ABCMdb

PMID: 12196520

See RH, Caday-Malcolm RA, Singaraja RR, Zhou S, Silverston A, Huber MT, Moran J, James ER, Janoo R, Savill JM, Rigot V, Zhang LH, Wang M, Chimini G, Wellington CL, Tafuri SR, Hayden MR
Protein kinase A site-specific phosphorylation regulates ATP-binding cassette A1 (ABCA1)-mediated phospholipid efflux.
J Biol Chem. 2002 Nov 1;277(44):41835-42. Epub 2002 Aug 23., [PubMed]

Sentences

No. Mutations Sentence Comment

37 ABCA1 p.Ser1042Ala ABCA1 p.Ser2054Ala PCR-based mutagenesis was performed on the ABCA1 gene to create the S1042A and S2054A mutants as described previously (30). Login to comment 39 ABCA1 p.Ser1042Ala ABCA1 p.Ser2054Ala The S1042A and S2054A mutations in ABCA1 were completely sequence-confirmed. Login to comment 40 ABCA1 p.Ser1042Ala ABCA1 p.Ser1042Ala ABCA1 p.Ser2054Ala ABCA1 p.Ser2054Ala The S1042A mutation was isolated by XhoI/XbaI restriction digestion and moved into the S2054A clone, thereby generating a S1042A/S2054A double mutant that was also sequence-confirmed. Login to comment 47 ABCA1 p.Ser1042Ala ABCA1 p.Ser2054Ala PCR-based mutagenesis was performed on the ABCA1 gene to create the S1042A and S2054A mutants as described previously (31). Login to comment 56 ABCA1 p.Ser1042Ala ABCA1 p.Ser1042Ala ABCA1 p.Ser2054Ala ABCA1 p.Ser2054Ala Polyclonal cell lines were also prepared by pooling at least 500 individual colonies from 293 Flip-in cells co-transfected with pOG44 and pcDNA5/FRT-ABCA1, pcDNA5/FRT-ABCA1 S1042A, pcDNA5/ FRT-ABCA1 S2054A, or pcDNA5/FTR-ABCA1 S1042A/S2054A cDNAs and selected for hygromycin resistance as described above. Login to comment 81 ABCA1 p.Ser1042Ala ABCA1 p.Ser2054Ala Pulse-chase labeling experiments were performed on polyclonal 293 Flip-in cells constitutively expressing wild-type ABCA1, S1042A, or S2054A. Login to comment 94 ABCA1 p.Ser1042Ala ABCA1 p.Ser1042Ala ABCA1 p.Ser2054Ala ABCA1 p.Ser2054Ala HeLa cells were transfected with GFP-ABCA1 wild-type, S1042A, S2054A, or S1042A/S2054A as described above. Login to comment 105 ABCA1 p.Ser1042Ala ABCA1 p.Ser1042Ala ABCA1 p.Ser2054Ala ABCA1 p.Ser2054Ala For each individual experiment, polyclonal cells expressing the pcDNA5/FRT wild-type ABCA1, S1042A, S2054A, or S1042A/S2054A mutants were plated in triplicate at a density of 80,000 per well in 24 well dishes and labeled with 10 ␮Ci per ml [3 H]choline (Amersham Biosciences) in 2 ml of DMEM with 1% fetal bovine serum for 24 h. Twenty-four h after labeling, cells were first washed with equilibration medium (DMEM containing 0.2% fatty acid-free bovine serum albumin) and then equilibrated in the same medium for 1 h. Login to comment 153 ABCA1 p.Ser2054Ala The S2054A mutant reduced PKA phosphorylation of GST-ABCA1 aa 1873 to 2261 by 95% as determined by liquid scintillation counting of the excised radioactive bands (Fig. 4, left panel, compare lanes 2 and 3). Login to comment 154 ABCA1 p.Ser2054Ala The S2054A mutant reduced PKA phosphorylation of GST-ABCA1 aa 1873 to 2261 by 95% as determined by liquid scintillation counting of the excised radioactive bands (Fig. 4, left panel, compare lanes 2 and 3). Login to comment 161 ABCA1 p.Ser1042Ala Moreover, GST-ABCA1 S1042A mutant reduced PKA phosphorylation by 80% (Fig. 6, left panel, lane 3), confirming that Ser-1042 is another PKA phosphorylation site on ABCA1. Login to comment 162 ABCA1 p.Ser1042Ala Moreover, GST-ABCA1 S1042A mutant reduced PKA phosphorylation by 80% (Fig. 6, left panel, lane 3), confirming that Ser-1042 is another PKA phosphorylation site on ABCA1. Login to comment 183 ABCA1 p.Ser1042Ala ABCA1 p.Ser1042Ala ABCA1 p.Ser2054Ala ABCA1 p.Ser2054Ala Polyclonal Flip-in 293 cells constitutively expressing wild-type ABCA1, S1042A, S2054A, or S1042A/S2054A mutants were then examined for their ability to efflux phospholipid to ApoA-I (8, 13, 14). Login to comment 184 ABCA1 p.Ser1042Ala ABCA1 p.Ser1042Ala ABCA1 p.Ser2054Ala ABCA1 p.Ser2054Ala Polyclonal Flip-in 293 cells constitutively expressing wild-type ABCA1, S1042A, S2054A, or S1042A/S2054A mutants were then examined for their ability to efflux phospholipid to ApoA-I (8, 13, 14). Login to comment 186 ABCA1 p.Ser1042Ala ABCA1 p.Ser2054Ala ABCA1 p.Ser2054Ala Compared with wild-type ABCA1, the S2054A mutant showed a 40% decrease (p Ͻ 0.01) in ApoA-I-dependent phospholipid efflux whereas a 50% reduction (p Ͻ 0.01) was observed for the S1042A/S2054A mutant (Fig. 7D). Login to comment 187 ABCA1 p.Ser1042Ala ABCA1 p.Ser1042Ala ABCA1 p.Ser1042Ala ABCA1 p.Ser2054Ala ABCA1 p.Ser2054Ala ABCA1 p.Ser2054Ala ABCA1 p.Ser2054Ala No significant difference in phospholipid efflux was observed between the S1042A mutant alone and wild-type ABCA1 cells or between the S2054A and S1042A/S2054A mutant cells (Fig. 7D). Login to comment 188 ABCA1 p.Ser1042Ala ABCA1 p.Ser1042Ala ABCA1 p.Ser2054Ala ABCA1 p.Ser2054Ala ABCA1 p.Ser2054Ala Comparison among the different mutants showed that of the two individual mutations, S2054A clearly had the greatest effect on phospholipid efflux. Login to comment 189 ABCA1 p.Ser1042Ala ABCA1 p.Ser2054Ala ABCA1 p.Ser2054Ala ABCA1 p.Ser2054Ala Additionally, efflux in cells expressing the combined mutant S1042A/S2054A was not significantly reduced compared with cells expressing S2054A alone, suggesting that Ser-2054 is the critical phosphorylated residue for phospholipid efflux (Fig. 7D). Login to comment 190 ABCA1 p.Ser1042Ala ABCA1 p.Ser2054Ala ABCA1 p.Ser2054Ala Additionally, efflux in cells expressing the combined mutant S1042A/S2054A was not significantly reduced compared with cells expressing S2054A alone, suggesting that Ser-2054 is the critical phosphorylated residue for phospholipid efflux (Fig. 7D). Login to comment 193 ABCA1 p.Ser1042Ala ABCA1 p.Ser1042Ala ABCA1 p.Ser2054Ala ABCA1 p.Ser2054Ala To determine whether ABCA1 phosphorylation affects ApoA-I binding, HeLa cells transiently expressing GFP-tagged wild-type ABCA1, S1042A, S2054A, or S1042A/S2054A were analyzed for Cy5-ApoA-I binding by dual-channel flow cytometric analysis. Login to comment 194 ABCA1 p.Ser1042Ala ABCA1 p.Ser1042Ala ABCA1 p.Ser1042Ala ABCA1 p.Ser1042Ala ABCA1 p.Ser2054Ala ABCA1 p.Ser2054Ala ABCA1 p.Ser2054Ala ABCA1 p.Ser2054Ala Whereas a non-functional mutant mouse ABCA1 defined previously (33) showed loss of ApoA-I binding compared with its wild-type counterpart ABCA1 (Fig. 8, p Ͻ; 0.001; compare AG and MM), no significant difference in ApoA-I binding was observed between cells expressing wild-type human ABCA1 or any of the phosphorylation mutants (Fig. 8, compare WT, S1042A, S2054A, and S1042A/S2054A). Login to comment 195 ABCA1 p.Ser1042Ala ABCA1 p.Ser1042Ala ABCA1 p.Ser2054Ala ABCA1 p.Ser2054Ala Whereas a non-functional mutant mouse ABCA1 defined previously (33) showed loss of ApoA-I binding compared with its wild-type counterpart ABCA1 (Fig. 8, p b0d; 0.001; compare AG and MM), no significant difference in ApoA-I binding was observed between cells expressing wild-type human ABCA1 or any of the phosphorylation mutants (Fig. 8, compare WT, S1042A, S2054A, and S1042A/S2054A). Login to comment 197 ABCA1 p.Ser1042Ala ABCA1 p.Ser2054Ala The decay constant for wild-type ABCA1 (about 3 h) was comparable with that found for either S1042A or S2054A alone (Table I). Login to comment 198 ABCA1 p.Ser1042Ala ABCA1 p.Ser2054Ala The decay constant for wild-type ABCA1 (about 3 h) was comparable with that found for either S1042A or S2054A alone (Table I). Login to comment 199 ABCA1 p.Ser2054Ala Therefore, the data indicate that the reduction in ApoA-I-dependent phospholipid efflux in the S2054A mutant cells is not because of defects in ApoA-I binding or because of decreased ABCA1 protein stability. Login to comment 200 ABCA1 p.Ser2054Ala Therefore, the data indicate that the reduction in ApoA-I-dependent phospholipid efflux in the S2054A mutant cells is not because of defects in ApoA-I binding or because of decreased ABCA1 protein stability. Login to comment 214 ABCA1 p.Ser2054Ala A, purified GST alone, GST-ABCA1 aa 1873 to 2261 wild-type, and GST-ABCA1 aa 1873 to 2261 S2054A mutant were incubated with PKA catalytic subunit and [␥- 32 P]ATP. Proteins were resolved by SDS-PAGE, and the dried gel was autoradiographed (left panel). Login to comment 239 ABCA1 p.Ser1042Ala Purified GST alone, GST-ABCA1 aa 1010 to 1171 wild-type, and GST-ABCA1 aa 1010 to 1171 S1042A mutant were incubated with PKA catalytic subunit and [␥- 32 P]ATP. Proteins were separated by SDS-PAGE followed by autoradiography of the gel (left panel). Login to comment 243 ABCA1 p.Ala1046Asp ABCA1 p.Arg2081Trp The phosphorylated Ser-1042 and Ser-2054 residues are near the ABCA1 mutations A1046D and R2081W, both of which have resulted in a clinical phenotype in humans with low high density lipoprotein cholesterol, low plasma ApoA-I, and low total cholesterol values (47- 49). Login to comment 259 ABCA1 p.Ser1042Ala ABCA1 p.Ser1042Ala ABCA1 p.Ser2054Ala ABCA1 p.Ser2054Ala C, 293 Flip-in cells constitutively expressing wild-type ABCA1, S1042A, S2054A, or S1042A/S2054A proteins were lysed, and 200 ␮g of protein was analyzed by SDS-PAGE followed by Western blotting with an anti-ABCA1 monoclonal antibody (AC10). Login to comment 261 ABCA1 p.Ser1042Ala ABCA1 p.Ser1042Ala ABCA1 p.Ser2054Ala ABCA1 p.Ser2054Ala D, polyclonal cells constitutively expressing wild-type ABCA1, S1042A, S2054A, or S1042A/S2054A were examined for total phospholipid efflux for 4 h in the presence or absence of ApoA-I as described under "Experimental Procedures." Login to comment 274 ABCA1 p.Ser1042Ala ABCA1 p.Ser2054Ala ABCA1 proteins Mean half-life Ϯ S.D. h Wild-type 3.0 Ϯ 0.4 S1042A 3.5 Ϯ 0.4 S2054A 2.9 Ϯ 0.3 ings show that kinases such as PKA can modulate ABCA1 function, and identification of these signaling molecules may provide leads to novel approaches for regulation of ABCA1 activity. Login to comment