CXXC5 is a ubiquitinated protein and is degraded by the ubiquitin-proteasome pathway.

CXXC5, as a member of the zinc-finger CXXC family proteins, interacts with unmodified CpG dinucleotides to modulate the expression of genes involved in cellular proliferation, differentiation, and death in physiology and pathophysiology. Various signaling pathways, including mitogenic 17β-estradiol (E2), contribute to the expression and synthesis of CXXC5. However, how signaling pathways modulate protein levels of CXXC5 in cells is largely unknown. We previously reported that some key regulators, including retinoblastoma 1 and E74-like ETS transcription factor 1, of the G1 to S phase transitions are involved in the expression of CXXC5 in estrogen-responsive MCF-7 cells, derived from a breast adenocarcinoma. We, therefore, predict that the synthesis of CXXC5 is regulated in a cell cycle-dependent manner. We report here that although E2 in synchronized MCF-7 cells augments both transcription and synthesis of CXXC5 in the G1 phase, CXXC5 protein levels are primarily mediated by ubiquitination independently of cell cycle phases. Utilizing the Ubiquitination approach, which is based on cellular biotinylation of ubiquitin, in HEK293FT cells derived from immortalized human embryonic kidney cells, followed by sequential immunoprecipitation coupled mass spectrometry analyses, we identified ubiquitinated lysine residues of CXXC5. We show in both MCF-7 and HEK293FT cells that the ubiquitinated lysine residues contribute to the degradation of CXXC5 through the ubiquitin-proteasome pathway.