Study Of The Regulation Of β-1, 4-GalT-Ⅰ By Protein Kinase B And The Association Of P110C With PAK1

Apoptosis is a form of altruistic cell suicide, which is involved in many physiological processes including tissue homeostasis, embryonic development, and immune response. Sugar chains on the glycoconjugates are completed by glycosyltransferases. Galactosyltransferase is a member of glycosyltransferase family. (1,4－galactosyltransferase 1((1,4GT 1) was identified as one of the first galactosyltransferase, which is the enzyme in the sugar chain synthesis, catalyzing the transfer of galactose from UDP-galactose to terminal N-acetylglucosamine forming Gal(1-->4GlcNAc structure. Previous study showed the association of (1,4GT1 with apoptosis. Protein kinase B (PKB) is a serine/threonine kinase, which was first cloned in 1991 and it is about 60kDa, plays an important role in cell survival when cells are exposed to different apoptotic stimuli. The p58PITSLRE was a serine/threonine kinase, which was identified as a component of semipurified galactosyltransferase. After that a series of isoforms of the p58 PITSLRE subfamily of protein kinases have been isolated and was named as PITSLRE protein kinases. The PITSLRE protein kinase isoforms function as effectors in an apoptotic signaling pathway and this activity is mediated by processed p110C isoform. In this paper we studied the regulation of β-1,4-GalT-I by protein kinase B and the association of p110C with PAK1.Here Lipofectamin was used to transfect a consistent active form of PKB (gagPKB) into SMMC 7721 cells to study the ability of the influence of this protein on proliferation and apoptosis of human hepatocellular carcinoma SMMC 7721 cells. Over expressing PKB/Akt promoted cells growth in serum culture, anchorage-indpendent growth in agarose with high efficiency, and was sufficient to promote the cells into the S phase of the cell cycle. Previous studies showed that the acceleration of cells entering S phase depended on the activity of cyclin E/cdk2 complex and the formation of cyclin E/cdk2 complex could be inhibited by p27kip1. In this study, it was found that p27kip1 expression was inhibited by PKB/Akt,so it suggests that the increasing percent of cells in S phase related to the decreasing expression of p27kip1. Furthermore, over-expression of PKB/Akt suppressed the apoptosis of cells induced by the detachment of the cells from extracelluar matrix(also known as anoikis). Integrin-linked kinase (ILK) could inhibit anoikis in IEC-18 epithelia cells by phosphorylating GSK-3 that is the first identified PKB/Akt substrate. It suggests that GSK-3 might participate in preventing SMMC 7721 cells anoikis mediated by PKB/Akt signaling pathway.PKB/Akt could promote proliferation and suppress apoptosis in cancer cells. Meanwhile, gag-PKB overexpression decreased the expression of β-1,4-GalT-I mRNA and its activities. However, the activities of cell surface (1,4GT1 were not changed. Because the level of Golgi (1,4-GalT-I is much higher than that of cell surface (1,4-GalT-I, we assumed that the total activity of (1,4-GalT-I we detected could stand for the activity of Golgi (1,4-GalT-I. In trans-Golgi complex, (1,4-GalT-I can transfer galactose from UDP-galactose to terminal N-acetylglucosamine forming a Galβ1→4GlcNAc structure, which is expressed on a variety of N-linked sugar chains of mammalian glycoprotein. Terminal galactose residues act as ligands of galectins, contactinhibin and hepatic lectin, thus involved in the regulation of apoptosis, cell growth and clearance of serum glycoproteins from the circulation. N-linked sugar chains of mammalian glycoprotein that expresss Galβ1→4GlcNAc structure were inevitably was influenced and then affected cell survival because of the alternation of (1,4-GalT-I mRNA expression and activity. The RCA-1 blot analysis was used to detect the alternation of cell surface membrane protein galactosylation between the gag-PKB transfected cells and mock-transfected cells. As shown in results, in Gag-PKB/7721 cells the expression of membrane protein doesn't change compared to that of control cells. The level of Galβ1→4GlcN...