ROLE OF UEV-1, AN INACTIVE VARIANT OF THE E2 UBIQUITIN-CONJUGATING ENZYMES, IN IN-VITRO DIFFERENTIATION AND CELL-CYCLE BEHAVIOR OF HT-29-M6INTESTINAL MUCOSECRETORY CELLS

By means of differential RNA display, we have isolated a cDNA correspo nding to transcripts that are down-regulated upon differentiation of t he goblet cell-like HT-29-M6 human colon carcinoma cell line, These tr anscripts encode proteins originally identified as CROC-1 on the basis of their capacity to activate transcription of c-fos, We show that th ese proteins are similar in sequence, and in predicted secondary and t ertiary structure, to the ubiquitin-conjugating enzymes, also known as E2, Despite the similarities, these proteins lack a critical cysteine residue essential for the catalytic activity of E2 enzymes and, in vi tro, they do not conjugate or transfer ubiquitin to protein substrates . These proteins constitute a distinct subfamily within the E2 protein family and are highly conserved in phylogeny from yeasts to mammals. Therefore, we have designated them UEV (ubiquitin-conjugating E2 enzym e variant) proteins, defined as proteins similar in sequence and struc ture to the E2 ubiquitin-conjugating enzymes but lacking their enzymat ic activity (HW/GDB-approved gene symbol, UBE2V), At least two human g enes code for UEV proteins, and one of them, located on chromosome 20q 13.2, is expressed as at least four isoforms, generated by alternative splicing, All human cell types analyzed expressed at least one of the se isoforms, Constitutive expression of exogenous human UEV in HT-29-M 6 cells inhibited their capacity to differentiate upon confluence and caused both the entry of a larger proportion of cells in the division cycle and an accumulation in G(2)-M, This was accompanied with a profo und inhibition of the mitotic kinase, cdk1, These results suggest that UEV proteins are involved in the control of differentiation and could exert their effects by altering cell cycle distribution.