Phosphorylation of a pest sequence in ABCA1 promotes calpain degradation and is reversed by ApoA-I.
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.
Martinez
Laurent O
LO
Division of Molecular Medicine, Department of Medicine, Columbia University, New York, New York 10032, USA. Lm2068@columbia.edu
Agerholm-Larsen
Birgit
B
Wang
Nan
N
Chen
Wengen
W
Tall
Alan R
AR
D064286:ATP Binding Cassette Transporter 1; D018528:ATP-Binding Cassette Transporters / Q000737:chemistry* / Q000378:metabolism; D000595:Amino Acid Sequence; D016632:Apolipoprotein A-I / Q000494:pharmacology*; D002154:Calpain / Q000378:metabolism*; D047390:Casein Kinase II; D017868:Cyclic AMP-Dependent Protein Kinases / Q000502:physiology; D006801:Humans; D008969:Molecular Sequence Data; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000502:physiology
C577836:ABCA1 protein, human; D064286:ATP Binding Cassette Transporter 1; D018528:ATP-Binding Cassette Transporters; D016632:Apolipoprotein A-I; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D002154:Calpain