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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    • "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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    • "Accordingly, it remains to be established whether cytosolic ATP depletion may increase the proteolytic susceptibility of p97 to Casp6-mediated proteolysis in vivo. In cells, p97 conformational state is likely influenced by multiple factors, including adaptor protein binding and posttranslational modifications such as ubiquitination (Hitchcock et al., 2001) and phosphorylation (Klein et al., 2005). Other studies have demonstrated that phosphorylation could modulate the susceptibility of a substrate to caspase-mediated processing, either positively or negatively. "
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    ABSTRACT: The valosin-containing protein (p97) is a ubiquitin-dependent ATPase that plays central roles in ubiquitin proteasome system (UPS)-mediated protein degradation pathways. p97 has been recently identified as a putative substrate of active Caspase-6 (Casp6) in primary human neurons. Since Casp6 is activated in mild cognitive impairment (MCI) and Alzheimer's disease (AD) patients' brains, the targeting of p97 by Casp6 may represent an important step that leads to UPS impairment in AD. Here, we show that p97 is a Casp6 substrate in vitro and in vivo. Casp6 cleavage of recombinant p97 generated two N-terminal fragments of 28 and 20 kDa, which were not generated by the other two effector caspases, Caspase-3 and Caspase-7. ATP binding to the D1 ATPase ring of p97 reduced the susceptibility of the N-domain to caspase-mediated proteolysis. Mass spectrometric analysis identified VAPD(179) as a Casp6 cleavage site within p97's N-domain. An anti-neoepitope serum immunohistochemically detected p97 cleaved at VAPD(179) in the cytoplasm of the cell soma and neurites of hippocampal neurons in MCI and AD. Overexpression of p97 (1-179) fragment, representing p97 cleaved at D179, impaired the degradation of model substrates in the ubiquitin-fusion degradation and the N-end rule pathways, and destabilized endogenous p97. Collectively, these results show that p97 is cleaved by Casp6 in AD and suggest p97 cleavage as an important mechanism for UPS impairment.
    The Journal of Neuroscience : The Official Journal of the Society for Neuroscience 04/2010; 30(17):6132-42. DOI:10.1523/JNEUROSCI.5874-09.2010 · 6.75 Impact Factor
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