Effect of p58(GTA) on beta-1,4-galactosyltransferase 1 activity and cell-cycle in human hepatocarcinoma cells

beta -1,4-galactosyltransferase 1 (beta1,4-GT 1) is the key enzyme transfer ring galactose to the terminal N-acetylglucosamine (GlcNAc) forming Gal bet a1 --> 4GlcNAc structure in the Golgi apparatus. In addition, it also serve s as a cell adhesion molecule by recognizing and binding to terminal GlcNAc of glycoconjugates on the adjacent cell surface and matrix through a subpo pulation of the enzyme distributed on the cell surface. Transient expressio n of the p58(GTA) protein kinase, which belongs to the p34cdc2-related supe rgene family, could enhance beta1,4-GT 1 total activity in COS cells. In th is study, the p58(GTA) interaction with beta1,4-GT 1 was confirmed using an in vitro assay with the TNT (R) Coupled Reticulocyte Lysate System. An exp ression vector containing p58(GTA) was stably transfected into 7721 cells, a human hepatocarcinoma cell line, expression was confirmed by Northern and Western blot analyses. The cells transfected with p58(GTA) (p58(GTA)/7721) contained 1.9 times higher total beta1,4-GT 1 activity and 2.6 times highe r cell-surface beta1,4-GT 1 activity than the mock transfected cells (pcDNA 3/7721). However, Ricinus communis agglutinin-I lectin blot analysis reveal ed that the enhanced beta1,4-GT 1 activity did not increase the Gal beta1 - -> 4GlcNAc groups on most of the membrane proteins in p58(GTA)/7721 cells. By flow cytometry analysis, it was found that the p58(GTA)/7721 cells were G2/M phase arrested, compared with the pcDNA3/7721 cells. These results sug gest that the p58(GTA) stable transfection into human hepatocarcinoma cells could enhance the two beta1,4-GT 1 subcellular pool activities independent ly and change its cell-cycle without modifying the beta -1,4-linked galacto se residues on most membrane proteins.