The specificity requirements for Pak-mediated phosphorylation of S338 were examined by substitution analysis of Raf-1 peptides and conserved region 3 (CR3) proteins. Phosphorylation was found to be very sensitive to alterations in amino acid side chains proximal to S338. Loss of N-terminal arginines resulted in decreased peptide phosphorylation while loss of these residues, as well as C-terminal glutamates and bulky C-terminal hydrophobic residues, decreased phosphorylation of the CR3 protein. Phosphorylation of Raf-1 on tyrosine 341 is significant in epidermal growth factor- and Src-mediated signaling, suggesting that cooperativity may exist between Pak and Src phosphorylation of Raf-1. Purified Pak and Src were found not to be cooperative in phosphorylating peptides or purified CR3 protein. However, the phosphorylation of Raf-1 S338 by Pak was increased in the presence of Src. The complexity of this signaling module could thus account for the different levels of Raf-1 activation required for fulfillment of different biological roles within the cell.