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G2-specific E3 ligase (G2E3) was originally identified in a global gene expressing profiling with the GenBank reference KIAA1333 when Crawford et al. investigated transcriptional control of gene expression in G2/M-phase. In addition, they studied gene expression after DNA damage treatment using γ-irradiation. They conducted a microarray analysis of HeLa cells after double thymidine block and identified genes with maximal expression in G2- and M-phase. Interestingly, a number of genes were down-regulated in response to γ-irradiation. One of the genes showing a maximal mRNA expression in G2/M-phase and decreased mRNA levels on γ-irradiation was G2E3.
G2E3 was proposed to have ubiquitin ligase activity since it consists of four domains which can act as E3 ligases. Three of these domains have similarity to both RING and PHD domains and the fourth C-terminal domain is a HECT domain. Nevertheless, HECT is catalytically inactive, but controls subcellular localization of G2E3. Reasons for lack of activity of the HECT domain are numerous: not matching amino acids in the E2 binding site and at the C-terminus of the HECT domain and poorly conserved residues surrounding the active site. Through an in vitro ubiquitination assay, PHD/RING2 and PHD/RING3 domain have been reported to possess ubiquitin ligase activity and catalyze Lys48-linked poly-ubiquitination.
Studies have found G2E3 to modulate cellular proliferation, survival and the DNA damage response (DDR). They show that G2E3 is regulated in a DNA damage responsive manner since G2E3 mRNA and protein levels are decreased upon DNA damage treatment. Knockdown of G2E3 resulted in p53-independent apoptosis and decreased proliferation. Moreover, researchers verified prior findings that G2E3 levels are maximal in G2-phase, arguing for a cell cycle-dependent regulation of G2E3. Therefore, they propose a model in which G2E3 acts as a pro-survival factor that protects normal cells against cell death. Upon DNA damage, G2E3 supports cells to go into apoptosis by its down-regulation (Figure 1).
Figure 1. Model of G2E3 affecting cellular survival.
The finding that apoptosis induced by G2E3 depletion is p53-independent raises the question of how G2E3 protects cells against apoptosis. The ubiquitin ligase was localized predominantly in the nucleus. Thus, G2E3 might not play a role in cytoplasmic and mitochondrial apoptotic processes. Conversely, G2E3 could influence transcription of apoptotic genes. Three domains of G2E3 have similarity to both PHD and RING domains. PHD domains infrequently have E3 activity, but bind methylated histones and are involved in chromatin-mediated gene regulation. It is not known whether G2E3 can bind chromatin and hence affect gene expression. This could be tested by performing a chromatin fractionation and exploring if G2E3 is enhanced in the chromatin-bound fraction. Furthermore, G2E3 could influence proteins which regulate the expression of apoptotic genes. The impact of p73 on translocation of Bax seems to be indirect as p73 localizes to the nucleus while inducing cell death. G2E3 is also a nuclear protein that could conduct its pro-survival role by inhibition of p73 or other nuclear proteins which indirectly affect apoptotic signaling in the cytoplasm.