HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE ADVANCED SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Mori-Konya, C. Articles by Kakizuka, A. Search for Related Content PubMed Articles by Mori-Konya, C. Articles by Kakizuka, A.

¹ The Laboratory of Functional Biology, Kyoto University Graduate School of Biostudies and Solution Oriented Research for Science and Technology (JST), Kyoto 606-8501, Japan
² The Laboratory of Frontier Science, Tokyo Metropolitan Institute of Medical Science, Tokyo 113-8613, Japan

	Abstract

Top Abstract Introduction Results Discussion Experimental procedures References

 
p97/valosin-containing protein (VCP) is a member of the AAA family proteins, which plays various important roles in cells by using its ATPase activity. But mechanism of regulating its ATPase activity is mostly unknown. We report here that VCP is highly modified throughout the protein via acetylation and phosphorylation. In addition to six previously identified phosphorylation sites, we identified at least 14 serines, 14 threonines, 6 tyrosines and 22 lysines as potential modification sites. Interestingly, these sites included Lys²⁵¹ and Lys⁵²⁴, which are very critical for the ATP binding in Walker A motif of D1 and D2 domains, respectively. It is notable that 16 sites are in the N-terminal region and 16 sites are clustered in D2 domain (from Pro⁶⁴⁶ to Gly⁷⁶⁵). Indeed, amino acid substitution of Lys⁶⁹⁶ and Thr⁷⁶¹ profoundly affect VCP ATPase activities. From these results, we propose that D2 domain acts as a VCP ATPase Regulatory domain or "VAR domain". VCP modifications including those in this VAR domain may endorse adaptive and multiple functions to VCP in different cell conditions such as in the cell cycle and with abnormal protein accumulation.

	Introduction

Top Abstract Introduction Results Discussion Experimental procedures References

 
p97/valosin-containing protein (VCP) is a member of the AAA (ATPases associated with diverse cellular activities) class of ATPase (Brunger & Delabarre 2003). VCP ordinarily exists as a homo hexamer, and most, if not all, hexamers make further complex with its adaptor proteins, such as p47 or Ufd1–Npl4 (Kondo et al. 1997; Meyer et al. 2000). Each VCP protomer is composed of four regions, the N-terminal, two ATPase (D1 and D2) and the C-terminal regions (Brunger & Delabarre 2003; Wang et al. 2004). The N-terminal region possesses binding activities to the adaptor proteins as well as ubiquitinated proteins (Brunger & Delabarre 2003). D1 and D2 ATPase regions are needed for maintenance of homo hexamer configuration and performing its cellular functions, respectively (Kobayashi et al. 2002; Wang et al. 2003). Indeed, D2 ATPase activities are much stronger than D1 ATPase activities. The C-terminal region of VCP has been reported to associate with some proteins, for example, Ufd3, and PGNase in a phosphorylation dependent manner (Zhao et al. 2007).

VCP is a very abundant protein that occupies approximately 1% of cytoplasmic proteins (Dalal & Hanson 2001). Accordingly, VCP has been proposed to perform many physiological cellular functions such as homotypic membrane fusions of the nucleus, Golgi apparatus and endoplasmic reticulum (ER), the protein degradation mediated by the ubiquitin-proteasome system as well as ER-associated protein degradation (ERAD) and the cell cycle control (Rabouille et al. 1995; Kondo et al. 1997; Zhang et al. 1999; Ye et al. 2001; Rabinovich et al. 2002; Cao et al. 2003). In order to participate in such different functions, VCP has been proposed to use different adapter proteins. For example, VCP uses p47 in membrane fusions, as well as Ufd1–Npl4 complex in protein degradations (Kondo et al. 1997; Meyer et al. 2000). Nevertheless, these observations could only partly explain the molecular mechanisms underlying the multifunction of VCP.

However, lines of evidence have shown that VCP is involved in the pathology of human neurodegenerative disorders (Hirabayashi et al. 2001; Kobayashi & Kakizuka 2003; Boeddrich et al. 2006; Boyault et al. 2006). We identified VCP as a binding protein of the MJD protein with expanded polyglutamine (Hirabayashi et al. 2001), which causes Machado–Joseph disease (MJD) (Kawaguchi et al. 1994), the most common inherited spinocerebellar ataxia. Immunochemical analysis showed that VCP co-localizes with nuclear inclusion bodies in polyglutamine diseases such as MJD and Huntington disease or Lewy bodies in Parkinson disease and dementia with Lewy bodies (Hirabayashi et al. 2001; Mizuno et al. 2003). Interestingly, recent functional analysis on VCP using cell culture system has revealed that VCP possesses both aggregate forming as well as clearing activities and, in both cases, its ATPase activities are essential (Kobayashi et al. 2007). VCP has also been identified as a genetic modifier of polyglutamine-induced eye degeneration in a Drosophila model of polyglutamine diseases (Higashiyama et al. 2002).

It is of note that over-expression of VCP(K524A), an ATPase activity-deficient VCP mutant in cultured cells induced ER-derived large vacuolization followed by cell death, demonstrating that ATPase activity of VCP is essential for cell survival (Hirabayashi et al. 2001; Kobayashi et al. 2002). Recombinant VCP has been shown to be inactivated easily via the oxidation of Cys⁵²² in VCP, suggesting that VCP inactivation may play important roles in the pathogenesis of oxidation-related disorders such as atherosclerosis and Parkinson disease (Noguchi et al. 2005). Furthermore, nine missense mutations in the VCP gene have recently been identified to cause inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia (IBMPFD), a dominantly inherited human genetic disorder affecting muscles, bones and the brain (Watts et al. 2004; Haubenberger et al. 2005; Forman et al. 2006), and these IBMPFD VCPs all appeared to possess elevated ATPase activities (Manno, A., Noguchi, M. and Kakizuka, A. unpublished observations).

These observations clearly demonstrate that certain single amino acid substitution or modification (Cys⁵²² oxidation, for example) can change VCP ATPase activities. However, continuous elevation or decrease of VCP ATPase activities by amino acid substitution may not be suitable for cells, which is typically observed in IBMPFD or VCP(K524A), respectively. In other words, yet unknown reversible regulatory mechanisms should exist to control its ATPase activity. One of possible mechanism would be phosphorylation or dephosphorylation. Indeed, several groups have identified phosphorylations at Ser⁷, Ser³⁵², Ser⁷⁴⁶, Ser⁷⁴⁸, Ser⁷⁸⁴ and Tyr⁸⁰⁵ in VCP (Egerton & Samelson 1994; Zhang et al. 1999; Klein et al. 2005; Livingstone et al. 2005; Rush et al. 2005; Villen et al. 2007).

Another potential mechanism would be acetylation or deacetylation. So far, most of the acetylated proteins that have been well investigated are nuclear proteins except for -tubulin (Verdin et al. 2003). Interestingly, acetylated -tubulin has been shown to be deacetylated by HDAC6 (Hubbert et al. 2002; Matsuyama et al. 2002; Zhang et al. 2003), and HDAC6 and VCP have been reported to physically interact each other (Seigneurin-Berny et al. 2001). In either phosphorylation or acetylation, the LC/MS/MS (liquid chromatography/tandem mass spectrometry) analysis is able to reveal the modifications with increasing molecular mass of 80 Da in serine, threonine and tyrosine, or 42 Da in lysine, respectively, in peptide fragments from purified proteins.

	Results

Top Abstract Introduction Results Discussion Experimental procedures References

 
Multiple iso-electric point forms of VCPs were produced by the baculovirus system

In order to prepare a large amount of VCP proteins, we chose the baculovirus system and expressed His-tagged mammalian VCP (His-VCP) in insect Sf-9 cells. After incubation for 2 days, the insect cells were lysed and His-VCP was purified via the Nickel columns. Resultantly, we were able to obtain approximately 0.5 to 1 mg His-VCP from 0.5 L culture. In order to evaluate the purity of the protein, we examined the purified His-VCP protein by SDS-PAGE, non-denatured PAGE and 2D-PAGE. As expected, the purified His-VCP migrated as a single band at the position predicted from its calculated molecular size of approximately 100 kDa by SDS-PAGE (Fig. 1A). VCP has been shown to exist as a homo-hexamer. Indeed, the purified His-VCP migrated as a single band at a position of approximately 600 kDa by non-denatured PAGE (Fig. 1B). The iso-electric point of VCP was calculated from its amino acid composition as 5.14. However, by 2D-PAGE, the purified His-VCP was detected as multiple dots with different iso-electric points, from pI 4.7 to pI 5.3 (Fig. 1C). Using HEK293T cells, we next examined iso-electric points of endogenous VCP via 2D-PAGE and immunoblotting, and observed at least four VCPs with different iso-electric points (Fig. 1D). In the literature, it has been shown that several amino acids in VCP are modified by phosphorylation, for example, Ser⁷, Ser³⁵², Ser⁷⁴⁶, Ser⁷⁴⁸, Ser⁷⁸⁴ and Tyr⁸⁰⁵ (Egerton & Samelson 1994; Zhang et al. 1999; Klein et al. 2005; Livingstone et al. 2005; Rush et al. 2005; Villen et al. 2007). These observations suggested the idea that VCP might be highly modified via post translational modifications at novel positions. In order to clarify this idea, we examined the His-VCP by using the molecular mass analysis.

View larger version (20K): [in this window] [in a new window]   Figure 1  Characterization of VCP protein. (A and B) His-VCP was expressed in insect Sf-9 cells, and purified using a nickel affinity gel. Purified His-VCP was analyzed by Western blotting with anti-penta His antibody (A) or non-denatured PAGE stained with SYPROR. Ruby (B). The positions of molecular weight markers were indicated on the left of the panels. Note that recombinant His-VCP migrated at the hexamer position as observed with endogenous VCP. (C) 2D-PAGE analysis of purified His-VCP. Purified His-VCP from Sf-9 cells was separated by 2D-PAGE using a pH 4–7 IPG strip, followed by SDS-PAGE, and the separated proteins were blotted on to a nitrocellulose filter. The blotted proteins were visualized with an anti-VCP antibody (upper panel) or silver staining (lower panel). The isoelectric points (pI) are indicated above the panels. (D) 2D-PAGE analysis of endogenous VCP in HEK293 cells. HEK293 cell lysate was separated by 2D-PAGE using a pH 5–6 IPG strip, and VCP protein was visualized with the anti-VCP antibody. The isoelectric points (pI) are indicated above the panel. (E) [H3] acetate-labeling of VCP in cultured cells. HEK293 cells were transfected with the expression vectors for indicated FLAG-VCPs. Forty-eight hours after transfection, the cells were incubated with [H3] acetate for 1 h. Then, FLAG-VCPs were immunoprecipitated, subjected to SDS-PAGE. Acetylations of FLAG-VCPs were visualized by autoradiography of a SDS-PAGE gel (upper panel). Expressed FLAG-VCPs were visualized by CBB staining (lower panel).

From the data obtained by the MALDI-TOF analysis, we identified several modified peptide fragments from His-VCP. These modified fragments predicted the existence of several potentially modified amino acid residues, including Ser²⁸², Ser²⁸⁴, Tyr⁴⁹⁵, Tyr⁷⁵⁵, Thr⁷⁶¹ and Ser⁷⁶⁵ as phosphorylation sites, and Lys⁶⁵¹ and Lys⁶⁹⁶ as acetylation sites. These amino acids did not overlap with previously reported modified amino acids. Although this protein was made from insect cells, these results suggested that VCP is a highly modified protein, and that many modification sites have remained unidentified.

VCP is composed of four regions, the N-terminal, two ATPase (D1 and D2) and the C-terminal regions (Fig. 2A). D1 and D2 regions have Walker A (WA) motifs as ATP binding sites, Walker B (WB) as ATP hydrolysis sites, and second regions of homology (SRH), followed by domains (Fig. 2A). It is notable that these potentially modified amino acids were mostly located in D2 domains (covering 646–765 amino acid residues), whose amino acid sequences were highly conserved among VCP homologs but of unknown functions. Indeed, Lys⁶⁹⁶, Tyr⁷⁵⁵, Thr⁷⁶¹, Ser⁷⁶⁵ and their surrounding amino acids are highly conserved among species (Fig. 2B).

View larger version (41K): [in this window] [in a new window]   Figure 2  Detected modification sites in D1 and D2 domains of VCP. A schematic structure of VCP protein. VCP is composed of the N-terminal, two ATPase (D1 and D2), and the C-terminal regions. The N-terminal region, which consists of Na and Nb domains, is required for cofactor and substrate binding. The positions of key elements in the ATPase domains, Walker A motif (WA), Walker B motif (WB), and the second region of homology (SRH), are depicted. Each ATPase domain (D1 and D2) is followed by the -helix rich region, which is termed as D1 and D2 domains, respectively. L: linker domain. (B) Possible modification sites in D1, linker, and D2 domains. The amino acid sequences of D1 and linker (amino acids residues from 371 to 480), and D2 (amino acids residues from 646 to 765) domains from human VCP and its orthologues are aligned. The green underlines indicate modified peptides detected by the MALDI-TOF analysis on His-VCP purified from Sf-9 cells. The peptides including Lys651 or Lys696 were detected as acetylated peptides, and the peptide including Tyr755, Thr761 and Ser765 as a phosphorylated peptide by the MALDI-TOF analysis. LC/MS/MS analyses were conducted many times to identify the modification sites in VCP protein from mammalian cells. The results of the analysis are displayed above the alignment. The residue numbers colored in red indicate detected modified amino acids by the analysis, and the corresponding amino acid residues are also colored in red. The frequencies of the detected times are shown under the residue numbers. The regions colored in pale blue indicate the peptides which had never been detected in all experiments, probably because of the less ionized properties of the peptides. Note that most of the modified amino acids in D2 domain are not conserved in NSF.

We then examined whether these modifications existed in VCP purified from mammalian cells. We again expressed FLAG-VCP in HEK293T cells, and the purified FLAG-VCPs were analyzed by LC/MS/MS analysis. The MS/MS spectrum (Fig. 3) matched on a database constructed from the human VCP sequence utilizing the SEQUEST program (Eng et al. 1994). The matching parameters were set up for picking up possible phosphorylation (+80) at all serine, threonine or tyrosine residues, and possible acetylation (+42) at lysine residues. For example, the program identified from the MS/MS spectrum (Xcorr score = 1.126, delta Cn = 0.198) a doubly charged ion at m/z 839.32 eluting at 22.62 min as that derived from the VCP peptide (Y*EMFAQT* LQQSR) containing both Tyr⁷⁵⁵ and Thr⁷⁶¹ being phosphorylated (see below). The peptide is juxtaposed with arginine, and thus was consistent to be a product of the trypsin digestion. Its calculated and measured molecular weights were 1517 and 1677.64, respectively. The difference was in good agreement with the peptide being phosphorylated at two positions. The reliability of this data was supported by the delta Cn score of 0.198; the delta Cn score > 0.1 is indicative of a high degree of relative confidence in a correct match of the peptide.

View larger version (18K): [in this window] [in a new window]   Figure 3  An analytical data of LC/MS/MS with VCP from HEK 293T cells. A tandem mass spectrum of a tryptic peptide from immunoprecipitated FLAG-VCP shows detection of phosphorylated Thr761 in VCP. HEK293T cells were transfected with an expression vector for FLAG-VCP, and were treated with 1 µM MG132 for 24 h. FLAG-VCP was immunoprecipitated, tryptic-digested, and analyzed by LC/MS/MS. The MS/MS spectrum was searched against a database constructed from the reported human VCP sequence utilizing SEQUEST program. Search parameters were modified to consider possible phosphorylation (+80) at all serine, threonine or tyrosine residues, possible oxidation (+16) at methionine, and possible acetylation (+42) at lysine. Matched b or y ions via automatic computational interpretation are displayed in bold letters in the sequence panel and the attribution of the strong peaks among them is indicated in the spectrum. The significant peaks that match to theoretical peaks interpreted by MS-Product <http://prospector.ucsf.edu/cgi-bin/msform.cgi?form=msproduct> are shown with numbers of the mass in the spectrum.

Creation of amino acid substitutions on Lys⁶⁹⁶ and Thr⁷⁶¹ in VCP

We then created and analyzed several VCP mutants, in which Lys⁶⁹⁶ and Thr⁷⁶¹ were substituted by different amino acids. For example, Lys⁶⁹⁶ was substituted by glutamine or arginine (referred to as VCP(K696Q) or VCP(K696R), respectively). We assumed that VCP(K696Q) would function as an acetylation-mimic form of VCP, whereas VCP(K696R) as a non-acetylated form of VCP. Thr⁷⁶¹ was substituted by glutamic acid or valine (referred to as VCP(T761E) or VCP(T761V), respectively). We assumed that VCP(T761E) would function as a phosphorylation-mimic form of VCP, whereas VCP(T761V) as a non-phosphorylated form of VCP.

Recombinant His-VCPs were expressed in insect Sf-9 cells with the baculovirus-mediated expression system. Each protein was purified using a Nickel gel. We examined the purified His-VCPs by SDS-PAGE and non-denatured PAGE. Purified proteins were all detected as a single band at the position of approximately 100 kDa by SDS-PAGE, and of approximately 600 kDa by non-denatured PAGE (data not shown), indicating that all of the VCP mutants were able to take hexameric conformation, as observed in wild-type VCP.

Characterization of the amino acid substitutions of Lys⁶⁹⁶ in VCP

We have previously reported that VCP is easily inactivated via oxidation of Cys⁵²² (Noguchi et al. 2005). To prevent such oxidation, 5 mM DTT was always added in the stock solution of 1 mg/mL His-VCP. We then used Martin–Doty method with minor modification for measuring ATPase activity of each purified VCP mutant (see Experimental procedures). As a result, ATPase activities of VCP (K696Q), an acetylation-mimic form of VCP, increased as compared with that of wild-type VCP, whereas ATPase activity of VCP(K696R), a non-acetylated form of VCP, dramatically decreased (Fig. 6A).

View larger version (13K): [in this window] [in a new window]   Figure 6  Kinetic properties of VCP ATPase activities by the amino acid substitution of Lys696. (A) Effects of ATP concentration on the ATPase activities of acetyl mimetic mutant, VCP(K696Q) or a non-acetyl mimetic mutant, VCP(K696R). Each of the His-tagged mutant or wild-type VCP was expressed in Sf-9 cells and purified. ATPase activities of each VCP were measured by Martin–Doty method with minor modifications (Chevalier et al. 1998). (B) Hanes–Woolf plots for determining kinetic parameters, Km and Vmax, of Lys696 substitution mutants. [ATP]/velocity was plotted against [ATP], using the kinetic data of (A). To determine kinetic parameters, triplicate experiments were carried out, and mean values and standard deviations were obtained. If not shown, error bars are smaller than the symbols. The kinetic data of (A) were fitted by nonlinear regression to the Michaelis–Menten model.

We next investigated ATPase activities of other purified VCP mutants, in which Thr⁷⁶¹ was substituted to valine or glutamic acid. VCP(T761V), a non-phosphorylated form, showed ATPase activity comparable to that of wild-type VCP (Fig. 7A). A plot of [ATP] vs. [ATP]/velocity gave a straight line (Fig. 7A). From this plot, estimated kinetic parameters of wild-type VCP and VCP (T761V) were as follows: K_m were 0.119 and 0.129 mM, and V_max were 233 and 225 pmol/min/µg, respectively.

View larger version (15K): [in this window] [in a new window]   Figure 7  Kinetic properties of VCP ATPase activities by the amino acid substitution on Thr761. (A) Effects of ATP concentration on the ATPase activities of a non-phospho-mimetic mutant, VCP(T761V). (B) Effects of ATP concentration on the ATPase activities of a phospho-mimetic mutant, VCP(T761E). His-tagged each mutant or wild-type VCP was expressed in Sf-9 cells and purified. ATPase activities of each VCP were measured by Martin–Doty method with minor modifications. Hanes–Woolf plots are shown (right panels). To determine kinetic parameters, triplicate experiments were carried out, and mean values and standard deviations were obtained. If not shown, error bars are smaller than the symbols. The kinetic data of right panels were fitted by nonlinear regression to the Michaelis–Menten model.

Potential interaction of phosphorylated Thr⁷⁶¹ with Arg⁷⁴¹ in the adjacent VCP protomer

From the 3D structure in the published data, Thr⁷⁶¹ is faced to Arg⁷⁴¹ in the adjacent VCP protomer, and the distance between Thr⁷⁶¹ and the adjacent Arg⁷⁴¹ was shown to be 6 Å (DeLaBarre & Brunger 2003). Furthermore, Arg⁷⁴¹ was conserved in various species (Fig. 2B). We thus hypothesized that phosphorylated Thr⁷⁶¹ may interact with the adjacent Arg⁷⁴¹ through an ionic bond, and that this interaction may contribute to the increase of VCP ATPase activity. Accordingly, we substituted Arg⁷⁴¹ into alanine on VCP(T761E). The resultant double substitution mutant was referred to as VCP(R741A-T761E). VCP(R741A-T761E) was detected as a single band at the position of approximately 100 kDa by SDS-PAGE, and of approximately 600 kDa by non-denatured PAGE (Fig. 8A). We then measured the ATPase activity of VCP(R741A-T761E) by the enzyme-coupling method. As expected, the increase of ATPase activity in VCP(T761E) was cancelled in VCP(R741A-T761E) (Fig. 8B). These results supported the idea that the interaction of phosphorylated Thr⁷⁶¹ and Arg⁷⁴¹ in the adjacent protomer through an ionic bond could render VCP an elevated ATPase activity.

View larger version (12K): [in this window] [in a new window]   Figure 8  Effects of possible inter-protomer interaction between Arg741 and phosphorylated Thr761 on VCP ATPase activities. (A) His-tagged VCP(R741A-T761E) or wild-type VCP was purified with the same procedure used in Fig. 1, and visualized with an anti-penta His antibody (left panel) or non-denatured PAGE with Ruby staining (right panel). (B) ATPase activities of VCP(R741A-T761E) were measured by an enzyme-coupling method (Tokuda et al. 1993). ATP concentration in the assay was 1 mM. Mean values and standard deviations were obtained from triplicate experiments. If not shown, error bars are smaller than the symbols. Note that ATPase activities of wild-type VCP and VCP(R741A-T761E) were indistinguishable. (Curve fitting was carried out using spline curve.)

	Discussion

Top Abstract Introduction Results Discussion Experimental procedures References

We have repeatedly shown that maintaining the ATPase activity of VCP is very important for cell survival (Hirabayashi et al. 2001; Kobayashi et al. 2002; Noguchi et al. 2005). In addition, we have observed that all tested IBMPFD VCPs possess elevated ATPase activities (Manno, A., Noguchi, M. and Kakizuka, A. unpublished observations), suggesting that continuous elevation of VCP ATPase activity is also problematic for cells. Thus, precise control of the ATPase activity of VCP should be very important for cells. Such precise control implies reversibility, and thus post-translational modifications are the most likely mechanism. VCP has been shown to be phosphorylated at least six sites, namely Ser⁷, Ser³⁵², Ser⁷⁴⁶, Ser⁷⁴⁸, Ser⁷⁸⁴ and Tyr⁸⁰⁵ (Egerton & Samelson 1994; Zhang et al. 1999; Klein et al. 2005; Livingstone et al. 2005; Rush et al. 2005; Villen et al. 2007), although it has not been shown at all whether these modifications affect the ATPase activities of VCP. We report here additional 34 and 22 sites in VCP as potential phosphorylation and acetylation sites, respectively (Supporting Tables S1–S4, Figs 2, 4, 5). All of the previously identified phosphorylation sites except for Ser⁷⁸⁴ and Tyr⁸⁰⁵ were included in the sites we identified. It is notable that the phosphorylation of Ser⁷⁸⁴ in VCP was identified from cells treated with -ray (Livingstone et al. 2005). As we did not treated cells with -ray, this may be a reason as to why we could not detect this phosphorylation. More importantly, however, we were not able to detect any C-terminal trypsin-digested fragments (downstream from Arg⁷⁶⁶) by our LC/MS/MS analysis, and thus fragments from the C-terminal region of VCP appeared to be very difficult to be ionized.

View larger version (42K): [in this window] [in a new window]   Figure 5  Detected modification sites in D1 and D2 ATPase domains of VCP. The amino acid sequences of D1 (amino acids residues from 208 to 370) and D2 (amino acids residues from 481 to 645) ATPase domains from human VCP and its orthologues are aligned. The results of LC/MS/MS analyses are displayed above the alignment. The residue numbers colored in red indicate detected modified amino acids by the analysis, and the corresponding amino acid residues are also colored in red. The frequencies of the detected times are shown under the residue numbers. The region colored in pale blue indicates the peptide that had never been detected in all experiments, probably because of the less ionized properties of the peptide. The conserved motifs are marked in the following ways. Single underline, Walker A motif. Double underline, Walker B motif. Dotted underline, SRH region. Green underlines, phosphorylated or acetylated peptides detected by the MALDI-TOF analysis.

In yeast, Cdc48, VCP yeast homologue, has been reported to move to nucleus by the phosphorylation of Tyr⁸⁰⁵ (Madeo et al. 1998). Recently, we have observed that mammalian VCP also shuttles between the nucleus and the cytoplasm (Koike, M. and Kakizuka, A. unpublished observations), and thus VCP may be acetylated in the nucleus like most of the other acetylated proteins. This possibility also remains to be tested.

Modifications appeared to occur throughout VCP; 16 sites in the N-terminal region, 15 in D1 region, 29 in D2 region. The N-terminal modification may contribute to its differential bindings or affinities to the partners as well as ubiquitinated proteins. D1 and D2 regions are further divided into ATPase domains (D1 and D2 ATPase domains) and -helical domains (D1 and D2 domains) (Fig. 2A). Thirteen modification sites were identified in D1 ATPase domain and two in D1 domain. It is notable that similar to the C-terminal fragments, some trypsin-digested fragments from D1 domain were hardly detected in our LC/MS/MS analysis (Fig. 5), and thus yet-unidentified modifications in D1 domain may exist. Among the 13 sites, three sites (Tyr²⁴⁴, Thr²⁴⁹ and Lys²⁵¹) are in or adjacent to Walker A motif, and thus their modifications may directly influence the ATP binding of D1 ATPase domain. Thirteen modification sites were identified in D2 ATPase domain and 16 in D2 domain. Among them, two sites (Lys⁵²⁴ and Thr⁵²⁵) are in Walker A motif, and thus their modifications may directly influence the ATP binding of D2 ATPase domain. Until now, VCP(K251A) and VCP(K524A) have been shown to lose D1 and D2 ATPase activities, respectively, because of their incapability of binding ATP (Kobayashi et al. 2002; Noguchi et al. 2005; Briggs et al. 2008).

D2 domain of VCP is highly conserved among different species, but its function is totally unknown. We identified at least 16 amino acids as potentially modified amino acids in this domain. NSF has been shown to act only in the membrane fusion, as opposed to the multifunction of VCP, suggesting that ATPase activities of VCP should be regulated in performing various functions. Because several modified amino acids in this D2 domain are surrounding D2 ATP-binding pocket (Fig. 9), it is reasonable to speculate that their modifications may affect the ATPase activity of VCP.

View larger version (56K): [in this window] [in a new window]   Figure 9  Structure of the ATP biding pocket in D2 domain of VCP. (A) A model structure of hexameric VCP protein, highlighting D1, D2 and the C-terminal regions. Two arrows, (b) and (c), indicate viewpoints of (B) and (C), respectively. (B, C) The crystal structure of D2 domain viewed from outside (B) or inside (C) of the protein, showing the proximity of Thr761 and Arg741 in the adjacent protomer. The indicated Arg741* and SRH* region are derived from the juxtaposing protomer. The distance between Thr761 and Arg741 in the adjacent protomer has been estimated 6 Å. The 1 and 2 indicate -helix regions from Ser683 to Ala694 and from Glu738 to Arg741, respectively. 1 and 2 helices are located at very close positions to Thr761 and Lys696, respectively, in the adjacent protomer, whose modifications are expected to change the shapes of the ATP-binding pockets made between the adjacent protomers. The yellow loops under 1 represent SRH regions. These structures are illustrated, based on the crystallographic data of VCP in complex with ADP/ADP-AlFx (accession number 1OZ4 in Protein data bank). ADP-AlFx is shown in ball and stick representation. The figure was prepared using Cn3D <http://www.ncbi.nlm.nih.gov/Structure/CN3D/cn3d.shtml>.

As a result of the amino acid substitution of Lys⁶⁹⁶, K_m and V_max of VCP(K696Q) were elevated as twice as those of wild-type VCP (Table 1). The elevation of K_m and V_max indicates the decrease of affinity to ATP and accelerated ATP hydrolysis, respectively. Thus, VCP(K696Q) was expected to function with approximately two times stronger ATPase activities than wild-type VCP when ATP was abundant. In contrast, VCP(K696R) showed very low or almost no measurable ATPase activities. These results indicate that acetylation and deacetylation of Lys⁶⁹⁶ can contribute to up- and down-regulation of VCP ATPase activity, respectively.

Regarding with the amino acid substitution of Thr⁷⁶¹, VCP(T761V) showed ATPase activity comparable to that of wild-type VCP. In contrast, VCP(T761E) was of K_m and V_max with approximately twice as much elevation as compared with wild-type VCP. As the amino acid sequences surrounding Thr⁷⁶¹ do not fit any consensus sequences that are known to be phosphorylated, kinases responsible for the Thr⁷⁶¹ phosphorylation were unable to be predicted at this moment. X-crystallographic data indicated that Thr⁷⁶¹ faced to Arg⁷⁴¹ in the adjacent VCP protomer, suggesting the formation of ionic bond between phosphorylated Thr⁷⁶¹ and Arg⁷⁴¹, which in turn would contribute to the ATPase activation. Indeed, the elevation of ATPase activities of VCP(T761E) was cancelled in VCP(R741A-T761E), a doubly substituted VCP mutant at Arg⁷⁴¹ and Thr⁷⁶¹.

These results indicate that phosphorylation of Thr⁷⁶¹ can create new ionic bond formation between phosphorylated Thr⁷⁶¹ and Arg⁷⁴¹ in the adjacent protomer. This interaction, in turn, is expected to create a more rigid conformation in the ATP binding pocket that is made between VCP protomers. This conformation change may hinder ATP to access the binding pocket, resulting in K_m elevation. In either case, precise molecular mechanisms underlying the observed ATPase activations, namely elevation of V_max, in VCP(K696Q) and VCP(T761E) remain to be elucidated.

In summary, we showed that VCP is a highly modified protein especially by phosphorylation and acetylation throughout the protein, and several modified amino acids are clustered in the previously called D2 domain (amino acid residues from 646 to 765). Among them, we showed that amino acid substitutions of Lys⁶⁹⁶ and Thr⁷⁶¹ by modification-mimic or non-modifiable amino acids can change VCP ATPase activities, which supports the idea that VCP ATPase activities are modulated by the post-translational modifications of phosphorylation and acetylation at least in this D2 domain. We thus propose that D2 domain of VCP is called "VCP ATPase Regulatory domain" or "VAR domain". Further analysis of post-translational modification in this VAR domain and other domains may also contribute to solve precise molecular bases underlying multifunction of VCP, and may provide hints to develop novel treatments in several human disorders in multiple organs such as bones, skeletal and cardio muscles, and neurons.

	Experimental procedures

Top Abstract Introduction Results Discussion Experimental procedures References

 
Plasmid and mutagenesis

Rat VCP cDNA was subcloned into pBluescriptII KS (+) (Stratagene) (pBS). Mutagenesis was carried out by PCR mediated method with Pfu turbo polymerase (Stratagene). pBS-VCP was used for the template. After PCR, the PCR products were digested with DpnI in order to select non-methylated PCR products. Then, mutated VCP cDNAs were subcloned into pFastBack HTa. Plasmids encoding FLAG-tagged wild-type VCP and mutated VCP were constructed in pFLAG-CMV^TM.-2 vector (SIGMA).

Antibodies

An anti-VCP antibody was developed by the standard procedures previously described (Hirabayashi et al. 2001; Kobayashi et al. 2002). A mouse monoclonal anti-penta His antibody (Qiagen) was purchased.

Expression and purification of proteins from Sf9 cells

His-tagged VCP was expressed in insect Sf-9 cells via the infection of recombinant baculoviruses. The recombinant baculoviruses were generated with Bac-to-Bac Baculovirus Expression Systems (Invitrogen Life Technologies). Cells were lysed in a phosphate buffer (50 mM sodium phosphate (pH 7.8), 300 mM NaCl, 1% Nonidet P-40, 1 mM ATP, 10 mM β-mercaptoethanol, 100 µg/mL 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF), 20 mM benzamidine and a protease inhibitor cocktails (AEBSF, aprotinin, E-64, leupeptin, pepstatin and pepstatinA) (Nacalai Tesque). The lysates were incubated with HIS-Select^TM. nickel affinity gel (SIGMA) for 1 h and washed with a washing buffer (50 mM sodium phosphate (pH 7.8), 300 mM NaCl and 10–50 mM imidazole gradient). The bound proteins were eluted with an elution buffer (50 mM sodium phosphate (pH 7.8), 300 mM NaCl and 150 mM imidazole). Then, the elution buffer was exchanged to a storage buffer (50 mM Tris–HCl (pH 8.0) and 20% glycerol) by dialysis with Spectra/Por Biotech regenerated cellulose dialysis membrane (Spectrum Laboratories, Inc.). Protein concentration was adjusted to 1 mg/mL, then the purified proteins were supplemented with 5 mM DTT and stored at –80 °C.

Western blotting and native-PAGE

Western blotting was carried out using a standard method. 0.5 µg protein was separated by SDS-PAGE with a 7.5% gel and transferred to a PVDF (polyvinylidene difluoride) membrane. The anti-penta His antibody (Qiagen) or the anti-VCP antibody was used for the primary antibody and a horseradish peroxidase-conjugated antibody (GE Healthcare) as the secondary antibody. After treatment with an ECL Western blotting Detection reagents (GE Healthcare), signals were detected with a luminescence image analyzer (LAS-1000 PLUS, Fuji Film). In Native-PAGE, 1 µg of protein was separated with 2%–15% gradient gel and the proteins were detected by SYPRO^R. Ruby (Molecular Probes).

Two-dimensional gel electrophoresis

The buffer of purified His-VCP was exchanged into the buffer containing 8 M urea, 4% CHAPS, 20 mM DTT, 0.5% appropriate IPG (immobilized pH gradient) Buffer, 1 mM NaF, 4 mM sodium orthovanadate, 5 mM β-glycerophosphate disodiumsalt hydrate, 1 mM sodium dihydrogen phosphate dihydrate and a protease inhibitor cocktails (Nacalai Tesque) by using ProbeQuant^TM. G-50 Micro Columns (GE Healthcare) following the manufacturer's protocol. The protein was subjected to 2D-PAGE using 18 cm, pI 4–7, linear immobilized pH gradient drystrips (GE Healthcare) in the first dimension and 18 cm x 8 cm 10% SDS-gels in the second dimension.

[H³]acetate-labeling in cultured cells

HEK293 cells were plated at the density of 3 x 10⁵ cells in a 6-well plate before transfection. HEK293 cells were transfected with 1.5 µg expression plasmids using Lipofectamine (Invitrogen) following the manufacturer's protocol. Forty-eight hours after the transfection, the cells were incubated with 7.4 MBq/mL of [H³]acetate (GE Healthcare) for 1 h. Soluble proteins were extracted with RIPA buffer (50 mM Tris–HCl (pH 8.0), 150 mM NaCl, 5 mM EDTA, 1% Nonidet P-40 and 1 mM DTT) containing a protease inhibitor cocktails (Nacalai Tesque) and 1 mM TSA (tricostatin A), a deacetylase inhibitor. For immunoprecipitation, the lysates were mixed with the anti-FLAG M2 affinity gel (SIGMA) for overnight at 4 °C. The immunocomplexes were washed with TBS (50 mM Tris–HCl (pH 7.4) and 150 mM NaCl) three times and 2 x SDS sample buffer was added. To check the levels of immunoprecipitated FLAG-VCPs, immunocomplexes were subjected to SDS-PAGE and stained with CBB (Coomassie Brilliant Blue). For fluorography, CBB-stained gel was fixed with fixing solution (isopropanol: water : acetic acid (25 : 65 : 10)) for 30 min and soaked in Amplify (GE Healthcare) for 30 min. The gel was dried and exposed on to an X-ray film (Fuji film).

LC/MS/MS analysis

Mammalian cell lines such as HEK293T cells were transfected with an expression vector for FLAG-VCP, and treated with or without 1 µM MG132, a proteasome inhibitor. FLAG-VCP was immunoprecipitated and was treated with 10% TCA (trichloroacetic acid), washed with acetone and dried. Next, the samples were treated with trypsin. The tryptic-digested peptides were analyzed by LC/MS/MS (Waters, 2795 separation module/Thermo Finnigan, LCQ Deca XP plus).

Measurement of ATPase activities of VCP

In measuring VCP ATPase activities, we used two methods, the enzyme-coupled method (Tokuda et al. 1993) and the Martin–Doty method (Chevalier et al. 1998). In the enzyme-coupled method, proteins were pre-incubated at 37 °C for 10 min, and then incubated with the ATPase assay buffer I (50 mM Tris–HCl (pH 9.0), 150 mM NaCl, 2 mM MgSO₄), 3 mM phosphoenol pyruvate, 1 mM ATP, 0.25 mM NADH, 1.0 unit of pyruvate kinase and 1.5 units of lactate dehydrogenase. The absorbance of NADH was measured at 340 nm for 20 min at 37 °C. In the Martin–Doty method, 500 µg of VCP protein was incubated in 20 µL of the ATPase assay buffer II (10 mM HEPES (pH 7.4), 25 mM KCl, 2.5 mM MgCl₂) with several concentrations of [-³²P]ATP, which were prepared from [-³²P]ATP (222TBq/mmol) (GE Healthcare) at 37 °C for 10 min. After incubation, the reaction was quenched by addition of 200 µL of 7% ice-cold TCA solution with 1 mM K₂HPO₄, and then 50 µL of solution A (3.75% ammonium molybdate, 0.02 M silicotungstic acid in 3 N H₂SO₄) and 300 µL of n-butyl acetate were added to the reaction. The samples were mixed well and centrifuged at 15 000 g for 5 min. Then, 200 µL aliquots from the upper organic phases were taken and their radioactivity was determined with a liquid scintillation counter for β-radiation, which determined the amounts of ³²P released.

[ATP]/velocity was plotted against [ATP] (Hanes–Woolf plot), and K_m and V_max were estimated from the intercept and the slope, respectively. The values obtained from all mutants were compared to those of wild-type VCP.

Amino acid sequence alignment

Amino acid sequences were obtained from UniProt or Flybase. Database entries used are as follows: Homo sapiens [P55072], Gallus gallus [Q5ZMU9], Xenopus laevis [P23787], Drosophila melanogaster [FBpp0087479], Caenorhabditis elegans [P54812], Arabidopsis thaliana [P54609], Saccharomyces cerevisiae [P25694], Schizosaccharomyces pombe [Q9P3A7], Thermoplasma acidophilum (VAT) [O05209], Homo sapiens (NSF) [P46459], Saccharomyces cerevisiae (SEC18) [P18759], Schizosaccharomyces pombe (SEC18) [Q9P7Q4]. Multiple alignment was carried out by CLUSTAL W <http://align.genome.jp>.

Statistical analysis

Each experiment was conducted at least three times with consistent results. The representative gel or blot from each experiment is presented in this study. Mean values and standard deviations were obtained from triplicate experiments.

View larger version (28K): [in this window] [in a new window]   Figure 4  Detected modification sites in the N-terminal region of VCP. The amino acid sequences of Na (amino acids residues from 1 to 110) and Nb (amino acids residues from 111 to 185) domains from human VCP and its orthologues are aligned. The results of LC/MS/MS analyses are displayed above the alignment. The residue numbers colored in red indicate detected modified amino acids by the analysis, and the corresponding amino acid residues are also colored in red. The frequencies of the detected times are shown under the residue numbers. The region colored in pale blue indicates the peptide that had never been detected in all experiments, probably because of the less ionized property of the peptides.

	Acknowledgements

	Footnotes

 
Communicated by: Eisuke Nishida

^aPresent address: Department of Degenerative Neurological Diseases, National Institute of Neuroscience, National Center of Neurology and Psychiatry, 4-1-1 Ogawahigashi, Kodaira, Tokyo 187-8502, Japan.

^bPresent address: Department of Pathology and Tumor Biology, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan.

^* Correspondence: kakizuka{at}lif.kyoto-u.ac.jp

	References

Top Abstract Introduction Results Discussion Experimental procedures References

 
Boeddrich, A., Gaumer, S., Haacke, A., et al. (2006) An arginine/lysine-rich motif is crucial for VCP/p97-mediated modulation of ataxin-3 fibrillogenesis. EMBO J. 25, 1547–1558.[CrossRef][Medline]

Boyault, C., Gilquin, B., Zhang, Y., Rybin, V., Garman, E., Meyer-Klaucke, W., Matthias, P., Muller, C.W. & Khochbin, S. (2006) HDAC6-p97/VCP controlled polyubiquitin chain turnover. EMBO J. 25, 3357–3366.[CrossRef][Medline]

Briggs, L.C., Baldwin, G.S., Miyata, N., Kondo, H., Zhang, X. & Freemont, P.S. (2008) Analysis of nucleotide binding to P97 reveals the properties of a tandem AAA hexameric ATPase. J. Biol. Chem. 283, 13745–13752.[Abstract/Free Full Text]

Brunger, A.T. & DeLaBarre, B. (2003) NSF and p97/VCP: similar at first, different at last. FEBS Lett. 555, 126–133.[CrossRef][Medline]

Cao, K., Nakajima, R., Meyer, H.H. & Zheng, Y. (2003) The AAA-ATPase Cdc48/p97 regulates spindle disassembly at the end of mitosis. Cell 115, 355–367.[CrossRef][Medline]

Chevalier, M., King, L. & Blond, S. (1998) Purification and properties of BiP. Methods Enzymol. 290, 384–409.[Medline]

Dalal, S. & Hanson, P.I. (2001) Membrane traffic: what drives the AAA motor? Cell 104, 5–8.[CrossRef][Medline]

DeLaBarre, B. & Brunger, A.T. (2003) Complete structure of p97/valosin-containing protein reveals communication between nucleotide domains. Nat. Struct. Biol. 10, 856–863.[CrossRef][Medline]

Egerton, M. & Samelson, L.E. (1994) Biochemical characterization of valosin-containing protein, a protein tyrosine kinase substrate in hematopoietic cells. J. Biol. Chem. 269, 11435–11441.[Abstract/Free Full Text]

Eng, J.K., McCormack, A.L. & Yates, J.R. (1994) An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database. J. Am. Soc. Mass Spectrom. 5, 976–989.[CrossRef]

Forman, M.S., Mackenzie, I.R., Cairns, N.J., et al. (2006) Novel ubiquitin neuropathology in frontotemporal dementia with valosin-containing protein gene mutations. J. Neuropathol. Exp. Neurol. 65, 571–581.[Medline]

Haubenberger, D., Bittner, R.E., Rauch-Shorny, S., Zimprich, F., Mannhalter, C., Wagner, L., Mineva, I., Vass, K., Auff, E. & Zimprich, A. (2005) Inclusion body myopathy and Paget disease is linked to a novel mutation in the VCP gene. Neurology 65, 1304–1305.[Abstract/Free Full Text]

Higashiyama, H., Hirose, F., Yamaguchi, M., Inoue, Y.H., Fujikake, N., Matsukage, A. & Kakizuka, A. (2002) Identification of ter94, Drosophila VCP, as a modulator of polyglutamine-induced neurodegeneration. Cell Death Differ. 9, 264–273.[CrossRef][Medline]

Hirabayashi, M., Inoue, K., Tanaka, K., Nakadate, K., Ohsawa, Y., Kamei, Y., Popiel, A.H., Sinohara, A., Iwamatsu, A., Kimura, Y., Uchiyama, Y., Hori, S. & Kakizuka, A. (2001) VCP/p97 in abnormal protein aggregates, cytoplasmic vacuoles, and cell death, phenotypes relevant to neurodegeneration. Cell Death Differ. 8, 977–984.[CrossRef][Medline]

Hubbert, C., Guardiola, A., Shao, R., Kawaguchi, Y., Ito, A., Nixon, A., Yoshida, M., Wang, X.F. & Yao, T.P. (2002) HDAC6 is a microtubule-associated deacetylase. Nature 417, 455–458.[CrossRef][Medline]

Kawaguchi, Y., Okamoto, T., Taniwaki, M., et al. (1994) CAG expansions in a novel gene for Machado–Joseph disease at chromosome 14q32.1. Nat. Genet. 8, 221–228.[CrossRef][Medline]

Klein, J.B., Barati, M.T., Wu, R., Gozal, D., Sachleben, L.R. Jr, Kausar, H., Trent, J.O., Gozal, E. & Rane, M.J. (2005) Akt-mediated valosin-containing protein 97 phosphorylation regulates its association with ubiquitinated proteins. J. Biol. Chem. 280, 31870–31881.[Abstract/Free Full Text]

Kobayashi, T. & Kakizuka, A. (2003) Molecular analyses of Machado–Joseph disease. Cytogenet. Genome. Res. 100, 261–275.[CrossRef][Medline]

Kobayashi, T., Manno, A. & Kakizuka, A. (2007) Involvement of valosin-containing protein (VCP)/p97 in the formation and clearance of abnormal protein aggregates. Genes Cells 12, 889–901.[Abstract/Free Full Text]

Kobayashi, T., Tanaka, K., Inoue, K. & Kakizuka, A. (2002) Functional ATPase activity of p97/valosin-containing protein (VCP) is required for the quality control of endoplasmic reticulum in neuronally differentiated mammalian PC12 cells. J. Biol. Chem. 277, 47358–47365.[Abstract/Free Full Text]

Kondo, H., Rabouille, C., Newman, R., Levine, T.P., Pappin, D., Freemont, P. & Warren, G. (1997) p47 is a cofactor for p97-mediated membrane fusion. Nature 388, 75–78.[CrossRef][Medline]

Livingstone, M., Ruan, H., Weiner, J., et al. (2005) Valosin-containing protein phosphorylation at Ser784 in response to DNA damage. Cancer Res. 65, 7533–7540.[Abstract/Free Full Text]

Madeo, F., Schlauer, J., Zischka, H., Mecke, D. & Frohlich, K.U. (1998) Tyrosine phosphorylation regulates cell cycle-dependent nuclear localization of Cdc48p. Mol. Biol. Cell 9, 131–141.[Abstract/Free Full Text]

Matsuyama, A., Shimazu, T., Sumida, Y., Saito, A., Yoshimatsu, Y., Seigneurin-Berny, D., Osada, H., Komatsu, Y., Nishino, N., Khochbin, S., Horinouchi, S. & Yoshida, M. (2002) In vivo destabilization of dynamic microtubules by HDAC6-mediated deacetylation. EMBO J. 21, 6820–6831.[CrossRef][Medline]

Meyer, H.H., Shorter, J.G., Seemann, J., Pappin, D. & Warren, G. (2000) A complex of mammalian ufd1 and npl4 links the AAA-ATPase, p97, to ubiquitin and nuclear transport pathways. EMBO J. 19, 2181–2192.[CrossRef][Medline]

Mizuno, Y., Hori, S., Kakizuka, A. & Okamoto, K. (2003) Vacuole-creating protein in neurodegenerative diseases in humans. Neurosci. Lett. 343, 77–80.[CrossRef][Medline]

Noguchi, M., Takata, T., Kimura, Y., Manno, A., Murakami, K., Koike, M., Ohizumi, H., Hori, S. & Kakizuka, A. (2005) ATPase activity of p97/valosin-containing protein is regulated by oxidative modification of the evolutionally conserved cysteine 522 residue in Walker A motif. J. Biol. Chem. 280, 41332–41341.[Abstract/Free Full Text]

Rabinovich, E., Kerem, A., Frohlich, K.U., Diamant, N. & Bar-Nun, S. (2002) AAA-ATPase p97/Cdc48p, a cytosolic chaperone required for endoplasmic reticulum-associated protein degradation. Mol. Cell. Biol. 22, 626–634.[Abstract/Free Full Text]

Rabouille, C., Levine, T.P., Peters, J.M. & Warren, G. (1995) An NSF-like ATPase, p97, and NSF mediate cisternal regrowth from mitotic Golgi fragments. Cell 82, 905–914.[CrossRef][Medline]

Rush, J., Moritz, A., Lee, K.A., Guo, A., Goss, V.L., Spek, E.J., Zhang, H., Zha, X.M., Polakiewicz, R.D. & Comb, M.J. (2005) Immunoaffinity profiling of tyrosine phosphorylation in cancer cells. Nat. Biotechnol. 23, 94–101.[CrossRef][Medline]

Seigneurin-Berny, D., Verdel, A., Curtet, S., Lemercier, C., Garin, J., Rousseaux, S. & Khochbin, S. (2001) Identification of components of the murine histone deacetylase 6 complex: link between acetylation and ubiquitination signaling pathways. Mol. Cell. Biol. 21, 8035–8044.[Abstract/Free Full Text]

Tokuda, H., Yamanaka, M. & Mizushima, S. (1993) Histidine residues are involved in translocation-coupled ATP hydrolysis by the Sec-A protein. Biochem. Biophys. Res. Commun. 195, 1415–1421.[CrossRef][Medline]

Verdin, E., Dequiedt, F. & Kasler, H.G. (2003) Class II histone deacetylases: versatile regulators. Trends Genet. 19, 286–293.[CrossRef][Medline]

Villen, J., Beausoleil, S.A., Gerber, S.A. & Gygi, S.P. (2007) Large-scale phosphorylation analysis of mouse liver. Proc. Natl. Acad. Sci. USA 104, 1488–1493.[Abstract/Free Full Text]

Wang, Q., Song, C. & Li, C.-C.H. (2003) Hexamerization of p97-VCP is promoted by ATP binding to the D1 domain and required for ATPase and biological activities. Biochem. Biophys. Res. Commun. 300, 253–260.[CrossRef][Medline]

Wang, Q., Song, C. & Li, C.-C.H. (2004) Molecular perspectives on p97-VCP: progress in understanding its structure and diverse biological functions. J. Struct. Biol. 146, 44–57.[CrossRef][Medline]

Watts, G.D., Wymer, J., Kovach, M.J., Mehta, S.G., Mumm, S., Darvish, D., Pestronk, A., Whyte, M.P. & Kimonis, V.E. (2004) Inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia is caused by mutant valosin-containing protein. Nat. Genet. 36, 377–381.[CrossRef][Medline]

Ye, Y., Meyer, H.H. & Rapoport, T.A. (2001) The AAA ATPase Cdc48/p97 and its partners transport proteins from the ER into the cytosol. Nature 414, 652–656.[CrossRef][Medline]

Zhang, Y., Li, N., Caron, C., Matthias, G., Hess, D., Khochbin, S. & Matthias, P. (2003) HDAC-6 interacts with and deacetylates tubulin and microtubules in vivo. EMBO J. 22, 1168–1179.[CrossRef][Medline]

Zhang, S.H., Liu, J., Kobayashi, R. & Tonks, N.K. (1999) Identification of the cell cycle regulator VCP (p97/CDC48) as a substrate of the band 4.1-related protein-tyrosine phosphatase PTPH1. J. Biol. Chem. 274, 17806–17812.[Abstract/Free Full Text]

Zhao, G., Zhou, X., Wang, L., Li, G., Schindelin, H. & Lennarz, W.J. (2007) Studies on peptide: N-glycanase-p97 interaction suggest that p97 phosphorylation modulates endoplasmic reticulum-associated degradation. Proc. Natl. Acad. Sci. USA 104, 8785–8790.[Abstract/Free Full Text]

Accepted: 14 January 2009

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Mori-Konya, C. Articles by Kakizuka, A. Search for Related Content PubMed Articles by Mori-Konya, C. Articles by Kakizuka, A.