p27Kip1 controls cell proliferation by binding to and regulating the activity of cyclin-dependent kinases (Cdks). Here we show that Cdk inhibition and p27 stability are regulated through direct phosphorylation by tyrosine kinases. A conserved tyrosine residue (Y88) in the Cdk-binding domain of p27 can be phosphorylated by the Src-family kinase Lyn and the oncogene product BCR-ABL. Y88 phosphorylation does not prevent p27 binding to cyclin A/Cdk2. Instead, it causes phosphorylated Y88 and the entire inhibitory 3(10)-helix of p27 to be ejected from the Cdk2 active site, thus restoring partial Cdk activity. Importantly, this allows Y88-phosphorylated p27 to be efficiently phosphorylated on threonine 187 by Cdk2 which in turn promotes its SCF-Skp2-dependent degradation. This direct link between transforming tyrosine kinases and p27 may provide an explanation for Cdk kinase activities observed in p27 complexes and for premature p27 elimination in cells that have been transformed by activated tyrosine kinases.

The apolipoprotein A5 gene (APOA5) has been repeatedly implicated in lowering plasma triglyceride levels. Since several studies have demonstrated that hyperinsulinemia is associated with hypertriglyceridemia, we sought to determine whether APOA5 is regulated by insulin. Here, we show that cell lines and mice treated with insulin down-regulate APOA5 expression in a dose-dependent manner. Furthermore, we found that insulin decreases human APOA5 promoter activity, and subsequent deletion and mutation analyses uncovered a functional E box in the promoter. Electrophoretic mobility shift and chromatin immunoprecipitation assays demonstrated that this APOA5 E box binds upstream stimulatory factors (USFs). Moreover, in transfection studies, USF1 stimulates APOA5 promoter activity, and the treatment with insulin reduced the binding of USF1/USF2 to the APOA5 promoter. The inhibition of the phosphatidylinositol 3-kinase (PI3K) pathway abolished insulin's effect on APOA5 gene expression, while the inhibition of the P70 S6 kinase pathway with rapamycin reversed its effect and increased APOA5 gene expression. Using an oligonucleotide precipitation assay for USF from nuclear extracts, we demonstrate that phosphorylated USF1 fails to bind to the APOA5 promoter. Taken together, these data indicate that insulin-mediated APOA5 gene transrepression could involve a phosphorylation of USFs through the PI3K and P70 S6 kinase pathways that modulate their binding to the APOA5 E box and results in APOA5 down-regulation. The effect of exogenous hyperinsulinemia in men showed a decrease in the plasma ApoAV level. These results suggest a potential contribution of the APOA5 gene in hypertriglyceridemia associated with hyperinsulinemia.Several epidemiological studies have established that, in addition to an elevated cholesterol level in low-density lipoprotein and reduced cholesterol level in high-density lipoprotein (HDL), hypertriglyceridemia is an independent risk factor for coronary heart diseases (12, 22). Moreover, hypertriglyceridemia is often associated with the metabolic syndrome that characterizes diabetes and obesity (21,35). Type 2 diabetes is frequently linked to hyperglycemia, hyperinsulinemia, and hypertriglyceridemia, and the leading cause of death for individuals with diabetes is cardiovascular diseases (34).The apolipoprotein A5 gene (APOA5) was identified through comparative sequence analysis of genomic DNA sequences and has been shown to be important in determining plasma triglyceride levels in mice and humans (42). This gene is mainly expressed in the liver and resides in HDL and very low density lipoprotein particles (42,59). It has been demonstrated that mice expressing a human APOA5 transgene showed a decrease in plasma triglyceride concentration to one-third the levels in control mice. Conversely, knockout mice lacking APOA5 had four times as much plasma triglycerides as controls. Moreover, adenoviral overexpression of APOA5 reduced serum levels of triglycerides and cholesterol in mice (60). Recent works focused on th...

Alterations in the expression of the recently discovered apolipoprotein A5 gene strongly affect plasma triglyceride levels. In this study, we investigated the contribution of APOA5 to the liver X receptor (LXR) ligandmediated effect on plasma triglyceride levels. Following treatment with the LXR ligand T0901317, we found that APOA5 mRNA levels were decreased in hepatoma cell lines. The observation that no down-regulation of APOA5 promoter activity was obtained by LXR-retinoid X receptor (RXR) co-transfection prompted us to explore the possible involvement of the known LXR target gene SREBP-1c (sterol regulatory element-binding protein 1c). In fact, we found that co-transfection with the active form of SREBP-1c down-regulated APOA5 promoter activity in a dose-dependent manner. We then scanned the human APOA5 promoter sequence and identified two putative E-box elements that were able to bind specifically SREBP-1c in gel-shift assays and were shown to be functional by mutation analysis. Subsequent suppression of SREBP-1 mRNA through small interfering RNA interference abolished the decrease of APOA5 mRNA in response to T0901317. Finally, administration of T0901317 to hAPOA5 transgenic mice revealed a significant decrease of APOA5 mRNA in liver tissue and circulating apolipoprotein AV protein in plasma, confirming that the described down-regulation also occurs in vivo. Taken together, our results demonstrate that APOA5 gene expression is regulated by the LXR ligand T0901317 in a negative manner through SREBP-1c. These findings may provide a new mechanism responsible for the elevation of plasma triglyceride levels by LXR ligands and support the development of selective LXR agonists, not affecting SREBP-1c, as beneficial modulators of lipid metabolism.Numerous epidemiological studies have revealed that, in addition to elevated low density lipoprotein and reduced high density lipoprotein levels, elevated plasma triglyceride (TG) 1 level is an independent risk factor for coronary heart disease (1, 2). Thus, understanding the regulation of genes that influence triglyceride levels is of prime interest and may aid in the development of therapies to reduce hypertriglyceridemia and the associated risk of atherosclerosis. The apolipoprotein gene family represents one class of molecules that affect plasma lipid levels. Apolipoprotein AV (apoAV) is a newly described member of the apolipoprotein family that was recently identified through human-mouse comparative sequence analysis (3).The APOA5 gene appears mainly expressed in the liver, where its gene product (apoAV) is exported into plasma and associates with high density lipoprotein and very low density lipoprotein particles. Studies in mice have revealed a crucial role of apoAV in plasma triglyceride metabolism with over-and underexpression in animals leading to decreased and increased triglycerides, respectively (3). Furthermore, in humans, several single nucleotide polymorphisms across the APOA5 locus were found to be significantly associated with high plasma triglyceride le...