Calmodulin Interacts With ATP Binding Cassette Transporter A1 to Protect From Calpain-Mediated Degradation and Upregulates High-Density Lipoprotein Generation
Objective— To investigate the interaction of ATP-binding cassette transporter A1 (ABCA1) with calmodulin in relation to its calpain-mediated degradation because many calpain substrates bind calmodulin to regulate cellular functions.
Methods and Results— The activity of ABCA1 is regulated through proteolysis by calpain. An immunoprecipitation and glutathione S-transferase pull-down assay revealed that ABCA1 directly binds calmodulin in a Ca2+-dependent manner. The cytoplasmic loop of ABCA1 contains a typical calmodulin binding sequence of 1-5-8-14 motifs (1245 to 1257 amino acids). The peptide of this region showed binding to calmodulin, and deletion of the 1-5-8-14 motif abolished this interaction. This motif is located near the ABCA1 Pro-Glu-Ser-Thr sequence, and the presence of calmodulin/Ca2+ protected the peptides from proteolysis by calpain. The knockdown of calmodulin by a specific small and interfering RNA increased the degradation of ABCA1 and decreased ABCA1 protein and apolipoprotein A-I–mediated lipid release. Surprisingly, calmodulin inhibitor W7 increased calmodulin binding to ABCA1 and protected it from calpain-mediated degradation, consistent with our previous finding that this compound increased apolipoprotein A-I–mediated cell cholesterol release.
Conclusion— Calmodulin directly binds and stabilizes ABCA1 in the presence of Ca2+ and increases the generation of high-density lipoprotein.
High-density lipoprotein (HDL) plays a central role in the catabolic pathway of cholesterol by transporting it from the peripheral tissues to the liver for conversion to bile acids. Therefore, it is thought to prevent accumulation of cholesterol in extrahepatic tissues and to act as an antiatherogenic factor.1 ATP-binding cassette transporter A1 (ABCA1) is a key rate-limiting protein for the generation of HDL in hepatocytes and peripheral cells; helical apolipoproteins, such as apolipoprotein A-I (apoA-I), interact with it to remove cellular phospholipid and cholesterol to form new HDL particles.1 ABCA1 is one of the target molecules that increases HDL and prevents cardiovascular disease.2,3
The expression of the ABCA1 gene is regulated by the liver X receptor through oxysterol as a signal of cellular cholesterol increase.4 Interestingly, it is also regulated by sterol-reacting element binding protein 2 in hepatocytes, perhaps to maintain cholesterol homeostasis for the body.5 The ABCA1 level is also modulated through proteolytic degradation by calpain, and the interaction with helical apolipoproteins to generate HDL stabilizes ABCA1 against this degradation.6–8 Several cellular factors also impeded the degradation by interacting with ABCA1.9,10 Pharmacological inhibition of ABCA1 degradation resulted in an increase of HDL biogenesis, an elevation of plasma HDL, and prevention of atherosclerosis.11
Many calpain substrates contain the Pro-Glu-Ser-Thr (PEST) sequence.12 ABCA1 also has a consensus PEST sequence in the cytoplasmic loop and a high PEST score; deletion of this sequence abolished the proteolysis by calpain.13 The half-life of ABCA1 is less than 2 hours in the absence of helical apolipoproteins, similar to other proteins with a PEST sequence.14
Calmodulin is an acidic Ca2+-binding protein and orchestrates many regulatory events through the interaction with cellular proteins. Calmodulin-binding proteins are classified into Ca2+-dependent and Ca2+-independent proteins, and calmodulin binds with high affinity to a relatively small α-helical region of many proteins.15 Many calmodulin-binding proteins are also substrates of calpain because most of the calmodulin-binding proteins contain a PEST sequence. Calmodulin was, indeed, found to regulate calapin-mediated proteolysis by binding to the substrates.12 Calmodulin binds to Ca2+-ATPase in a Ca2+-dependent manner at the calmodulin-binding sequence to complete its calpain-mediated cleavage to activate it while EGTA protected it from this cleavage, perhaps by inducing the optimum conformation.16 Phosphocholine cytidylyltransferase, the enzyme essential for phosphatidylcholine biosynthesis, is degraded by calpain. Both the PEST sequence and a calmodulin binding site are identified in this enzyme, and calmodulin directly binds to and stabilizes phosphocholine cytidylyltransferase against calpain-mediated degradation.17
Because ABCA1 contains a conserved PEST sequence, calmodulin is potentially involved in its proteolytic degradation. In the present study, we investigated the regulation of ABCA1 degradation in relation to calmodulin. We identified a conserved Ca2+-dependent calmodulin binding site near the PEST sequence in ABCA1 and demonstrated protection by calmodulin of ABCA1 against calpain-mediated proteolysis.
Calmodulin and calmodulin-agarose were obtained from Sigma; rabbit anti–calmodulin monoclonal antibody, Upstate; and control rabbit IgG and protein agarose A/G beads, Santa Cruz Biotechnology. Purified μ-calpain was purchased from Calbiochem; and cycloheximide and calmodulin inhibitor W7, Wako. Monoclonal antibody against human ABCA1 (MABI98-7) was generated in rats at MAB Institute against the C-terminus peptide of human ABCA1 (CNFAKDQSDDDHLKDLSLHKN).18 Human apoA-I was prepared from fresh human plasma.19
Cell Culture and Immunoprecipitation
BALB/3T3 and THP1 cells were incubated in the medium (a 1:1 mixture of Dulbecco’s modified Eagle medium and Ham F12 medium [DF medium] and RPMI1640 medium, respectively) with 10% FBS at 37°C in 5% CO2.
Cell pellets were harvested with sonication in the 50-mmol/L Tris-hydrochloride buffer (pH 8.0) containing 150-mmol/L NaCl, 5-mmol/L EDTA, and protease inhibitor cocktail (Sigma). Insoluble debris was removed by centrifugation at 12 000 g for 2 minutes at 4°C. The supernatant was collected; and its protein, 500 μg, was rotated with the anti-ABCA1 or anticalmodulin antibody overnight at 4°C and incubated with agarose A/G beads. Proteins bound to the beads were eluted by the SDS-PAGE sample buffer and analyzed by immunoblotting using specific antibodies.
Calmodulin-Agarose Binding Assay
Cell lysate as 250 μg of protein was incubated with 30 μL of 50% calmodulin-agarose bead suspension in Tris-hydrochloride saline for 3 hours at 4°C with 1-mmol/L EGTA or with the various concentrations of calcium chloride. Protein bound to the beads was eluted with the SDS-PAGE sample buffer and analyzed by Western immunoblotting using the anti-ABCA1 antibody.
Glutathione S-Transferase–Fusion Protein and Glutathione S-Transferase Pull-Down Assays
Expression vectors of the peptides corresponding to 1213 to 1349 and to 1264 to 1349 amino acid residue numbers of ABCA1, representing the PEST sequences with and without calmodulin (CaM) recognition sequence 1244 to 1257, respectively (CaM-PST and PST respectively), were prepared by introducing the EcoRI/XhoI fragment from enhanced green fluorescent protein–ABCA1 expression vector into pGEX-5X-1 vector (Amersham Biosciences). The expression of glutathione S-transferase (GST)–CaM–PST and GST-PST fusion proteins was induced in Escherichia coli BL-21 DE3 (Nippon Gene) and purified from cellular lysates by glutathione-Sepharose chromatography, solubilized with 50-mmol/L Tris-hydrochloride containing 10-mmol/L glutathione.
GST-CaM-PST and GST-PST proteins, 2 μg each, prepared as previously described,20 were incubated with 2 μg of purified calmodulin at 30°C for 45 minutes, followed by incubation with 25 μL of immobilized glutathione agarose beads at 4°C for 1 hour. After the incubation and washing of beads 4 times, protein was eluted with the SDS sample buffer and then immunoblotted by the anticalmodulin antibody.
Calpain-Mediated Proteolysis Assay and Calpain Activity In Vivo
The cell lysate was incubated with 0.5 to 3.0 μg of μ-calpain at 32°C for 1 hour. Alternatively, GST-CaM-PST or GST-PST protein and cell lysates were incubated with 1.5 μg of purified μ-calpain in the same manner. This reaction was discontinued by the SDS sample buffer, and the product was analyzed by electrophoresis in 15% SDS-PAGE. Proteins were stained by Coomassie brilliant blue or transferred to a polyvinylidene difluoride membrane for immunoblotting by anti-GST or anti-ABCA1 antibody.
The cells were pretreated with appropriate compounds for 3 hours and incubated with synthetic calpain substrate t-butoxycarbonyl-Leu-Met-chloromethylaminocoumarin (BOC-LM-CMAC) (Molecular Probes) for 20 min. After washing with PBS twice, fluorescence of the reaction product was examined in situ with a fluorescent plate reader (model FL600; Bio-Tek Inc) with excitation at 360 nm and emission at 410 nm.
Control of Intracellular Ca2+ Concentration
BALB/3T3 cells were preincubated with Ca2+-free Hanks’ solution containing EGTA, 1 mmol/L, and 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetoxymethyl ester, 100 μmol/L, for 30 minutes; washed twice; and cultured in the conditions of various extracellular Ca2+ concentrations (0, 10, 50, 100, and 1500 μmol/L calcium chloride) for 60 minutes, as previously reported.21
Small and Interfering RNA
Mouse calmodulin small and interfering RNA (CUU UCU CCC UAU UCG UAA TT for sense and UUA UCG AAU AGG GAG AAA GCT for antisense) and scramble control were obtained from Ambion and transfected into BALB/3T3 cells using Lipofectamine 2000 transfection reagents (Invitrogen), according to the protocol provided by the manufacturer. The cells were used for the experiments after 48-hour preincubation.
Clearance of ABCA1 Protein and Labeling and Tracing ABCA1 for Endocytosis
Cells were preincubated in DF medium containing 0.02% BSA in the presence of 100-μg/mL cycloheximide for 30 minutes, and cellular ABCA1 protein was analyzed by Western blotting at the indicated times.6
Cell surface proteins were biotinylated with sulfosuccinimidyl 2-(biotinamid)-rthyl-1, 3-dithiopropionate (Pierce) for 1 hour at 4°C, as previously described.8 To trace the labeled surface ABCA1 for internalization, biotinylation of the cell surface protein was cleaved with 50 mmol/L reduced glutathione (Sigma), and the uncleaved biotinylated ABCA1 was determined as the internalized portion.8
The release of cholesterol and phospholipid from BALB/3T3 cells was measured after incubation with 10-μg/mL apoA-I in DF medium containing 0.1% BSA for 16 hours, as previously described.22 Statistical analysis was performed by Wilcoxon signed rank test.
ABCA1-Calmodulin Interaction in a Ca2+-Dependent Manner
The interaction of ABCA1 with calmodulin was examined in the lysate of BALB/3T3 cells. ABCA1 was degraded by μ-calpain in a dose-dependent manner (Figure 1A). The interaction of ABCA1 with calmodulin was demonstrated by coimmunoprecipitation with the anti-ABCA1 antibody and with the anti-calmodulin antibody, whereas knockdown of each protein abolished these apparent cross interactions (Figure 1B). Binding of the membrane ABCA1 to calmodulin-agarose beads required Ca2+ showing the interaction to be Ca2+ dependent (Figure 1C).
The interaction of ABCA1 with calmodulin was further examined in cultured cells in the medium containing various Ca2+ concentrations or in the presence of calcium-chelating reagents. Chelating of calcium ion increases ABCA1 protein (Figure 1D, left, and E) as the result of a decease of calpain activity (Figure 1F). On the other hand, precipitation of calmodulin with ABCA1 increased with calcium (Figure 1D, right). The chelating treatment markedly decreased this association and increased ABCA1 as much as other reagents to block ABCA1 degradation (Figure 1E, bottom).
Calmodulin Recognition Motif in the Cytoplasmic Loop of ABCA1
Database analysis identified the PEST sequence in the cytoplasmic loop of ABCA1 (Figure 2A). The Ca2+-dependent calmodulin-binding motifs 1-5-8-14 were found in the cytoplasmic loop of ABCA1 as 1244 to 1257 (Figure 2B), located near the PEST sequence (1283 to 1306). To analyze whether this motif binds calmodulin in a Ca2+-dependent manner, GST-fusion peptides of the ABCA1 cytoplasmic loop were generated with and without this motif (GST-CaM-PST and GST-PST, respectively) (Figure 2A and C). The GST pull-down assay indicated that the GST-CaM-PST protein interacts with calmodulin in the presence of Ca2+, but the GST-PST protein does not (Figure 2D). The 1-5-8-14 motif in the cytoplasmic loop of ABCA1 was shown to bind to calmodulin in the presence of Ca2+.
Calmodulin Binding Protects ABCA1 Against Calpain-Mediated Degradation
The reactivity of GST-CaM-PST protein to calpain was examined in vitro (Figure 3A). GST-CaM-PST protein (44 kDa) was completely degraded by calpain in the absence of calmodulin. In the presence of calmodulin and Ca2+, GST-CaM-PST protein was partially degraded; 34- and 28-kDa peptides were identified as limited cleavage products. These results were confirmed by immunoblotting with an anti-GST antibody (Figure 3B). For GST-CaM-PST alone, a 26-kDa band was detected as the GST protein copurified in the sample. Preincubation with calmodulin resulted in limited proteolytic digestion of GST-CaM-PST by calpain to produce 34- and 28-kDa peptides; GST-CaM-PST was almost completely degraded without the pretreatment. The results demonstrated that calmodulin binds to ABCA1 in the presence of Ca2+ by the 1-5-8-14 motif, and this reaction leads to limited calpain-mediated digestion of ABCA1 in vitro.
Calmodulin Modulates Stability of ABCA1 and Its Activity In Vivo
The intracellular roles of calmodulin in regulation of ABCA1 expression were investigated. Knockdown of calmodulin resulted in decrease of ABCA1 as the total (Figure 4A, left) and cell surface (Figure 4A, right), without changing intracellular calpain activity (data not shown). The degradation of ABCA1 was accelerated when calmodulin was knocked down (Figure 4B and C) because it decreased to 20% of the original level in the knockdown cells after 120 minutes while it remained 50% in the control. It was recently reported that ABCA1 is degraded by calpain after its endocytosis,8 so that the rate of endocytosis of ABCA1 was examined and found unchanged by calmodulin knockdown (Figure 4C). Finally, the activity of ABCA1 was evaluated by measuring the release of cellular cholesterol and phospholipid by apoA-I. Figure 4D shows that the lipid releases were both decreased by knockdown of calmodulin (cholesterol, 2.45±0.31 versus 1.70±0.32; phospholipid, 4.92±0.48 versus 3.99±0.18, mean±SD). The results indicated that ABCA1 turnover was significantly accelerated in the calmodulin-knockdown cells; this resulted in decreased HDL biogenesis.
Binding to ABCA1, but Not “Activity” of Calmodulin, Modulates Stability of ABCA1
We reported that calmodulin inhibitor W7 increased the apoA-I–mediated release of cellular phospholipid and cholesterol.23 W7 increased ABCA1 protein in total cell (Figure 5A and B) and cell surface (Figure 5B). When the cell lysate was incubated with μ-calpain, ABCA1 decreased along with the increase of the 120-kDa band that reacts to anti-ABCA1 C-terminus peptide antibody (Figure 5C, left), consistent with the view that ABCA1 is cleaved at the region near the PEST sequence and calmodulin binding site. When the cells were preincubated with 10-μmol/L W7, ABCA1 increased and became resistant to the calpain-mediated degradation (Figure 5C, right, and Figure 5D). Under the condition that W7 increased ABCA1 by inhibiting calpain-mediated degradation, the apoA-I–mediated cellular lipid release increased in a dose-dependent manner, consistent with our previous work23 (Figure 5E). W7 increased the association of calmodulin with ABCA1 in a dose-dependent manner (Figure 5F). The results demonstrated that the physical interaction of calmodulin with ABCA1, but not the “activity” of calmodulin, protects ABCA1 from proteolysis by calpain. No further increase of ABCA1 was observed by inhibiting calpain in the presence of W7, and W7 was not effective when calmodulin was knocked down (Figure 6A).
We demonstrated the following: (1) calmodulin interacts with ABCA1 in a Ca2+-dependent manner and (2) this interaction inhibits calpain-mediated ABCA1 degradation and is involved in the regulation of its activity. These events are likely to take place at the calmodulin interaction site in the cytoplasmic loop near the PEST sequence and are interestingly enhanced by calmodulin inhibitor W7 (Figure 6B). Many cellular proteins with a rapid turnover contain the PEST sequence, including ABCA1. Its half-life is less than 2 hours unless degradation suppressors are present, and deletion of the PEST sequence results in retardation of the degradation.13 The PEST sequence is highly hydrophilic, easily accessible by calpain, and identified in many of calmodulin binding proteins.12 The binding of calmodulin induces conformational change in some calmodulin binding proteins and protects their calpain cleavage sites,24 consistent with the current finding with ABCA1.
Previously, it was found that calmodulin inhibitor W7 increased the apoA-I–dependent cell cholesterol release,23 apparently being contradictory to the current finding that calmodulin binding protects ABCA1 and upregulates its activity. Surprisingly, W7 increased calmodulin binding to ABCA1 and inhibited its calpain-mediated degradation. Therefore, the physical interaction of calmodulin with ABCA1 may interfere with its proteolysis; however, it is unrelated to other regular activities of calmodulin. Thus, previous data with W723 are consistent with the current observation. This view was supported by the findings that calpeptin does not further increase ABCA1 in the presence of W7 and that the effects of W7 were abolished by knockdown of calmodulin. It remains unknown why W7 increases calmodulin binding to ABCA1 despite its calmodulin inhibition.
We reported that ABCA1 in the plasma membrane is internalized by endocytosis and hydrolyzed by calpain, presumably in the endosome.8 The calpain inhibitor, calpeptin, increased ABCA1 and apoA-I–mediated lipid release,13,25 but did not influence ABCA1 endocytosis.8 When surface ABCA1 interacts with a helical apolipoprotein, such as apoA-I, it becomes resistant to calpain and is recycled to increase surface ABCA1. Consequently, inhibition of ABCA1 endocytosis decreases its degradation and increases it in the surface to enhance HDL biogenesis, by such compounds as cytochalasin D8 or spiroquinone and diphenoquinone, oxidized products of probucol.11 The current findings (Figure 4) showed that knockdown of calmodulin did not influence ABCA1 endocytosis, so that inhibition of ABCA1 degradation by calmodulin is likely the result of ABCA1 resistant to calpain.
However, a role of the PEST sequence in ABCA1 degradation is unclear. Deletion of the sequence prevented ABCA1 internalization, increased cell surface ABCA1,13 and decreased cholesterol efflux from late endosomes,26 suggesting that the PEST sequence associates with ABCA1 internalization despite the fact that it is unrelated to any internalization motif. These findings may agree with the observation that ABCA1 removes cholesterol from the late endosome27; however, they are inconsistent with many other reports8,28,29 that ABCA1 generates HDL particles mainly at the plasma membrane.
Phosphorylation of the PEST sequence reportedly promoted calpain-mediated degradation of ABCA1.30 Phosphorylation of PEST sequences generates a strong negative charge at the region that may bind calcium; this may locally activate calpains.31 The dephosphorylation-mimicking form of ABCA1 by deleting the PEST sequence became insensitive to the apoA-I treatment against the calpain-mediated proteolysis,30 supporting this hypothesis. On the other hand, previous reports showed that ABCA1 can be phosphorylated by protein kinases A,32 C,25 and CK233 in relation to regulation of ABCA1 functions. CK2 phosphorylation sites are the Thr1242, Thr1243, and Ser1255 residues in the cytoplasmic loop domain of ABCA1; dephosphorylation of these residues increased the functional activity of ABCA1 but did not significantly affect ABCA1 turnover.33 The calmodulin binding site of ABCA1 is located at 1244 to 1257, at a close or overlapping position to a CK2 phosphorylation site, so that the phosphorylation by CK2 may have interference through calmodulin binding.
In conclusion, ABCA1 is regulated by its calcium-dependent interaction with calmodulin. The mechanism is apparently by direct interaction between the 2 molecules but not through the regular mechanism for the activity of calmodulin.
Sources of Funding
This study was supported by grants-in-aid from The Ministries of Education, Science, Technology, Culture and Sports and of Health, Welfare and Labor of Japan; and by the Program for Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation.
Dr Yokoyama is involved in the establishment of a venture company (HYKES Laboratories).
Received on: September 10, 2009; final version accepted on: April 6, 2010.
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