Phosphorylation of a Pest Sequence in ABCA1 Promotes Calpain Degradation and Is Reversed by ApoA-I

Abstract

ATP-binding cassette transporter A1 (ABCA1), the defective molecule in Tangier disease, mediates the apoAI-dependent efflux of excess cholesterol from cells. We recently showed that ABCA1 proteolysis by calpain was dependent on a PEST sequence in the cytoplasmic region of ABCA1 and was reversed by apoA-I interaction with ABCA1. We show here that phosphorylation of ABCA1 in HEK293 cells was reduced by 63 ± 2.4% after removal of the PEST sequence (ABCA1delPEST) or by incubation of cells with apoAI (58 ± 3.3%). By contrast, ABCA1delPEST showed no further decrease of phosphorylation upon apoAI treatment. To assess the hypothesis that PEST sequence phosphorylation could regulate ABCA1 calpain proteolysis, we mutagenized S/T residues in the PEST sequence and identified Thr-1286 and Thr-1305 as constitutively phosphorylated residues. The ABCA1-T1286A/T1305A mutant was not degraded by calpain and was not further stabilized upon apoA-I treatment. The T1286A/T1305A mutant showed a 3.1-fold increase in cell surface expression and a 2.3-fold increase of apoAI-mediated cholesterol efflux compared with wild type ABCA1. In conclusion, we propose a mechanism of regulation of ABCA1 cell surface expression and function in which the interaction with apoA-I results in dephosphorylation of the ABCA1 PEST sequence and thereby inhibits calpain degradation leading to an increase of ABCA1 cell surface expression. ATP-binding cassette transporter A1 (ABCA1), the defective molecule in Tangier disease, mediates the apoAI-dependent efflux of excess cholesterol from cells. We recently showed that ABCA1 proteolysis by calpain was dependent on a PEST sequence in the cytoplasmic region of ABCA1 and was reversed by apoA-I interaction with ABCA1. We show here that phosphorylation of ABCA1 in HEK293 cells was reduced by 63 ± 2.4% after removal of the PEST sequence (ABCA1delPEST) or by incubation of cells with apoAI (58 ± 3.3%). By contrast, ABCA1delPEST showed no further decrease of phosphorylation upon apoAI treatment. To assess the hypothesis that PEST sequence phosphorylation could regulate ABCA1 calpain proteolysis, we mutagenized S/T residues in the PEST sequence and identified Thr-1286 and Thr-1305 as constitutively phosphorylated residues. The ABCA1-T1286A/T1305A mutant was not degraded by calpain and was not further stabilized upon apoA-I treatment. The T1286A/T1305A mutant showed a 3.1-fold increase in cell surface expression and a 2.3-fold increase of apoAI-mediated cholesterol efflux compared with wild type ABCA1. In conclusion, we propose a mechanism of regulation of ABCA1 cell surface expression and function in which the interaction with apoA-I results in dephosphorylation of the ABCA1 PEST sequence and thereby inhibits calpain degradation leading to an increase of ABCA1 cell surface expression. Atherosclerosis, the major cause of death in industrialized societies (1Assmann G. Carmena R. Cullen P. Fruchart J.C. Jossa F. Lewis B. Mancini M. Paoletti R. Circulation. 1999; 100: 1930-1938Crossref PubMed Scopus (104) Google Scholar), is initiated by the deposition of lipoprotein cholesterol in the artery wall and its subsequent uptake by macrophages, giving rise to cholesterol-engorged foam cells (2Williams K.J. Tabas I. Curr. Opin. Lipidol. 1998; 9: 471-474Crossref PubMed Scopus (299) Google Scholar). Elevated levels of HDL 1The abbreviations used are: HDL, high density lipoprotein; ABCA1, ATP-binding cassette transporter A1; PEST, proline-glutamic acid-serine-threonine; apo, apolipoprotein; PBS, phosphate-buffered saline; CK2, protein kinase CK2 (formerly caseine kinase II); PKA, protein kinase A; DMEM, Dulbecco's modified Eagle's medium; LXR, liver X receptor.1The abbreviations used are: HDL, high density lipoprotein; ABCA1, ATP-binding cassette transporter A1; PEST, proline-glutamic acid-serine-threonine; apo, apolipoprotein; PBS, phosphate-buffered saline; CK2, protein kinase CK2 (formerly caseine kinase II); PKA, protein kinase A; DMEM, Dulbecco's modified Eagle's medium; LXR, liver X receptor. are associated with a decrease in atherosclerosis that is believed to be due in part to the ability of HDL and its apolipoproteins to remove cholesterol from foam cells (3DeLamatre J. Wolfbauer G. Phillips M.C. Rothblat G.H. Biochim. Biophys. Acta. 1986; 875: 419-428Crossref PubMed Scopus (44) Google Scholar, 4Sviridov D. Nestel P. Atherosclerosis. 2002; 161: 245-254Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 5Tall A.R. Costet P. Wang N. J. Clin. Invest. 2002; 110: 899-904Crossref PubMed Scopus (186) Google Scholar). A breakthrough in this area of research has been the identification of mutations in the ATP-binding cassette transporter A1 (ABCA1) (6Brooks-Wilson A. Marcil M. Clee S.M. Zhang L.H. Roomp K. van Dam M. Yu L. Brewer C. Collins J.A. Molhuizen H.O. Loubser O. Ouelette B.F. Fichter K. Ashbourne-Excoffon K.J. Sensen C.W. Scherer S. Mott S. Denis M. Martindale D. Frohlich J. Morgan K. Koop B. Pimstone S. Kastelein J.J. Hayden M.R. Nat. Genet. 1999; 22: 336-345Crossref PubMed Scopus (1496) Google Scholar, 7Bodzioch M. Orso E. Klucken J. Langmann T. Bottcher A. Diederich W. Drobnik W. Barlage S. Buchler C. Porsch-Ozcurumez M. Kaminski W.E. Hahmann H.W. Oette K. Rothe G. Aslanidis C. Lackner K.J. Schmitz G. Nat. Genet. 1999; 22: 347-351Crossref PubMed Scopus (1338) Google Scholar, 8Rust S. Rosier M. Funke H. Real J. Amoura Z. Piette J.C. Deleuze J.F. Brewer H.B. Duverger N. Denefle P. Assmann G. Nat. Genet. 1999; 22: 352-355Crossref PubMed Scopus (1258) Google Scholar) as the genetic defect in Tangier disease, a disorder characterized by very low plasma HDL levels, defective apolipoprotein-mediated phospholipid, and cholesterol efflux from cells (9Francis G.A. Knopp R.H. Oram J.F. J. Clin. Invest. 1995; 96: 78-87Crossref PubMed Scopus (372) Google Scholar), macrophage foam cell accumulation in various tissues, including arteries and susceptibility to atherosclerosis (10Schaefer E.J. Zech L.A. Schwartz D.E. Brewer Jr., H.B. Ann. Intern. Med. 1980; 93: 261-266Crossref PubMed Scopus (174) Google Scholar). The phenotype of Tangier Disease is consistent with the proposed function of ABCA1 as a cell surface transporter that promotes the efflux of cellular phospholipid and cholesterol to lipid-poor apolipoproteins, a process that constitutes the initial step in HDL formation (11Hara H. Yokoyama S. J. Biol. Chem. 1991; 266: 3080-3086Abstract Full Text PDF PubMed Google Scholar, 12Young S.G. Fielding C.J. Nat. Genet. 1999; 22: 316-318Crossref PubMed Scopus (117) Google Scholar). Thus, the up-regulation of ABCA1 expression may provide a key to HDL formation and promotion of foam cell cholesterol efflux. The cellular expression of ABCA1 is highly regulated both on transcriptional and post-transcriptional levels (13Wang N. Tall A.R. Arterioscler. Thromb. Vasc. Biol. 2003; 23: 1178-1184Crossref PubMed Scopus (217) Google Scholar). The turnover of ABCA1 protein is rapid with a half-life of less than 1 h in murine macrophage-like cells and differentiated THP-1 cells (14Oram J.F. Lawn R.M. Garvin M.R. Wade D.P. J. Biol. Chem. 2000; 275: 34508-34511Abstract Full Text Full Text PDF PubMed Scopus (468) Google Scholar, 15Arakawa R. Yokoyama S. J. Biol. Chem. 2002; 277: 22426-22429Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar). Recently we showed that ABCA1 protein degradation is regulated by a PEST sequence in the cytoplasmic region of ABCA1 and mediated by calpain protease (16Wang N. Chen W. Linsel-Nitschke P. Martinez L.O. Agerholm-Larsen B. Silver D.L. Tall A.R. J. Clin. Invest. 2003; 111: 99-107Crossref PubMed Scopus (242) Google Scholar). Interestingly, the interaction of ABCA1 with extracellular apoA-I inhibits calpain protease degradation in a PEST sequence-dependent fashion and thereby increases ABCA1 protein level at the cell surface (16Wang N. Chen W. Linsel-Nitschke P. Martinez L.O. Agerholm-Larsen B. Silver D.L. Tall A.R. J. Clin. Invest. 2003; 111: 99-107Crossref PubMed Scopus (242) Google Scholar). Arakawa and Yokoyama (15Arakawa R. Yokoyama S. J. Biol. Chem. 2002; 277: 22426-22429Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar) independently discovered the stabilization of ABCA1 by apoA-I and suggested its degradation by a thiol protease. In the present study, we attempted to elucidate the nature of the signal by which apoA-I can reverse the PEST-dependent ABCA1 degradation by calpain. We first observed that apoA-I promotes PEST sequence dephosphorylation. It has been reported that calpain-mediated degradation can be induced by the phosphorylation of a target protein (17Shen J. Channavajhala P. Seldin D.C. Sonenshein G.E. J. Immunol. 2001; 167: 4919-4925Crossref PubMed Scopus (94) Google Scholar), which led us to the hypothesis that the ABCA1-PEST sequence phosphorylation might regulate ABCA1 calpain proteolysis. We identified Thr-1286 and Thr-1305 as constitutively phosphorylated sites in the ABCA1-PEST sequence and then examined the critical role of the Thr-1286 and Thr-1305 phosphorylation sites in the process of ABCA1 degradation by calpain. Chemicals and Reagents—Human apoA-I (BIODESIGN International, Saco, ME) was dialyzed against PBS. M2 anti-FLAG antibody was from Sigma (St. Louis, MO); the polyclonal ABCA1[159-F11] antibody, directed to the amino acid sequence of mouse and human ABCA1 (CLTSFLQDEKVKESYV), was kindly provided by Dr. R. Barbaras (INSERM U563, Toulouse, France). A protease inhibitor mixture set was from Roche Applied Science (Indianapolis, IN). Phosphatase inhibitor mixture set, purified μ-calpain, and calpeptin were from Calbiochem-Novabiochem Corp. (San Diego, CA). All cell culture reagents were form Invitrogen. All other reagents (analytical grade) were from Sigma (St. Louis, MO). Plasmid Constructs and Cell Transfection—mABCA1-FLAG, deleted of the PEST sequence (amino acids 1283–1306), was constructed as described previously (18Wang N. Silver D.L. Costet P. Tall A.R. J. Biol. Chem. 2000; 275: 33053-33058Abstract Full Text Full Text PDF PubMed Scopus (501) Google Scholar). mABCA1-FLAG mutation constructs on Thr-1286, Ser-1296, Ser-1302, and The-1305 to Ala were generated by PCR using Pfu polymerase (Stratagene) and confirmed by sequencing. mABCA1-FLAG mutation constructs were further called: MutAAAA for mutation to Ala on T1286A/S1296A/S1302A/T1305A, MutTAAT for mutation to Ala on S1296A/S1302A, and MutASSA for mutation to Ala on T1286A/T1305A. HEK293 cells were grown in 6-, 12-, or in Dulbecco's modified Eagle's then as previously described (16Wang N. Chen W. Linsel-Nitschke P. Martinez L.O. Agerholm-Larsen B. Silver D.L. Tall A.R. J. Clin. Invest. 2003; 111: 99-107Crossref PubMed Scopus (242) Google Scholar). 6-, 12-, and were with and of were from by with after with 1 of The cells were and to by incubation for h at in with removal of cells by with PBS, the cells were further for in To ABCA1 protein cells were with the The after cells were used for phosphorylation and of with HEK293 cells or mouse macrophages, were with and in for 1 h at were then with for h at cells were with as and then at with as previously described (16Wang N. Chen W. Linsel-Nitschke P. Martinez L.O. Agerholm-Larsen B. Silver D.L. Tall A.R. J. Clin. Invest. 2003; 111: 99-107Crossref PubMed Scopus (242) Google Scholar) in the of and mouse macrophages, the cell were first with protein then were with of by a incubation with protein HEK293 cells with constructs the the cell were with of anti-FLAG both cells the were then with the and in of were a and to associated with the ABCA1 was with a were generated by with for 1 h and with or mouse antibody in were with then 1 h with or antibody in PBS. with protein were with The of the were by Cell constructs the were HEK293 cells in The cells were first with at for cells were with at the of cell was with anti-FLAG at and the were in of were a and to Cell surface ABCA1 was by with and then ABCA1 protein was with anti-FLAG were as in a (18Wang N. Silver D.L. Costet P. Tall A.R. J. Biol. Chem. 2000; 275: 33053-33058Abstract Full Text Full Text PDF PubMed Scopus (501) Google Scholar). HEK293 cells were by in The cells were with and to in at for 1 The cells were then with acid with or apoA-I at for The was and of cellular and cholesterol was by The cell were in and the cellular cholesterol was by The cholesterol efflux was as the cellular of constructs the were HEK293 cells in The cells were with and on for cells were by of in and on for the cells were with DMEM, and then purified in was at the and for at cells were by of 1 of with ABCA1 protein was by using the anti-FLAG antibody, and the of the were by of the ABCA1 PEST HEK293 cells were with then with or apoA-I in was and by and The level of phosphorylation was from the of in due to in with apoA-I in a and in the phosphorylation of in a decrease of phosphorylation of ± The phosphorylation of ABCA1 was reduced by removal of the PEST sequence ± and in to wild type ABCA1 incubation with apoA-I not in further in phosphorylation and apoA-I reduced the phosphorylation level of to that for ABCA1delPEST to increase ABCA1 protein level for not for ABCA1delPEST a consistent with that showed that stabilization of ABCA1 by apoA-I the PEST sequence (16Wang N. Chen W. Linsel-Nitschke P. Martinez L.O. Agerholm-Larsen B. Silver D.L. Tall A.R. J. Clin. Invest. 2003; 111: 99-107Crossref PubMed Scopus (242) Google Scholar). in of ABCA1 in led to in this at To provide further for the apoA-I on ABCA1 phosphorylation in mouse were with an ABCA1 expression then with from incubation with of apoA-I for h ABCA1 phosphorylation to the in HEK293 cells. The ABCA1 PEST on results reduced phosphorylation of ABCA1 after removal of the PEST sequence led us to phosphorylation sites the PEST the PEST sequence ABCA1 from various The PEST sequence phosphorylation Thr-1286, Ser-1296, Ser-1302, and We generated in which and both or both residues were to and were on HEK293 cells or the S/T MutAAAA and MutASSA showed a in phosphorylation level to that observed for MutTAAT showed phosphorylation to wild type ABCA1 for at as with the and after protein ± for ± for ± for ABCA1delPEST and no decrease for phosphorylation using constructs of Thr-1286 or Thr-1305 showed that both to phosphorylation not Thus, that Thr-1286 and Thr-1305 are phosphorylation sites the ABCA1 PEST of residues and ABCA1 mABCA1-FLAG to Ala on to Ala on or to Ala on T1286A/T1305A were in of cells of were with as described then ABCA1 protein was with antibody, on and to ABCA1 was by then ABCA1 protein was with The PEST ABCA1 Cell and examined the of the phosphorylation on the cell surface expression and function of ABCA1. which has been to the PEST phosphorylation sites the cell surface ABCA1 protein level as on the constitutively phosphorylated sites and showed an increase of cell surface ABCA1 protein to a level to ABCA1delPEST increase as compared with ± for ± for ± for In to on cell surface was a and less increase in of MutAAAA and MutASSA in cell a on cell surface ABCA1 as previously for the PEST mutant (16Wang N. Chen W. Linsel-Nitschke P. Martinez L.O. Agerholm-Larsen B. Silver D.L. Tall A.R. J. Clin. Invest. 2003; 111: 99-107Crossref PubMed Scopus (242) Google Scholar). We previously that of the PEST sequence results in cell surface of ABCA1 with a increase in (16Wang N. Chen W. Linsel-Nitschke P. Martinez L.O. Agerholm-Larsen B. Silver D.L. Tall A.R. J. Clin. Invest. 2003; 111: 99-107Crossref PubMed Scopus (242) Google Scholar). We the stabilization of ABCA1 cell surface protein that from mutation of the PEST sequence residues was with an increase of ABCA1 protein The increase of cell surface ABCA1 protein with MutAAAA and MutASSA was associated with a than increase of cellular cholesterol efflux as compared with the ABCA1 and a level to the ABCA1delPEST as compared with ± for ± for ± for In contrast, the with PEST phosphorylation not increase cellular cholesterol efflux to apoA-I as compared with ± for is consistent with the of cell surface expression observed with this mutant The PEST ABCA1 by and by that apoA-I the PEST sequence to a degradation (16Wang N. Chen W. Linsel-Nitschke P. Martinez L.O. Agerholm-Larsen B. Silver D.L. Tall A.R. J. Clin. Invest. 2003; 111: 99-107Crossref PubMed Scopus (242) Google Scholar), that PEST sequence phosphorylation could regulate ABCA1 calpain proteolysis. To this we using a calpain in we confirmed that ABCA1 protein cell surface expression was for MutASSA than for the calpeptin no on cell surface MutASSA protein a increase was observed for increase upon calpeptin ± for ± for to show that PEST sequence phosphorylation ABCA1 degradation by cells were and then with purified a of calpain this in degradation decrease upon calpain ± as reported previously (16Wang N. Chen W. Linsel-Nitschke P. Martinez L.O. Agerholm-Larsen B. Silver D.L. Tall A.R. J. Clin. Invest. 2003; 111: 99-107Crossref PubMed Scopus (242) Google Scholar). was no degradation of that calpain proteolysis on the phosphorylation of the PEST we previously observed that apoA-I increases the ABCA1 protein level in with reduced phosphorylation of the ABCA1 PEST sequence 1 and N. Chen W. Linsel-Nitschke P. Martinez L.O. Agerholm-Larsen B. Silver D.L. Tall A.R. J. Clin. Invest. 2003; 111: 99-107Crossref PubMed Scopus (242) Google Scholar), we examined the of apoA-I on the ABCA1 mutant for the PEST sequence phosphorylation sites We observed that the ability of apoA-I to increase cell surface protein level in cells was for MutASSA upon apoA-I ± for ± for that apoA-I increases ABCA1 protein level in a process dependent on the PEST sequence phosphorylation CK2 and of the PEST of of the PEST sequence of ABCA1, we that Thr-1286 and Thr-1305 CK2 and phosphorylation sites J. Biol. Chem. 1991; 266: Full Text PDF PubMed Google Scholar). To CK2 and could be in the PEST sequence we examined the of the CK2 (17Shen J. Channavajhala P. Seldin D.C. Sonenshein G.E. J. Immunol. 2001; 167: 4919-4925Crossref PubMed Scopus (94) Google Scholar), and a B. Denis M. L. Marcil M. Jr., J. J. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar), on ABCA1 in of cells with for h not phosphorylation of or or or incubation to ABCA1 phosphorylation not that CK2 not phosphorylation of the PEST sequence of ABCA1, or of the ABCA1 of cells with inhibitor for h a decrease of the phosphorylation decrease as compared with the ± we observed that inhibitor not cell surface expression of ABCA1 not is consistent with B. Denis M. L. Marcil M. Jr., J. J. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, R.H. S. A. J. R. Zhang L.H. Wang M. G. Hayden M.R. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar) that promotes ABCA1 phosphorylation and subsequent phospholipid efflux in an fashion to ABCA1 protein we observed a decrease in the phosphorylation of MutASSA upon decrease as with the ± which that is not in the phosphorylation of the PEST sequence of ABCA1 on other ABCA1 phosphorylation sites as suggested by B. Denis M. L. Marcil M. Jr., J. J. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). By and residues to we provide for the phosphorylation of residues and the PEST sequence of ABCA1. Interestingly, phosphorylation of residues is by incubation with extracellular in a process that the PEST-dependent stabilization of ABCA1 by apoA-I (16Wang N. Chen W. Linsel-Nitschke P. Martinez L.O. Agerholm-Larsen B. Silver D.L. Tall A.R. J. Clin. Invest. 2003; 111: 99-107Crossref PubMed Scopus (242) Google Scholar). that apoA-I stabilization of ABCA1 is mediated by reduced PEST sequence which in to calpain proteolysis of ABCA1. of the PEST phosphorylation sites to cell surface expression and of ABCA1, that of kinase or at the PEST sequence could a to up-regulation of cellular ABCA1 that phosphorylation of the PEST sequence has a key role in the stabilization of ABCA1 by The and of apoA-I induced dephosphorylation of the PEST sequence the of apoA-I on stabilization of ABCA1 (16Wang N. Chen W. Linsel-Nitschke P. Martinez L.O. Agerholm-Larsen B. Silver D.L. Tall A.R. J. Clin. Invest. 2003; 111: 99-107Crossref PubMed Scopus (242) Google Scholar) and of the residues that phosphorylation to apoA-I on cell surface of ABCA1 and were not to the of apoA-I that the of apoA-I on ABCA1 stabilization were mediated by PEST The phosphorylation defective mutant was to calpain and showed no increase in level in the of calpain that apoA-I phosphorylation of PEST residues and thereby calpain proteolysis. The mechanism the of extracellular apoA-I to in ABCA1 PEST phosphorylation is on the stabilization of ABCA1 suggested that the mechanism a in ABCA1 by of apoA-I to ABCA1, or a in the to phospholipid efflux. A mutant showed levels of of apoA-I phospholipid efflux J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar, A.R. S. T. R. G. F. M. N. T. M. Yokoyama S. K. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). mutant was not stabilized by apoA-I that efflux is for ABCA1 stabilization (16Wang N. Chen W. Linsel-Nitschke P. Martinez L.O. Agerholm-Larsen B. Silver D.L. Tall A.R. J. Clin. Invest. 2003; 111: 99-107Crossref PubMed Scopus (242) Google Scholar). we could not the that apoA-I to this mutant in an an Thus, a of ABCA1 by of or a to phospholipid efflux could to of a kinase or at the PEST sequence we the that apoA-I or phospholipid efflux a that to kinase or It has been previously that phosphorylation by protein kinase CK2 the sequence promotes calpain-mediated degradation of by the of calpain to the PEST sequence (17Shen J. Channavajhala P. Seldin D.C. Sonenshein G.E. J. Immunol. 2001; 167: 4919-4925Crossref PubMed Scopus (94) Google Scholar). in to the mechanism by which PEST can of M. Full Text PDF PubMed Scopus Google Scholar), is no on the mechanism by which PEST calpain on target the PEST sequence a using a role of in PEST sequence recently reported that ABCA1 was constitutively phosphorylated in on R.H. S. A. J. R. Zhang L.H. Wang M. G. Hayden M.R. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar) and human B. Denis M. L. Marcil M. Jr., J. J. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar) and in HEK293 cells R.H. S. A. J. R. Zhang L.H. Wang M. G. Hayden M.R. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). In both protein kinase A has been showed to be in the ABCA1 phosphorylation and R.H. S. A. J. R. Zhang L.H. Wang M. G. Hayden M.R. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar) suggested by in kinase that and in the of ABCA1, are major phosphorylation sites for the reported that ABCA1 phosphorylation by on regulate cholesterol and phospholipid efflux not apoA-I or ABCA1 protein we confirmed that ABCA1 is constitutively phosphorylated in mouse and HEK293 cells. We observed that phosphorylation of ABCA1 was by after mutation of Thr-1286 and Thr-1305 residues in the PEST sequence of ABCA1. the of phosphorylation level after mutation of Thr-1286 and Thr-1305 is consistent with other sites of phosphorylation the PEST as and In with the phosphorylation on that not ABCA1 protein R.H. S. A. J. R. Zhang L.H. Wang M. G. Hayden M.R. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar), we showed by that the phosphorylation on Thr-1286 and Thr-1305 residues in the PEST sequence of ABCA1, ABCA1 protein and thereby ABCA1 cell surface levels and confirmed that is in the phosphorylation of ABCA1 on a than the PEST on and as previously suggested R.H. S. A. J. R. Zhang L.H. Wang M. G. Hayden M.R. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). in of the PEST the of mutation of the phosphorylation sites was to increase cell surface ABCA1 with a increase in ABCA1 in cell that PEST sequence-dependent degradation ABCA1 in the plasma could of calpain to plasma ABCA1. is that calpain of ABCA1 to of an that is in removal of ABCA1 from the plasma It is to the of the on cell surface and cholesterol efflux that efflux is mediated by cell surface ABCA1. other of both apoA-I and ABCA1 S. A. 1999; 96: PubMed Scopus Google Scholar, E. C. Kaminski W.E. Bottcher A. G. Drobnik W. A. O. Diederich W. Langmann T. T. Rothe G. Lackner K.J. G. Schmitz G. Nat. Genet. 2000; PubMed Scopus Google Scholar, A. J. R. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar), and further be to the of cellular cholesterol efflux and of ABCA1. in ABCA1 and as as in with Tangier disease, that macrophage efflux is (10Schaefer E.J. Zech L.A. Schwartz D.E. Brewer Jr., H.B. Ann. Intern. Med. 1980; 93: 261-266Crossref PubMed Scopus (174) Google Scholar, D. L. S. T. M. Arterioscler. Thromb. Vasc. Biol. 2002; 22: PubMed Scopus Google Scholar, C. G. N. Clee S.M. N. J.C. Fruchart J.C. B. Hayden M.R. J. Clin. Invest. 2002; 110: PubMed Scopus Google Scholar). of ABCA1 expression mediated by is associated with N. A. T. N. D. T. 2003; PubMed Scopus Google Scholar) is by liver and J.J. G. J. I. B. 2000; PubMed Scopus Google Scholar, H. A. J.J. J.C. L. S. Wang S. M. B. 2000; PubMed Scopus Google Scholar). Thus, to increase ABCA1 are The stabilization of ABCA1 by apoA-I in (16Wang N. Chen W. Linsel-Nitschke P. Martinez L.O. Agerholm-Larsen B. Silver D.L. Tall A.R. J. Clin. Invest. 2003; 111: 99-107Crossref PubMed Scopus (242) Google Scholar) and to the of apoA-I or A. S. W. T. F. T. M. H. M. K. T. S. Arterioscler. Thromb. Vasc. Biol. 1995; PubMed Scopus Google Scholar, G. C. M. N. R. G. F. Arterioscler. Thromb. Vasc. Biol. 1998; PubMed Scopus Google Scholar, R.M. S.M. 1991; PubMed Scopus Google Scholar, C.J. S. A. PubMed Scopus Google Scholar). stabilization mechanism might provide a to target ABCA1. could be as or that the of apoA-I to ABCA1. The present results a that decrease phosphorylation of the residues the PEST sequence of ABCA1. are a of and in the and of kinase in the of C. H. O. G. W. 2002; PubMed Scopus Google Scholar) the for this in