Molecular basis for the differential use of glucose and glutamine in cell proliferation as revealed by synchronized HeLa cells

Abstract

During cell division, the activation of glycolysis is tightly regulated by the action of two ubiquitin ligases, anaphase-promoting complex/ cyclosome-Cdh1 (APC/C-Cdh1) and SKP1/CUL-1/F-box protein-β-transducin repeat-containing protein (SCF-β-TrCP), which control the transient appearance and metabolic activity of the glycolysispromoting enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, isoform 3 (PFKFB3). We now demonstrate that the breakdown of PFKFB3 during S phase occurs specifically via a distinct residue (S 273 ) within the conserved recognition site for SCF-β-TrCP. Glutaminase 1 (GLS1), the first enzyme in glutaminolysis, is also targeted for destruction by APC/C-Cdh1 and, like PFKFB3, accumulates after the activity of this ubiquitin ligase decreases in mid-to-late G1. However, our results show that GLS1 differs from PFKFB3 in that its recognition by APC/C-Cdh1 requires the presence of both a Lys-GluAsn box (KEN box) and a destruction box (D box) rather than a KEN box alone. Furthermore, GLS1 is not a substrate for SCF-β-TrCP and is not degraded until cells progress from S to G2/M. The presence of PFKFB3 and GLS1 coincides with increases in generation of lactate and in utilization of glutamine, respectively. The contrasting posttranslational regulation of PFKFB3 and GLS1, which we have verified by studies of ubiquitination and protein stability, suggests the different roles of glucose and glutamine at distinct stages in the cell cycle. Indeed, experiments in which synchronized cells were deprived of either of these substrates show that both glucose and glutamine are required for progression through the restriction point in mid-tolate G1, whereas glutamine is the only substrate essential for the progression through S phase into cell division.

Spatial Distribution of Cellular Function: The Partitioning of Proteins between Mitochondria and the Nucleus in MCF7 Breast Cancer Cells

“…The present results suggest further investigation of the functional roles of glycolytic enzymes in mitochondria and the nucleus is needed, especially in the light of indications that they concentrate in the MAM region of mitochondria 84 and that mitochondrial restructuring and bioenergetic plasticity are also important for stem cell differentiation/regression. 96 Both PKM2 97,98 and PFKFB3 74 have recently been proposed as "master" proteins for controlling the metabolic changes involved in hypoxia, proliferation and cancer, but a substantial body of other literature and the present results suggest that much more complicated networks with feedback loops involving many glycolysis, TCAcycle, OxPhos and signaling proteins may be involved. Furthermore hypoxia causes dynamic changes in protein subcellular location involving functions that go far beyond changes in glycolysis and include TCA-cycle, OxPhos and chromatin remodeling proteins among many others.…”

“…Very recently, connections to the cell cycle have been established for nuclear FBP1 72 and PGAM1 in the nucleus of cancer cells has been observed. 73 This is also the case for fructose-2,6-biphosphatase 3 (PFKFB3) 74 which, although not a direct participant in the glycolysis enzymatic cascade, has major effects on glycolysis through the allosteric regulator fructose 2,6-bisphosphate that apparently include variations in cellular energy source (glutamine vs glucose) as a function of the cell cycle in cancer cells. 74,75 There are scattered experimental reports of further functions of these and other glycolytic enzymes in the nucleus, mitochondria and elsewhere in cells (Supporting Information Table 5).…”

“…73 This is also the case for fructose-2,6-biphosphatase 3 (PFKFB3) 74 which, although not a direct participant in the glycolysis enzymatic cascade, has major effects on glycolysis through the allosteric regulator fructose 2,6-bisphosphate that apparently include variations in cellular energy source (glutamine vs glucose) as a function of the cell cycle in cancer cells. 74,75 There are scattered experimental reports of further functions of these and other glycolytic enzymes in the nucleus, mitochondria and elsewhere in cells (Supporting Information Table 5). Furthermore, GPI, HK1 and TKT have been detected in the nucleus in large scale antibody screening of subcellular protein locations 76 (Supporting Information Table 5).…”

Mitochondrial Sirtuins and Their Relationships with Metabolic Disease and Cancer

“…Consistently, many cancer cells are ''glutamine addicted,'' and require exogenous glutamine to support survival and proliferation (95). For example, cell cycle progression in HeLa cells is absolutely dependent on glutamine (29). Jeong et al showed that genotoxic stress, which arrests cell cycle progression to allow DNA damage repair, induces SIRT4 expression, which, in turn, represses mitochondrial glutamine metabolism (71).…”

Metabolic control of the cell cycle

“…1A). 10 Whether this relates to the use of glutamine for the production of biomass, redox homeostasis and/or energy requires further study.…”