ABCMdb

ABCA1 p.Ser2054Ala

Predicted by SNAP2:	A: N (78%), C: N (66%), D: D (75%), E: D (80%), F: D (80%), G: D (59%), H: D (75%), I: D (75%), K: D (80%), L: D (75%), M: D (71%), N: D (59%), P: D (85%), Q: D (71%), R: D (80%), T: D (63%), V: D (66%), W: D (85%), Y: D (80%),
Predicted by PROVEAN:	A: N, C: D, D: D, E: D, F: D, G: D, H: D, I: D, K: D, L: D, M: D, N: N, P: D, Q: D, R: D, T: N, V: D, W: D, Y: D,

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Publications
[hide] Protein kinase A site-specific phosphorylation reg... J Biol Chem. 2002 Nov 1;277(44):41835-42. Epub 2002 Aug 23. 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., [PMID:12196520]

Abstract [show]
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 kinase in 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.

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Sentences [show]
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
153 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
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
199 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 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
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
154 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
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
200 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