Akt-mediated valosin-containing protein 97 phosphorylation regulates its association with ubiquitinated proteins
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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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; 2013:183421. DOI:10.1155/2013/183421
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ABSTRACT: Salt-inducible kinase 2 (SIK2) is an important regulator of cAMP response element-binding protein (CREB)-mediated gene expression in various cell types and is the only AMP-activated protein kinase (AMPK) family member known to interact with the p97/VCP ATPase. Previously, we have demonstrated that SIK2 could regulate autophagy when proteasomal function is compromised. Here we report that physical and functional interactions between SIK2 and p97/VCP underlie the regulation of ERAD. SIK2 co-localizes with p97/VCP in the ER membrane and stimulates its ATPase activity through direct phosphorylation. While the expression of wild-type recombinant SIK2 accelerated the degradation and removal of ERAD substrates, the kinase-deficient variant conversely had no effect. Furthermore, down-regulation of endogenous SIK2 or mutation of the SIK2 target site on p97/VCP led to impaired degradation of ERAD substrates and disruption of ER homeostasis. Collectively, these findings highlight a mechanism by which the interplay between SIK2 and p97/VCP contributes to the regulation of ERAD in mammalian cells.Journal of Biological Chemistry 10/2013; 288(47). DOI:10.1074/jbc.M113.492199 · 4.60 Impact Factor
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ABSTRACT: LaCoO3, Co3O4 and a mix of them, prepared by two different methods, were studied for the CO + NO reaction under two different gas mixtures. Under the binary gas mixture, we have shown two types of reduction reaction of NO: at low temperature, N2O is formed, and at high temperature N2 is formed. On Co3O4, we have observed the decomposition of N2O in the medium temperature range. Under the complete gas mixture, no NO decomposition was observed whatever the catalyst. But for the perovskite LaCoO3 obtained from simultaneous precipitation, containing 4.5% of potassium, we have pointed out an adsorption/desorption phenomenon, which can be very interesting in the application of NO storage.