(ITPK1) by reversible lysine acetylation

Regulation of inositol 1,3,4-trisphosphate 5/6-kinase (ITPK1) by reversible lysine acetylation Chunfen Zhanga, Philip W. Majerusa,b, and Monita P. Wilsona,1 a

Departments of Internal Medicine and bBiochemistry, Washington University School of Medicine, St. Louis, MO 63110

Contributed by Philip W. Majerus, December 2, 2011 (sent for review November 4, 2011)

The enzyme inositol 1,3,4-trisphosphate 5/6-kinase (ITPK1) catalyzes the rate-limiting step in the formation of higher phosphorylated forms of inositol in mammalian cells. Because it sits at a key regulatory point in the inositol metabolic pathway, its activity is likely to be regulated. We have previously shown that ITPK1 is phosphorylated, a posttranslational modification used by cells to regulate enzyme activity. We show here that ITPK1 is modified by acetylation of internal lysine residues. The acetylation sites, as determined by mass spectrometry, were found to be lysines 340, 383, and 410, which are all located on the surface of this protein. Overexpression of the acetyltransferases CREB-binding protein or p300 resulted in the acetylation of ITPK1, whereas overexpression of mammalian silent information regulator 2 resulted in the deacetylation of ITPK1. Functionally, ITPK1 acetylation regulates its stability. CREB-binding protein dramatically decreased the half-life of ITPK1. We further found that ITPK1 acetylation down-regulated its enzyme activity. HEK293 cells stably expressing acetylated ITPK1 had reduced levels of the higher phosphorylated forms of inositol, compared with the levels seen in cells expressing unacetylated ITPK1. These results demonstrate that lysine acetylation alters both the stability as well as the activity of ITPK1 in cells. inositol kinase ∣ phosphorylation ∣ inositol metabolism

P

rotein acetylation is a widespread reversible covalent modification, transferring acetyl groups from acetyl CoA to either the α-amino (Nα) group of N-terminal residues or to the ε-amino group (Nε) of internal lysine residues at specific sites (1). The process is mediated by acetyltransferases, of which there are several families. Similarly, deacetylation is brought about by enzymes which belong to one of several different classes of deacetylases. Lysine acetylation was first discovered as a posttranslational modification of histones in the early 1960s and has long been considered to be a direct regulator of chromatin structure and function (2–4). Following the initial discovery of histone acetylation, extensive studies over the past four decades have not only identified the enzymes that catalyze reversible acetylation, the protein lysine acetyltransferases and deacetylases (HDACs), but have also identified many nonhistone substrates (5, 6). Like protein phosphorylation, acetylation of specific lysine residues in proteins can have numerous functional consequences including regulation of protein–DNA interactions, enzyme activity, protein stability, subcellular localization, and specific functional complex formation (e.g., protein–protein interactions) (7, 8). Numerous studies have shown that the dynamic processes of acetylation and deacetylation play central roles in diverse physiological processes including gene expression, metabolism, and regulation of life span (9, 10). ITPK1 plays a pivotal role in inositol metabolism. This enzyme is conserved from plants to animals, to Entamoeba histolytica (11– 14). The formation of inositol 1,3,4,6-tetrakisphosphate by ITPK1 represents the rate-limiting step in the formation of the higher phosphorylated forms of inositol in mammalian cells (15). Most recently, we have shown that mice producing reduced levels of ITPK1 develop neural tube defects with incomplete penetrance (16, 17). 2290–2295 ∣ PNAS ∣ February 14, 2012 ∣ vol. 109 ∣ no. 7

In this report, we show that ITPK1 can be acetylated by the acetyltransferases CREB-binding protein (CBP) and p300 both in vivo and in vitro and can be deacetylated by mammalian silent information regulator 2 (SIRT1). Acetylation of ITPK1 decreases its enzyme activity and protein stability, and inhibits the synthesis of higher phosphorylated forms of inositol polyphosphates in the inositol signaling pathway. Thus, ITPK1 is regulated in several ways by acetylation. Results ITPK1 is Acetylated by CBP and p300 in Vivo. The related proteins

p300 and CBP are transcriptional coactivators that act with other factors to regulate gene expression (18–20) and play roles in many cell-differentiation and signal transduction pathways (21–23). Both proteins have intrinsic histone-acetyltransferase activity (24, 25). To test whether ITPK1 could be acetylated by either CBP or p300, we first used transient transfection assays. Previous studies of protein acetylation showed that maximum induction of protein acetylation requires inhibition of both class I (HDAC I) and class III (SIRT1) deacetylase activities by treatment with trichostatin A (TSA) (for HDAC I) and nicotinamide (Nia) for SIRT1 (24). In these experiments, unless indicated otherwise, 2 μM TSA and 5 mM Nia were added to cells 6 h before harvest and included during protein purification. As indicated in Fig. 1A (lane 3), acetylated ITPK1 was found in cells cotransfected with ITPK1 and CBP, when cells were treated with TSA and Nia. There was no detectable acetylated protein in cells transfected with ITPK1 alone (lane 1). When treated with TSA and Nia, in the absence of CBP, ITPK1 was not detected to be acetylated (Fig. 1A, lane 2). ITPK1 was also acetylated in vivo by p300 (Fig. S1). In vivo acetylation of ITPK1 was also detected by 3 H-acetate labeling. ITPK1 was transfected alone or cotransfected with CBP into HEK293 cells, incubated with 3 H-acetate, affinity-purified, and subjected to autoradiography. A band corresponding to 3 H-acetylated ITPK1 can be seen when CBP is cotransfected with ITPK1 (Fig. 1B, lane 2). Thus, these data indicate that ITPK1 can be acetylated by CBP in vivo. ITPK1 is Acetylated by CBP/P300 in Vitro. N-terminal FLAG-tagged ITPK1 was expressed in SF9 cells and purified using FLAG-agarose beads. Reactions containing 10 μg of ITPK1 were incubated with 5 mM acetyl coenzyme A (AcCoA) and 500 ng of recombinant CBP (1,319–1,710) for 2 h at 30 °C. Products were resolved using SDS-PAGE and protein acetylation was detected by Western blotting using an anti-acetyl lysine antibody. As shown in Fig. 1C, ITPK1 was acetylated by CBP in vitro. Note that in the absence of CBP, a faint band is visible upon incubation of ITPK1 with AcCoA. Author contributions: C.Z., P.W.M., and M.P.W. designed research; C.Z. and M.P.W. performed research; C.Z., P.W.M., and M.P.W. analyzed data; and C.Z. wrote the paper. The authors declare no conflict of interest. 1

To whom correspondence should be addressed. Email: [email protected].

This article contains supporting information online at www.pnas.org/lookup/suppl/ doi:10.1073/pnas.1119740109/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1119740109

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Fig. 1. CBP acetylates ITPK1 both in vitro and in vivo. (A) ITPK1 acetylation in transiently transfected HEK293 cells, treated with TSA and Nia. Immunoprecipitated ITPK1 was subjected to SDS-PAGE and Western blotting, probed with anti-acetylated lysine antibody (Upper) and anti-FLAG antibody (Lower). (B) Detection of ITPK1 in vivo acetylation by 3H-acetate labeling. Autoradiograph of 3H-acetate labeled samples (Upper). Western blotting with antiFLAG antibody serves as a loading control (Lower). (C) Acetylation of ITPK1 in an in vitro acetylation assay. (D). Determination of the half-life for acetylation of ITPK, data were fitted as ½CoASH ¼ 42.8 − 38.8 × eð−t∕67.6Þ.

Fig. 2. SIRT1 deacetylates ITPK1 both in vitro and in vivo. (A) In vivo deacetylation of ITPK1 by SIRT1. HEK293 cells were cotransfected with FLAG–ITPK1 and CBP with or without varying amount of SIRT1. After immunoprecipitation (IP) with anti-FLAG M2 beads, the IP products were subjected to SDSPAGE and Western blot analyses were done using antibody against either acetylated lysine (Upper) or FLAG (Lower). (B) In vitro deacetylation of ITPK1. Purified ITPK1, NADþ , and recombinant SIRT1 were incubated at 30 °C for the indicated times followed by SDS-PAGE and Western blotting with anti-acetylated lysine (Upper) and anti-FLAG antibody (Lower).

In vitro acetylation of ITPK1 was measured using a colorimetric assay (Enzo Life Science) according to the manufacturer’s instructions. CBP, purified FLAG-tagged ITPK1, and AcCoA were incubated at 30 °C for 1 h. Protein acetylation releases coenzyme A with free sulfhydryl group (CoASH) which reacts with 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) to release 2-nitro-5-thiobenzoic acid, which is measured at a wavelength of 412 nm. The rate of acetylation of in vitro ITPK1 was determined by fitting the data for a single exponential rate (Fig. 1D) with a half-life of 40.8 min, which is similar to the in vivo turnover rates reported for histone acetylation (26). Because there are no deacetylases present in this assay, the leveling off of the rate likely represents the stoichiometric acetylation of the three lysine residues.

sites using site-directed mutagenesis, so mass spectrometry was performed to identify them. Acetylated ITPK1 was purified from HEK293 cells following cotransfection of FLAG-tagged ITPK1 with CBP, then digested with trypsin and analyzed by liquid chromatography–electrospray ionization MS/MS. Three acetylated lysine containing peptides were identified (Fig. 3A). The acetylated lysine residues in ITPK1 were determined to be K340, K383, and K410. The MS spectra of the acetylated peptides are shown in Figs. S2–S4.

SIRT1 Deacetylates ITPK1 both in Vivo and in Vitro. We next investi-

gated the deacetylation of ITPK1. Among the four major classes of histone deacetylases (HDAC), SIRT1 belongs to class III HDAC, differentiated from class I and II enzymes in that their activity depends on NADþ (27). Recently, considerable attention has been given to SIRT1 due to its role in multiple diverse processes including metabolism, development, stress response, neuroprotection, hormone responses, and apoptosis (28, 29). SIRT1 deacetylates histones and other substrates such as p53, forkhead box O, and nuclear factor κB (30–32). To determine whether or not SIRT1 can deacetylate ITPK1, FLAG-tagged ITPK1 was cotransfected with CBP in the presence or absence of SIRT1. After immunoprecipitation with anti-FLAG M2 beads, ITPK1 acetylation was detected by Western blotting with anti-acetyl lysine antibody. As shown in Fig. 2A, cotransfection of HEK293 cells with ITPK1 and CBP results in ITPK1 acetylation. However, in the presence of SIRT1, ITPK1 was deacetylated and the level of deacetylation correlated with the amount of SIRT1 added (lanes 3–6). Furthermore, deacetylation was inhibited in the presence of the inhibitors TSA and Nia (lane 2). In vitro deacetylation of ITPK1 was performed by incubating recombinant SIRT1 with purified acetylated FLAG-ITPK1 at 30 °C for the indicated times followed by SDS-PAGE and Western blotting. The acetylated ITPK1 was obtained by cotransfecting HEK293 cells with FLAGITPK1and CBP and purified using anti-FLAG M2 beads. As shown in Fig. 2B, ITPK1 can be deacetylated by SIRT1 in vitro. Identification of ITPK1 Acetylation Sites. Acetyltransferases acetylate specific sites on their target proteins. ITPK1 has a total of 20 lysines, which makes it very difficult to identify the acetylation Zhang et al.

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Fig. 3. Identification of ITPK1 acetylation sites. (A) Mass-spectrometry identified the ITPK1 acetylation sites to be K340, K383, and K410. (B) Comparison of the acetylation level of wild-type ITPK1 with that of site-directed mutants of ITPK1. HEK293 cells were transfected with ITPK1 alone (lane 1) or cotransfected with ITPK1 (wild-type or mutants) and CBP. Western blot analysis of immunoprecipitated ITPK1 probed with anti-acetylated lysine antibody (Upper) and anti-FLAG antibody (Lower). (C) Schematic representation of the ITPK1 protein and its truncated mutants. PNAS ∣

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Confirmation of ITPK1 Acetylation Sites in Cells. To confirm the ITPK1 acetylation sites identified by mass spectrometry, the three lysines were mutated to either alanine or arginine. These residues cannot be acetylated and arginine retains the positive charge. Constructs were made containing single, double, and triple mutations. Each construct was transfected into HEK293 cells for expression, and were immunoprecipitated using FLAG M2 beads. The total amount of each protein was detected by blotting with an anti-FLAG antibody, and their acetylation level was determined using an anti-acetylated lysine antibody. First, single mutations were made on all three lysines, then double mutation constructs K340,383A, K340,410A, K340,383R, and K340, 410R were constructed, and the triple mutants K340,383,410A and K340,383, 410R were also made. Both single and double mutants showed decreased ITPK1 acetylation (Fig. 3B, lanes 3–7 and 9–13). Furthermore, upon expression of the triple mutants, no acetylation is observed (Fig. 3B, lanes 8 and 14), indicating that all three lysines are subject to acetylation. The results were further confirmed using ITPK1 truncated mutants. The NΔ30C110 mutant, which lacks both the N-terminal 30 amino acids and the C-terminal 110 amino acids, contains no potentially acetylated lysine sites and was not acetylated (Fig. S5).

was added to the cultures, and the amount of ITPK1 was determined by immunoblotting (Fig. 4). As shown in Fig. 4A, wild-type ITPK1 was found to be a stable protein (t1∕2 > 8 h), whereas wildtype ITPK1 in the CBP-transfected cells was rapidly degraded (Fig 4B) (t1∕2 < 2 h). The data from these two blots are quantified in Fig. 4C. The triple mutant K340,383,410R has a half-life similar to that of the wild-type protein (Fig. 4D, Left). When this triple mutant is cotransfected with CBP, no increase is seen in its degradation (Fig. 4D, Right). These results suggest that the acetylation at lysines 340, 383, and 410 destabilizes ITPK1. Effect of ITPK1 Acetylation on Its Enzyme Activity. It is difficult to analyze the functional differences between the unacetylated form and the acetylated form of one protein as both states are present in cells. In order to test whether or not acetylation of ITPK1 can modulate its activity, we needed to obtain a purified preparation of acetylated ITPK1; a two-step strategy was employed to purify acetylated ITPK1 from human cells. First, total ITPK1 protein was purified from HEK293 cell extracts, transiently transfected with ITPK1 and CBP, then the acetylated ITPK1 was isolated using an anti-acetyl lysine column (Fig. 5A). The acetylated form will bind to this column and the unacetylated ITPK1 will not. As indicated in Fig. 5B, unacetylated ITPK1 can be separated from the acetylated ITPK1 fractions using the anti-acetyl lysine antibody column (lane 1 versus lane 3). The unacetylated protein remains in the flow-through (lane 1), shown next to a control, consisting of unacetylated ITPK1 (lane 2), and the acetylated ITPK1 binds to the column (lane 3). We then compared the enzymatic activity of the two forms of ITPK1, and found that nonacetylated ITPK1 is threefold more active than the acetylated enzyme (Fig. 5C). The ratio of products produced by ITPK1 phosphorylation of Insð1;3;4ÞP3 , Insð1;3;4;6ÞP4 , and Insð1;3;4;5ÞP4 is unchanged when ITPK1 is acetylated (Fig. S8).

Regulation of ITPK1 Stability by Acetylation. To gain insights into the role of ITPK1 acetylation, we tested whether or not ubiquitination was altered following ITPK1 acetylation. FLAG-ITPK1 alone or with HA-ubiquitin was transfected into HEK293 cells with or without CBP. Cells were harvested 36 h after transfection, and cell extracts were immunoprecipitated with anti-FLAG monoclonal antibody M2 beads. Proteins were resolved by SDSPAGE and analyzed by Western blotting with anti-HA antibody to detect ubiquitination, and anti-FLAG antibody to detect ITPK1 protein level. No significant difference in the ubiquitination of ITPK1 was observed when ITPK1 was acetylated (Fig. S6). In addition, the subcellular localization of ITPK1 was not affected by its acetylation (Fig. S7). As there are a number of reports that acetylation regulates protein stability, we next examined whether the stability of ITPK1 is affected by acetylation. HeLa cells were transiently transfected with wild-type ITPK1, or the triple mutant K340,383,410R with or without CBP. After the transfected cells had been treated with 2 μM TSA plus 5 mM Nia for 6 h, cycloheximide (CHX), a protein synthesis inhibitor,

Down-Regulation of the Synthesis of Higher Inositol Phosphates by ITPK1 Acetylation. As the enzyme that catalyzes the rate-limiting

step in the synthesis of higher phosphorylated forms of inositol, the activity of ITPK1 is tightly regulated, and its acetylation may affect in vivo levels of inositol phosphates. Fig. 6 shows the results of HPLC analysis of soluble inositol phosphates isolated from HEK293 cells stably expressing ITPK1 following metabolic labeling with 3 H-inositol for 72 h. Representative examples of HPLC

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Fig. 4. ITPK1 stability is decreased by acetylation. HeLa cells were transfected with ITPK1 alone (A) or cotransfected with ITPK1 and CBP (B). CHX (150 μg∕mL) was added into the medium for the indicated times. The level of ITPK1 was detected by Western blotting with anti-FLAG antibody. Levels of β-actin are shown as a loading control. (C) Quantification of blots shown in A and B. Transfection with ITPK1 alone (○) and with ITPK1 and CBP (▪) is shown. (D) HeLa cells were transfected with the triple mutant K340,383,410R alone or cotransfected with K340,383,410R and CBP, with CHX added to the medium for the indicated times. Stability of ITPK1 is measured by Western blotting with β-actin as a loading control. 2292 ∣


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Fig. 5. Lysine acetylation decreases ITPK1 enzymatic activity. (A) Schematic representation of the purification steps used to separate acetylated ITPK1 from unacetylated ITPK1. (B) Western blot analyses of separated proteins were performed with anti-FLAG antibody (Lower) and acetyl-lysine antibody (Upper). (C) Comparison of the enzymatic activity of acetylated ITPK1 to that of the unacetylated protein.

profiles are shown in Fig. S9. Upon tetracycline induction, ITPK1 increases the levels of inositol phosphates in cells. When CBP was transiently transfected in these cells, ITPK1 induction with tetracycline resulted in remarkably diminished levels of the higher phosphorylated forms of inositol. As the activity of ITPK1 regulates the levels of these metabolites (15), it is not unexpected that a posttranslational modification such as acetylation would affect this activity.

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Fig. 6. Quantification of soluble 3H-labeled inositol polyphosphates separated using anion exchange HPLC chromatography. HEK293 cells stably expressing ITPK1 were grown in medium containing 3H-inositol for 3 d, and induced with tetracycline for 2 d in the presence and absence of transfection with CBP. The soluble inositol-containing extracts were separated using a Whatman Partisphere 5 strong anion exchange HPLC column. Counts incorporated are normalized to protein level.

Zhang et al.

Reagents and Chemicals. All chemicals and reagents, unless noted otherwise, were purchased from Sigma-Aldrich. Recombinant CBP (1,319–1,710) (BMLSE452) and recombinant SIRT1 (BML-SE239) were purchased from Enzo Life Science. Antibodies. Mouse monoclonal anti-FLAG epitope antibody was from Sigma. Rabbit polyclonal antibody to acetyl lysine ab21623 and rabbit polyclonal antibody to β-actin ab1808 were from Abcam. DNA Constructs and Cell Culture. A FLAG peptide fusion construct of human ITPK1 was generated by adding the FLAG peptide DNA sequences to the C terminus of ITPK1 followed by a stop codon in the pcDNA4/TO plasmid (InPNAS ∣


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Discussion Our lab has elucidated the pathway leading to the synthesis of InsP6 in human cells, and ITPK1 catalyzes the rate-limiting step by phosphorylating Insð1;3;4ÞP3 to both Insð1;3;4;5ÞP4 and

Insð1;3;4;6ÞP4 . It is likely that posttranslational modification of ITPK1 plays a role in controlling the activity of this key cellular regulator. To gain insight into the function of posttranslational modification, we have shown that ITPK1 is phosphorylated in vivo (33). Here, we have shown that ITPK1 is acetylated on lysine residues both in vivo and in vitro. We have identified three lysines as major acetylation sites by mass spectrometry analysis and sitedirected mutagenesis. The three lysines on ITPK1 that were found to be acetylated were K340, K383, and K410, which are all located on the surface of ITPK1, as determined by the crystal structure (14). This location makes them easily accessible to acetylases and deacetylases. To elucidate the biological role of ITPK1 acetylation, we analyzed the effects of acetylation on subcellular localization of ITPK1 and ubiquitination of ITPK1. We observed no differences in either localization or ubiquitination between the acetylated and unacetylated forms of ITPK1. We found that acetylation decreases the stability of ITPK1 in COS-7 cells and in HeLa cells. Cotransfection of ITPK1 and CBP reduces the stability of ITPK1. In HeLa cells transfected with the ITPK1 mutant K340,383,410R or cotransfected with the K340,383,410R mutant and CBP, the half-life of these mutants is similar to that of unacetylated ITPK1. We further found that acetylated ITPK1 displayed significantly lower enzyme activity and the synthesis of the higher phosphorylated forms of inositol was decreased. These results are consistent with the decreased stability of ITPK1. The stability of several proteins have been reported to be regulated by acetylation (34, 35). In most cases, acetylation stabilizes the protein by the ubiquitinproteasome system. Treatment of cells with the proteasome inhibitor MG132 did not alter the level of ITPK1 (Fig. S10), indicating that it is likely not degraded via this pathway. It is currently unclear what proteases are responsible for ITPK1 degradation following acetylation. It remains to be shown whether ITPK1 is acetylated constitutively in vivo, or is acetylated in a regulated fashion in response to some signal(s). It is also unknown whether acetylation and deacetylation occur in cells through the actions of multiple acetyltransferases and deacetylases, as has been shown to be the case for a number of other proteins (36). It will be of interest to determine whether or not acetylation has an effect on any of the cellular processes that are regulated by either a substrate or a product of ITPK1. For example, it has been shown that inositol 3,4,5,6-tetrakisphosphate, a product of ITPK1 dephosphorylation of IP5 , regulates chloride channels in several cell types (37–40). Because acetylation of ITPK1 alters the level of this inositol polyphosphate isomer in cells, this may in turn affect chloride flux. In only a handful of cases has protein stability been shown to be regulated by acetylation. In some instances, acetylation of a protein increases its stability, for example E2F1 (41), the estrogen receptor α isoform (42), and the nuclear hormone receptor Nur77 (43). As is shown here with ITPK1, the stability of other proteins decreases when they are acetylated, for example SV40 large T antigen (44) and the Escherichia coli exoribonuclease RnaseR (45). Further studies are needed to elucidate the mechanism by which acetylation regulates ITPK1 stability and enzymatic activity.

vitrogen). Site-directed mutants were constructed by using the QuikChange site-directed mutagenesis kit (Stratagene). For production of truncated mutants of ITPK1, DNA sequences corresponding to the indicated regions of human ITPK1 were amplified by PCR and subcloned into FLAG-pcDNA4/TO. All constructs were verified by DNA sequencing. HeLa cells and HEK293 cells were maintained in culture using 10% fetal bovine serum in Dulbecco’s modified Eagle’s medium. Unless noted, transfection was conducted by using Lipofectamine 2000 (Invitrogen). Stably transfected HEK293 TRex cells expressing ITPK1 were prepared as described in SI Material and Methods. 3 H-Acetate Labeling. Cells were radiolabeled 48 h after transfection and were pretreated with 2 μM TSA and 5 mM Nicotinamide for 6 h and CHX for 2 h to prevent protein synthesis. Cells were labeled in DMEM containing 1 mCi∕mL 3 H-acetate (Moravek Biochemicals), 25 μM CHX, 2 μM TSA, and 5 mM nicotinamide at 37 °C for 2 h. Immunoprecipitated proteins were separated by 12% SDS-PAGE. Gels were treated with En3 Hance®, dried, and exposed X-ray film for 3 wk prior to exposure.

ITPK1 Acetylation in Vivo and in Vitro. For transient transfection, HEK293 cells were transfected with ITPK1 alone or cotransfected with ITPK1 and CBP. Cells were lysed 36 h after transfection in 100 mm NaCl, 50 mm Tris•HCl pH 7.3, 10% glycerol, 0.2% Triton X-100 containing Complete Protease Inhibitor (Roche Diagnostics), 2 μM TSA, and 5 mM nicotinamide. Cell extracts were incubated with anti-FLAG M2 beads at 4 °C overnight. Beads were washed three times with lysis buffer, and bound proteins were eluted with 100 μg∕mL FLAG peptide (Sigma) for 2 h. Western blotting was done using anti-FLAG antibody and a rabbit polyclonal antibody against acetyl lysine. The in vitro acetylation assay (colorimetric acetyltransferase assay) was performed following the manufacturer’s instructions with some modifications. FLAG-tagged ITPK1 was expressed in SF9 cells and purified using FLAGagarose. Assays (50 μL) were done with 50 mM Hepes, pH 7.9, 0.1 mM EDTA, 50 μg∕mL BSA, 0.5 μg CBP, 2 mM acetyl CoA, and 5 μg ITPK1. The reaction mix was incubated at 30 °C for varying times (from 0 to 120 min) and the reaction was quenched with 100 μL 3.2 M guanidinium HCl, 100 mM Na2 HPO4 ∕ NaH2 PO4 , pH 6.8. The reaction mixtures were subjected to SDS-PAGE or used 1. Kouzarides T (2000) Acetylation: A regulatory modification to rival phosphorylation? EMBO J 19:1176–1179. 2. Phillips DM (1963) The presence of acetyl groups of histones. Biochem J 87:258–263. 3. Allfrey VG, Faulkner R, Mirsky AE (1964) Acetylation and methylation of histones and their possible role in the regulation of RNA synthesis. Proc Natl Acad Sci USA 51:786–794. 4. Allfrey VG, Mirsky AE (1964) Structural modifications of histones and their possible role in the regulation of RNA synthesis. Science 144:559 . 5. Sterner DE, Berger SL (2000) Acetylation of histones and transcription-related factors. Microbiol Mol Biol Rev 64:435–459. 6. Yang XJ, Seto E (2008) Lysine acetylation: Codified crosstalk with other posttranslational modifications. Mol Cell 31:449–461. 7. Batta K, Das C, Gadad S, Shandilya J, Kundu TK (2007) Reversible acetylation of non histone proteins: Role in cellular function and disease. Subcell Biochem 41:193–212. 8. Arif M, Selvi BR, Kundu TK (2010) Lysine acetylation: The tale of a modification from transcription regulation to metabolism. Chembiochem 11:1501–1504. 9. Falbo KB, Shen X (2009) Histone modifications during DNA replication. Mol Cell 28:149–154. 10. Guan KL, Xiong Y (2011) Regulation of intermediary metabolism by protein acetylation. Trends Biochem Sci 36:108–116. 11. Wilson MP, Majerus PW (1996) Isolation of inositol 1,3,4-trisphosphate 5/6-kinase, cDNA cloning and expression of the recombinant enzyme. J Biol Chem 271:11904–11910. 12. Wilson MP, Majerus PW (1997) Characterization of a cDNA encoding Arabidopsis thaliana inositol 1,3,4-trisphosphate 5/6-kinase. Biochem Biophys Res Commun 232:678–681. 13. Field J, Wilson MP, Mai Z, Majerus PW, Samuelson J (2000) An Entamoeba histolytica inositol 1,3,4-trisphosphate 5/6-kinase has a novel 3-kinase activity. Mol Biochem Parasitol 108:119–123. 14. Miller GJ, Wilson MP, Majerus PW, Hurley JH (2005) Specificity determinants in inositol polyphosphate synthesis: Crystal structure of inositol 1,3,4-trisphosphate 5/6-kinase. Mol Cell 18:201–212. 15. Verbsky JW, Chang SC, Wilson MP, Mochizuki Y, Majerus PW (2005) The pathway for the production of inositol hexakisphosphate in human cells. J Biol Chem 280:1911–1920. 16. Wilson MP, et al. (2009) Neural tube defects in mice with reduced levels of inositol 1,3,4-trisphosphate 5/6-kinase. Proc Natl Acad Sci USA 106:9831–9835. 17. Majerus PW, Wilson DB, Zhang C, Nicholas PJ, Wilson MP (2010) Expression of inositol 1,3,4-trisphosphate 5/6-kinase (ITPK1) and its role in neural tube defects. Adv Enzyme Regul 50:365–372. 18. Eckner R, et al. (1994) Molecular cloning and functional analysis of the adenovirus E1A-associated 300-kD protein (p300) reveals a protein with properties of a transcriptional adaptor. Genes Dev 8:869–884.

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for the in vitro colorimetric assay. Released CoASH was detected following the addition of 50 μL DTNB at an absorbance of 412 nm. Protein Stability and ITPK1 Enzyme Activity Assays. FLAG-tagged ITPK1 alone or with CBP, its mutants alone or with CBP were transfected into HEK293 cells. After 24 h, the cells were treated with cycloheximide (150 μg∕mL) for indicated times, harvested, and lysed in 100 μL lysis buffer (100 mm NaCl, 50 mm Tris•HCl pH 7.3, 10% glycerol, 0.2% Triton X-100). Equal amounts of protein were separated by SDS-PAGE and analyzed by Western blotting with antiFLAG antibody and anti-β-actin antibody as a loading control. Enzyme activity assays were done as previously described (11). HPLC of Inositol Phosphates. Stable cell lines expressing ITPK1 and its mutants were grown to 60% confluence in six-well plates. Cells were labeled for 72 h in 80% inositol-free DMEM medium (Millipore), 9% complete DMEM medium, 10% dialyzed tetracycline-free FBS, 2 mM glutamine, 10 μCi 3 H-inositol/ mL, 1X inositol-free vitamin mix (4 mg∕mL pantothenic acid, 4 mg∕mL choline chloride, 4 mg∕mL folic acid, 4 mg∕mL nicotinamide, 4 mg∕mL pyridoxine, 0.4 mg∕mL riboflavin, and 4 mg∕mL thiamine), 1.75 mg∕mL glucose, and 1X minimal essential medium amino acid (Invitrogen), induced with 200 ng∕mL tetracycline for 48 h, lysed in 0.5 mL methanol∶0.5 M HCl (2∶1), and extracted with 1 mL chloroform. The aqueous phase was dried, suspended in water, and applied to a 250 × 4.6 mm Adsorbosphere strong anion exchange column (Alltech/Applied Science). Inositol phosphates were eluted as described (11). Prior to extraction, 10% of each sample was retained for protein assay. 3 H-inositol counts incorporated in each sample were normalized to total protein. ACKNOWLEDGMENTS. The authors thank Dr. Cheryl Lichti and Dr. Reid Townsend of the Proteomic Mass Spectrometry Facility at Washington University in St. Louis for mass-spectrometry analysis to identify the ITPK1 acetylation sites. This work was supported by National Institutes of Health Grants HL-16634-43 (to P.W.M.) and DK075618 (to D.B.W.) and a Children’s Discovery Institute Grant MD-II-2009-174 (to M.P.W.). 19. Lundblad JR, Kwok RP, Laurance ME, Harter ML, Goodman RH (1995) Adenoviral E1Aassociated protein p300 as a functional homologue of the transcriptional co-activator CBP. Nature 374(6517):85–88. 20. Arany Z, Newsome D, Oldread E, Livingston DM, Eckner R (1995) A family of transcriptional adaptor proteins targeted by the E1A oncoprotein. Nature 374(6517):81–84. 21. Eckner R, Yao TP, Oldread E, Livingston DM (1996) Interaction and functional collaboration of p300/CBP and bHLH proteins in muscle and B-cell differentiation. Genes Dev 10:2478–2490. 22. Kamei Y, et al. (1996) A CBP integrator complex mediates transcriptional activation and AP-1 inhibition by nuclear receptors. Cell 85:403–414. 23. Chakravarti D, et al. (1996) Role of CBP/P300 in nuclear receptor signalling. Nature 383 (6595):99–103. 24. Ogryzko VV, Schiltz RL, Russanova V, Howard BH, Nakatani Y (1996) The transcriptional coactivators p300 and CBP are histone acetyltransferases. Cell 87:953–959. 25. Alcendor RR, Kirshenbaum LA, Imai S, Vatner SF, Sadoshima J (2004) Silent information regulator 2α, a longevity factor and class III histone deacetylase, is an essential endogenous apoptosis inhibitor in cardiac myocytes. Circ Res 95:971–80. 26. Waterborg JH, Kapros T (2002) Kinetic analysis of histone acetylation turnoverand Trichostatin A induced hyper- and hypoacetylation in alfalfa. Biochem Cell Biol 80:279–293. 27. Imai S, Armstrong CM, Kaeberlein M, Guarente L (2000) Transcriptional silencing and longevity protein Sir2 is an NAD-dependent histone deacetylase. Nature 403:795–800. 28. Kim D, et al. (2007) SIRT1 deacetylase protects against neurodegeneration in models for Alzheimer’s disease and amyotrophic lateral sclerosis. EMBO J 26:3169–3179. 29. Michan S, Sinclair D (2007) Sirtuins in mammals: Insights into their biological function. Biochem J 404:1–13. 30. Vaziri H, et al. (2001) hSIR2(SIRT1) functions as an NAD-dependent p53 deacetylase. Cell 107:149–159. 31. Motta MC, et al. (2004) Mammalian SIRT1 represses forkhead transcription factors. Cell 116:551–563. 32. Yeung F, et al. (2004) Modulation of NF-kappaB-dependent transcription and cell survival by the SIRT1 deacetylase. EMBO J 23:2369–2380. 33. Sun Y, Wilson MP, Majerus PW (2002) Inositol 1,3,4-trisphosphate 5/6-kinase associates with the COP9 signalosome by binding to CSN1. J Biol Chem 277:45759–45764. 34. Giandomenico V, Simonsson M, Gronroos E, Ericsson J (2003) Coactivator-dependent acetylation stabilizes members of the SREBP family of transcription factors. Mol Cell Biol 23:2587–2599. 35. Caron C, Boyault C, Khochbin S (2005) Regulatory cross-talk between lysine acetylation and ubiquitination: Role in the control of protein stability. Bioessays 27:408–415. 36. Yang XJ (2004) Lysine acetylation and the bromodomain: A new partnership for signaling. Bioessays 26:1076–1087. 37. Vajanaphanich M (1994) Long-term uncoupling of chloride secretion from intracellular calcium levels by Ins(3,4,5,6)P4. Nature 371:711–714.

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