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

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 glycolysis-promoting 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 (S273) 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-Glu-Asn 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-to-late G1, whereas glutamine is the only substrate essential for the progression through S phase into cell division.