Regulation Of The Expression And Activity Of β1, 4-Galactosyltransferase Ⅰ By Focal Adhesion Kinase

β1,4-Galactosyltransferase I(GalT) participates in both glycoconjugates and cellular interactions. GalT' s role in lamellipodia formation and cell migration on laminin is associated with a transient phosphorylation of focal adhesion kinase (FAR) and a consequent reorganization of the actin cytoskeleton and focal adhesions. They both are localized to focal adhesion contacts, and both are associated with cytoskeleton. FAK has been implicated in playing an important role in signal transduction pathways regulated by integrin-mediated cell adhesion to ECM. Activation of FAK contributes to a variety of biological functions such as cell adhesion and spreading, cell migration, cell survival and apoptosis, and cell cycle regulation and proliferation. As a receptor of laminin, GalT is involved in various biological activities of laminin, including promotion of cell adhesion, growth, migration, differentiation, neurite outgrowth, sperm-egg interactions and tumor metastases. Gl/S checkpoint regulation proteins such as p!6 control GalT mRNA transcription and GalT surface and total activity. However, the effect of FAK on GalT is still unknown. So we transfected wild type FAK (wtFAK) and different FAK mutants into NIH3T3 cell line, measured GalT gene expression by Northern blot hybridization, and evaluated its activity. It was found that wtFAK and FAKY576F up-regulated GalT gene expression and its surface activity, while dominant negative mutant FAKY397F down-regulated them. At the same time, we used ricinus communis agglutinin (RCA)-I lectin staining to demonstrate its binding reactions. We found that wtFAK and FAKY576F bound stronger, while FAKY397F bound weaker than the control. By flow cytometry analysis, it was found that FAK promoted Gl/S transition while FAKY397F inhibited this step compared with the control NIH3T3 cells. It was found that wtFAK and FAKY576F enhanced the expression of cyclin D1, decreased the expression of p16, while FAKY397F played a role that is contrary to them. We alsofound that wtFAK inhibited the expression of cyclin D3 and p21, while FAKY397F and FAKY576F increased them. In our lab, it was found that cell cycle suppressor gene p16 transfected into A549 cell line, caused Gl phase arrested, suppressed the GalT gene expression and its activity. However, we found that cell surface GalT activity were increased while no obvious change in total GalT activity regulated by wtFAK. It was indicated from the results that more cell cycle factors involved in the effect of FAK on the expression and activity of GalT. These results also implictated that cell cycle specific protein regulating G1/S phase transition, such as Rb~E2F complex, E2F-DP complex, should involved in the regulation of GalT mRNA transcription. It was reported that cyclin D1 but not p21 played a critical role in cell cycle regulated by FAK. Furthermore, it was found that antisense cyclin Dl inhibited GalT mRNA. Cotransfection of GalT promoter/luciferase reporter and antisense cyclin Dl decreased the luciferase reporter activity obviously, indicating that the differences in mRNA levels might be regulated through promoter function. Gl/S checkpoint regulation proteins control GalT mRNA transcription. The results indicate that FAK regulated the expression of GalT and its activity in NIH3T3 cells may contribute to the effect of FAK on the cell cycle.The elevated expression of surface GalT characteristic of highly metastatic cells contributes to their invasive phenotype in vitro and to their metastatic phenotype in vivo. Our above study indicated that FAK regulated the expression of GalT and its activity in NIH3T3 cells may contribute to the effect of FAK on the cell cycle. Y397 has been identified as the major site of FAK autophosphorylation and the binding site for the SH2 domains of Src, phosphatidylinositide 3-kinase (PI3K), and other signal proteins. Inhibition of cell cycle progression by the dominant negative FAK mutant required the Y397, suggesting a role for Src and/or PI3K relating to regulating the process. FAK/Src association and...