Physical and Functional Interaction of Acyl-CoA-binding Protein with Hepatocyte Nuclear Factor-4α

Abstract

Although acyl-CoA-binding protein (ACBP) has been detected in the nucleus, the physiological significance of this observation is unknown. As shown herein for the first time, ACBP in the nucleus physically and functionally interacted with hepatocyte nuclear factor-4 alpha (HNF-4 alpha), a nuclear binding protein that regulates transcription of genes involved in both lipid and glucose metabolism. Five lines of evidence showed that ACBP bound HNF-4 alpha in vitro and in the nucleus of intact cells. (i) ACBP interaction with HNF-4 alpha elicited significant changes in secondary structure. (ii) ACBP and HNF-4 alpha were coimmunoprecipitated by antibodies to each protein. (iii) Double immunolabeling and laser scanning confocal microscopy (LSCM) of rat hepatoma cells and transfected COS-7 cells significantly colocalized ACBP and HNF-4 alpha within the nucleus and in the perinuclear region close to the nuclear membrane. (iv) LSCM fluorescence resonance energy transfer determined an intermolecular distance of 53 A between ACBP and HNF-4 alpha in rat hepatoma cell nuclei. (v) Immunogold electron microscopy detected ACBP within 43 A of HNF-4 alpha. These interactions were specific since ACBP did not interact with Sp1 or glucocorticoid receptor in these assays. The functional significance of ACBP interaction with HNF-4 alpha was evidenced by mammalian two-hybrid and transactivation assays. ACBP overexpression in COS-7 or rat hepatoma cells enhanced transactivation of an HNF-4 alpha-dependent luciferase reporter plasmid by 3.2- and 1.6-fold, respectively. In contrast, cotransfection with antisense ACBP expression vector inhibited transactivation. LSCM of the individual triple fluorescent-labeled (HNF-4 alpha, ACBP, and luciferase) rat hepatoma cells showed a high correlation (r2, 0.936) between the level of luciferase and the level of ACBP expression. In summary, ACBP physically interacted with HNF-4 alpha in vitro and in intact cells, although ACBP expression level directly correlated with HNF-4 alpha-mediated transactivation in individual cells.