Prior studies provide data supporting the notion that ATP binding cassette transporter A1 (ABCA1) promotes lipid efflux to extracellular acceptors in a two-step process: first, ABCA1 mediates phospholipid efflux to an apolipoprotein, and second, this apolipoprotein-phospholipid complex accepts free cholesterol in an ABCA1-independent manner. In the current study using RAW264.7 cells, ABCA1-mediated free cholesterol and phospholipid efflux to apolipoprotein A-I (apoA-I) were tightly coupled to each other both temporally and after treatment with ABCA1 inhibitors. The time course and temperature dependence of ABCA1-mediated lipid efflux to apoA-I support a role for endocytosis in this process. Cyclodextrin treatment of RAW264.7 cells partially inhibited 8Br-cAMP-induced efflux of free cholesterol and phospholipid to apoA-I. ABCA1-expressing cells are more sensitive to cell damage by highdose cyclodextrin and vanadate, leading to increased lactate dehydrogenase leakage and phospholipid release even in the absence of the acceptor apoA-I. Finally, we could not reproduce a two-step effect on lipid efflux using conditioned medium from ABCA1-expressing cells pretreated with cyclodextrin. Cellular expression of ATP binding cassette transporter A1 (ABCA1) promotes the efflux of both free cholesterol (FC) and phospholipids (PLs) to extracellular acceptors such as apolipoprotein A-I (apoA-I); however, the mechanism of this efflux is not understood. Fielding et al. (1) proposed a two-step mechanism in which ABCA1 mediates PL efflux to apoA-I, which in turn can then pick up FC in an ABCA1-independent autocrine or paracrine manner. This conclusion was based on two types of experiments: 1 ) PL efflux from vascular smooth muscle cells to apoA-I is less sensitive to vanadate inhibition than FC efflux, and 2 ) medium containing apoA-I that is conditioned on smooth muscle cells can lead to FC efflux from vascular endothelial cells that do not express ABCA1 (1). Wang et al. (2) provided evidence for the two-step pathway by demonstrating that 1 ) a 30 min pretreatment of ABCA1-expressing cells with 20 mM 2-hydroxypropyl-␤ -cyclodextrin reduced FC efflux to apoA-I without reducing PL efflux, and 2 ) medium containing apoA-I that is conditioned on cyclodextrin-pretreated ABCA1-expressing cells could lead to FC efflux from cells that do not express ABCA1. These experiments and others from Chimini and colleagues (3, 4), who demonstrated that ABCA1 could mediate phosphatidylserine translocase activity, have led to the notion that the primary activity of ABCA1 is the assembly of PL onto acceptors and that FC efflux follows passively by a mechanism not dependent on ABCA1.We demonstrate here that FC and PL efflux to apoA-I is concurrent in the RAW264.7 murine macrophage cell line, in which ABCA1 expression is inducible by cAMP analogs (5, 6). ABCA1-mediated lipid efflux has delayed kinetics and is abolished at room temperature, results that are consistent with the need for endocytosis and vesicular trafficking for efflux to occur. We...

The following two theories for the mechanism of ABCA1 in lipid efflux to apolipoprotein acceptors have been proposed: 1) that ABCA1 directly binds the apolipoprotein ligand and then facilitates lipid efflux and 2) that ABCA1 acts as a phosphatidylserine (PS) translocase, increasing PS levels in the plasma membrane exofacial leaflet, and that this is sufficient to facilitate apolipoprotein binding and lipid assembly. Upon induction of ABCA1 in RAW264.7 cells by cAMP analogues there was a moderate increase in cell surface PS as detected by annexin V binding, whereas apoAI binding was increased more robustly. Apoptosis induced large increases in annexin V and apoAI binding; however, apoptotic cells did not efflux lipids to apoAI. Annexin V did not act as a cholesterol acceptor, and it did not compete for the cholesterol acceptor or cell binding activity of apoAI. ApoAI binds to ABCA1-expressing cells, and with incubation at 37°C apoAI is co-localized within the cells in ABCA1-containing endosomes. Fluorescent recovery after photobleaching demonstrated that apoAI bound to ABCA1-expressing cells was relatively immobile, suggesting that it was bound either directly or indirectly to an integral membrane protein. Although ABCA1 induction was associated with a small increase in cell surface PS, these results argue against the notion that this cell surface PS is sufficient to mediate cellular apoAI binding and lipid efflux.The ATP binding cassette (ABC) 1 family of proteins serves to pump a diverse set of molecules out of cells. ABCA1, the Tangier disease gene, is required for cholesterol and phospholipid efflux to lipid-free apolipoproteins (for review see Ref. 1). The exact mechanism of this lipid efflux pump and the location of the lipid transfer and assembly onto apolipoprotein acceptors have yet to be determined definitively. It has been shown previously that ABCA1 expression leads to increased cell surface binding and uptake of apoAI (2). Protein cross-linking studies have shown that cell surface ABCA1 is closely associated with exogenously added apoAI (3, 4). Thus, one theory of ABCA1 action is that the apolipoprotein ligand is bound directly to the ABCA1 receptor, which can then mediate lipid assembly to form a nascent lipoprotein. An alternative theory of ABCA1 function has been put forth by Chimini and coworkers (5, 6). Phosphatidylserine (PS) is normally asymmetrically distributed on the plasma membrane such that most of it is sequestered on the inner leaflet. Increased levels of PS in the exofacial leaflet have been demonstrated in ABCA1 transiently transfected cells, thus providing evidence that ABCA1 may function as a PS translocase to pump PS from the cytoplasmic leaflet to the exofacial leaflet (5).This activity is referred to as "floppase" activity to distinguish it from the "flippase" activity that pumps PS from the outer to inner plasma membrane leaflets (7). These authors propose that PS translocase activity is sufficient to lead to apoAI binding to and lipid efflux from ABCA1-expressing cells and that...

Growth factors activate Raf-1 by engaging a complex program, which requires Ras binding, membrane recruitment, and phosphorylation of Raf-1. The present study employs the microtubule-depolymerizing drug nocodazole as an alternative approach to explore the mechanisms of Raf activation. Incubation of cells with nocodazole leads to activation of Pak1/2, kinases downstream of small GTPases Rac/Cdc42, which have been previously indicated to phosphorylate Raf-1 Ser 338 . Nocodazole-induced stimulation of Raf-1 is augmented by co-expression of small GTPases Rac/Cdc42 and Pak1/2. Dominant negative mutants of these proteins block activation of Raf-1 by nocodazole, but not by epidermal growth factor (EGF). Thus, our studies define Rac/ Cdc42/Pak as a module upstream of Raf-1 during its activation by microtubule disruption. Although it is Ras-independent, nocodazole-induced activation of Raf-1 appears to involve the amino-terminal regulatory region in which the integrity of the Ras binding domain is required. Surprisingly, the Raf zinc finger mutation (C165S/C168S) causes a robust activation of Raf-1 by nocodazole, whereas it diminishes Ras-dependent activation of Raf-1. We also show that mutation of residues Ser 338 to Ala or Tyr 340 -Tyr 341 to Phe-Phe immediately amino-terminal to the catalytic domain abrogates activation of both the wild type and zinc finger mutant Raf by both EGF/4␤-12-O-tetradecanoylphorbol-13-acetate and nocodazole. Finally, an in vitro kinase assay demonstrates that the zinc finger mutant serves as a better substrate of Pak1 than the wild type Raf-1. Collectively, our results indicate that 1) the zinc finger exerts an inhibitory effect on Raf-1 activation, probably by preventing phosphorylation of 338 SSYY 341 ; 2) such inhibition is first overcome by an unknown factor binding in place of Ras-GTP to the amino-terminal regulatory region in response to nocodazole; and 3) EGF and nocodazole utilize different kinases to phosphorylate Ser 338 , an event crucial for Raf activation.The proto-oncogene raf-1, first identified as a cellular counterpart of the oncogene v-raf, encodes a serine/threonine protein kinase. Raf-1 is ubiquitously expressed and plays an important role in cell proliferation and differentiation (1). The mechanism by which Raf-1 is activated by growth factors is still incompletely understood, although it is known to be preassembled as a complex with 14-3-3 and heat shock proteins 90/50 (2-6). It involves multiple steps including Ras-GTP binding, membrane recruitment, and phosphorylation.Raf-1 consists of an amino-terminal regulatory domain and a carboxyl-terminal kinase domain. The amino-terminal moiety of Raf-1 exerts an inhibitory effect on the catalytic activity, since amino-terminal truncations lead to progressive increases in its transforming ability (7,8). The amino-terminal regulatory region of Raf-1 contains a Ras binding domain (RBD) 1 and a cysteine-rich zinc finger domain (CRD), both of which participate in binding to Ras. The first interaction engages Raf RBD ranging from ...

The present study characterizes the interaction between the Raf-1 kinase domain and MEK1 and examines whether the magnitude of their interaction correlates to the ability of Raf to phosphorylate MEK1. Here we show that the minimal domain required for the Raf kinase activity starts from tryptophan 342.

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