Study On The Pro-apoptotic Effect Of Beta 1,4-galactosyltransferase 1 And Its Effect On CDK11～(p58)-mediated Apoptosis

The enzyme β1,4-galactosyltransferasel (β1,4GT1) is one of the most exhaustively studied glycosyltransferases. It resides in two distinct subcellular compartments, the trans-Golgi network, where it is one of the key enzymes involved in the biosynthesis of complex-type oligosaccharide, and the plasma membrane, where it serves as an adhesion molecule and participates in a number of cellular interactions. Surface-localized glycosyltransferase would form a stable adhesive bond with its glycoside substrate in the extracellular matrix or on an adjacent cell surface. It participates in cellular interactions, including neurite extension, cell growth, sperm-egg interaction, cell spreading, and migration.Many experiments have suggested that altering the expression of cell surface β1,4GTl modulates cell growth and apoptosis. The mRNA level of β1,4GTl is greatly increased during the apoptosis of SMMC-7721 hepatocarcinoma cells induced by cycloheximide (CHX). Meanwhile, overexpression of β1,4GT1 enhances the SMMC-7721 hepatocarcinoma cell susceptibility to apoptosis. Cell surface β1,4GT1 interacts with multiple ligands and cytoskeletons. Evidence is presented suggesting that cell surface β1,4GTl interacts with epidermal growth factor receptor (EGFR), which directly affects cell proliferation rate. Binding of EGFR to its cognate ligands leads to autophosphorylation of receptor tyrosine kinase and subsequent activation of signal transduction pathways that are involved in regulating cellular proliferation, differentiation, and survival. This suggests that EGFR may participate in the apoptotic process enhanced by β1,4GTl.Overexpressing the complete cytoplasmic and transmembrane domains of the long form of the β1,4GT1 protein, referred to as the truncated long β1,4GTl (TL) protein, which displaces the endogenous cell surface β1,4GTl from its cytoskeletal attachment, led to decreased apoptosis induced by CHX in SMMC-7721 cells. This suggests that cell surface β1,4GTl promotes apoptosis induced by CHX. We found that long β1,4GTl could interact with EGFR in vitro. To determine how long pl,4GTl affect EGFR pathway SMMC-7721 cells were transiently transfected with long β1,4GTl and were induced by CHX. Results indicated that long β1,4GTlinhibited EGFR phosphorylation at TyrlO68. It also inhibited PKB/Akt phosphorylation at Serl36 and Serll2, Erk and JNKs phosphorylation. The Bcl-2 family members regulate apoptosis in a positive and negative fashion. Some of the Bcl-2 family proteins are regulated by PKB/Akt, Erk and JNKs. In this experiment Bcl-2 protein level and phosphorylation at Ser70 was decreased by long pi,4GTl. Translocation of pro-apoptotic protein Bad and Bax to the mitochondria increased in long pl,4GTl-overexpressing cells during apoptosis. The changes of Bcl-2, Bad and Bax were consistent with that of PKB/Akt, Erk and JNKs. Thus we conclude that long P1,4GT1 promotes apoptosis by inhibiting EGFR mediated signaling pathway. The downstream molecular of EGFR, PKB/Akt, Ras-MAPK and JNKs were also inactivated. The balance of pro- and anti-apoptotic proteins of Bcl-2 family was disturbed and the permeability of mitochondria membrane was increased.Cyclin-dependent kinase 11 (CDK11; also named PITSLRE) is part of the large family of p34cdc2-related kinases whose functions appear to be linked with cell cycle progression, tumorigenesis and apoptotic signaling. The mechanism that CDKllp58 induces apoptosis is not clear. Previously work has shown that CDKllp58 could phosphorylate pi,4GTl protein and enhance its activity. It also interacted with pi,4GTl. pi,4GTl has been suggested to promote apoptosis in some cells. So P1,4GT1 might participate in apoptosis induced by CDK1 lp58.In this study, we demonstrated that ectopically expressed pi,4GTl increased CDK1 lp58-mediated apoptosis induced by CHX. In contrast, RNAi-mediated knockdown of pi,4GTl effectively inhibited apoptosis induced by CHX in CDKllp58-overexpressing cells. For example, the cell morphological and nuclear changes were reduced; the loss of cell viability was prevented and the number of cells in sub-Gl phase was decreased. Knockdown of pi,4GTl also inhibited the release of cytochrome c from mitochondria and caspase-3 processing. Therefore the cleavage of CDKllp58 by caspase-3 was lessened. In CDK1 lp58-overexpressing cells the protein level of Bcl-2 decreased and the translocation of Bad and Bax increased. This suggests that CDKllp58 promotes apoptosis by regulating Bcl-2 family protein in SMMC-7721 cells induced by CHX. Knockdown of pl,4GTl also reversed the changes of Bcl-2 family protein regulated by CDKllp58. We proposed that pi,4GTl activated by CDKllp58 might contribute to the pro-apoptotic effect of CDKllp58 and CDKllp58 promotes apoptosis by mitochondria mediated pathway. This may represent a new mechanism of CDKllp58 in CHX-induced apoptosis in SMMC-7721...