Phosphoproteomic Analysis Of Pyruvate Dehydrogenase In Response To Environmental Stress

Abstract Pyruvate dehydrogenase is a mitochondrial enzyme that regulates the flux of carbohydrates into oxidative phosphorylation. The activity of PDH is largely regulated by reversible phosphorylation. The pyruvate dehydrogenase kinases add inhibitory phosphorylations to PDH on the E1α subunit at positions 232, 293, and 300. Interestingly, there is a family of 4 related PDHK genes with overlapping affinity for the 3 phosphorylation sites. PDHK1 and PDHK3 have been identified as hypoxia-inducible at the transcriptional level, and PDHK1 has been shown to be important for the growth of model tumors. This work attempts to more carefully define the functions of PDHK1 and 3 using CRISPR technology in colorectal cancer lines in vitro and genetically engineered mice in vivo. Using antibodies specific for the phosphorylated sites on E1α, we find that PDHK1 is essential for the phosphorylation of the 232 site in response to hypoxia and partially responsible for the hyperphosphorylation of 293 and 300. The level of phosphorylation in response to hypoxia is also sensitive to nutrients in the environment, with high levels of glucose enhancing E1α phosphorylation in hypoxia. Analysis of the number of phosporylations on individual E1α molecules using isoelectric focusing shows a range of events, with some molecules having 2 or 3 phosphorylations, while a large fraction of the E1α remains unphosphorylated in hypoxia. This type of pattern suggests a non-random phosphorylation of the multiple sites. Analysis of PDH activity supports the in vitro findings that any single phosphorylation event is sufficient to inhibit PDH complex activity. Preliminary mitochondrial oxygen consumption experiments show that colorectal cancer cells are less dependent on PDHK1 for regulating mitochondrial function compared to published data in pancreatic and head and neck cancer. Finally, analysis of PDH phosphorylation in mice supports a role for PDHK1 in the regulation of PDH activity in response to fasting and blood glucose levels. In conclusion, regulation of PDH in normal and cancerous colonocytes appears to be sensitive to environmental oxygen and nutrients. PDHK1 is the PDHK family member with the greatest influence on the addition of non-random inhibitory phosphorylation events. These in vitro findings will be analyzed with respect to the findings obtained using the new PDHK1 fl/fl villin Cre mouse. Citation Format: Wendi O'Neill, Tereza Golias, Martin Benej, Nicholas Denko. Phosphoproteomic analysis of pyruvate dehydrogenase in response to environmental stress [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5467. doi:10.1158/1538-7445.AM2017-5467