Apolipoprotein A-I Activates Protein Kinase Cα Signaling to Phosphorylate and Stabilize ATP Binding Cassette Transporter A1 for the High Density Lipoprotein Assembly

ATP-binding cassette transporter A1 (ABCA1), a molecule mediating free cholesterol efflux from peripheral tissues to apoAI and high density lipoprotein (HDL), inhibits the formation of lipid-laden macrophage/foam cells and the development of atherosclerosis. ERK1/2 are important signaling molecules regulating cellular growth and differentiation. The ERK1/2 signaling pathway is implicated in cardiac development and hypertrophy. However, the role of ERK1/2 in the development of atherosclerosis, particularly in macrophage cholesterol homeostasis, is unknown. In this study, we investigated the effects of ERK1/2 activity on macrophage ABCA1 expression and cholesterol efflux. Compared with a minor effect by inhibition of other kinases, inhibition of ERK1/2 significantly increased macrophage cholesterol efflux to apoAI and HDL. In contrast, activation of ERK1/2 reduced macrophage cholesterol efflux and ABCA1 expression. The increased cholesterol efflux by ERK1/2 inhibitors was associated with the increased ABCA1 levels and the binding of apoAI to cells. The increased ABCA1 by ERK1/2 inhibitors was due to increased ABCA1 mRNA and protein stability. The induction of ABCA1 expression and cholesterol efflux by ERK1/2 inhibitors was concentration-dependent. The mechanism study indicated that activation of liver X receptor (LXR) had little effect on ERK1/2 expression and activation. ERK1/2 inhibitors had no effect on macrophage LXR␣/␤ expression, whereas they did not influence the activation or the inhibition of the ABCA1 promoter by LXR or sterol regulatory element-binding protein (SREBP). However, inhibition of ERK1/2 and activation of LXR synergistically induced macrophage cholesterol efflux and ABCA1 expression. Our data suggest that ERK1/2 activity can play an important role in macrophage cholesterol trafficking.Development of atherosclerotic lesions in coronary arteries is an underlying cause of coronary heart disease. Lipid-laden macrophage/foam cells are a prominent part of atherosclerotic lesions (1). Cellular cholesterol content in macrophages is determined by uptake and efflux of cholesterol (2). The type A scavenger receptor and type B scavenger receptor (CD36) mediate the binding and internalization of modified low density lipoprotein (LDL), 3 thus, demonstrating pro-atherogenic properties (3, 4). In contrast, type BI scavenger receptor and ATPbinding cassette transporter A1 (ABCA1) can mediate cellular free cholesterol efflux to extracellular high density lipoprotein (HDL) or lipid-free apolipoprotein AI (apoAI) thereby inhibiting the development of atherosclerosis (5, 6). Compared with bi-directional cholesterol transport across cellular membranes that are mediated by type BI scavenger receptor (7, 8), ABCA1 stimulates free cholesterol efflux from macrophages and other peripheral cell types to apoAI and/or HDL by using the energy from ATP hydrolysis (9). The binding of free cholesterol and phospholipids to apoAI also leads to the generation of nascent HDL (10). ABCA1 can also mediate cholesterol efflux to exog...

Section: Discussionmentioning

confidence: 99%

Inhibition of ERK1/2 and Activation of Liver X Receptor Synergistically Induce Macrophage ABCA1 Expression and Cholesterol Efflux

Zhou

Yin

Guo

et al. 2010

“…The functional significance of the conserved sequences downstream of the NBDs is still unknown; they might have a regulatory function, as proposed for other transporters (12). Phosphorylation by protein kinase A or C has been demonstrated for several ABC transporters, including cystic fibrosis transmembrane conductance regulator (13), P-glycoprotein (14), and recently ABCA1 (15)(16)(17). According to See et al (15) serines 1042 and 2054 in NBD are phosphorylated by PKA in vitro and may potentially regulate ABCA1 function (15).…”

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confidence: 99%

“…Mutation of a PKA phosphorylation site in ABCA1 impaired apoAI-mediated release of cellular lipid (15). According to Yamauchi et al (16) ABCA1 phosphorylation is, furthermore, critical for the stability and integrity of the transporter (16). PKC ␣ is activated by apoAI, and it subsequently phosphorylates and stabilizes ABCA1.…”

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confidence: 99%

“…According to See et al (15) serines 1042 and 2054 in NBD are phosphorylated by PKA in vitro and may potentially regulate ABCA1 function (15). Yamauchi et al (16) report that apoAI activates PKC, resulting in phosphorylation and stabilization of ABCA1 (16). According to Martinez et al (17), the potential phosphorylation sites for protein kinase 2 (CK2) and PKA in the ABCA1-PEST sequence, threonine 1286 and 1305, respectively, are constitutively phosphorylated and protect ABCA1 against calpain-mediated degradation.…”

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confidence: 99%

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Phosphorylation by Protein Kinase CK2 Modulates the Activity of the ATP Binding Cassette A1 Transporter

Roosbeek

Peelman

Verhee

et al. 2004

In a previous characterization of the ABCA subfamily of the ATP-binding cassette (ABC) transporters, we identified potential protein kinase 2 (CK2) phosphorylation sites, which are conserved in eukaryotic and prokaryotic members of the ABCA transporters (Peelman, F., Labeur, C., Vanloo, B., Roosbeek, S., Devaud, C., Duverger, N., Denefle, P., Rosier, M., Vandekerckhove, J., and Rosseneu, M. (2003) J. Mol. Biol. 325, 259 -274). These phosphorylation residues are located in the conserved cytoplamic R1 and R2 domains, downstream of the nucleotide binding domains NBD1 and NBD2. To study the possible regulation of the ABCA1 transporter by CK2, we expressed the recombinant cytoplasmic domains of ABCA1, NBD1؉R1 and NBD2؉R2. We demonstrated that in vitro ABCA1 NBD1؉R1, and not NBD2؉R2, is phosphorylated by CK2, and we identified Thr-1242, Thr-1243, and Ser-1255 as the phosphorylated residues in the R1 domain by mass spectrometry. We further investigated the functional significance of the threonine and serine phosphorylation sites in NBD1 by site-directed mutagenesis of the entire ABCA1 followed by transfection into Hek-293 Tet-Off cells. The ABCA1 flippase activity, apolipoprotein AI and AII binding, and cellular phospholipid and cholesterol efflux were enhanced by mutations preventing CK2 phosphorylation of the threonine and serine residues. This was confirmed by the effect of specific protein kinase CK2 inhibitors upon the activity of wild type and mutant ABCA1 in transfected Hek-293 Tet-Off cells. The activities of the mutants mimicking threonine phosphorylation were close to that of wild type ABCA1. Our data, therefore, suggest that besides protein kinase A and C, protein kinase CK2 might play an important role in vivo in regulating the function and transport activity of ABCA1 and possibly of other members of the ABCA subfamily.The role of the ABCA1 1 transporter, a member of the subfamily A of the ATP binding cassette transporters, in the efflux of cellular phospholipids and cholesterol has become well established (2). Several studies link ABCA1 mutations to impaired cellular cholesterol and phospholipid efflux characteristic of Tangier disease and high density lipoprotein-deficiency patients (3-5). Expression of WT and mutant ABCA1 in cultured cells demonstrated the correlation between the level of expression and activity of the ABCA1 transporter, the extent of binding to the apolipoprotein AI (apoAI) acceptor and the efflux of cellular lipid (6).Human ABC transporters consist of a cytoplasmic nucleotide binding domain (NBD), which binds and hydrolyzes ATP, and of a membrane-spanning domain through which the substrate is translocated (7-8). Besides these elements, regulatory domains with putative phosphorylation sites were described in several human transporters (7). We carried out an extensive analysis of the subfamily A of the ABC transporters and showed that this subfamily consists of 13 human ABCA and of many eukaryotic and prokaryotic ABCA homologues (1). Multiple alignments of the subfamily A transporters de...

“…On the other hand, stabilization of ABCA1 can be an alternative mechanism to regulate ABCA1 activity, such as protection of ABCA1 protein by helical apolipoprotein (26 -28) and enhancement of its degradation by unsaturated fatty acid (29) or overloaded cholesterol (30). Previous studies have suggested that ABCA1 phosphorylation is involved in its stabilization (31,32). A PDZ adaptor protein ␣1-syntrophin was shown to stabilize ABCA1 (33).…”

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confidence: 99%

Probucol Inactivates ABCA1 in the Plasma Membrane with Respect to Its Mediation of Apolipoprotein Binding and High Density Lipoprotein Assembly and to Its Proteolytic Degradation

Chengai

Tsujita

Hayashi

et al. 2004

Self Cite

Probucol has been shown to inhibit the release of cellular lipid by helical apolipoprotein and thereby to reduce plasma high density lipoprotein. We attempted to explore the underlying mechanism for this effect in human fibroblast WI-38. Probucol inhibited the apoA-Imediated cellular lipid release and binding of apoA-I to the cells in a dose-dependent manner. It did not influence cellular uptake of low density lipoprotein, transport of cholesterol to the cell surface whether de novo synthesized or delivered as low density lipoprotein, and overall cellular content of cholesterol, although biosynthesis of lipids from acetate was somewhat increased. Probucol did not affect the mRNA level of ABCA1, and ABCA1 was recovered along with marker proteins for plasma membrane regardless of the presence of probucol. However, the protein level of ABCA1 increased, and the rate of its decay in the presence of cycloheximide was slower in the probucol-treated cells. ABCA1 in the probucol-treated cells was resistant to digestion by calpain but not by trypsin. We concluded that probucol inactivates ABCA1 in the plasma membrane with respect to its function in mediating binding of and lipid release by apolipoprotein and with respect to proteolytic degradation by calpain.Cholesterol is an essential molecule for animal cells to maintain and regulate function and structure of the biomembrane. It is synthesized in most somatic cells, whereas its catabolic site is limited to the liver and to the steroidogenic cells except for partial hydroxylation in some somatic cells. Accordingly, cholesterol is removed from the cells and transported to the liver for its conversion to bile acids, and this is one of the essential events in cholesterol homeostasis for the body and for the cells (1). High density lipoprotein (HDL) 1 is believed to play a central role in this system, and this is thought to be one of the antiatherogenic characteristics of HDL. This reaction takes place through at least two distinct mechanisms: 1) physicochemical release of cholesterol from the cell surface, which is driven by cholesterol esterification on HDL, and 2) the apolipoprotein-mediated pathway to remove cellular cholesterol and phospholipid to generate new HDL particles (2). HDL thus plays a central role in both mechanisms.Apolipoprotein-dependent cellular cholesterol release is absent in fibroblasts from patients with Tangier disease (3, 4), and mutations in the gene encoding the ATP-binding cassette transporter A1 (ABCA1) are the underlying cause of this disease (5-9). On the other hand, in vitro overexpression of functional ABCA1 in the cells (10, 11) and induction of ABCA1 expression by cyclic AMP analogues (12, 13) or by the ligands for the liver X receptor or retinoid X receptor (14, 15) enhanced the release of cellular cholesterol and phospholipid by apolipoprotein. The transgenic mice for ABCA1 had a significant increase in plasma HDL (16,17). These results indicate that this protein is a regulating factor for the plasma HDL level through generation of HDL b...