Protein Kinase A Site-specific Phosphorylation Regulates ATP-binding Cassette A1 (ABCA1)-mediated Phospholipid Efflux*

ATP-binding cassette A1 (ABCA1) is a key mediator of cholesterol and phospholipid efflux to apolipoprotein particles. We show that ABCA1 is a constitutively phosphorylated protein in both RAW macrophages and in a human embryonic kidney cell line expressing ABCA1. Furthermore, we demonstrate that phosphorylation of ABCA1 is mediated by protein kinase A (PKA) or a PKA-like kinasein vivo. Through site-directed mutagenesis studies of consensus PKA phosphorylation sites and in vitro PKA kinase assays, we show that Ser-1042 and Ser-2054, located in the nucleotide binding domains of ABCA1, are major phosphorylation sites for PKA. ApoA-I-dependent phospholipid efflux was decreased significantly by mutation of Ser-2054 alone and Ser-1042/Ser-2054 but was not significantly impaired with Ser-1042 alone. The mechanism by which ABCA1 phosphorylation affected ApoA-I-dependent phospholipid efflux did not involve either alterations in ApoA-I binding or changes in ABCA1 protein stability. These studies demonstrate a novel serine (Ser-2054) on the ABCA1 protein crucial for PKA phosphorylation and for regulation of ABCA1 transporter activity.

[1]  P. Cohen,et al.  The origins of protein phosphorylation , 2002, Nature Cell Biology.

[2]  B. McManus,et al.  ABCA1 mRNA and Protein Distribution Patterns Predict Multiple Different Roles and Levels of Regulation , 2002, Laboratory Investigation.

[3]  M. Hayden,et al.  Pivotal role of ABCA1 in reverse cholesterol transport influencing HDL levels and susceptibility to atherosclerosis. , 2001, Journal of lipid research.

[4]  J. Sasaki,et al.  Novel mutations in ABCA1 gene in Japanese patients with Tangier disease and familial high density lipoprotein deficiency with coronary heart disease. , 2001, Biochimica et biophysica acta.

[5]  A. Tall,et al.  ATP-binding Cassette Transporter A1 (ABCA1) Functions as a Cholesterol Efflux Regulatory Protein* , 2001, The Journal of Biological Chemistry.

[6]  H. Rigneault,et al.  Specific Docking of Apolipoprotein A-I at the Cell Surface Requires a Functional ABCA1 Transporter* , 2001, The Journal of Biological Chemistry.

[7]  T. Hudson,et al.  Common Genetic Variation in ABCA1 Is Associated With Altered Lipoprotein Levels and a Modified Risk for Coronary Artery Disease , 2001, Circulation.

[8]  J. Genest,et al.  Cellular cholesterol efflux is modulated by phospholipid-derived signaling molecules in familial HDL deficiency/Tangier disease fibroblasts. , 2001, Journal of lipid research.

[9]  R. Pulido,et al.  The Tumor Suppressor PTEN Is Phosphorylated by the Protein Kinase CK2 at Its C Terminus , 2001, The Journal of Biological Chemistry.

[10]  A. Tall,et al.  Tangier disease as a test of the reverse cholesterol transport hypothesis. , 2000, The Journal of clinical investigation.

[11]  M. Hayden,et al.  Age and residual cholesterol efflux affect HDL cholesterol levels and coronary artery disease in ABCA1 heterozygotes. , 2000, The Journal of clinical investigation.

[12]  R. Lawn,et al.  ABCA1 Is the cAMP-inducible Apolipoprotein Receptor That Mediates Cholesterol Secretion from Macrophages* , 2000, The Journal of Biological Chemistry.

[13]  C. Fielding,et al.  A two-step mechanism for free cholesterol and phospholipid efflux from human vascular cells to apolipoprotein A-1. , 2000, Biochemistry.

[14]  A. Tall,et al.  Specific Binding of ApoA-I, Enhanced Cholesterol Efflux, and Altered Plasma Membrane Morphology in Cells Expressing ABC1* , 2000, The Journal of Biological Chemistry.

[15]  J. Mcneish,et al.  The Correlation of ATP-binding Cassette 1 mRNA Levels with Cholesterol Efflux from Various Cell Lines* , 2000, The Journal of Biological Chemistry.

[16]  A. Tall,et al.  Sterol-dependent transactivation of the ABC1 promoter by the liver X receptor/retinoid X receptor. , 2000, The Journal of biological chemistry.

[17]  B. Zinman,et al.  Common and rare ABCA1 variants affecting plasma HDL cholesterol. , 2000, Arteriosclerosis, thrombosis, and vascular biology.

[18]  A. Horwitz,et al.  Identification of cAMP analogue inducible genes in RAW264 macrophages. , 2000, Biochimica et biophysica acta.

[19]  A. Hackam,et al.  Inhibiting Caspase Cleavage of Huntingtin Reduces Toxicity and Aggregate Formation in Neuronal and Nonneuronal Cells* , 2000, The Journal of Biological Chemistry.

[20]  Yannick Hamon,et al.  ABC1 promotes engulfment of apoptotic cells and transbilayer redistribution of phosphatidylserine. , 2000, Nature Cell Biology.

[21]  A. Vaughan,et al.  The Tangier disease gene product ABC1 controls the cellular apolipoprotein-mediated lipid removal pathway. , 1999, The Journal of clinical investigation.

[22]  T. Langmann,et al.  The gene encoding ATP-binding cassette transporter 1 is mutated in Tangier disease , 1999, Nature Genetics.

[23]  J. Piette,et al.  Tangier disease is caused by mutations in the gene encoding ATP-binding cassette transporter 1 , 1999, Nature Genetics.

[24]  C. Sensen,et al.  Mutations in ABC1 in Tangier disease and familial high-density lipoprotein deficiency , 1999, Nature Genetics.

[25]  L. Reuss,et al.  Phosphorylation of P-glycoprotein by PKA and PKC modulates swelling-activated Cl- currents. , 1999, American journal of physiology. Cell physiology.

[26]  A. Nairn,et al.  Regulation of CFTR Cl- ion channels by phosphorylation and dephosphorylation. , 1999, Advances in second messenger and phosphoprotein research.

[27]  S. Yokoyama,et al.  Selective down-regulation by protein kinase C inhibitors of apolipoprotein-mediated cellular cholesterol efflux in macrophages. , 1997, Biochemistry.

[28]  J. Oram,et al.  High density lipoproteins stimulate mitogen-activated protein kinases in human skin fibroblasts. , 1997, Arteriosclerosis, thrombosis, and vascular biology.

[29]  H. Brewer,et al.  Decreased reverse cholesterol transport from Tangier disease fibroblasts. Acceptor specificity and effect of brefeldin on lipid efflux. , 1997, Arteriosclerosis, thrombosis, and vascular biology.

[30]  J. Glavy,et al.  Identification of the in Vivo Phosphorylation Sites for Acidic-directed Kinases in Murine mdr1b P-glycoprotein* , 1997, The Journal of Biological Chemistry.

[31]  B. Verrier,et al.  ABC1, an ATP Binding Cassette Transporter Required for Phagocytosis of Apoptotic Cells, Generates a Regulated Anion Flux after Expression in Xenopus laevis Oocytes* , 1997, The Journal of Biological Chemistry.

[32]  A S Verkman,et al.  Identification of protein kinase A phosphorylation sites on NBD1 and R domains of CFTR using electrospray mass spectrometry with selective phosphate ion monitoring , 1996, Protein science : a publication of the Protein Society.

[33]  I. Verma,et al.  Constitutive phosphorylation of IkappaBalpha by casein kinase II occurs preferentially at serine 293: requirement for degradation of free IkappaBalpha , 1996, Molecular and cellular biology.

[34]  C. Higgins,et al.  Protein Kinase C-mediated Phosphorylation Does Not Regulate Drug Transport by the Human Multidrug Resistance P-glycoprotein* , 1996, The Journal of Biological Chemistry.

[35]  G. Bren,et al.  Casein kinase II phosphorylates I kappa B alpha at S-283, S-289, S-293, and T-291 and is required for its degradation , 1996, Molecular and cellular biology.

[36]  I. Pastan,et al.  Characterization of Phosphorylation-defective Mutants of Human P-glycoprotein Expressed in Mammalian Cells (*) , 1996, The Journal of Biological Chemistry.

[37]  G. Francis,et al.  Defective removal of cellular cholesterol and phospholipids by apolipoprotein A-I in Tangier Disease. , 1995, The Journal of clinical investigation.

[38]  J. Riordan,et al.  cAMP-dependent Protein Kinase-mediated Phosphorylation of Cystic Fibrosis Transmembrane Conductance Regulator Residue Ser-753 and Its Role in Channel Activation (*) , 1995, The Journal of Biological Chemistry.

[39]  F. Denizot,et al.  Cloning of two novel ABC transporters mapping on human chromosome 9. , 1994, Genomics.

[40]  E. Nieves,et al.  Identification of the major phosphorylation domain of murine mdr1b P-glycoprotein. Analysis of the protein kinase A and protein kinase C phosphorylation sites. , 1993, The Journal of biological chemistry.

[41]  J. Riordan,et al.  Protein kinase A (PKA) still activates CFTR chloride channel after mutagenesis of all 10 PKA consensus phosphorylation sites. , 1993, The Journal of biological chemistry.

[42]  B. Neel,et al.  Solubilization and purification of enzymatically active glutathione S-transferase (pGEX) fusion proteins. , 1993, Analytical biochemistry.

[43]  P. Greengard,et al.  Phosphorylation of the cystic fibrosis transmembrane conductance regulator. , 1992, The Journal of biological chemistry.

[44]  M. Welsh,et al.  Phosphorylation of the R domain by cAMP-dependent protein kinase regulates the CFTR chloride channel , 1991, Cell.

[45]  E. Krebs,et al.  Consensus sequences as substrate specificity determinants for protein kinases and protein phosphatases. , 1991, The Journal of biological chemistry.

[46]  A. Mendez,et al.  Protein kinase C as a mediator of high density lipoprotein receptor-dependent efflux of intracellular cholesterol. , 1991, The Journal of biological chemistry.

[47]  K. Klinger,et al.  Expression and characterization of the cystic fibrosis transmembrane conductance regulator , 1990, Nature.

[48]  S. Yokoyama,et al.  A Japanese family with high density lipoprotein deficiency. , 1983, Atherosclerosis.