extfieldsofstudy: Medicine Biology

text:
The Dynamic Alterations of H2AX Complex during DNA Repair Detected by a Proteomic Approach Reveal the Critical Roles of Ca2+/Calmodulin in the Ionizing Radiation-induced Cell Cycle Arrest*S

By using DNA nuclease digestion and a quantitative “dual tagging” proteomic approach that integrated mass spectrometry, stable isotope labeling, and affinity purification, we studied the histone H2AX-associating protein complex in chromatin in mammalian cells in response to ionizing radiation (IR). In the non-irradiated control cells, calmodulin (CaM) and the transcription elongation factor facilitates chromatin transcription (FACT) were associated with H2AX. Thirty minutes after exposing cells to IR the CaM and FACT complexes dissociated, whereas two DNA repair proteins, poly(ADP-ribose) polymerase-1 and DEAH box polypeptide 30 isoform 1, interacted with H2AX. Two hours and 30 min after exposure, none of the above proteins were in the complex. H2B, nucleophosmin/B23, and calreticulin were associated with H2AX in both non-irradiated and irradiated cells. The results suggest that the H2AX complex undergoes dynamic changes upon induction of DNA damage and during DNA repair. The genuine interactions between H2AX and H2B, nucleophosmin/B23, calreticulin, poly(ADP-ribose) polymerase-1, and CaM under each condition were validated by immunoprecipitation/Western blotting and mammalian two-hybrid assays. Because multiple Ca2+-binding proteins were found in the H2AX complex, the roles of Ca2+ were examined. The results indicate that Ca2+/CaM plays important roles in regulating IR-induced cell cycle arrest, possibly through mediating chromatin structure. The dataset presented here demonstrates that sensitive profiling of the dynamics of functional cellular protein-protein interactions can successfully lead to the dissection of important metabolic or signaling pathways.

The basic structural unit of chromatin is the nucleosome, which consists of a histone octamer (two copies of each H2A, H2B, H3, and H4) around which is coiled 1.7 turns of 168-bp DNA stabilized by a fifth histone, H1. Controlled nuclease digestion results in a stable subnucleosomal "core particle," which contains 146-bp DNA coiled around the histone octamer. Except for histone H4, each histone is in a family of subtypes. Of particular interest are the histone subtypes called "replacement histones." Unlike the major histone subtypes, which are synthesized in S phase of the cell cycle, replacement histones are synthesized through the cell cycle and in terminally differentiated cells (1). H2AX is such a replacement histone, which exists in all cell types, and accounts for 2-25% of the total cellular H2A (2,3). Accumulating evidence has shown that H2AX is a key factor in the repair of DNA double strand breaks (DSBs) 1 (4). Recent studies suggest that H2AX may be a major source of genome instability (5). H2AX is rapidly phosphorylated (␥-H2AX) following exposure of cells to IR, which induces DSBs, and forms IR-induced foci at the damage sites (2,4). It was proposed that ␥-H2AX may serve as a docking site for other DNA damage repair/ signaling proteins to bind in the vicinity of DNA lesions (6). However, despite major advances achieved primarily by immunofluorescence and immunochemical techniques, important questions regarding the role of H2AX in chromatin in DNA damage/repair remain to be elucidated. For example, what are the factors involved in the formation of H2AX IR-induced foci prior to and after the induction of the DSBs?
Extraction of nuclear proteins from mammalian cells with buffers containing high salt and detergent has become a standard procedure. However, a recent study designed to profile RNA polymerase II-interacting proteins has failed to identify any known transcription or elongation factors and illustrates the limitations of the salt extraction approach in characterizing chromatin-related protein complexes (7). To obtain H2AX-interacting proteins from insoluble chromatin, we used nuclease to digest chromatin. The advantage of this approach is that soluble intact protein complexes containing histone H2AX can be obtained without using high salt conditions.
Due to the high sensitivity, high specificity, and the capability of high throughput, mass spectrometry has emerged as a powerful tool to efficiently and systematically identify proteins in biological samples (8,9). To identify the components of H2AX complexes, we used a mass spectrometry based dual-tagging proteomic approach (Fig. 1). The bait protein H2AX is epitope-tagged for affinity isolation of the complex (epitope tagging), and in parallel the whole proteome of the cells expressing the epitope-tagged H2AX at physiologically relevant levels is labeled with deuterium-labeled heavy amino acids (isotope tagging). In mass spectrometric measurements, the heavy amino acids incorporated in the cellular proteins provide "in-spectra" quantitative markers so that proteins in a complex with H2AX can be unambiguously identified after a single step affinity purification (10). In the present study, by using DNA nuclease digestion of nuclei and the dual tagging strategy, we identified the proteins that associate with H2AX in chromatin in mammalian cells both before and after IR to monitor the dynamics of the complex during DNA repair.

EXPERIMENTAL PROCEDURES
Plasmids, Stable Cell Line, and Cell Culture-The coding sequence of H2AX (2) was cloned into the BamHI and XhoI sites of a retroviral vector, pMIR-DFT, with a double FLAG tag at the N terminus. The plasmids containing the tag alone or FLAG-H2AX were transfected into 293T cells with the calcium phosphate method, and the cells were selected in DMEM supplemented with 10% fetal bovine serum, 1% penicillin and streptomycin, and 1.2 g/ml puromycin. 293T is an SV40 T antigen-expressing variant of the 293 adenovirus-transformed human embryonic kidney cell line. The cell line offers many advantages such as high transfection yield, can be easily grown in suspension culture, and is widely used for various biological studies (9,11). Whereas the cells containing the tag alone (control cells) were cultured in regular DMEM, the cells containing FLAG-tagged H2AX (FLAG-H2AX cells) were maintained in DMEM containing Leu-d 3 to isotopically label the proteome (Fig. 1A).
Irradiation of Cells, Nuclei Isolation, and Nuclease Digestion-Cells were irradiated with a ␥-ray 137 Cs source (Mark I model 68A) at 3.7 Gy/min. Approximately 8 ϫ 10 8 FLAG-H2AX cells as well as the control cells were harvested and washed twice with cold PBS, and then equal numbers of each cell were mixed. The mixed cells were lysed in 5 packed cell pellet volumes of lysis buffer (10 mM Hepes-NaOH, pH 7.9, 10 mM KCl, 1.5 mM MgCl 2 , and 0.5 mM ␤-mercaptoethanol) supplemented with protease inhibitor mixture (Roche Applied Science) and phosphatase inhibitors (1 mM sodium orthovanadate, 10 mM sodium fluoride, and 10 mM ␤-glycerophosphate). The nuclei were isolated by centrifuging the lysate at 1000 ϫ g for 10 min at 4°C and washed twice with 10 ml of digestion buffer (10 mM Tris-HCl, pH 8.0, 7.5 mM MgCl 2 , 0.75 mM CaCl 2 , and 0.5 mM ␤-mercaptoethanol) plus the protease and phosphatase inhibitors. The nuclei were then digested with 500 units/ml DNase I (Roche Applied Science) at 37°C for 50 min, lysed in nuclei lysis buffer (10 mM Tris-HCl, pH 7.6, 5 mM EDTA, 0.05% Nonidet P-40, and 0.5 mM ␤-mercaptoethanol) plus the protease and phosphatase inhibitors, and cleared by centrifugation. Completion of the digestion was checked with a 2% agarose gel.
Protein Purification-Following completion of the chromatin digestion, glycerol and NaCl were added to the cleared lysate to the final concentrations of 20% and 150 mM, respectively, and the nuclear extract was incubated with 200 l of M2 anti-FLAG beads (Sigma) at 4°C for 2 h. The beads were then washed four times with 4 ml of washing buffer (10 mM Tris-HCl, pH 7.6, 150 mM NaCl, 20% glycerol, 1 mM EDTA, 0.05% Nonidet P-40, and 0.5 mM ␤-mercaptoethanol) plus the protease and phosphatase inhibitors. The bound proteins were eluted with a buffer containing 250 g/ml 3ϫ FLAG peptide (Sigma). The eluted proteins were concentrated with trichloroacetic acid precipitation and then separated by 4 -20% SDS-PAGE. After staining with Coomassie Brilliant Blue, the whole lane of gel was cut into 25 slices for LC-MS/MS analysis.
LC-MS/MS Analysis and Database Searching-In-gel digestion and LC-MS/MS analysis were performed as described previously (12). The MS/MS data for each sample were searched against the NCBInr (January 20, 2004) protein sequence database downloaded from the National Center for Biotechnology Information under the species restriction of Homo sapiens using the in-house licensed Mascot searching program (version 2.0). The Leu-d 3 modification was added in the configuration file and selected as variable modifications in the database searching. The parameters for database searching were as follows: (i) 0.2-Da mass error tolerance for both MS and MS/MS, (ii) tryptic enzyme specificity with a maximum of two missed cleavages, and (iii) the following variable modifications: acetylation at peptide N terminus, phosphorylation on tyrosine/serine/threonine, and oxidation on methionine.
Immunoblotting Analysis-293T cells (7 ϫ 10 7 cells/assay) were left untreated or irradiated with 30 Gy, recovered at 37°C at the times indicated in each specific experiment, and harvested for nuclei isolation/digestion, immunoprecipitation, and Western blotting. The procedures for nuclei isolation/digestion and immunoprecipitation were essentially the same as described under "Irradiation of Cells, Nuclei Isolation, and Nuclease Digestion" and "Protein Purification" except that buffer volumes in each step were reduced ϳ10-fold.
Mammalian Two-hybrid Analysis-Vectors encoding Gal4 DNAbinding domain (BD) and transcription activation domain (AD) were from a mammalian two-hybrid assay kit (BD Biosciences). The Gal4 GFP reporter plasmid was kindly provided by Dr. Toshi Shioda (Massachusetts General Hospital Cancer Center). The coding sequences of H2AX and CaM were inserted into the BD and AD vectors, respectively, and the two constructs were co-transfected into 293T cells with the Gal4 GFP reporter plasmid (2 g of each plasmid in a 60-mm plate). The negative control was performed by co-transfection of 293T cells with the Gal4 GFP reporter plasmid and the BD and AD constructs in which the BD and AD were fused with two proteins that do not interact. The expression of GFP was analyzed by both flow cytometry and Western blotting.
Cell Cycle Analysis-The exponentially growing cells in DMEM were mock-or ␥-ray-irradiated and immediately replated. For the Ca 2ϩ treatments, CaCl 2 was added to the culture medium to the indicated concentrations immediately after replating. After allowing cells to recover at 37°C for the indicated period of time, the cells were harvested, washed, fixed, and stained with propidium iodide, and cellular fluorescence was measured by using a FACSCalibur flow cytometer (BD Biosciences). To detect the phosphorylated histone H3 in mitosis, cells were analyzed as described previously (13), and 10,000 events were recorded for each measurement.

The Dual Tagging Proteomic Strategy for Protein Complex
Identification-The schematic of our quantitative dual tagging proteomic approach for identifying proteins associated with H2AX is illustrated in Fig. 1A, and representative results are shown in Fig. 1B. The stable cells expressing tag alone and the stable cells expressing the FLAG-H2AX are grown in the "light" and "heavy" medium, respectively. After affinity purification of H2AX using the beads conjugated with anti-FLAG antibody and LC-MS/MS analysis of the bound proteins, all of the Leu-containing peptides appear as pairs in the MS spectrum with one set of peaks from the stable isotope-labeled cells and the other set of peaks from the unlabeled cells. The intensity ratios of the paired peaks reflect the binding profiles of the parent protein to the bait H2AX (Fig. 1).
The Epitope-tagged H2AX Is Incorporated into Chromatin-To determine whether a tag added to the N terminus of H2AX would affect the correct packing and function of H2AX in the nucleosome, we first determined the extraction profiles with increasing concentrations of NaCl of (i) the FLAG-H2AX from the FLAG-H2AX cells and (ii) the endogenous H2AX from the parental 293T cells (Fig. 2A). The results showed that the FLAG-tagged and endogenous H2AX had similar extraction profiles. Both were resistant to NaCl extraction, suggesting that the tagged H2AX has tightly bound within chromatin. Then we digested nuclei with micrococcal nuclease, immunoprecipitated the digested nuclear extract with anti-FLAG beads, and analyzed the DNA fragment size released by the digestion (Fig.  2B). Whereas, as expected, no immunoprecipitates were found for the parental 293T cells, the immunoprecipitates from the FLAG-H2AX cells produced two DNA bands of approximate 150 and 300 bp, which corresponded to the DNA sizes of core particle and two closely adjacent core particles. This result indicates that the tagged H2AX is correctly packed into the core particle. Lastly we mock-and ␥-ray-treated (30 Gy) the FLAG-H2AX cells and determined the phosphorylation states of both the tagged and endogenous H2AX using anti-␥-H2AX (Fig. 2C). As for its endogenous counterpart, the tagged H2AX was phosphorylated after IR, suggesting that it responds properly to the induction of DSBs. Furthermore the FLAG-H2AX was expressed at levels similar to those of its endogenous counterpart (Fig. 3), and the cells expressing the FLAG-H2AX showed a growth rate and morphology similar to those of the parental cells, indicating that the expression of the FLAG-tagged H2AX has no effect on the phenotype of the parental cells. Collectively the above results strongly suggest that the FLAG-tagged H2AX, like its endogenous counterpart, is correctly packaged into the nucleosome and functions properly in chromatin.

FIG. 1. Identification of H2AX-interacting proteins by a quantitative dual tagging proteomic approach.
A, strategy to identify H2AXinteracting proteins. The control cells are cultured in regular DMEM (green dish, unlabeled), and the FLAG-H2AX cells are cultured in the Leu-d 3 -containing medium (red dish, labeled). Equal numbers of unlabeled and labeled cells are mixed, and their nuclei are isolated from the total lysate. After digestion and lysis of the nuclei, the resulting soluble nuclear proteins are affinity-purified with anti-FLAG beads. Proteins eluted from the beads are separated by SDS-PAGE and digested with trypsin, and the resulting peptides are analyzed by mass spectrometry. Whereas the ratios of the labeled:unlabeled peak intensity for the nonspecific binding proteins are around 1, the ratios for the proteins that specifically bind to H2AX are significantly larger than 1. B, representative MS spectra showing the nonspecific and specific binding to H2AX: panel a, nonspecific binding; panel b, specific binding.
The Assessments of Chromatin Digestion-We used nuclease DNase I to digest chromatin to obtain soluble intact H2AX complexes. In such an approach, complete digestion of chro-matin is essential for the identification of proteins associated with H2AX directly. For this reason, we tested chromatin digestion conditions extensively. As shown in Fig. 4, when the partially digested chromatin was immunoprecipitated with anti-FLAG beads, Western blotting with anti-H2AX antibody exhibited two bands, one at approximate 14 kDa corresponding to the endogenous H2AX ( . We assume that the endogenous H2AX in the partially digested chromatin was not from the same nucleosome as the tagged H2AX because the two copies of H2A do not interact directly in the same nucleosome (14). Further analysis showed that when the completely digested chromatin was used for immunoprecipitation, H2B could be detected ( Fig. 4C) but not H3 or H4 (data not shown). Collectively these results strongly suggest that under the complete digestion conditions established in this study, no intact nucleosomes are present in the digested nuclear extract, and the H2AX-H2B dimer is the major stable form of H2AX in solution. Therefore in this study we refer to the H2AX-interacting proteins as those associated with H2AX directly or indirectly through H2B or other closely associated proteins.
Identification of H2AX-interacting Proteins in the Non-irradiated Control Cells-We first characterized the H2AX complex isolated from the cells without IR exposure. Of the 79 proteins identified, 13 proteins were selectively enriched with the anti-FLAG beads by a factor of at least 1.5 (Table I). Reproducibility was assessed primarily according to the method described by Blagoev et al. (15), that is by (i) deter-

FIG. 2. The FLAG-tagged H2AX is incorporated into chromatin.
A, FLAG-tagged H2AX shows resistance to NaCl extraction similar to that of its endogenous counterpart. Parental 293T and the FLAG-H2AX cells were lysed in PBS plus 0.1% Triton X-100 and 0 -2 M NaCl, and the total cell lysate was fractionated into supernatant and pellet by centrifugation. Proteins from each fraction were resolved by SDS-PAGE and detected by Western blotting (WB). The results showed that the FLAG-tagged and endogenous H2AX had similar extraction profiles, indicating that the FLAG-tagged H2AX has tightly bound within chromatin. B, FLAG-H2AX-containing mono-and dinucleosome core particles harbor the DNA fragments with expected sizes. Nuclei from parental 293T and the FLAG-H2AX cells (1.8 ϫ 10 7 cells/each) were digested with micrococcal nuclease and immunoprecipitated (IP) with anti-FLAG beads. After extraction/precipitation, the DNA was separated by 2% agarose gel and visualized with ethidium bromide staining. The immunoprecipitates from FLAG-H2AX cells (lane 4) produced two DNA bands of approximately 150 and 300 bp, which corresponded to the expected DNA sizes of core particle and two closely adjacent core particles, indicating that the FLAGtagged H2AX was correctly packed into the core particle. C, FLAGtagged H2AX is phosphorylated similarly to its endogenous counterpart upon IR. Parental 293T and the FLAG-H2AX cells were mock-or ␥-ray-irradiated and lysed in 2ϫ sample buffer. The proteins were then resolved by SDS-PAGE and detected by Western blotting. The induction of phosphorylation of FLAG-tagged H2AX by irradiation (lane 2) is shown. mining the variability of ratios for different peptides of the same protein, (ii) determining the variability of ratios for different spectra of the same peptide across the eluted chromatographic profile, (iii) determining the variability of ratios of the same protein detected in different gel fractions (i.e. the same protein appeared in different gel bands; see Supplemental Table I), and (iv) performing independent runs for some of the fractions. The results from the above analyses led to a cutoff value of enrichment-fold of 1.5 for Table I and the first  part (30 min section) of Table II and 1.3 for the second part (2.5 h section) of Table II. A smaller cutoff value was set for the  second part of Table II (2.5 h section) due to these data being more uniform, which was reflected by smaller standard deviations (compare the standard deviations in the 2.5 h section in Table II with those in Table I and those in the 30 min section in Table II). H2B and H4 were among the histones identified. Because no intact nucleosomes were found in the digested nuclear extract (Fig. 4), the H4 in the H2AX complex probably resulted from free H4. The crystal structure of the nucleosome core particle shows that the histone octamer is composed of two copies of each H2A-H2B dimer and the (H3-H4) 2 tetramer formed by H3-H3 "histone fold" interactions (14). Each H2A-H2B dimer interacts with the tetramer through the H2B-H4 histone fold. Consistent with the direct interactions of H2A and H2B in the structure, the enrichment ratio for H2B was clearly different from the ratio for H4 (Table I) whose interaction is mediated through H2B (14).
CaM is a Ca 2ϩ -dependent regulatory protein, and its target enzymes are among important regulators such as protein kinases and phosphatases. Calreticulin is also a Ca 2ϩ -binding protein. Although calreticulin is mainly located in the endoplasmic reticulum, it is also found in the nucleus and can bind to histones (16). BiP is a chaperone protein localized in the endoplasmic reticulum, and its function is highly regulated by Ca 2ϩ . It has been reported that BiP binds to denatured histones in vitro (16).
The structure-specific recognition protein 1 and the chromatin-specific transcription elongation factor are the two components of the transcription elongation factor FACT. The two proteins form a stable heterodimer, which is required for transcription elongation when chromatin is used as a template (17). In vitro biochemical studies with purified proteins have shown that FACT interacts with the H2A-H2B dimer of the nucleosome and forms a FACT-H2A-H2B complex. It facilitates transcription elongation by displacing the H2A-H2B dimer from the nucleosome (18). In this study, both subunits of the FACT were identified, suggesting that FACT may play a crucial role in DNA damage repair or transcription in vivo.
Nucleophosmin/B23 is a nuclear phosphoprotein and is more abundant in cancer cells than in normal resting cells. UV radiation triggers an immediate up-regulation of nucleophosmin/B23 expression in mammalian cells, suggesting that nucleophosmin/B23 may be involved in the acute response of cells to environmental stress (19). It has been reported that nucleophosmin/B23 binds to histone proteins in HeLa cells (20).
Identification of H2AX-interacting Proteins in the Irradiated Cells-Previous reports have shown that ϳ30 min after the exposure of cells to IR, H2AX was strongly phosphorylated FIG. 4. The assessments of the experimental conditions for chromatin digestion. Nuclei were isolated from cells and digested with DNase I. FLAG-tagged H2AX and its associated proteins were then immunoprecipitated (IP) and visualized with Western blotting (WB). A, partial versus complete digestion of chromatin. After digestion of chromatin with DNase I, the resulting soluble nuclear extract was checked by 2% agarose gel stained with ethidium bromide. The difference between partial digestion (lane 1) and complete digestion (lane 2) with regard to the amount of DNA detected is shown. B, Western blot analyses of the partially and completely digested chromatin. Partially and completely digested chromatin from FLAG-H2AX cells was immunoprecipitated with anti-FLAG beads and Western blotted with the antibodies indicated (note that a weak band around 25 kDa in lane 3 was a contamination). The difference between partially digested chromatin (lane 1), which contained both FLAGtagged H2AX and endogenous H2AX, and the completely digested chromatin (lane 2), which contained only FLAG-H2AX, is shown. The difference indicates that the partially digested chromatin contained intact polynucleosome core particles, whereas the completely digested chromatin did not (see text for details). C, H2B is complexed with H2AX after the complete digestion of chromatin. After complete digestion, immunoprecipitation with anti-FLAG beads was performed, and the immunoprecipitates were probed with anti-H2B. and formed IR-induced foci around the DSB site (4). We analyzed the H2AX protein complex to identify the factors associating with H2AX in this dynamic phase. Thirty minutes after IR, four proteins that were found to interact with H2AX from the non-irradiated cells, H2B, calreticulin precursor, nucleophosmin/B23, and BiP (Table I), were again identified to DnaK-type molecular chaperone HSPA5 precursor 1.33 0.08 3 a Only the proteins with two or more peptides matched are listed. b Fold of enrichment was calculated as the ratio of Leu-d 3 -labeled peptide to unlabeled peptide. c Standard deviation was determined from multiple peptides except for proteins H2B and H2BE from the cells recovered for 2.5 h whose enrichments were estimated from a single Leu-containing peptide. NA, not applicable. complex with H2AX (Table II). The repeated identification of these four proteins on one hand suggests that these proteins form stable complexes with H2AX and on the other hand confirms the reproducibility of our experimental approach. In addition to the four proteins, two proteins involved in DNA damage and repair, PARP-1 and the DEAH box polypeptide 30 isoform 1, were identified (Table II). Interestingly CaM and FACT, which were shown to be associated with H2AX in the non-irradiated cells (Table I), had dissociated from H2AX after IR. PARP-1 is the enzyme that uses NAD ϩ as a substrate to catalyze the transfer of ADP-ribose to a variety of nuclear protein acceptors. The most poly(ADP-ribosyl)ated proteins in vivo are PARP-1 itself and histones H1 and H2B (21). It has been shown that PARP-1 plays important roles in DNA damage repair and in transcription (22,23). Although histones are the preferred targets in vivo and in vitro (21), the stable histone-PARP-1 complex has not been reported. DEAH box polypeptide 30 isoform 1 is a member of the DEAH helicase family, which plays important roles in basal transcription, DNA repair, and chromosome transmission (24). A member of the DEAH helicase family has been shown to directly interact with the highly conserved C-terminal BRCT repeat of the tumor suppressor BRCA1, and the disruption of this interaction leads to defects in DNA repair and progression of breast and ovarian cancer (25). BRCA1 was reported to co-localize with H2AX at DSB sites (4,26). However, to our knowledge no physical interaction between BRCA1 and H2AX has been observed. The identified association between H2AX and DEAH box polypeptide 30 isoform 1 suggests that the DNA damage-induced co-localization of BRCA1 and H2AX may be mediated by members of the DEAH helicase family. The next step was to profile the H2AX complex when cells were irradiated and allowed to recover at 37°C for a longer period of time, 2.5 h. As shown in Table II, histone H2B, calreticulin precursor, and nucleophosmin/B23 were reproducibly identified. At this time point, DNA repair proteins, PARP-1 and the DEAH box polypeptide 30 isoform 1, were found to be dissociated from H2AX. One interesting phenomenon was that two histone H2B isoforms, gi͉4504263 and gi͉184086, were identified to join the H2AX complex. The two H2B isoforms (gi͉4504263 and gi͉184086) are distinguished from the bulk H2B (gi͉28173554) by peptide EIQTAVR-LLLPGELAK. The two H2B isoforms (gi͉4504263 and gi͉184086) are distinguished from one another by two unique peptides, KESYSVYVYK for gi͉4504263 and KESYSIYVYK for gi͉184086 (Supplemental Table I).  (Tables I and II), Western blotting showed that the two H2AX partners, nucleophosmin/B23 and calreticulin, were coprecipitated with H2AX under all three conditions (Fig. 5A). However, there were some inconsistencies between the proteomic and the Western data. The Western data showed that when cells were irradiated and allowed to recover for 2.5 h more nucleophosmin/B23 and less calreticulin were associated with H2AX than those under the other two conditions (Fig. 5A, compare lane 6 with lanes 2 and 4). Proteomic data showed that the two proteins had similar isotopic enrichment ratios under three different treatment conditions (Tables I and  II). A possible explanation is that for the mass spectrometric analysis we had mixed the labeled and unlabeled protein lysates together before affinity purification, and some in vitro binding or exchange might have occurred between the unlabeled proteins and the labeled FLAG-H2AX protein complexes. There were two possible mechanisms: (i) the components of the H2AX complex, which were Leu-d 3 -labeled, might exchange with the unlabeled counterparts in the mixed protein lysate or (ii) the labeled H2AX protein complex might bind additional unlabeled components in addition to its existing ones (i.e. increasing the copy numbers of the components). Both reactions would lead to lower labeled-to-unlabeled ratios (i.e. the "Enrichment-fold" in Tables I and II) in the mass spectrometric analysis, which have been observed in this experiment (Tables I and II) and another report that used similar experimental strategy (15). The fact that the proteins that specifically associate with bait protein have significantly higher labeled-to-unlabeled ratios than those for nonspecific bound proteins, which were observed in this study and the other report (15), suggests that the unlabeled proteins cannot reach a copy number similar to the existing labeled components of the protein complex under defined experimental conditions. In short, the reason for the inconsistency between proteomic data and Western data is that the isotopic ratios in the mass spectrometric analysis may not reflect the stoichiometry profile of the protein complex as Western blotting indicates. We also tested for PARP-1, which the proteomic data had shown to be associated with H2AX when the cells were irradiated and allowed to recover for 30 min but not FIG. 6. Ca 2؉ partially relieves the IR-induced G 2 arrest. A, Ca 2ϩ partially relieves the IR-induced G 2 arrest. The cells in the exponential growth phase in DMEM were mock-or ␥-ray-irradiated and immediately replated. For the Ca 2ϩ treatments, CaCl 2 was added to the culture medium to a final concentration of 15 mM immediately after replating. After allowing cells to recover for the indicated period of time, the cells were harvested for analysis by flow cytometry.  c with panel b). B, Ca 2ϩ enhances the G 2 -to-M transition of the irradiated cells. The cells were grown, irradiated, Ca 2ϩ -treated as in A, and allowed to recover for 30 min. The cells were then harvested and costained with propidium iodide and antibody to phospho-histone H3 (Ser-10) as described (13). under the other two conditions (Tables I and II). The immunoprecipitation and Western blot analysis results (Fig. 5A) were consistent with the proteomic data. The H2AX-H2B interaction in the non-irradiated control cells has been demonstrated in Fig. 4C. To ensure that similar amounts of target protein H2AX were loaded for each treatment, the Western blot filter was also probed with anti-H2AX. As expected, the tagged H2AX was present in similar amounts for all three treatments (Fig. 5A, compare lanes 2, 4, and 6).

Immunochemical and Mammalian Two-hybrid Analyses Validate the Proteomic Data-In agreement with the proteomic data
We analyzed the interaction between H2AX and CaM with a mammalian two-hybrid system with the expression of the reporter GFP detected by flow cytometry and Western blotting (Fig. 5, B and C). There was no GFP expression in the non-transfected 293T cells (Fig. 5, B, lane 1, and C, lane 1). Co-transfection of 293T cells with the Gal4 GFP reporter plasmid and the constructs encoding fusion proteins Gal4 BD-H2AX and AD-CaM resulted in the expression of GFP in 13.6% of the total cells (Fig. 5, B, lane 3, and C, lane 3) relative to the negative control of 3.9% (Fig. 5, B, lane 2, and C, lane  2). These results suggest that H2AX interacts with CaM in mammalian cells in situ in agreement with our proteomic data (Table I).
Cell Cycle Is Arrested at the G 2 Phase after IR, and the Elevated Extracellular Ca 2ϩ Partially Relieves the Arrest-After observing the dynamic alterations of the components of the H2AX complex upon IR, we investigated how chromatin structure and cell cycle checkpoint responded to IR. The results from both 4Ј,6-diamidino-2-phenylindole staining of the cellular DNA and the salt extraction analysis of nuclei showed that chromatin was condensed after cells were exposed to IR (Supplemental Fig. 1); this has also been observed in HeLa cells (27). We then examined the effects of IR on cell growth and division. Whereas the non-irradiated cells continued to grow from about 50% confluency to almost full confluency in 24 h, the irradiated cells stopped dividing and appeared larger than the non-irradiated cells, suggesting that the IR had induced cell cycle arrest (Supplemental Fig. 2). We then analyzed the cell cycle by flow cytometry. As shown in Fig. 6A, panel a, the non-irradiated cells exhibited a similar profile of heterogeneous log phase distribution in the 12-h period. When irradiated with 8 Gy of ␥-ray, cells gradually accumulated in the G 2 /M phase (Fig. 6A, compare panel b with panel  a), indicating IR-induced G 2 /M arrest. Because multiple Ca 2ϩbinding/signaling proteins were identified to be involved in the dynamics of the H2AX complex during DNA repair (Tables I  and II), we asked whether Ca 2ϩ binding/signaling was involved in the IR-induced G 2 /M arrest. To test this speculation, we examined the effects of Ca 2ϩ on cell cycle progression of the irradiated cells. As shown in Fig. 6A, panel c, when CaCl 2 was added to the culture medium of the irradiated cells immediately after IR, to increase the Ca 2ϩ concentration from 1.8 mM (the original Ca 2ϩ concentration in culture medium) to 15 mM, the IR-induced accumulation of cells in the G 2 /M phase was significantly reduced (Fig. 6A, compare panel c   with panel b). For control purposes, we also tested the effects of elevated Ca 2ϩ concentration (15 mM) on non-irradiated cells. As shown in Fig. 6A, panel d, no obvious effects of Ca 2ϩ on the cell phase distribution of the untreated cells were observed (Fig. 6A, compare panel d with panel a).
To further examine the effects of Ca 2ϩ on cell cycle progression, we evaluated the transition of cells from the G 2 to M phase (Fig. 6B). In Fig. 6B, propidium iodide was used to monitor the cellular DNA content (y axis), and the antibody to histone H3, which is phosphorylated exclusively during mitosis, was used to identify the mitotic cells from the G 2 cells (x axis). Compared with the non-irradiated cells, 30 min after IR the transition from the G 2 to M phase was reduced significantly in the irradiated cells (Fig. 6B, compare panel b with  panel a). When CaCl 2 was added to the culture medium of the irradiated cells, the IR-induced reduction in the G 2 -to-M transition was restored (Fig. 6B, compare panel c with panel b). We did not observe any significant effects of elevated extracellular Ca 2ϩ concentration on the G 2 -to-M transition of the non-irradiated cells (Fig. 6B, compare panel d with panel a).
Collectively these results suggest that Ca 2ϩ is involved in the G 2 -to-M transition in mammalian cells, and IR-induced G 2 arrest may involve Ca 2ϩ binding/signaling. Ca 2ϩ Promotes the Proliferation of the Irradiated Cells-We also examined the effects of Ca 2ϩ on cell growth and division. The exponentially growing cells were irradiated (8 or 30 Gy), and equal numbers of cells were plated on 60-mm plates. CaCl 2 was added to the culture medium to the indicated concentrations, and the cells were allowed to grow at 37°C. After 2 days, viable cells were counted using trypan blue exclusion assay (Sigma). Fig. 7 shows that in contrast to the somewhat inhibitory effects of elevated extracellular Ca 2ϩ concentration (over ϳ5 mM) on non-irradiated cell numbers, increasing extracellular Ca 2ϩ concentrations resulted in increased viable cell numbers for the irradiated cells, and the responses were dose-dependent. DISCUSSION By combining DNA nuclease digestion of chromatin, which releases intact protein complexes from the insoluble chromatin, and a quantitative dual tagging proteomic strategy, which identifies real time specific protein-protein interactions, we observed dynamic changes in the H2AX protein complex in response to the induction of DSBs. One noteworthy observation is that multiple Ca 2ϩ -binding/signaling proteins were found in the H2AX protein complexes (Table I). This result led us to examine whether Ca 2ϩ /CaM is involved in the IR-induced G 2 arrest and cell proliferation. The results demonstrate that the elevated extracellular Ca 2ϩ partially relieves the IR-induced G 2 arrest (Fig. 6A), enhances the IR-affected G 2to-M transition (Fig. 6B), and promotes cell division of the irradiated cells (Fig. 7). It has been established that entry into mitosis requires the activation of the mitotic cyclin-dependent kinase Cdc2 through the dephosphorylation of phosphoryl-ated Thr-14 and Tyr-15 by the mitosis-promoting phosphatase Cdc25c (28). In response to DNA damage, the checkpoint kinases Chk1 and Chk2 inactivate Cdc25c by phosphorylating Ser-216, leading to the G 2 arrest (29). The results observed in this study (Fig. 6) suggest that Ca 2ϩ -dependent factors may also be involved in the IR-induced G 2 checkpoint in addition to Chk1 and Chk2. CaM-dependent protein kinase II is a CaM-dependent protein kinase and can phosphorylate Cdc25c on Ser-216 in vitro (30,31). It was proposed that CaM-dependent protein kinase II controls the G 2 checkpoint through the phosphorylation of Cdc25c on Ser-216 (30,31). However, the results in this study argue against this hypothesis because if CaM-dependent protein kinase II plays a major role in controlling the G 2 checkpoint, it is difficult to explain the observation that the elevated extracellular Ca 2ϩ promotes the G 2 -to-M transition in the irradiated cells (Fig. 6). Therefore, a mechanism other than CaM-dependent protein kinase II must be responsible for the Ca 2ϩ / CaM-dependent regulation of the cell cycle progression in mammalian cells.
CaM is the primary intracellular Ca 2ϩ receptor and is universally required for cell proliferation in eukaryotes (32). In this study, we found that CaM associated with H2AX in the nonirradiated cells but dissociated in the irradiated cells. In vitro biochemical studies have shown that CaM interacts with histones and forms a CaM-histone complex (33,34). The interaction between CaM and histones is both Ca 2ϩ -and charge density-dependent, and the removal of Ca 2ϩ and phosphorylation of histones cause dissociation of the complex (33). It has also been shown that the binding of CaM to histones dramatically promotes the dephosphorylation of histones by phosphatase (34). The results in Fig. 6 suggest that IR induced the decreased level of intracellular Ca 2ϩ or CaM. Consistent with this finding, it was reported that IR resulted in dramatic reductions in CaM expression in multiple cell lines (35). Perhaps the IR-induced rapid phosphorylation of H2AX (2, 4) and the IR-repressed expression of CaM (35) contributed to the dissociation of the CaM-H2AX complex in vivo (Tables I and  II). Because H2AX is a structural component of chromatin, it is possible that changes in H2AX complex structure and H2AX phosphorylation after IR result in chromatin condensation (Supplemental Fig. 1) (27). On the other hand, it was reported that H2AX Ϫ/Ϫ cells exposed to low doses of irradiation exhibited mild G 2 arrest (36,37). Furthermore most of the factors in the checkpoint signaling pathway were reported to be localized at the DNA damage sites in chromatin (38). Several lines of recent evidence support the hypothesis that chromatin structure plays crucial roles in DNA repair and checkpoint signaling (39 -43). We propose that in mammalian cells the repression of CaM expression and the phosphorylation of H2AX are important cellular responses to the induction of DSBs. These changes, along with other histone modifications and chromatin remodeling, may trigger the reconfiguration of chromatin structure and hence regulate cell cycle progression and DNA repair. According to this model, the elevated extracellular Ca 2ϩ is required to compensate the IR-repressed level of intracellular CaM for the maintenance of chromatin structure (32).
Multiple repair/signaling proteins are recruited to DSB sites (6). Among those factors, NBS1, 53BP1, and MDC1 have been shown to physically interact with ␥-H2AX (26,44,45). In yeast, an ATP-dependent chromatin-remodeling complex, INO80, was shown to interact with ␥-H2AX (42). The absence of those proteins in the list of proteins we identified here suggests that the use of more cells for complex purification and employment of higher sensitivity mass spectrometry techniques for protein detection may lead to identification of additional H2AX-interacting partners. It should be pointed out that previous relevant studies usually used many more cells than used in this study. For example, 4.25 ϫ 10 10 cells were used to purify a histone deacetylase-dependent corepressor complex (46) in comparison with 8 ϫ 10 8 cells used in the present study.
In the present study, to obtain soluble intact H2AX protein complexes, we used nuclease DNase I to cleave chromatin every 10 bp of DNA under mild conditions. To identify those proteins associated with intact nucleosomes, micrococcal nuclease can be used for the digestion. For the identification of the protein-protein interactions, many relevant studies used overexpression of the proteins of interest, which may affect the cell phenotypes. The tandem affinity purification approach was developed to isolate complexes in high purity, but it requires multiple steps of affinity purification to reduce contaminating proteins (47). The repetitive washings often result in loss of weak or transient protein interactions of biological relevance. In our dual tagging approach for complex compo- FIG. 7. Ca 2؉ promotes the division of the irradiated cells. The cells in the exponential growth phase were mock-or ␥-ray-irradiated (8 or 30 Gy), and immediately equal numbers of cells were replated in 60-mm plates. CaCl 2 was added to the culture medium to the indicated concentrations, and the cells were grown at 37°C for 2 days. Viable cells were counted using trypan blue exclusion assay. Note that increasing extracellular Ca 2ϩ concentration of the irradiated cells resulted in increased viable cell numbers. nent profiling, a stable cell line is first established that expresses a tagged bait protein whose expression level is comparable to that of the endogenous counterpart. The technology then integrates the capabilities of natural complex formation, epitope affinity isolation, and in-spectra quantitative measurements to distinguish systematically those interacting proteins from a large nonspecific binding background. Sensitive and unambiguous identification of genuine components relies on the isotope ratio in MS spectra, not on the purity of the complexes through multistep purifications. This approach should be useful in other areas such as identification of transcription mediators/factors.
In summary, using DNA nuclease digestion of chromatin and a quantitative dual tagging proteomic system we identified several proteins that have been shown to interact with members of the histone family, including H2B, CaM, two components of FACT, BiP, calreticulin, and nucleophosmin/ B23. We also identified several novel binding partners of H2AX under the defined conditions. That is, we showed that DNA repair proteins PARP-1 and DEAH box polypeptide 30 isoform 1 interacted with H2AX when cells were exposed to ␥-ray and allowed to recover for a relatively short period of time (e.g. 30 min). More importantly, we demonstrated that the H2AX protein complex undergoes dynamic changes upon induction of DNA damage and during DNA repair. Finally we characterized the biological functions for part of the identified proteins, i.e. the Ca 2ϩ -binding/signaling proteins. The results demonstrated that Ca 2ϩ /CaM played important roles in regulating IR-induced cell cycle arrest. Our data support the hypothesis that chromatin structure may play crucial roles in DNA repair and checkpoint signaling (39 -43).


extfieldsofstudy: Biology Medicine

s2fieldsofstudy: Biology Chemistry

text:
A Revised Model for AMP-activated Protein Kinase Structure

The 5′-AMP-activated protein kinase (AMPK) is a master sensor for cellular metabolic energy state. It is activated by a high AMP/ATP ratio and leads to metabolic changes that conserve energy and utilize alternative cellular fuel sources. The kinase is composed of a heterotrimeric protein complex containing a catalytic α-subunit, an AMP-binding γ-subunit, and a scaffolding β-subunit thought to bind directly both the α- and γ-subunits. Here, we use coimmunoprecipitation of proteins in transiently transfected cells to show that the α2-subunit binds directly not only to the β-subunit, confirming previous work, but also to the γ1-subunit. Deletion analysis of the α2-subunit reveals that the C-terminal 386-552 residues are sufficient to bind to the β-subunit. The γ1-subunit binds directly to the α2-subunit at two interaction sites, one within the catalytic domain consisting of α2 amino acids 1-312 and a second within residues 386-552. Binding of the α2 and the γ1-subunits was not affected by 400 μm AMP or ATP. Furthermore, we show that the β-subunit C terminus is essential for binding to the α2-subunit but, in contrast to previous work, the β-subunit does not bind directly to the γ1-subunit. Taken together, this study presents a new model for AMPK heterotrimer structure where through its C terminus the β-subunit binds to the α-subunit that, in turn, binds to the γ-subunit. There is no direct interaction between the β- and γ-subunits.

The 5Ј-AMP-activated protein kinase (AMPK) 2 is a master metabolic regulator; it is activated by high intracellular AMP/ ATP ratios to respond to diminishing cellular energy (1,2) and by the adipokines leptin (3) and adiponectin (4). AMPK activation because of metabolic stress leads to serine/threonine phosphorylation of multiple targets reducing energy consuming processes, and increasing energy generating pathways, such as fatty acid oxidation (5). Furthermore, metformin, a drug in current clinical use to treat type II diabetes promotes glucose uptake at least in part through activation of AMPK (6,7); how this activation occurs is still unclear. AMPK is reported to exist as a heterotrimer consisting of an ␣-catalytic subunit and two non-catalytic ␤and ␥-subunits. In mammals there are two ␣-subunits, three ␥-subunits, and two ␤-subunits (␣1, ␣2, ␥1, ␥2, ␥3, ␤1, and ␤2). The ␥-subunits each contain four tandem cystathionine ␤-synthase (CBS) domains that together bind two molecules of AMP and allow the kinase complex to sense adenine nucleotide levels. The ␤-subunits have been reported to serve a scaffolding function for the ␣and ␥-subunits as well as target the complex to intracellular sites such as the cell membrane through myristoylation of its N terminus (8,9), and to intracellular glycogen through a glycogen binding domain (10). Binding of AMP to AMPK leads to phosphorylation of the ␣-subunit at Thr-172 by a family of upstream kinases, including LKB1 (11,12) and increases the activity of AMPK by 50 -100fold (13,14).
AMPK is functionally related to the yeast Snf1 (sucrose nonfermenting 1) protein kinase complex which plays a major role in regulating the nutritional stress response in Saccharomyces cerevisiae (2,15). The Snf1 complex is also composed of three subunits; Snf1 (␣-subunit), Snf4 (␥-subunit), and one of Sip1, Sip2, or Gal83 (␤-subunits), which share limited primary amino acid sequence homology with their mammalian counterparts. Two regions in the yeast ␤-subunit homologs, the KIS (kinase interacting sequence) and ASC (association with Snf1 complex) domains interact with the ␣and ␥-subunits, respectively, in a yeast two-hybrid assay (16) implicating them as potential scaffolds. Initial characterization of the rat AMPK-␤subunit showed that, when synthesized in rabbit reticulocyte lysates, it bound individually to both the ␣and ␥-subunits, supporting a model where the ␤-subunit acts as a scaffold for the ␣and ␥-subunits (17).
The KIS and ASC domains present in the three yeast ␤-subunits (Sip1/Sip2/Gal83) were applied to the mammalian AMPK-␤-subunits by sequence homology, and thus similar functional conservation of binding the ␣and ␥-subunits was inferred. This model was challenged recently when a ␤1-subunit mutant missing the KIS region was still able to co-immunoprecipitate with the ␣1-subunit (18) and with the identification of a glycogen binding domain overlapping the KIS domain (10). It was subsequently shown that the C-terminal 186 -270 residues of AMPK-␤1 were sufficient to tether the ␣and ␥-subunits (19). Another layer of complexity was uncovered by elucidation of the crystal structure of the yeast Snf1 catalytic domain. The catalytic domain of Snf1 was found to form dimers in solution and inhibit kinase activity (20). Furthermore, FLAG and HA epitope-tagged versions of full length Snf1 co-immunoprecipitated together and many residues that mediate the dimer interaction are conserved across species. These data suggest that AMPK may exist in a stoichiometry other than 1:1:1 (␣:␤:␥) with additional subunit interactions potentially involved in activation or repression of the kinase complex. These recent discoveries involving AMPK subunit interactions, particularly the role of the ␤-subunits, has led us to undertake a detailed examination of the subunit relationships of the AMPK heterotrimer. Here, we show that the full length AMPK-␤-subunit does not bind directly to the ␥1-subunit. Furthermore, we identify two interactions between the ␣and ␥-subunits, one of which is independent of the ␤-subunit. This study provides evidence for a new model of AMPK heterotrimer structure where the ␤-subunit binds to the ␣-subunit which, in turn, binds the ␥-subunit but with no direct interaction between the ␤and ␥-subunits (Fig. 1).

EXPERIMENTAL PROCEDURES
Materials-HEK-293T cells were purchased from American Type Culture Collection. Anti-Myc tag 9B11 mouse monoclonal and Anti-AMPK-␣ were purchased from Cell Signaling Technology (Danvers, MA). Anti-FLAG M2 mouse monoclonal purchased from Sigma-Aldrich. Anti-HA rat monoclonal antibody purchased from Roche Applied Science (Indianapolis, IN). Anti-AMPK-␤ rabbit antibody was purchased from Upstate Cell Signaling Solutions (Charlottesville, VA).
Expression Constructs-Oligonucleotide primers used to amplify cDNAs for the AMPK expression constructs are detailed in Table 1. The coding sequence for mouse AMPK-␤2 was amplified from total RNA extracted from mouse heart using an RNeasy kit (Qiagen, Valencia, CA) followed by a One-Step RT-PCR (Invitrogen, Carlsbad, CA) reaction and cloning into pCR4Blunt-Topo vector (Invitrogen). The cDNA was then cloned in-frame into a C-terminal FLAG expression vector, pCMV-Tag4A (Stratagene). Construction of the other AMPK-␤ expression plasmids used the full-length ␤2 cDNA as PCR template with primers listed in Table 1 and blunt-cloned into pCR4Blunt-Topo followed by subcloning into pCMV-Tag4A (C-term FLAG), pCMV-Tag5A (C-term Myc), or pCMV-Tag2A (N-term FLAG) with restriction sites in the primers ( Table 1). The Myc-AMPK-␣2 constructs were amplified from the rat cDNA for AMPK-␣2 (14) with primers from Table 1 and were contributed by Dr. Tsu-Shuen Tsao. For the AMPK-␥1-HA construct, the HA tag was incorporated on the reverse primer and the cDNA amplified from a mouse E10.5 cDNA library. The PCR product was blunt end cloned into pCR4Blunt-Topo, followed by cloning into the pCMV-Tag4A expression construct. A stop codon was incorporated prior to the FLAG sequence in pCMV-Tag4A. GFP was expressed from pEGFP-N1 (Clontech, Mountain View, CA).
Western Blotting-Cells were collected and disrupted in lysis buffer (1ϫ phosphate-buffered saline, 1% Nonidet P-40, 0.5% FIGURE 1. A new model for AMPK structure. A diagram of the structure of heterotrimeric AMPK based on the indicated references is depicted on the left. A new model for AMPK structure is proposed on the right based on data presented here: the ␣-subunit binds both ␤and ␥-subunits with no direct interaction between ␤and ␥-subunits. Alpha (␣), beta (␤), and gamma (␥) subunits are labeled in each model.

TABLE 1 Oligonucleotide primers used for cloning of AMPK expression vectors
Mammalian expression plasmids used in this study are listed in the first column. Oligonucleotide primers used to amplify specific cDNAs are listed in the second column, forward (Fwd) and reverse (Rev). All primers are displayed in the 5Ј to 3Ј orientation. DNA coding for the Myc and HA tags are in bold. Restriction endonuclease sites incorporated on the primers are underlined and listed in the third column. Species from which cDNA was derived is listed in the fourth column. sodium deoxycholate, 0.1% SDS, and complete protease inhibitor mixture (Roche Applied Science)). Protein concentrations of lysates were determined using D C Protein Assay (Bio-Rad). 40 g of total protein lysate was electrophoresed on 4 -12% NuPage BisTris gradient gels (Invitrogen) followed by semi-dry transfer to nitrocellulose. Membranes were blocked in 5% nonfat dry milk in 50 mM Tris-Cl, 150 mM NaCl, 0.1% Tween 20 (TBST) overnight before incubation with primary antibodies for 1-3 h. Membranes were washed three times in TBST for 10 min each followed by incubation with anti-mouse, anti-rabbit, or anti-rat horseradish peroxidase-conjugated secondary antibodies (Amersham Biosciences). The membranes were washed three times in TBST, developed with Western Lightning Reagent Chemiluminescent detection (PerkinElmer Life Sciences, Boston, MA) and exposed to x-ray film.

␤2-FLAG
Immunoprecipitations-Five hundred micrograms of total protein lysate was brought to a volume of 1 ml in lysis buffer and incubated with FLAG or Myc antibodies at 1:1000 at 4°C on a rotator overnight. 50 l of a 1:1 slurry of protein A/G-conjugated beads (Pierce) was added to the immunoprecipitation reactions and rotated for an additional 2 h. Immunoprecipitates were pelleted at 2500 rpm and washed three times with 0.5 ml of lysis buffer. Samples were boiled in LDS sample buffer for 4 min and centrifuged at 14,000 rpm for 4 min to pellet the beads. Supernatants were Western-blotted as described above.
Cell Culture and Transfection-HEK-293T cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, L-glutamine, and penicillin/streptomycin in a 37°C incubator at 5% CO 2 . For transfections, cells were split to ϳ70% confluency and transfected the following day. Lipofectamine 2000 transfection reagent (Invitrogen) was used with 2 g of DNA per expression construct in 10-cm tissue culture plates per manufacturer's protocol. Cells were harvested 24 -48-h post-transfection.

AMPK-␤ Binds Directly to AMPK-␣2 but Not To AMPK-␥1-
To examine the binding properties of the mammalian AMPK-␤-subunit, expression constructs were created and tagged with a FLAG epitope at the C terminus to avoid disrupting the N-terminal myristoylation site. Additionally, a mutant AMPK-␤2 construct was created that comprised only the first 203 residues, effectively deleting the ASC domain (residues 204 -271). These AMPK-␤2 constructs were used to test for binding to the Myc-AMPK-␣2 subunit by co-expression in 293T cells followed by immunoprecipitation of ␤2 with anti-FLAG antibody and Western blotting for the AMPK-␣2 subunit using the anti-Myc antibody. AMPK-␣2 subunit bound to full-length AMPK-␤2 ( Fig. 2A, top blot, lane 4) but not to AMPK-␤2-(1-203) ( Fig. 2A, top blot, lane 5). As expected, the Myc-␣2 band was not present in lysate from cells expressing GFP ( Fig. 2A, top blot, lane 6). Western blotting for FLAG indicated that immunoprecipitation with FLAG antibody was successful ( Fig. 2A, bottom blot, lanes 4 and 5). The same AMPK-␤2 constructs were used to test for direct binding of the ␤2-subunit to the AMPK-␥1-subunit in a similar co-IP assay. In contrast to the AMPK-␣2 subunit, the AMPK-␥1-HA subunit did not bind to full-length AMPK-␤2-FLAG protein (Fig. 2B, top blot, lane 4), and as anticipated, ␥1-HA did not bind to the ␤2-(1-203) mutant deleted for the ASC domain (Fig. 2B, top blot, lane 5). AMPK-␤2-FLAG and ␤2-(1-203)-FLAG proteins were both present in the immunoprecipitate (Fig. 2B, bottom blot, lanes 4 and 5) indicating that these results were not caused by unsuccessful AMPK-␤2 immunoprecipitation. To examine the interaction of the ␤-subunit with the ␣and ␥-subunits when expressed together, a triple transfection in 293T cells was performed using either full-length or ASC deleted ␤2-FLAG subunits together with full-length Myc-␣2 and ␥1-HA. Following FLAG-IP against the tagged ␤-subunits, the presence of the Myc-␣2 and ␥1-HA subunits in the immunoprecipitate was assessed by Western blot. Consistent with the results in Fig. 2A, the ␣2-subunit was coimmunoprecipitated with the ␤2-FLAG subunit (Fig. 2C, top blot, lane 4). Importantly, the ␥1-subunit was also present in the ␤-subunit IP (Fig. 2C, middle blot, lane 4) in contrast to the result seen when expressing the ␤2and ␥1-subunits alone. The ␤2-(1-203) deletion mutant did not bind either the ␣2or ␥1-subunit, consistent with previous results (Fig. 2C, lane 5). Similar results were obtained when AMPK-␤1-FLAG and ␤1-(1-203)-FLAG subunits were used (data not shown). These data indicate that the AMPK-␤2 subunit does not bind directly to the ␥1-subunit. Fig. 2C suggests that the ␤2-subunit does bind to the ␣2-subunit which, in turn, binds directly to the ␥1-subunit; below we show direct binding of ␣2to ␥1-subunit (Fig. 4).
To ensure that placement of the FLAG epitope at the ␤2 C terminus did not cause disruption of the ␥1-subunit binding site, an N-terminal FLAG tagged ␤2-subunit expression construct was created (Table 1 and Fig. 3) and used in a similar co-immunoprecipitation assay. Furthermore, a FLAG-␤2 (71-271) expression construct was also generated ( Table 1) to express ␤-subunit protein similar to one used in an earlier AMPK subunit interaction study that found that the ␤-subunit bound to both ␣and ␥-subunits (17). Expression vectors for the full-length ␤2-subunit with either an N-or C-terminal FLAG tag and the deletion mutants FLAG-␤2 (71-271) or ␤2-(1-203)-FLAG were transfected in 293T cells along with the ␥1-HA vector together with, or without, the Myc-␣2 expression vector (Fig. 3, left). Lysates prepared from these transfected cells were Western-blotted with anti-FLAG, Myc, and HA antibodies (Fig. 3A, top, middle, bottom, respectively), showing that all of the AMPK subunits were expressed. Immunoprecipitation with FLAG antibody was performed on these lysates and blotted with antibodies against Myc for the Myc-␣2 subunit, HA for the ␥1-HA subunit, and AMPK-␣, for endogenous AMPK-␣ (Fig. 3B). In the absence of the Myc-␣2 subunit, little ␥1-HA co-immunoprecipitated with the ␤-subunit; whereas upon overexpression of the Myc-␣2 subunit, a much stronger ␥1-HA (ϳ100-fold) co-immunoprecipitated with the ␤-subunit (Fig. 3B, lanes 1 and 2). This result was comparable to that observed for C-terminal FLAG-tagged AMPK-␤2 (Fig. 3B,  lanes 3 and 4). The FLAG-␤2 (71-271) N-terminal deletion did not co-immunoprecipitate the ␥1-HA subunit unless Myc-␣2 subunit was co-expressed (Fig. 3B, lanes 5 and 6). Consistent with the results in Fig. 2, the ASC domain deleted ␤2-(1-203) did not co-immunoprecipitate either the ␣2or ␥1-subunits (Fig. 3B, lanes 7 and 8). A very small amount of AMPK-␥1-HA was also detected in immunoprecipitates that did not contain exogenous Myc-␣2 subunit (Fig. 3B, middle blot, lanes 1, 3, and  5). However, endogenous AMPK-␣ is also present in these immunoprecipitates as shown by re-blotting the membrane with antibody that recognizes both the endogenous and overexpressed AMPK-␣ subunit (Fig. 3B, bottom blot, lanes 1, 3, and  5). Western blotting of lysate from GFP-transfected cells did not react with anti-FLAG, Myc, or HA antibodies (Fig. 3A, lane  9), and only show the background immunoglobulin bands from the immunoprecipitate (Fig. 3B, lane 9). These data further indicate that in contrast to previous studies, the ␤2-subunit does not bind directly to the ␥1-subunit.

DISCUSSION
These experimental data present three major points that cannot be explained by the existing model for assembly of mammalian AMPK (Fig. 1, left). First, we show that AMPK-␣2 does not bind to the KIS domain of AMPK-␤, but instead to the C-terminal ASC domain of AMPK-␤. The full-length ␤2-subunit binds to the ␣2-subunit, but the ␤2-(1-203) mutant, which lacks only the ASC domain, does not (Fig. 2). This result indicates that the C-terminal ASC domain of the ␤-subunit is critical for binding the AMPK-␣2-subunit, and is consistent with results that show that the isolated C terminus of the ␤1-subunit is sufficient to bind to the ␣1-subunit (18,19). It is therefore unlikely that our lack of ␣-subunit binding to the ␤2-(1-203) mutant are caused by a general disruption of the ␤-subunit structure. Our result supports the abandonment of the KIS nomenclature as proposed in the study by Hudson et al. (18). Second, there are at least two binding sites on the ␣2-subunit for the ␥1-subunit. Utilizing a series of AMPK-␣2 deletion mutants with the ␥1-subunit in co-immunoprecipitation experiments, distinct binding sites for the ␥1-subunit were mapped to residues 1-312 and 386 -552 of AMPK-␣2 (Fig. 4). Third, the AMPK-␤-subunit does not bind directly to the AMPK-␥1-subunit despite its reported role as a scaffold to bind directly both the ␣and ␥-subunits. This negative result is based on immunoprecipitation of the ␤-subunit followed by Western blotting for the ␥1-subunit. Only upon co-expression of the ␣2-subunit, are all three subunits bound together in the immunoprecipitate (Fig. 2). Through deletion analysis of the ␣2-subunit, we showed that residues 386 -552 of the ␣2-subunit were sufficient to bind to both the ␤and ␥-subunits (Fig.  5). These findings demonstrate that the catalytic ␣-subunit, not the ␤-subunit, binds both of the non-catalytic AMPK subunits. The results of these experiments led us to propose a new model for AMPK where the ␤-subunit binds to the ␣-subunit, which in turn binds the ␥-subunit (Fig. 1, right).
In the existing model for AMPK heterotrimer structure, the ␤-subunit plays a central role in bringing the ␣and ␥-subunits together. With this in mind, we designed ␤-subunit expression constructs to control for potential artifactual effects of the epitope tags. We originally cloned a C-terminal FLAG epitope tag on the ␤-subunits because a previous report indicated that modification of the N terminus leads to a loss of myristoylation on glycine at position two (8). To ensure that the ␤-subunit C-terminal FLAG epitope tag itself did not disrupt binding to the ␥-subunit, we performed similar experiments with an N-terminal FLAG-tagged ␤2-subunit, despite concerns about altering myristoylation. Both N-terminal and C-terminal FLAG-tagged ␤2-subunits bound the ␣2but not the ␥1-subunit, indicating that the effects we observed were not caused by modification of the ␤-subunit C terminus (Fig. 3). Furthermore, an N-terminal 70 amino acid-deleted AMPK-␤2-(71-271) subunit still bound to the ␣2-subunit, and not the ␥1-subunit. This particular deletion mutant was chosen based on a previous study performed by Woods et al. (17) to examine subunit interactions. In that study radiolabeled AMPK subunits were prepared from rabbit reticulocytes and immunoprecipitated with one another. They used a 70-amino acid N-terminal-deleted  ␤-subunit to distinguish it from the AMPK-␥1-subunit because they are both 38 kDa in size and found that the ␤-subunit bound to the ␣and ␥-subunits individually and together in a heterotrimer. Our results do not support binding of the ␤-subunit to the ␥1-subunit independent of the ␣-subunit even when employing a similar ␤2-(71-271) mutant protein. The discrepancy observed may be caused by other differences between the programmed rabbit reticulocyte system versus the 293T overexpression system used in this study. The ␤2-FLAG immunoprecipitations in Fig. 3 show a very small amount of ␥1-HA co-immunoprecipitating with the ␤2-subunit; however, re-blotting with antibody against AMPK-␣ clearly shows that endogenous AMPK-␣ is also present in the immunoprecipitate (Fig. 3B, bottom blot). Our interpretation of this result is that the small amount of ␥1-HA seen in the immunoprecipitate is caused by binding to the endogenous AMPK-␣ expressed in 293T cells, and its subsequent interaction with the AMPK-␤2-FLAG subunit. While we do not observe direct binding of the ␤-subunit to the ␥1-subunit, we have not tested whether or not the ␥2or ␥3-subunits can directly bind the ␤-subunit.
Utilizing a series of AMPK-␣2 deletion mutants we determined that the C-terminal 167 residues of AMPK-␣2 are sufficient to bind to both AMPK-␤2 and AMPK-␥1. This region was previously identified to be critical for AMPK-␣1 interaction with the ␤-subunit (21). However, in contrast to the report by Dyck et al., our finding is that the AMPK-␤ and ␥-subunits do not interact with one another unless AMPK-␣ is present, specifically the C terminus of AMPK-␣ to act as a bridge between the ␤and ␥-subunits. The differences between these results may be due to the level of overexpression and/or the difference in cell type used for the subunit interaction experiments (293T versus COS7). Furthermore, our experiments show that the catalytic domain of AMPK-␣2, and thus catalytic activity, is not required for forming a complex with the ␤and ␥-subunits, similar to conclusions made by Dyck et al. (21) using a kinase inactive AMPK-␣1 mutant.
A significant finding of our work is that the ␣2and ␥1-subunits are able to interact independently of the ␤-subunit, and that there are at least two interaction sites on the ␣-subunit for the ␥1-subunit. The catalytic domain of AMPK-␣2 (residues 1-312) and the C-terminal 167 residues of AMPK-␣2-(386 -552) were both able to bind to AMPK-␥1. An important distinction between these two reactions is that the AMPK-␤ subunit did not bind to the ␣2-(1-312) catalytic domain but did bind to the C-terminal ␣2-(386 -552) subunit (Fig. 4). This clearly demonstrates that AMPK-␤ is not required to join the ␣and ␥-subunits together as the current AMPK assembly model suggests. At this point, we do not know if the two ␥-subunit binding sites on full-length AMPK-␣ are mutually exclusive. If they are not, then it is possible that AMPK contains more than one of each subunit rather than existing as a heterotrimer with 1:1:1 stoichiometry. This hypothesis is further supported by a recent report detailing the ability of the yeast Snf1 catalytic domain to form homodimers (20).
Because the ␥1-subunit binds AMP and coordinates energy sensing, we sought to determine if the interactions we observed between the ␣and ␥-subunits are modulated by either 400 M AMP or 400 M ATP, a concentration of nucleotide expected to be saturating for binding to the enzyme (22). Our experiments showed that at this concentration, AMP and ATP did not affect the interaction of either the catalytic (1-312) or C-terminal (386 -552) ␣2-domains to the ␥1-HA subunit suggesting that their binding is not dependent on AMPK activation (Fig. 6). Furthermore, the ␣2-(386 -552) C-terminal deletion does not contain the Thr-172 residue, critical for activation via phosphorylation by the upstream kinase LKB1, yet this construct still binds to the ␥1-subunit. This demonstrates that this particular interaction is independent of the activation state of AMPK.
Understanding how mammalian AMPK is assembled is of great importance given its role as a master metabolic regulator. The identification of a family of upstream kinases able to phosphorylate and activate AMPK along with a newly discovered link to calcium dependent signaling (23)(24)(25) increases the complexity of AMPK regulation and activity. An accurate picture of how AMPK is assembled is essential to understanding its many functions. Based on the results presented here, we propose a new model for AMPK assembly where the ␤-subunit does not function as a "scaffold" but interacts with the ␣-subunit which in turn binds to the ␥-subunit (Fig. 1). The ␣-subunits have a clear function as the catalytic subunit of AMPK, and the ␥-subunits have an apparent role in sensing and binding AMP through their four CBS domains (22). Our new ␤-␣-␥ assembly model clearly introduces a new question, principally what is the function(s) of the ␤-subunit? One potential answer might be that instead of acting as a scaffold, the ␤-subunit targets the kinase to subcellular locations. For example, myristoylation of the ␤-subunit aids in localizing AMPK to cellular membranes (8,9), and a glycogen binding domain localizes it to a site of a major energy store (10). The physiological importance of these properties and how they are regulated is currently unclear and awaits analysis in vivo with animal models. While AMPK has been an attractive drug target for many years, metformin has already been shown to mediate some of its beneficial effects through AMPK. Exactly how metformin activates AMPK is still unclear. An accurate AMPK model is critical for rationale design of therapeutics that can modulate AMPK function, and highlights the importance of our findings here.