Tyr-301 Phosphorylation Inhibits Pyruvate Dehydrogenase by Blocking Substrate Binding and Promotes the Warburg Effect

Jun Fan,Hee-Bum Kang,Changliang Shan,Shannon Elf,Ruiting Lin,Jianxin Xie,Ting-Lei Gu,Mike Aguiar,Scott Lonning,Tae-Wook Chung,Martha Arellano,Hanna J Khoury,Dong M Shin,Fadlo R Khuri,Titus J Boggon,Sumin Kang,Jing Chen

doi:10.1074/jbc.m114.593970

Abstract

The mitochondrial pyruvate dehydrogenase complex (PDC) plays a crucial role in regulation of glucose homoeostasis in mammalian cells. PDC flux depends on catalytic activity of the most important enzyme component pyruvate dehydrogenase (PDH). PDH kinase inactivates PDC by phosphorylating PDH at specific serine residues, including Ser-293, whereas dephosphorylation of PDH by PDH phosphatase restores PDC activity. The current understanding suggests that Ser-293 phosphorylation of PDH impedes active site accessibility to its substrate pyruvate. Here, we report that phosphorylation of a tyrosine residue Tyr-301 also inhibits PDH α 1 (PDHA1) by blocking pyruvate binding through a novel mechanism in addition to Ser-293 phosphorylation. In addition, we found that multiple oncogenic tyrosine kinases directly phosphorylate PDHA1 at Tyr-301, and Tyr-301 phosphorylation of PDHA1 is common in EGF-stimulated cells as well as diverse human cancer cells and primary leukemia cells from human patients. Moreover, expression of a phosphorylation-deficient PDHA1 Y301F mutant in cancer cells resulted in increased oxidative phosphorylation, decreased cell proliferation under hypoxia, and reduced tumor growth in mice. Together, our findings suggest that phosphorylation at distinct serine and tyrosine residues inhibits PDHA1 through distinct mechanisms to impact active site accessibility, which act in concert to regulate PDC activity and promote the Warburg effect.

Highlights

Current understanding of mitochondrial pyruvate dehydrogenase (PDH) inhibition involves Ser-293 phosphorylation that impedes active site accessibility
Our findings suggest that phosphorylation at distinct serine and tyrosine residues inhibits PDHA1 through distinct mechanisms to impact active site accessibility, which act in concert to regulate pyruvate dehydrogenase complex (PDC) activity and promote the Warburg effect
FGFR1 Inhibits PDHA1 via Phosphorylation at Tyr-301— Our phosphoproteomics studies [11, 12] and multiple proteomics-based studies performed by our collaborators at Cell Signaling Technology revealed that, in addition to its upstream kinase PDK1 [12] and phosphatase PDP1 [11, 13], PDHA1 is phosphorylated at a group of tyrosine residues in human cancer cells (Fig. 1A)

Summary

Background

Current understanding of mitochondrial PDH inhibition involves Ser-293 phosphorylation that impedes active site accessibility. We report that phosphorylation of a tyrosine residue Tyr-301 inhibits PDH ␣ 1 (PDHA1) by blocking pyruvate binding through a novel mechanism in addition to Ser-293 phosphorylation. Phosphorylation of Ser-293 was suggested to impede active site accessibility of PDH to its substrate pyruvate [7] It is not clear whether other types of post-translational modifications such as tyrosine phosphorylation and lysine acetylation are involved in PDH regulation in mammalian cells. We report that phosphorylation of PDHA1 at tyrosine residue Tyr-301 is common in EGF-stimulated cells as well as diverse human cancer cells, which inhibits PDHA1 by blocking pyruvate binding through a novel molecular mechanism in addition to Ser-293 phosphorylation, and promotes the Warburg effect

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION