Pim-1L Protects Cell Surface–Resident ABCA1 From Lysosomal Degradation in Hepatocytes and Thereby Regulates Plasma High-Density Lipoprotein Level

Abstract

Objective-ATP-binding cassette transporter A1 (ABCA1) exerts an atheroprotective action through the biogenesis of highdensity lipoprotein in hepatocytes and prevents the formation of foam cells from macrophages. Controlling ABCA1 is a rational approach to improving atherosclerotic cardiovascular disease. Although much is known about the regulatory mechanism of ABCA1 synthesis, the molecular mechanism underpinning its degradation remains to be clearly described. Approach and Results-ABCA1 possesses potential sites of phosphorylation by serine/threonine-protein kinase Pim-1 (Pim-1). Pim-1 depletion decreased the expression of cell surface-resident ABCA1 (csABCA1) and apolipoprotein A-I-mediated [ 3 H]cholesterol efflux in the human hepatoma cell line HepG2, but not in peritoneal macrophages from mice. In vitro kinase assay, immunoprecipitation, and immunocytochemistry suggested phosphorylation of csABCA1 by the long form of Pim-1 (Pim-1L). Cell surface biotinylation indicated that Pim-1L inhibited lysosomal degradation of csABCA1 involving the liver X receptor β, which interacts with csABCA1 and thereby protects it from ubiquitination and subsequent lysosomal degradation. Cell surface coimmunoprecipitation with COS-1 cells expressing extracellularly hemagglutinin-tagged ABCA1 showed that Pim-1L-mediated phosphorylation of csABCA1 facilitated the interaction between csABCA1 and liver X receptor β and thereby stabilized the csABCA1-Pim-1L complex. Mice deficient in Pim-1 kinase activity showed lower expression of ABCA1 in liver plasma membranes and lower plasma high-density lipoprotein levels than control mice. Conclusions-Pim-1L protects hepatic csABCA1 from lysosomal degradation by facilitating the physical interaction between csABCA1 and liver X receptor β and subsequent stabilization of the csABCA1-Pim-1L complex and thereby regulates the circulating level of high-density lipoprotein. Our findings may aid the development of high-density lipoproteintargeted therapy. Katsube et al Protection of Hepatic csABCA1 by Pim-1L 2305mechanism underlying the degradation of ABCA1 remains elusive. Although in vitro and animal studies using inhibitors of the protease calpain and proteasome suggest that they cleave and degrade ABCA1, [16][17][18][19] the molecular entity of calpain and the molecular mechanism of the degradation of ABCA1 involving proteasome have not yet been clearly described. On the basis of the report by Hozoji et al 20 that LXRβ interacts with and post-translationally modulates ABCA1, we recently found that LXRβ protects the cell surface-resident ABCA1 (csABCA1) from ubiquitination through its physical interaction with csABCA1 and not through its transcriptional activity, which prevents lysosomal degradation of csABCA1 through the endosomal sorting complex required for transport (ESCRT) system. 21,22 This mechanism mediates ABCA1 degradation independently of the pathway involving calpain and proteasome. To gain further insight into this finding, the current study focuses on phosphorylation because phosphory...

Temporary sequestration of cholesterol and phosphatidylcholine within extracellular domains of ABCA1 during nascent HDL generation

“…To examine whether ECDs of ABCA1 were released to the medium by trypsin, BHK cells expressing ABCA1-207HA, in which HA epitope was inserted in ECD1 at position 207, were established. This insertion was reported to have no effects on subcellular localization or functions of ABCA1 17 , 24 , 25 . Then, the slot blot analyses were performed to detect fragments of ECD1 by using anti-HA antibody (Fig.…”

GLUT6 is a lysosomal transporter that is regulated by inflammatory stimuli and modulates glycolysis in macrophages

“…Isolation of mice peritoneal macrophages (MPMs) was performed as previously described . Preparation of bone marrow derived macrophages (BMDM) was conducted as previously described . Briefly, bone marrow cells were isolated from the femurs and tibias of mice and flushed with sterile PBS using a pair of 25‐gauge needle and syringe.…”

mTORC1 suppresses PIM3 expression via miR-33 encoded by the SREBP loci

“…Consistent with their expression requiring transcriptional induction of the SREBP1/2 loci, the majority of miR-33 targets that have been characterized to date are involved in fatty acid and cholesterol metabolism 27 , 32 , 62 . Given that the PIM kinases are believed to have a high degree of functional redundancy, it is interesting to note that PIM1 appears to stabilize the cholesterol transporter ABCA1, which is also a canonical miR-33 target, providing a rationale for PIM targeting by miR-33 63 . While many canonical PIM targets are involved in cell survival, recent work has also implicated them in various metabolic pathways, including glycolysis and mitochondrial biogenesis 64 – 67 .…”