ABCMdb

ABCA1 p.Ser1042Ala

None has been submitted yet.

Publications

PMID: 12196520 [PubMed] See RH et al: "Protein kinase A site-specific phosphorylation regulates ATP-binding cassette A1 (ABCA1)-mediated phospholipid efflux."

No. Sentence Comment

37 PCR-based mutagenesis was performed on the ABCA1 gene to create the S1042A and S2054A mutants as described previously (30). Login to comment
39 The S1042A and S2054A mutations in ABCA1 were completely sequence-confirmed. Login to comment
40 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 PCR-based mutagenesis was performed on the ABCA1 gene to create the S1042A and S2054A mutants as described previously (31). Login to comment
56 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 Pulse-chase labeling experiments were performed on polyclonal 293 Flip-in cells constitutively expressing wild-type ABCA1, S1042A, or S2054A. Login to comment
94 HeLa cells were transfected with GFP-ABCA1 wild-type, S1042A, S2054A, or S1042A/S2054A as described above. Login to comment
105 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
161 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 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 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 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 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 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 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 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
197 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
239 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
259 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 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 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
162 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
184 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
190 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
195 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
198 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