Akt-mediated Valosin-containing Protein 97 Phosphorylation Regulates Its Association with Ubiquitinated Proteins

Department of Biochemistry and Molecular Biology, University of Louisville, Louisville, Kentucky, United States
Journal of Biological Chemistry (Impact Factor: 4.57). 10/2005; 280(36):31870-81. DOI: 10.1074/jbc.M501802200
Source: PubMed

ABSTRACT 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.

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    • "Upon DNA damage, Cdc48 is phosphorylated at Ser784 by DNA-PK and accumulates at sites of DNA lesion [44], suggesting that Ser784 may be the key to DNA damage-triggered signaling. Under sustained hypoxia in PC-12 cells, Akt kinase can phosphorylate Cdc48 on Ser352, Ser746, and Ser748 [45], which leads to markedly reduced association between Cdc48 and ubiquitylated protein. Furthermore, the phosphomimetic form of Cdc48 on Thr761 exhibits elevated ATPase activity [46]. "
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    ABSTRACT: Cdc48 (also called VCP and p97) is an abundant protein that plays essential regulatory functions in a broad array of cellular processes. Working with various cofactors, Cdc48 utilizes its ATPase activity to promote the assembly and disassembly of protein complexes. Here, we review key biological functions and regulation of Cdc48 in ubiquitin-related events. Given the broad employment of Cdc48 in cell biology and its intimate ties to human diseases (e.g., amyotrophic lateral sclerosis), studies of Cdc48 will bring significant insights into the mechanism and function of ubiquitin in health and diseases.
    09/2013; 2013(4):183421. DOI:10.1155/2013/183421
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    • "Klein and colleagues [37] have demonstrated that p97/CDC-48 is phosphorylated by the anti-apoptotic affinity-regulating kinase (Akt). Akt is another important mediator of cell survival and cell proliferation [47]. "
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    ABSTRACT: P97/CDC-48 is a prominent member of a highly evolutionary conserved Walker cassette - containing AAA+ ATPases. It has been involved in numerous cellular processes ranging from the control of protein homeostasis to membrane trafficking through the intervention of specific accessory proteins. Expression of p97/CDC-48 in cancers has been correlated with tumor aggressiveness and prognosis, however the precise underlying molecular mechanisms remain to be characterized. Moreover p97/CDC-48 inhibitors were developed and are currently under intense investigation as anticancer drugs. Herein, we discuss the role of p97/CDC-48 in cancer development and its therapeutic potential in tumor cell biology.
    Cancer letters 05/2013; 337(1). DOI:10.1016/j.canlet.2013.05.030 · 5.62 Impact Factor
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    • "Given VCP's wide-ranging activities, it comes as no surprise that its post-translational modification would impact equally diverse cellular functions (see Fig. 3 for a list of reported VCP modifications). For example , it was demonstrated that the chaperone could be phosphorylated at S352, S745 and S747 by Akt under growth hormone activation or in response to hypoxia [177] [178]. VCP phosphorylation has also been shown to take place during cold acclimation [179], sperm capacitation [180] and transitional ER assembly via Jak2 [181]. "
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    ABSTRACT: Molecular chaperones and their associated cofactors form a group of highly specialized proteins that orchestrate the folding and unfolding of other proteins and the assembly and disassembly of protein complexes. Chaperones are found in all cell types and organisms, and their activity must be tightly regulated to maintain normal cell function. Indeed, deregulation of protein folding and protein complex assembly is the cause of various human diseases. Here, we present the results of an extensive review of the literature revealing that the post-translational modification (PTM) of chaperones has been selected during evolution as an efficient mean to regulate the activity and specificity of these key proteins. Because the addition and reciprocal removal of chemical groups can be triggered very rapidly, this mechanism provides an efficient switch to precisely regulate the activity of chaperones on specific substrates. The large number of PTMs detected in chaperones suggests that a combinatory code is at play to regulate function, activity, localization, and substrate specificity for this group of biologically important proteins. This review surveys the core information currently available as a starting point towards the more ambitious endeavor of deciphering the "chaperone code".
    Biochimica et Biophysica Acta 02/2013; 1829(5). DOI:10.1016/j.bbagrm.2013.02.010 · 4.66 Impact Factor
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