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Originally published In Press as doi:10.1074/jbc.M501802200 on July 18, 2005

J. Biol. Chem., Vol. 280, Issue 36, 31870-31881, September 9, 2005
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Akt-mediated Valosin-containing Protein 97 Phosphorylation Regulates Its Association with Ubiquitinated Proteins*

Jon B. Klein{ddagger}§¶||**, Michelle T. Barati{ddagger}, Rui Wu¶, David Gozal{ddagger}{ddagger}§§, Leroy R. Sachleben, Jr.{ddagger}{ddagger}, Hina Kausar¶, John O. Trent§**, Evelyne Gozal{ddagger}{ddagger}§§, and Madhavi J. Rane§¶¶

From the {ddagger}Core Proteomics Laboratory, the §Department of Biochemistry and Molecular Biology, and the Department of Medicine, University of Louisville, Louisville, Kentucky 40202, the ||Veterans Affairs Medical Center, Louisville, Kentucky 40206, and the {ddagger}{ddagger}Department of Pediatrics, the §§Department of Pharmacology, and the **James Graham Brown Cancer Center, University of Louisville, Louisville, Kentucky 40202

Received for publication, February 17, 2005 , and in revised form, July 15, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Hypoxia is a common environmental stress that influences signaling pathways and cell function. Previous studies from our laboratory have identified significant differences in cellular responses to sustained or intermittent hypoxia with the latter proving more cytotoxic. We hypothesized that differences in susceptibility of neurons to intermittent (IH) and sustained hypoxia (SH) are mediated by altered Akt signaling. SH, but not IH, induced a significant increase in Akt activation in rat CA1 hippocampal region extracts compared with room air controls. Akt immunoprecipitations followed by proteomic analysis identified valosin-containing protein (VCP) as an Akt-binding protein. In addition, VCP expression and association with Akt was enhanced during SH, and this association was decreased upon phosphoinositide 3-kinase/Akt pathway blockade with LY294002. Active recombinant Akt phosphorylated recombinant VCP in vitro. Site-directed mutagenesis studies identified Ser352, Ser746, and Ser748 as Akt phosphorylation sites on VCP. In addition, rat CA1 hippocampal tissue exposed to SH exhibited an acidic pI shift of VCP. Protein phosphatase 2A treatment inhibited this acidic shift consistent with SH-induced phosphorylation of VCP in vivo. PC-12 cells transfected with active Akt, but not dominant negative Akt or vector, induced VCP expression and an acidic shift in VCP pI, which was inhibited by protein phosphatase 2A treatment. Furthermore, VCP association with ubiquitinated proteins was demonstrated in vector-transfected PC-12 cell lysates, whereas active Akt-transfected cells demonstrated a marked decrease in association of VCP with ubiquitinated proteins. We concluded that Akt phosphorylates VCP in vitro and in vivo, and VCP phosphorylation releases it from ubiquitinated substrate protein(s) possibly allowing ubiquitinated protein(s) to be degraded by the proteosome.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Most mammalian neurons are sensitive to hypoxia to a varying degree. However, the mechanisms underlying this heterogeneous sensitivity are not well understood. Heterotopic differences in hypoxic sensitivity are now well established in the brain during conditions of oxygen deprivation such as hypoxic ischemia and the oxidant stress that follows reperfusion or malonate-induced mitochondrial metabolic disruption (1-4). Previous studies from our laboratory have identified significant differences in the hypoxic susceptibility of the CA1 and CA3 hippocampal regions, with CA1 vulnerability being particularly prominent in response to intermittent mild hypoxic episodes (5-7). These findings suggest that the CA3 region is relatively resistant to hypoxia when compared with the CA1 region, and CA1 regional vulnerability to hypoxia varies depending on the mode, severity, and duration of the hypoxic insult (8-10). The mechanisms underlying the greater susceptibility of the CA1 region to intermittent hypoxia (IH)1 than to sustained hypoxia (SH) are unknown. We have recently shown in PC-12 cells that different signaling pathways are involved in sustained and intermittent hypoxia-induced cell death. Identifying these pathways may contribute to our understanding of differential brain susceptibility to sustained and intermittent hypoxia (11). In the present study, we found that in rat CA1, SH but not IH induces significant increase in Akt activation compared with room air (RA) control, suggesting that differences in Akt signaling maybe implicated in the differences in hypoxia sensitivity in IH versus SH. Proteomic analysis of Akt immunoprecipitates from RA, SH, and IH CA1 samples was performed to identify differences in the assembly of Akt-binding partners among different conditions. These studies demonstrated that SH but not IH increased expression and association of endoplasmic reticulum (ER)-proteasome interacting protein, velosin-containing protein 97 (VCP) with Akt, compared with RA-exposed animals. Additionally, VCP was identified as an Akt substrate in vitro and in vivo thereby potentially implicating the VCP-Akt complex in the regulation of protein quality control during SH.

VCP is a member of the AAA (ATPases associated with various cellular activities) family and is divided into N (residues 1-187), D1 (residues 209-460), D2 (residues 481-761), and C (residues 742-806) domains, and two linkers, N-D1 linker (residues 188-208) and D1-D2 linker (residues 461-480). The N domain is involved in substrate and cofactor binding; D1 is required for oligomerization, and both D1 and D2 domains contribute to the ATPase activity. Intact ATPase activity is required for various biological functions of VCP. VCP is involved in a variety of cellular processes, including altering morphology of nuclear and Golgi membranes, transcriptional regulation, membrane fusion, cell cycle control, stress response, programmed cell death, B and T cell activation, protein degradation, ER-associated degradation, and the dislocation of ubiquitinated proteins from the ER (12-23). The ubiquitin-proteasome system is responsible for the constitutive degradation of most cellular proteins (24-26). The ubiquitin-proteasome system ubiquitinates its substrates and targets them for degradation by the 26 S proteasome. VCP plays a role in mediating ER-associated protein degradation by interacting with potential proteasome substrates before being degraded by the proteasome. VCP has been shown to associate physically with ubiquitinated I{kappa}B{alpha} and the 26 S proteasome and to target I{kappa}B{alpha} to the proteasome for degradation (15, 27). VCP has been shown to associate with a number of proteins, including Duf, UIP-5, Ataxin-3, 26 S proteosome, GP78, dorfin, PI4K II, clathrin, and others (15, 28-34). Several lines of evidence suggest that binding of alternative cofactors determines the specific role of VCP. Thus far, a number of VCP cofactors have been identified and regulate various VCP-mediated functions (35-39). Here we identify VCP as an Akt-binding protein and as an Akt substrate, suggesting a role for Akt in the regulation of VCP function. Tyrosine phosphorylation of VCP has been documented previously and suggested to play a role in membrane fusion and presentation of polyubiquitinated proteins to the proteosome and in the nuclear localization of VCP during the late G1 phase in Saccharomyces cerevisiae (13, 27, 40). VCP tyrosine phosphorylation has also been shown to regulate cell proliferation and apoptosis, suggesting it to be a pro-proliferation or anti-apoptotic protein (41). Moreover, VCP tyrosine dephosphorylation has been shown to destabilize VCP/ER membrane association thereby promoting ER transitional assembly (42).

VCP has been shown to target proteins to the 26 S proteosome. It is unclear if VCP further modifies ubiquitinated proteins, but it is known that once these proteins are targeted at the proteosome, VCP dissociates from ubiquitinated proteins. The exact mechanism of VCP release from ubiquitinated protein is unclear. Our current data demonstrate that VCP Ser/Thr phosphorylation releases it from ubiquitinated substrate protein(s) possibly allowing ubiquitinated protein(s) to be degraded by the proteosome.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Active recombinant Akt was obtained from Upstate%20Biotechnology">Upstate Biotechnology Inc. (Lake Placid, NY). Anti-phospho-Ser473-Akt, anti-pleckstrin homology domain Akt, and anti-Akt antisera were obtained from Cell Signaling Inc (Beverly, MA). Mouse isotype control and VCP antisera were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Protein A-Sepharose was obtained from Pharmingen. Histone H2B was obtained from Roche Applied Science. The synthetic AKTide-2T inhibitory peptide (ARKRERTYSFGHHA) and scrambled peptide (HAKEAYGHARRPRA) were obtained from the Macromolecular Structure Analysis Facility at the University of Kentucky (Lexington, KY).

Animals—Animal experimental protocols were approved by the University of Louisville Institutional Animal Care and Use Committee and are in agreement with the National Institutes of Health guide for the care and use of laboratory animals. Adult male Sprague-Dawley rats (weights175-225 g) were purchased from Charles River Breeding Laboratories (Wilmington, MA).

Hypoxic Exposures—Hypoxic exposures were conducted in four identical commercially designed chambers (30 x 20 x 20 inches) that can accommodate up to 12 rats each and were operated under a 12-h light-dark cycle (Oxycycler model A44XO, Biospherix, Redfield, NY). Ambient CO2 in the chamber was constantly monitored and maintained at <0.03% by servo-controlled adjustments in overall chamber ventilation, and humidity was maintained at <40%. Animals were exposed to either room air (RA), SH (8% O2), or IH (8% O2 to 21% O2 alternating every 6 min).

CA1 Sample Preparation—Animals were anesthetized with pentobarbital (intraperitoneal 50 mg/kg) and decapitated. Brains were dissected on ice to retrieve the hippocampal formation bilaterally, and the CA1 and CA3 regions of the hippocampus were then dissected as described previously (7, 43). Tissues corresponding to the CA1 region harvested from either normoxic or hypoxic animals were homogenized on ice with a tissue tearer in 300 µl of sample buffer (58 mM dithiothreitol, 65 mM CHAPS, 7 M urea, 1.9 M thiourea, 1.75% pH 3-10 carrier ampholytes). The mixture was then mixed for 1 h and centrifuged at 18,000 x g for 10 min. Protein concentration was determined in the soluble supernatant, using the RC-DC-Bio-Rad protein assay (Bio-Rad) to avoid interference of ampholytes and reducing agents present in the buffer, with accurate protein determination. In addition, an equal amount of proteins from each sample was separated on a Tris/glycine SDS-polyacrylamide gel and stained with Coomassie Blue staining to confirm visually the accuracy of the assay.

PC-12 Cell Culture and Transfection—PC-12 cells were cultured on collagen-coated plates in RPMI 1640 medium (Invitrogen) supplemented with 1% antibiotic/antimycotic (Invitrogen), 10% fetal calf serum (Hyclone), and 5% heat-inactivated (30 min at 56 °C) horse serum (Hyclone). Cells were exposed normoxia (21% O2, 5% CO2, balanced N2) or to a defined profile of sustained hypoxia (0.1% O2, 5% CO2, balanced N2), using a custom-designed computer-controlled incubator chamber attached to an external O2/CO2 computer-driven controller (Biospherix, Redfield, NY), for 6 h unless otherwise stated. Chamber oxygen, nitrogen, and carbon dioxide levels were continuously monitored and adjusted according to the programmed profile. Media oxygen content was monitored with a fiberoptics oxygen sensor (Ocean Optics, Dunedin, FL). Oxygen levels in the air-phase as well as in the media were monitored and recorded as described previously (11).

Transient Transfection of PC-12 Cells—PC-12 cells were plated on collagen-coated 6-well trays each day prior to performing transfections, to reach 60% confluence. On the day of transfection, PC-12 cells were washed with serum-free RPMI 1640 medium. One µg of appropriate cDNA was transfected into PC-12 cells using geneporter reagent according to the manufacturer's protocol (Gene Therapy Systems). Twenty-four h after transfection, PC-12 cells were lysed in 100 µl of Akt lysis buffer containing 20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100 (v/v), 0.5% Nonidet P-40 (v/v), 1 mM EDTA, 1 mM EGTA, 20 mM sodium orthovanadate, 10 µM p-nitrophenol phosphate, 20 mM NaF, 5 mM PMSF, 21 µg/ml aprotinin, and 5 µg/ml leupeptin, and proteins concentrations were determined. Protein lysates (100 µg) were diluted to 160 µl in urea/thiourea rehydration buffer (Genomic Solutions) and subjected to two-dimensional PAGE as described above. Alternatively, protein lysates (50 µg) were subjected to one-dimensional PAGE and immunoblot analysis.

Akt Immunoprecipitation Assays—Tissue lysates were prepared in Akt lysis buffer containing 20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100 (v/v), 0.5% Nonidet P-40 (v/v), 1 mM EDTA, 1 mM EGTA, 20 mM sodium orthovanadate, 10 µM p-nitrophenol phosphate, 20 mM NaF, 5 mM PMSF, 21 µg/ml aprotinin, and 5 µg/ml leupeptin. Following centrifugation at 15,000 x g for 15 min at 4 °C, cleared lysates were incubated with 20 µl of anti-Akt pleckstrin homology domain agarose beads or with mouse isotype control antibody beads as described previously (44). Immunoprecipitated proteins were eluted with 160 µl of urea/thiourea buffer (Genomics Solutions) and subjected to two-dimensional PAGE followed by MALDI-MS analysis as described previously (7). Alternatively, immunoprecipitated proteins were eluted with 40 µl of 2x Laemmli dye. Samples were boiled for 2 min; beads were precipitated by a quick spin in a picofuge, and 40 µl supernatant containing eluted proteins was subjected to one-dimensional PAGE and immunoblot analysis with anti-VCP antibody (1:200 dilution).

VCP Immunoprecipitation Assay—Transfected PC-12 lysates were prepared in Akt lysis buffer containing 20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100 (v/v), 0.5% Nonidet P-40 (v/v), 1 mM EDTA, 1 mM EGTA, 20 mM sodium orthovanadate, 10 µM p-nitrophenol phosphate, 20 mM NaF, 5 mM PMSF, 21 µg/ml aprotinin, and 5 µg/ml leupeptin. Following centrifugation at 15,000 x g for 15 min at 4 °C, cleared lysates (40 µg) were incubated with 1 µg of rabbit anti-goat VCP antibody or with 1 µg of goat isotype control antibody overnight at 4 °C with shaking as described previously (44). The next day protein A-Sepharose beads (20 µl) were added to the lysates and incubated further at 4 °C with shaking. Antibody-conjugated protein A-Sepharose beads were washed three times, and immunoprecipitated proteins were eluted with 40 µl of 2x Laemmli dye. Samples were boiled for 2 min; beads were precipitated by a quick spin in a picofuge, and 40 µl of supernatant containing eluted proteins was subjected to SDS-PAGE and immunoblot analysis with anti-ubiquitin antibody (1:1000 dilution).

Immunoblot Analysis—Immunoblotting procedures of immunoprecipitates were performed as described previously (44). PC-12 cells were pretreated with 50 µM LY294002, a PI3K inhibitor for 1 h prior to subjecting cells to sustained hypoxia (0.1% O2 for 6 h) or RA treatments. After the treatments, PC-12 cells were lysed with 100 µl of Akt lysis buffer. Lysates were subjected to protein determination (DC, Bio-Rad) and 50 µg of protein was subjected to 10% SDS-PAGE and immunoblot analysis with anti-pAktSer473 (1:1000, Santa Cruz Biotechnology), anti-Akt (1:1000, Santa Cruz Biotechnology), and anti-actin antibody as described previously (44).

Subcloning of VCP into pRSET and Generation of Recombinant VCP—VCP was excised from pCMX-VCP (kind gift from Dr. Akira Kakizuka) with restriction enzyme KpnI and ligated into KpnI site of pRSET-B (Invitrogen) vector. HindIII restriction digestion was performed to confirm 5' to 3' orientation of the gene. Positive clone was confirmed by DNA sequencing. Recombinant VCP protein was expressed and isolated from BL21pLysSDE3 strain of Escherichia coli as described previously (44).

Site-directed Mutagenesis of VCP—VCP serine 352 mutation to alanine (S352A), S746A, S748A, and a double mutant S746A/S748A were carried out using the transformer site-directed mutagenesis kit from BD Biosciences according to manufacturer's instructions. The mutant primers used to generate the mutants were as follows: S352A, 5'-GACCCAACGCCATTGACCC-3'; S746A, 5'-GCCCGACGTGCTGTCAGCG-3'; S748A, 5'-CGACGTTCTGTCGACGATAATGAC-3'; S746A/S748A, 5'-CGACGTGCTGTCGCCGATAATG-3', and the selection primer for pRSETB-VCP (mutating the BglII site and creating a new PvuI site) was 5'-GAGCTCGCGATCGGCAGCTG-3'. Cloning and mutation were confirmed by DNA sequencing. Expression of pRSET-VCP, pRSET-VCPS352A, pRSET-VCPS746A, pRSET-VCPS748A, and pRSET-VCPS746A/S748A plasmids was carried out in BL21(DE3)pLysS chemically competent E. coli cells, and protein was purified using the ProBond Purification System (Invitrogen).

Recombinant Active Akt Kinase Phosphorylation of VCP—Active recombinant Akt (400 ng) was incubated in 30 µl of reaction mixture containing 20 µM ATP, 5 µCi of [{gamma}-32P]ATP, 5 or 10 µg of recombinant VCP and Akt kinase buffer (20 mM HEPES, 10 mM MgCl2, 10 mM MnCl2) and in the presence and absence of 200 µg of scrambled peptide or AKTide-2T, a competitive inhibitor of Akt (44). The reaction was carried out at room temperature for 2 h and terminated by adding 6 µl of 6x Laemmli buffer. Proteins were resolved by SDS-PAGE, and gels were imaged by autoradiography. The gels were also stained with colloidal Coomassie Blue dye (Genomics Solutions) to ensure equal loading of proteins used in the assay.

Phosphorylation of Wild Type and Mutant VCP Proteins by Active Recombinant Akt—Active recombinant Akt (400 ng) was incubated in 30 µl of reaction mixture containing 20 µM ATP, 5 µCi of [{gamma}-32P]ATP, 10 µg of recombinant VCP wild type or various VCP mutants, and Akt kinase buffer (20 mM HEPES, 10 mM MgCl2, 10 mM MnCl2). The reaction was carried out at room temperature for 2 h and terminated by adding 6 µl of 6x Laemmli buffer. Proteins were resolved by SDS-PAGE, and gels were imaged by autoradiography. The gels were also stained with colloidal Coomassie Blue dye (Genomics Solutions) to ensure equal loading of proteins used in the assay.

Akt Kinase Assay from RA, SH, and IH CA1 Tissue Lysates—RA, SH, and IH CA1 tissue lysates were subjected to anti-Akt immunoprecipitation. Immunoprecipitated Akt was subjected to an in vitro Akt kinase assay using histone H2B as substrate as described previously (44).

GST-Akt Pull-down Assay with Recombinant VCP—Recombinant VCP protein (150 ng) was added to 20 µl of GST or 20 µl of GST-Akt-Sepharose in the presence of 50 µl of Akt kinase buffer. The beads were incubated at 4 °C for 3 h with rotation. The beads were briefly spun down in a picofuge to precipitate the beads. Supernatant was discarded. The beads were washed three times with 100 µl of Akt kinase buffer, and VCP protein was eluted by adding 40 µl of 2x Laemmli dye. The samples were boiled for a minute and spun down briefly in a picofuge to precipitate the beads. Supernatants were subjected to 10% SDS-PAGE and immunoblotted with rabbit anti-goat VCP (1:200).

Two-dimensional PAGE—RA, SH, and IH CA1 lysates were subjected to isotype control antibody or anti-Akt immunoprecipitation, and the antibody conjugated beads were washed with Krebs+, and proteins were eluted by either adding 40 µl of 2x Laemmli dye or by adding 155 µl of urea/thiourea rehydration buffer (Genomic Solutions). Proteins eluted with 2x Laemmli dye were subjected to one-dimensional PAGE, whereas proteins eluted with urea/thiourea rehydration buffer were separated by two-dimensional PAGE. For two-dimensional PAGE IPG strips, pH 3-10 (Invitrogen), were rehydrated overnight with protein samples. Proteins were separated by isoelectric focusing using the ZOOM IPG Runner (Invitrogen) with a maximal voltage of 2000 V and 50 µA per gel. Following isoelectric focusing, IPG strips were incubated twice in equilibration buffer I (6 M urea, 130 mM dithiothreitol, 30% glycerol, 45 mM Tris base, 1.6% SDS, 0.002% bromphenol blue; Genomic Solutions) and once in equilibration buffer II (6 M urea, 135 mM iodoacetamide, 30% glycerol, 45 mM Tris base, 1.6% SDS, 0.002% bromphenol blue; Genomic Solutions) for 10 min. Equilibrated IPG strips were applied to 4-12% BisTris gradient gels (Invitrogen), and proteins were separated in the second dimension using NuPAGE MES/SDS buffer (Invitrogen) at 200 V for 40 min. Following electrophoresis, gels were fixed in 40% methanol and 10% acetic acid for 30 min and stained with colloidal Coomassie Blue (Genomic Solutions).

Trypsin Digestion and Mass Spectrometry—Stained protein spots from Coomassie Blue-stained gels were excised from gels and subjected to in-gel trypsin digestion as described in detail by Thongboonkerd et al. (45). Trypsin-generated peptides were applied by a thin film-spotting procedure for MALDI-MS analysis using {alpha}-cyanohydroxycinnamic acid as the matrix on stainless steel targets, as described previously (45). Mass spectral data were obtained using a TOF-Spec 2E (Micromass) and a 337-nm N2 laser at 20-35% power in the reflector mode. Spectral data were obtained by averaging 10 spectra, each of which was the composite of 10 laser firings. Mass axis calibrations were accomplished using peaks from tryptic auto-hydrolysis. Peptide masses obtained by MALDI-MS were analyzed using the Mascot search engine by comparison to the NCBI protein data base. A probability-based MOWSE (MOlecular Weight SEarch) score greater than 74 was assumed to indicate a significant match that was not a random event.

PP2A Treatments—For PP2A treatments appropriate CA1 tissue lysate (100 µg) or transfected PC-12 lysates (100 µg) were incubated with 5 µl of recombinant active PP2A (0.1 units/µl, Upstate%20Biotechnology">Upstate Biotechnology, Inc.) at 30 °C for 1 h as described previously (46). The samples were diluted in urea/thiourea rehydration buffer (Genomics Solutions) to a final volume of 155 µl. These lysates were subjected to two-dimensional PAGE as described above.

Molecular Modeling—The crystal structure of activated Akt (Protein Data Bank entry 1O6L [PDB] ) was docked with the crystal structure of VCP (Protein Data Bank entry 1OZ4 [PDB] ), using GRAMM software (47-49). The 100 generated docked structures were evaluated for post-translational modification sites and close contacts of Akt with VCP. Identification of potential Akt recognition sites on VCP was performed using the scansite tool (scansite.mit.edu). Scansite is a peptide library-based searching algorithm, accessible over the internet that identifies sequence motifs (50). GRAMM analysis was performed as described previously (51).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Hypoxia Induces Akt Phosphorylation and Activation in CA1 Region of the Brain—To determine whether Akt is differentially activated in CA1 by SH and IH, Sprague-Dawley rats were exposed to RA, IH, and SH for 6 h followed by dissection of the CA1 region of the brain and generation of tissue lysates. CA1 tissue lysates were subjected to one-dimensional or two-dimensional PAGE immunoblot analysis with anti-Akt and anti-Ser(P)473Akt antibody. We selected the 6-h time point for hypoxia treatments, as we have previously shown that 6-h IH regulated proteins in the CA1 region, including structural proteins, proteins related to apoptosis, chaperone proteins, and proteins involved in the cellular metabolic pathways (7). As shown in Fig. 1A, immunoblotting of proteins separated by one-dimensional PAGE demonstrated that SH induced marked increase in Ser(P)473-Akt levels, compared with RA control, without altering total Akt expression, whereas IH induced a modest increase. Concurring with the results obtained by one-dimensional PAGE analysis, two-dimensional PAGE Ser(P)473-Akt immunoblot analysis demonstrated an acidic pI shift in SH, and to a lesser extent in IH as compared with the RA sample, suggestive of Akt phosphorylation. Furthermore, in vitro Akt kinase activity confirmed our immunoblot findings, with significantly increased Akt kinase activity in SH but not in IH (p < 0.05, n = 3). In addition, the difference in SH-versus IH-induced Akt kinase activity was found to be statistically significant (p < 0.05, n = 3). Thus, Akt kinase activity correlated with the changes observed in Akt phosphorylation for each of the experimental conditions. These results suggest that increased resistance of the CA1 region to SH when compared with IH may be attributed in part to SH-induced enhanced Akt kinase activity.



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  		FIG. 1.
Hypoxia induces Akt phosphorylation and activation in CA1 region of the brain. CA1 lysates from Sprague-Dawley rats were exposed to RA, IH (8% O2 to 21% O2 alternating every 6 min), or SH (8% O2) for 6 h and subjected to one-dimensional PAGE and immunoblot (IB) analysis with anti-Akt and anti-phospho-Ser473Akt antibody (n = 3) (A). B, two-dimensional PAGE and immunoblotting with anti-Akt and anti-phospho-Ser473Akt antibody (n = 3). C, CA1 tissue lysates from RA, SH, and IH samples were subjected to anti-Akt immunoprecipitation followed by an in vitro Akt kinase assay using histone H2B as substrate. These results demonstrated that SH but not IH significantly increased Akt kinase activity (p < 0.05, n = 3). Additionally, the difference in SH-versus IH-induced Akt kinase activity was found to be statistically significant (p < 0.05, n = 3).

 
PI3K Pathway Regulates SH-induced Akt Phosphorylation in PC-12 Cells—In vitro kinase and phosphorylation studies in the CA1 demonstrated enhanced Akt phosphorylation and activation during SH compared with IH. To confirm SH-induced Akt phosphorylation observed in the CA1, we sought to examine the effect of SH-induced Akt phosphorylation in PC-12 cells. PC-12 cells were subjected to RA or SH (0.1% O2) for 3, 6, 9, and 24 h, lysed, and subjected to 10% SDS-PAGE and immunoblot analysis with anti-Akt, anti-actin, and anti-Ser(P)473-Akt antibody. These data demonstrated that maximal Akt phosphorylation occurred at 6 h; thus, this time point was selected for all experiments that followed (data not shown). To determine the role of PI3K in SH-induced Akt phosphorylation in PC-12 cells, PC-12 cells were pretreated with LY294002 (50 µM) or with vehicle (Me2SO) for 1 h in RA and then subjected to either RA or to SH for 6 h. Proteins from PC-12 cell lysates were separated by 10% SDS-PAGE and immunoblotted with anti-Ser(P)473-Akt antibody and total anti-Actin antibody as a loading control. Fig. 2A shows that SH significantly increased Akt-Ser473 phosphorylation compared with RA control and that LY294002 treatment reduced both basal and SH-induced Akt-Ser473 phosphorylation (Fig. 2A, p < 0.05, n = 3). A representative blot from three separate experiments demonstrated increased Akt-Ser473 phosphorylation in response to SH, which was inhibited by pretreatment with LY294002 (Fig. 2B, top panel). Equal loading of samples was demonstrated by stripping and reprobing the blot with anti-actin antibody (Fig. 2B, bottom panel). These results indicate that PI3K mediates SH-induced Akt-Ser473 phosphorylation in PC-12 cells.

Akt-binding Proteins in SH-treated CA1 Lysates—The observed differences in Akt phosphorylation and activation in SH versus IH CA1 lysates prompted us to identify potential Akt-binding partners present in SH-treated rat CA1 tissue lysates. To this end, 6-h SH CA1 tissue lysates were subjected to isotype control or anti-Akt immunoprecipitation. Immunoprecipitated proteins were resolved by either one-dimensional or two-dimensional PAGE. Proteins identified in colloidal Coomassie Blue-stained gels were excised and subjected to in-gel trypsin incubation and MALDI-MS. Proteins were identified by peptide mass fingerprinting analysis. Fifty gel spots were subjected to analysis, with 12 gene products identified from one-dimensional gels and 28 gene products from two-dimensional gels. We further performed bioinformatic analysis to help differentiate between proteins within the Akt complex and likely Akt kinase substrates. The amino acid sequences of the putative Akt-binding partners were analyzed for Akt recognition motifs using scansite software (scansite.mit.edu). scansite is a peptide library-based searching algorithm, accessible over the internet that identifies sequence motifs (50, 52). Akt-binding protein bands from SH CA1 lysates identified from one-dimensional PAGE (Fig. 3A) are numbered, and their corresponding identities, NCBI accession numbers, estimated molecular sizes, matched peptides, percent coverage on the protein identified, and number of predicted Akt recognition sequences are shown in Table I, and protein spots identified from two-dimensional PAGE (Fig. 3B) are shown in Table II. A probability-based MOWSE score greater than 74 was assumed to indicate a significant match for all proteins listed unless otherwise stated. Of note, VCP was identified as an Akt-binding partner both in the one-dimensional and two-dimensional PAGE analysis of Akt immunoprecipitates, prompting us to further this association investigate.


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  		TABLE I
MALDI-MS identification of candidate Akt-binding partners from one-dimensional-PAGE

 


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  		TABLE II
MALDI-MS identification of candidate Akt-binding partners from two-dimensional-PAGE

 



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  		FIG. 2.
PI3K pathway regulates SH-induced Akt phosphorylation in PC-12 cells. PC-12 cells pretreated with 50 µM LY294002 (LY) (PI3K inhibitor) or with vehicle (Me2SO) and exposed to RA and SH were subjected to immunoblot (IB) analysis with anti-actin and anti-Akt-Ser473 antisera. A, quantitative analysis of these data demonstrate that SH significantly increased Akt-Ser473 phosphorylation, which was markedly inhibited by pretreatment with PI3K inhibitor LY294002 (p < 0.05, n = 3). B, representative blot from three separate experiments demonstrated increased Akt-Ser473 phosphorylation in response to SH, which was inhibited by pretreatment with LY294002. Equal loading of samples was demonstrated by stripping and reprobing the blot with anti-actin antibody (n = 3).

 
Association of Akt and VCP—By having shown association between Akt/VCP in SH-treated CA1 tissue lysates (Tables I and II), we sought to determine the status of Akt/VCP association in RA and IH lysates compared with SH-treated CA1 lysates. RA, SH, and IH rat CA1 lysates were subjected to isotype control or Akt immunoprecipitation, and proteins were separated by one-dimensional PAGE. Gels were fixed and stained with colloidal Coomassie dye, and a 97-kDa protein was excised from all Akt immunoprecipitates. Excised bands were trypsin-digested and identified by MALDI-MS and peptide mass fingerprinting analysis as VCP. Fig. 4A demonstrates the presence of VCP in Akt immunoprecipitates but not isotype control immunoprecipitates. Additionally, enhanced Akt/VCP association was detected in SH-treated samples compared with RA and IH-treated samples. This enhanced association of Akt/VCP in SH was confirmed by subjecting isotype control or Akt immunoprecipitates from RA and SH CA1 tissue lysates to two-dimensional PAGE and MALDI-MS and peptide mass fingerprinting analysis. Fig. 4B demonstrates the presence of VCP in Akt immunoprecipitates but not isotype control immunoprecipitates. Moreover, enhanced Akt/VCP association was detected during SH treatment, compared with RA control. To determine whether this enhanced Akt/VCP association during SH was dependent on Akt activation, O2-sensitive rat pheochromocytoma (PC-12) cells were pretreated with LY294002 followed by exposure to RA or 6-h SH (a time point of optimal Akt phosphorylation). PC-12 cell lysates were immunoprecipitated with isotype control or anti-Akt antibody and immunoblotted with anti-VCP antibody. Fig. 4C demonstrates that during SH, Akt/VCP association was markedly enhanced compared with RA control and was inhibited by pretreatment with 50 µM LY294002 (PI3K inhibitor). These results suggest a role for Akt activation in the enhanced association of Akt/VCP during SH.

Molecular Modeling Identifies Putative Akt Recognition Motifs and Binding Sites on VCP—To characterize further the potential relationship between Akt and VCP, a key ER stress protein, with a key role in the cellular response to hypoxia (53), molecular modeling of a putative Akt and VCP interaction was performed. As shown in Fig. 5A, the crystal structure of activated Akt (Protein Data Bank entry 1O6L [PDB] ), colored in yellow, was docked with the crystal structure of valosin (Protein Data Bank entry 1OZ4 [PDB] ; colored in cyan) using GRAMM. The 100 generated docked structures were evaluated for post-translational modification sites and close contacts of Akt with valosin. An Akt kinase recognition motif was identified on valosin at residues 739-753, shown in red CPK format in Fig. 5A. Most interestingly, the web-based scansite software identified two residues in this area of VCP, Ser746 as a low stringency Akt site and Ser748 as a medium stringency Akt site. Additionally, scansite identified VCP Ser352 as a third Akt phosphorylation site. Furthermore, points of contact between VCP and Akt include Akt residues 242-249, 344-348, 351, 370, 372-373 and the Akt kinase activity domain encompassing residues 152-409. This was consistent with the association of the valosin recognition site with the Akt kinase domain in the docked complex.



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  		FIG. 3.
Akt-binding proteins in SH-treated CA1 lysates. SH CA1 tissue lysates were subjected to isotype control or anti-Akt immunoprecipitation (IP). A, immunoprecipitated proteins were resolved by one-dimensional PAGE. Akt-binding protein bands from SH CA1 lysates identified from one-dimensional PAGE are numbered. These protein bands were excised from the gel trypsin, digested, and identified by MALDI-MS and peptide mass fingerprinting analysis. The numbered proteins and their corresponding identities, NCBI accession numbers, estimated molecular sizes, and number of predicted Akt recognition sequences are shown in Table I. B, immunoprecipitated proteins were resolved by two-dimensional PAGE. Akt-binding protein spots were identified by MALDI-MS as described above. The numbered proteins and their corresponding identities, NCBI accession numbers, estimated molecular sizes, and number of predicted Akt recognition sequences are shown in Table II.

 
It should be noted that the predictive value of the scansite software is limited and should be used only as an indicator of Akt motifs. A comprehensive review of this topic by Lawlor and Alessi (54) indicates that "low stringency" Akt motifs predicted by scansite are frequently biologically significant, whereas a functional role cannot be demonstrated for some high stringency sites.

Valosin-containing Protein Directly Associates with Akt—To determine whether Akt directly associates with VCP, recombinant VCP was precipitated with GST or GST-Akt-Sepharose and immunoblotted with anti-VCP antibody as described under "Materials and Methods." Fig. 5B demonstrates association of recombinant VCP with GST-Akt but not with GST-Sepharose (Fig. 5B, top panel). The loading control for GST and GST-Akt beads is demonstrated by Coomassie Blue staining of the gel (Fig. 5B, bottom panel). These results demonstrate direct protein-protein interaction between Akt and VCP.



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  		FIG. 4.
SH induces enhanced association of Akt and VCP. CA1 lysates from Sprague-Dawley rats exposed to 6 h of RA, SH, and IH were subjected to isotype control or anti-Akt immunoprecipitation (IP) as described under "Materials and Methods." A, immunoprecipitated proteins were separated by one-dimensional PAGE, and a 97-kDa protein from Akt immunoprecipitates, but not from isotype control immunoprecipitates, was identified as VCP by MALDI-MS analysis. Enhanced association of VCP/Akt was detected in Akt immunoprecipitates from SH-treated CA1 lysates compared with RA- or IH-treated lysates. B, immunoprecipitated proteins were separated by two-dimensional PAGE, and a 97-kDa protein with a pI of around 5.1 from Akt immunoprecipitates, but not from isotype control immunoprecipitates, was identified as VCP by MALDI-MS analysis. Enhanced association of VCP/Akt was detected in Akt immunoprecipitates from SH-treated CA1 lysates compared with RA- or IH-treated lysates. C, PC-12 cells were exposed to RA or 6 h of SH (a time point of optimal Akt phosphorylation) in the presence and absence of a PI3K inhibitor, LY294002 (50 µM). PC-12 cell lysates were subjected to isotype control or Akt immunoprecipitation (IP), followed by immunoblot (IB) analysis with anti-VCP antibody (n = 2).

 
Valosin-containing Protein Is an Akt Substrate—Akt immunoprecipitation, molecular modeling, and bioinformatic analysis with the scansite tool were all consistent with the hypothesis that VCP is an Akt substrate. To test this hypothesis, VCP cDNA was subcloned into a His-tagged pRSET (Invitrogen) vector, and recombinant VCP protein was generated. As shown in Fig. 6A, recombinant VCP protein was immunoblotted with anti-VCP antibody. Recombinant VCP protein (5 or 10 µg) in kinase buffer was subjected to an in vitro kinase assay in the presence or absence of active recombinant Akt (400 ng; Upstate%20Biotechnology">Upstate Biotechnology, Inc.). To determine whether VCP directly interacts with Akt, we performed a GST and GST-Akt pull-down assay. Fig. 6B demonstrates that recombinant VCP directly interacts with GST-Aktwt but not with GST-Sepharose. Active recombinant Akt phosphorylated recombinant VCP in a dose-dependent manner, whereas no VCP phosphorylation was detected in the absence of Akt. To further demonstrate specificity of Akt-mediated VCP phosphorylation, recombinant VCP protein (10 µg) was subjected to an in vitro Akt kinase assay in the presence of a 200 µM scrambled peptide or 200 µM AKTide-2T, which is a competitive inhibitor of Akt (44, 51). Fig. 6C shows that active recombinant Akt phosphorylated VCP in the presence of a scrambled peptide but not in the presence of AKTide-2T. Colloidal Coomassie staining of kinase gels demonstrated that equal amounts of recombinant VCP were added to every condition. These results suggest that VCP is an in vitro Akt substrate.



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  		FIG. 5.
Molecular modeling and in vitro GST pull-down assay demonstrate direct protein-protein interaction between Akt and VCP. A, the crystal structure of activated Akt (Protein Data Bank entry 1O6L [PDB] ), colored in yellow, was docked with the crystal structure of valosin (Protein Data Bank entry 1OZ4 [PDB] ), and colored in cyan, using GRAMM. The 100 generated docked structures were evaluated for post-translational modification sites and close contacts of Akt with valosin. Molecular modeling along with scansite analysis predicted Akt/VCP-binding sites on each other and Akt phosphorylation sites on VCP. B, GST or GST-Akt-Sepharose beads were subjected to a GST pull-down assay with recombinant VCP. Precipitated protein was separated by 10% SDS-PAGE and immunoblotted with anti-VCP antibody as described under "Materials and Methods" (n = 3). Results demonstrate direct association between Akt and VCP.

 
Identification of Akt Phosphorylation Sites on VCP—As we demonstrated that VCP is an Akt substrate in vitro, we performed bioinformatics search to identify putative Akt phosphorylation sites on VCP using web-based scansite software. Molecular modeling of Akt/VCP identified Akt kinase recognition motif on VCP at residues 739-753, and scansite software identified Ser352, Ser746, and Ser748 on VCP as putative Akt phosphorylation sites. To generate pRSETB-VCPS352A, pRSETB-VCPS746A, pRSETB-VCPS748A, and pRSETB-VCPS746A/S748A mutants, pRSETB-VCP cDNA construct was subjected to site-directed mutagenesis reactions. These VCP wild type and mutant cDNAs were expressed in bacterial cells, and recombinant proteins of each were generated. These proteins were subjected to in vitro Akt kinase assay using active recombinant Akt and various recombinant VCP proteins as described under "Materials and Methods." Fig. 6D demonstrates that active recombinant Akt phosphorylates wild type VCP to a greater extent as compared with S352A, S746A, and S748A mutants. The double mutant S746A/S748A is phosphorylated to an even lesser extent as compared with the single mutants. No phosphorylation of VCP was detected in the absence of active Akt kinase. These data collectively suggest that Ser352, Ser746, and Ser748 are all Akt phosphorylation sites on VCP.

SH Induces VCP Expression and a Phosphorylation-induced Acidic pI Shift in VCP—By having shown that SH induced enhanced Akt phosphorylation in CA1 and PC-12 cell lysates (Figs. 1 and 2) and that active recombinant Akt phosphorylated VCP in vitro (Fig. 6), we sought to determine the effect of hypoxia on VCP phosphorylation in RA-, SH-, and IH-treated CA1 tissue lysates. RA, SH, and IH CA1 tissue lysates were subjected to two-dimensional PAGE and immunoblot analysis with anti-VCP antibody. Certain post-translational modifications such as phosphorylation add negative charge(s) on a protein causing them to migrate toward the acidic end on a two-dimensional gel because of a reduction in the isoelectric point (pI) of the protein. Fig. 7A represents a VCP two-dimensional immunoblot of RA-, SH-, and IH-treated CA1 tissue lysates. SH but not IH induced increased expression of VCP compared with RA samples. Additionally, VCP exhibited an acidic shift in pI under SH treatment, whereas IH treatment resulted in decreased abundance of the most acidic spot (Fig. 7A). To determine whether this acidic shift in pI was due to phosphorylation, RA, SH, and IH CA1 tissue lysates were treated with PP2A as described under "Materials and Methods." PP2A-treated RA, SH, and IH CA1 tissue lysates were subjected to two-dimensional PAGE and immunoblotted with anti-VCP antibody. Fig. 7B demonstrates that PP2A treatment of RA, SH, and IH CA1 tissue lysate resulted in identical VCP distribution in all the samples. These results suggest that VCP is marginally phosphorylated under basal conditions and is markedly phosphorylated during SH and to a lesser extent during IH treatment. These results collectively suggest that VCP is strongly phosphorylated in the presence of SH compared with IH or RA samples. However, these studies do not directly demonstrate a role of Akt in mediating VCP phosphorylation.



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  		FIG. 6.
VCP is an Akt substrate. A, immunoblot analysis of recombinant VCP protein with anti-VCP antibody. A single band of appropriate VCP molecular weight (97 kDa) was detected. B, autoradiograph of SDS-PAGE following addition of recombinant VCP protein (5 or 10 µg) and [{gamma}-32P]ATP in the presence and absence of active recombinant Akt (400 ng) (n = 3). C, autoradiograph of SDS-PAGE following addition of active recombinant Akt (400 ng) to recombinant VCP (10 µg) and [{gamma}-32P]ATP in the presence of scrambled peptide (200 µM) or AKTide-2T (200 µM) (n = 3). D, autoradiograph of SDS-PAGE following addition of active recombinant PKB/Akt (400 ng) to recombinant VCP wild type and mutants (10 µg) and [{gamma}-32P]ATP. No VCP phosphorylation was detected in the absence of active recombinant Akt (n = 3).

 



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  		FIG. 7.
SH induces VCP expression and a phosphorylation-induced acidic pI shift in VCP. CA1 lysates from Sprague-Dawley rats exposed to RA, SH (8%), and IH (8% O2 to 21% O2 alternating every 6 min) for 6 h were subjected to two-dimensional PAGE and immunoblotted with anti-VCP antibody as described under "Materials and Methods." A, two-dimensional PAGE VCP immunoblot (IB) of RA, SH, and IH CA1 tissue lysate (n = 3). B, two-dimensional PAGE VCP immunoblot of RA, SH, and IH CA1 tissue lysate treated with PP2A, a Ser/Thr phosphatase as described under "Materials and Methods."

 



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  		FIG. 8.
PC-12 cells transfected with constitutively active Akt induce VCP phosphorylation. PC-12 cells were transfected with vector, AktCA, and AktDN constructs as described under "Materials and Methods." A, anti-c-Myc (top panel), anti-Akt (middle panel), and anti-actin antisera (bottom panel) one-dimensional immunoblot analysis of vector-, AktCA-, and AktDN-transfected PC-12 lysates (n = 3). B, two-dimensional PAGE VCP immunoblot of vector-, AktCA-, and AktDN-transfected PC-12 lysates (n = 3). C, two-dimensional PAGE VCP immunoblot (IB) of vector- and AktCA-transfected PC-12 lysates treated with active PP2A (n = 3).

 
Expression of Constitutively Active Akt in PC-12 Cells Induces an Acidic pI Shift in VCP—To determine directly a role of Akt in mediating VCP phosphorylation, we transiently transfected vector, c-Myc-tagged myristoylated, and constitutively active Akt (AktCA), or c-Myc-tagged dominant negative Akt (AktDN) (Upstate%20Biotechnology">Upstate Biotechnology, Inc.) in PC-12 cells. Transfected cells were lysed and subjected to one-dimensional PAGE and immunoblotted with anti-c-Myc, anti-Akt, and anti-actin antisera to document efficient transfection. Anti-c-Myc immunoblots demonstrate successful transfection of AktCA and AktDN constructs in PC-12 cells (Fig. 8A, top panel). The overexpression of Akt in AktCA- and AktDN-transfected cells was demonstrated by anti-Akt immunoblotting (Fig. 8A, middle panel), and equal loading of proteins was verified by anti-actin immunoblots (Fig. 8A, bottom panel). To determine the effect of AktCA and AktDN overexpression on VCP expression and phosphorylation, vector-, AktCA-, and AktDN-transfected PC-12 lysates were subjected to two-dimensional PAGE and immunoblotted with anti-VCP antibody. Fig. 8B demonstrates an increase in VCP expression and an acidic shift of VCP from AktCA-transfected cells compared with vector- or AktDN-transfected PC-12 cell lysates. To determine whether this increased acidic shift in pI of VCP results from Akt-mediated VCP phosphorylation, AktCA- and vector-transfected PC-12 lysates were treated with PP2A prior to two-dimensional PAGE VCP immunoblot analysis. Fig. 8C demonstrates that PP2A treatment of vector- and AktCA-transfected PC-12 cell lysate resulted in identical VCP distribution in both samples. These results suggest that VCP is basally phosphorylated and that transfection of AktCA results in enhanced phosphorylation of VCP in PC-12 cells.

Serine/Threonine Phosphorylation of VCP Markedly Inhibits Its Association with Ubiquitinated Proteins—It is known that VCP targets ubiquitinated proteins at the proteosome. However, the mechanism of VCP release from ubiquitinated proteins is unclear. To determine whether Akt-mediated VCP phosphorylation results in its release from ubiquitinated proteins, PC-12 cells were transfected with vector and active Akt (AktCA) constructs. Transfected cell lysates were subjected to isotype control antibody or anti-VCP immunoprecipitation and were immunoblotted with anti-ubiquitin antibody. Immunoprecipitated proteins were also immunoblotted with anti-VCP to demonstrate equal immunoprecipitation in every case. Fig. 9 demonstrates that a number of proteins associate with unphosphorylated VCP in vector-transfected cells. Association of phosphorylated VCP with ubiquitinated proteins was markedly inhibited in AktCA-transfected cells (Fig. 9, 3rd lane, top panel). Equal VCP immunoprecipitation was demonstrated by immunoblot analysis with anti-VCP antibody (Fig. 9, bottom panel). In addition, transfected cell lysates (vector and AktCA) were subjected to 10% SDS-PAGE and immunoblotted with anti-ubiquitin antibody to determine whether overexpression of AktCA inhibited protein ubiquitination globally. Furthermore, transferred gels were stained with Coomassie Blue stain to demonstrate equal protein load of each sample. Our results demonstrate that overexpression of AktCA did not inhibit protein ubiquitination, and the amount of ubiquitinated proteins was equal between vector- and active Akt-transfected cells (data not shown). Collectively, these results demonstrate a role for VCP phosphorylation in regulating its interaction with ubiquitinated proteins.



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  		FIG. 9.
Serine/threonine phosphorylation of VCP markedly inhibits its association with ubiquitinated proteins. PC-12 cells were transfected with vector and AktCA. Transfected cells were subjected to goat isotype control antibody or anti-VCP immunoprecipitation (IP) and followed by immunoblot (IB) analysis with anti-ubiquitin antibody and with anti-VCP antibody to demonstrate equal immunoprecipitation in every sample. These results demonstrate association of ubiquitinated proteins with unphosphorylated VCP in vector-transfected cells, whereas this association was markedly inhibited when VCP is phosphorylated in AktCA-transfected cells (n = 3).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Episodic hypoxia, as occurs in obstructive sleep apnea, induces significant neuronal damage leading to impairments of neurocognitive, respiratory, and cardiovascular functions (5, 55-59). Most mammalian neurons are sensitive to hypoxia to a varying degree (3, 9, 10). However, the mechanisms underlying this heterogeneous sensitivity are not well understood. Regional variability in hypoxic sensitivity has been reported in the brain during conditions of oxygen deprivation such as hypoxic ischemia, malonate-induced mitochondrial metabolic disruption (2), and during oxidant stress following ischemia (4). As an example of selective vulnerability within a well defined brain structure, the hippocampus contains two regions that clearly differ in their susceptibility to hypoxia. For example, severe hypoxia for 3 h in organotypic hippocampal slice cultures increased delayed neuronal cell death in the CA1 hippocampal region but failed to do so in the CA3 region (60). Additionally, within the CA1 region, mechanisms underlying increased susceptibility to mild IH and concomitant adaptation to SH of similar magnitude are unknown. We have recently shown in PC-12 cells that caspase-dependent and caspase-independent pathways are implicated in IH- and SH-induced cell death respectively (11). However, distinct signal transduction pathways initiated by IH versus SH require further investigation. Identifying IH- and SH-initiated kinase cascades may contribute to our understanding of differential brain susceptibility to IH and SH.

Akt is a serine/threonine kinase and has been shown to regulate cell survival responses in a variety of cell types and in rat brain slices (44, 61, 62). The mechanisms underlying Akt inhibition of apoptosis include phosphorylation of pro-apoptotic Akt substrates resulting in phosphorylation-dependent binding with 14-3-3 proteins, sequestration, and inactivation of substrate proteins (63-65). Hence, we determined whether differential susceptibility to IH versus SH in the CA1 region was mediated by altered Akt signaling.

The present study shows that in the CA1 region of the hippocampus, SH but not IH leads to increased Akt kinase activity and increased Akt binding of several proteins compared with RA controls. This phenomenon strongly suggests a mechanistic role for Akt and its effectors/binding partners in the delayed apoptotic cell death induced by SH when compared with IH in this hippocampal region. Indeed, increased amounts of two proteins, namely VCP and GRP78, were identified in Akt immunoprecipitates from CA1 lysates obtained from SH-exposed animals when compared with RA- and IH-exposed animals. A common characteristic of these two proteins is their role in cellular ER stress response and ER stress-induced apoptosis (53, 66-70). Additionally, six proteins (VCP, ubiquitin hydrolase, 26 S proteosome, clathrin, Hsc70, and H+-transporting ATPase) that also co-precipitated with Akt have been shown to play a role in the ubiquitin/proteasomal protein degradative pathway (27, 34, 71, 72). These data strongly suggest that SH induces increased association of Akt with proteins that regulate the ER stress response, the unfolded protein response, and proteasomal degradation and that such associations may delay neuronal cell death. A lesser association of these proteins with Akt during IH is consistent with no significant induction of Akt kinase activity during IH and with increased neuronal cell loss. Furthermore, the identification of 14-3-3 as an Akt-binding partner in CA1 SH immunoprecipitates was not entirely surprising. Akt substrates have been shown to associate with 14-3-3 in a phosphorylation-dependent manner, and we have demonstrated previously that 14-3-3{zeta} associates with Akt and is an Akt substrate (73). However, the role of 14-3-3{zeta} in the cellular response to SH and/or IH remains to be elucidated.

Given that during SH, Akt associated with proteins regulating ER stress response, the unfolded protein response, and proteasomal degradation and the role of VCP in ER stress response, unfolded protein response, proteasomal degradation, membrane fusion, transcriptional activation, cell cycle control, and apoptosis, we chose to further evaluate the Akt/VCP interaction. Molecular modeling of Akt/VCP identified the Akt kinase recognition motif on VCP at residues 739-753, and scansite software identified Ser352, Ser746, and Ser748 on VCP as putative Akt phosphorylation sites. In addition, we demonstrated that VCP/Akt association was dependent on Akt activation as VCP/Akt association was enhanced in PC-12 cells at 6 h SH, a time point of maximal Akt activation, and was decreased by inhibition of PI3K/Akt pathway in the presence of LY294002. In addition, VCP/Akt association was enhanced in the CA1 from animals exposed to SH rather than IH, correlating with increased Akt activity.

Based on these observations, we hypothesized that VCP is an Akt substrate. To examine this hypothesis, we expressed VCP as a recombinant protein, and we demonstrated the ability of active recombinant Akt to phosphorylate VCP in the presence of a scrambled peptide or Akt inhibitor peptide, AKTide-2T. As expected, AKTide-2T, but not the scrambled peptide, blocked active recombinant Akt-mediated in vitro phosphorylation of VCP. Site-directed mutagenesis studies identified Ser352, Ser746, and Ser748 as Akt phosphorylation sites on VCP. Additionally, in vivo VCP phosphorylation was demonstrated by two-dimensional PAGE VCP immunoblot analysis of RA-, SH-, and IH-treated CA1 tissue lysates. An acidic pI shift, suggestive of phosphorylation, was detected in CA1 of SH-exposed animals compared with RA controls, whereas IH treatment decreased the abundance of the most acidic spot. PP2A treatment inhibited this acidic shift. These results collectively demonstrate VCP phosphorylation in vitro and in vivo. Furthermore, a role for Akt in regulating VCP expression and phosphorylation was demonstrated in transfected PC-12 cells. Constitutively active Akt, but not dominant negative Akt, induced VCP expression. Moreover, transfection of AktCA, but not vector or AktDN, caused acidic pI shift of VCP, which was inhibited in the presence of PP2A. These results demonstrate Akt-mediated in vivo VCP phosphorylation in transfected PC-12 cells.

VCP has been shown previously to be tyrosine-phosphorylated by p34 cdc2 kinase and cAMP-activated boar sperm tyrosine kinase (35, 74). In addition, VCP has been shown to be a substrate of the band 4.1-related protein-tyrosine phosphatase PTPH1 (36). In the mammalian system, VCP is one of the first proteins to be tyrosine-phosphorylated during stimulation of T cells. Tyrosine phosphorylation of VCP has also been suggested to play a role in membrane fusion and presentation of poly-ubiquitinated proteins to the proteosome and in the nuclear localization of VCP during the late G1 phase in S. cerevisiae (13, 27, 40). VCP tyrosine dephosphorylation has been shown to destabilize VCP/ER membrane association thereby promoting ER transitional assembly (42). It is interesting to note that tyrosine phosphorylation of VCP does not alter its ATPase activity; hence it is currently unclear how tyrosine phosphorylation regulates various VCP functions.

In the present study we demonstrated for the first time Akt-mediated serine phosphorylation on VCP. We hypothesized that similarly to what occurs with various chaperone proteins, VCP serine phosphorylation may result in the release of ubiquitin-tagged proteins chaperoned by VCP to the proteosome to be targeted for degradation. The current study tested this hypothesis and demonstrated association of ubiquitinated proteins with unphosphorylated VCP in vector-transfected PC-12 cells, whereas this association was markedly inhibited when VCP was phosphorylated in constitutively active Akt-transfected cells.

In summary, in contrast with IH, exposure to SH is not associated with marked increases in neuronal apoptosis in the hippocampus, which correlates with increased Akt phosphorylation and activation as well as with increased Akt/VCP association. Additionally, Akt-mediated VCP phosphorylation results in the release of ubiquitinated proteins, possibly for their degradation at the proteosome.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants HL66358 (to J. B. K.), HL69932 (to D. G.), and HL074296 (to E. G.), the Kentucky Challenge for Excellence Trust Fund, an American Heart Association postdoctoral fellowship (to M. T. B.), and a scientist development grant from the American Heart Association (to M. J. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

¶¶ To whom correspondence should be addressed: University of Louisville, 570 S. Preston St., Baxter I Bldg. South-102C, Louisville, KY 40202. Tel.: 502-852-0014; Fax.: 502-852-4384; E-mail: mrane{at}louisville.edu.

1 The abbreviations used are: IH, intermittent hypoxia; ER, endoplasmic reticulum; RA, room air; SH, sustained hypoxia; PP2A, protein phosphatase 2A; VCP, valosin-containing protein 97; PI3K, phosphoinositide 3-kinase; AktCA, c-Myc-tagged myristoylated and constitutively active Akt; AktDN, c-Myc-tagged myristoylated dominant negative Akt; MALDI-MS, matrix-assisted laser desorption ionization-mass spectrometry; PC-12, O2-sensitive rat pheochromocytoma cells; PMSF, phenylmethylsulfonyl fluoride; MES, 4-morpholineethanesulfonic acid; GST, glutathione S-transferase; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol. Back


   ACKNOWLEDGMENTS
 
We thank Sanchita Sen for technical assistance.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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