Effect of p58GTA 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 transferring galactose to the terminal N-acetylglucosamine (GlcNAc) forming Galbeta3-->4GlcNAc structure in the Golgi apparatus. In addition, it also serves as a cell adhesion molecule by recognizing and binding to terminal GlcNAc of glycoconjugates on the adjacent cell surface and matrix through a subpopulation of the enzyme distributed on the cell surface. Transient expression of the p58GTA protein kinase, which belongs to the p34cdc2-related supergene family, could enhance beta1,4-GT 1 total activity in COS cells. In this study, the p58GTA interaction with beta1,4-GT 1 was confirmed using an in vitro assay with the TNT Coupled Reticulocyte Lysate System. An expression vector containing p58GTA was stably transfected into 7721 cells, a human hepatocarcinoma cell line, expression was confirmed by Northern and Western blot analyses. The cells transfected with p58GTA (p58GTA/7721) contained 1.9 times higher total beta1,4-GT 1 activity and 2.6 times higher cell-surface beta1,4-GT 1 activity than the mock transfected cells (pcDNA3/7721). However, Ricinus communis agglutinin-I lectin blot analysis revealed that the enhanced beta1,4-GT1 activity did not increase the Galbetal-->4GlcNAc groups on most of the membrane proteins in p58GTA/7721 cells. By flow cytometry analysis, it was found that the p58GTA/7721 cells were G2/M phase arrested, compared with the pcDNA3/7721 cells. These results suggest that the p58GTA stable transfection into human hepatocarcinoma cells could enhance the two beta1,4-GT1 subcellular pool activities independently and change its cell-cycle without modifying the beta-1,4-linked galactose residues on most membrane proteins.
Zhang
S W
SW
Gene Research Center, Medical Center of Fudan University (Former Shanghai Medical University), Shanghai, PR China.
Xu
S L
SL
Cai
M M
MM
Yan
J
J
Zhu
X Y
XY
Hu
Y
Y
Gu
J X
JX
D000818:Animals; D015152:Blotting, Northern; D019556:COS Cells; D006528:Carcinoma, Hepatocellular; D002453:Cell Cycle; D018844:Cyclin-Dependent Kinases; D004789:Enzyme Activation; D005434:Flow Cytometry; D005690:Galactose / Q000378:metabolism; D005700:Galactosyltransferases / Q000235:genetics / Q000378:metabolism*; D006023:Glycoproteins / Q000032:analysis; D006801:Humans; D037102:Lectins / Q000378:metabolism; D008565:Membrane Proteins / Q000032:analysis; D037121:Plant Lectins; D011494:Protein Kinases / Q000235:genetics / Q000502:physiology*; D017346:Protein Serine-Threonine Kinases; D012333:RNA, Messenger / Q000096:biosynthesis; D014162:Transfection; D014407:Tumor Cells, Cultured
D006023:Glycoproteins; D037102:Lectins; D008565:Membrane Proteins; D037121:Plant Lectins; D012333:RNA, Messenger; C017707:Ricinus communis agglutinin-1; D005700:Galactosyltransferases; C119701:beta-1,4-galactosyltransferase I; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; C447612:CDK11a protein, human; D018844:Cyclin-Dependent Kinases; D005690:Galactose