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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¶||**, Michelle T. Barati¶, Rui Wu¶, David Gozal, Leroy R. Sachleben, Jr., Hina Kausar¶, John O. Trent**, Evelyne Gozal, and Madhavi J. Rane¶¶¶

From the 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 Department of Pediatrics, the Department of Pharmacology, and the ^**James Graham Brown Cancer Center, University of Louisville, Louisville, Kentucky 40202

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.

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

^* 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.

^¶¶ 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.

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