Abstract
Dysregulation of lipid metabolism is common in cancer cells, but the underlying mechanisms are poorly understood. Sterol regulatory element-binding proteins (SREBPs) stimulate lipid biosynthesis through transcriptional activation of lipogenic enzymes. However, SREBPs' roles and potential interacting partners in cancer cells are not fully defined. Using a biochemical approach, we found here that pyruvate kinase M2 (PKM2) physically interacts with the nuclear form of SREBP-1a (nBP1a), by binding to amino acids 43-56 in nBP1a. We also found that PKM2 activates SREBP target gene expression and lipid biosynthesis by stabilizing nBP1a proteins. Using a competitive peptide inhibitor to block the formation of the SREBP-1a/PKM2 complex, we observed that this blockade inhibited lipogenic gene expression. Of note, nBP1a phosphorylation at Thr-59 enhanced the binding to PKM2 and promoted cancer cell growth. Moreover, we show that PKM2 phosphorylates Thr-59 in vitro Lastly, in human patients with hepatocellular carcinoma, nBP1a phosphorylation at Thr-59 was negatively correlated with clinical outcomes. Together, our results reveal that nBP1a/PKM2 interaction activates lipid metabolism genes in cancer cells and that Thr-59 phosphorylation of SREBP-1a plays an important role in cancer cell proliferation.
Highlights
Dysregulation of lipid metabolism is common in cancer cells, but the underlying mechanisms are poorly understood
Our results reveal that nuclear form of SREBP-1a (nBP1a)/pyruvate kinase M2 (PKM2) interaction activates lipid metabolism genes in cancer cells and that Thr-59 phosphorylation of SREBP-1a plays an important role in cancer cell proliferation
These results suggest a model in which PKM2 interaction with nuclear SREBP-1a increases SREBP target gene expression and subsequent lipid biosynthesis in cancer cells, which is at least in part correlated with cancer cell proliferation
Summary
Protein/protein interaction is one of the key regulatory mechanisms in biology. To better understand the regulation of SREBP-1a in the nucleus, we overexpressed a FLAG-tagged nuclear form of SREBP-1a (FLAG–nBP1a) in HEK293T cells and screened for novel SREBP-1a– binding proteins by co-immunoprecipitation (co-IP) of nuclear extracts (Fig. 1A). Semi-quantitative analyses of immunoblotting data indicate that PKM2 knockdown or T59A mutation accelerated the degradation of nBP1a (Fig. 3G) Together, these results indicate that Thr-59 phosphorylation is a novel mechanism for enhancing the nBP1a protein stability. We performed phosphopeptide competition analyses in immunohistochemistry assays of HepG2 cells (data not shown) and liver samples from patients with HCC (Fig. 4B) to further confirm its specificity Using this antibody, we examined the p-Thr-59 –BP1 levels in both tumors and the adjacent normal tissues from a total of 90 patients with HCC by immunohistochemistry. We found that the p-Thr-59 –BP1 levels were significantly correlated with histologic grade (p ϭ 0.008) and TNM stage (p ϭ 0.030) (Table 1 and Fig. 4, F and G). sing a Cox multivariate model, we found that the p-Thr-59 –BP1 level was an indepen-
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