Phosphorylation of p97(VCP) and p47 in vitro by p34cdc2 kinase

Mayr, Petra; Allan, Viki; Woodman, Philip

doi:10.1016/s0171-9335(99)80055-7

Cited by 19 publications

(14 citation statements)

References 59 publications

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“…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)(36)(37)(38)(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.…”

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confidence: 66%

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

Klein

Barati

et al. 2005

Journal of Biological Chemistry

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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. 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-

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confidence: 66%

“…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).…”

Section: Expression Of Constitutively Active Akt In Pc-12 Cells Inducmentioning

confidence: 93%

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

Klein

Barati

et al. 2005

Journal of Biological Chemistry

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“…Because our in vitro binding assay used Arabidopsis cytosol as a source of AtCDC48 and adapter proteins, a third possibility is that the observed ATP hydrolysis requirement reflects posttranslational phosphorylation of AtCDC48. It has been previously shown that Cdc48p/p97 activity is regulated by phosphorylation (Madeo et al, 1998;Mayr et al, 1999;Lavoie et al, 2000), however, an ATP requirement for binding of purified Cdc48p/ p97 and p47 to syntaxin 5 in vitro (Rabouille et al, 1998) would argue against this model. These models can be tested through the use of our in vitro SYP31 binding assay using purified AtCDC48 adapters and through the in vitro and in vivo analysis of AtCDC48 ATPase mutants.…”

Section: Discussionmentioning

confidence: 99%

Characterization of AtCDC48. Evidence for Multiple Membrane Fusion Mechanisms at the Plane of Cell Division in Plants

Dickey²,

et al. 2002

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The components of the cellular machinery that accomplish the various complex and dynamic membrane fusion events that occur at the division plane during plant cytokinesis, including assembly of the cell plate, are not fully understood. The most well-characterized component, KNOLLE, a cell plate-specific soluble N-ethylmaleimide-sensitive fusion protein (NSF)-attachment protein receptor (SNARE), is a membrane fusion machine component required for plant cytokinesis. Here, we show the plant ortholog of Cdc48p/p97, AtCDC48, colocalizes at the division plane in dividing Arabidopsis cells with KNOLLE and another SNARE, the plant ortholog of syntaxin 5, SYP31. In contrast to KNOLLE, SYP31 resides in defined punctate membrane structures during interphase and is targeted during cytokinesis to the division plane. In vitro-binding studies demonstrate that AtCDC48 specifically interacts in an ATP-dependent manner with SYP31 but not with KNOLLE. In contrast, we show that KNOLLE assembles in vitro into a large approximately 20S complex in an Sec18p/NSF-dependent manner. These results suggest that there are at least two distinct membrane fusion pathways involving Cdc48p/p97 and Sec18p/NSF that operate at the division plane to mediate plant cytokinesis. Models for the role of AtCDC48 and SYP31 at the division plane will be discussed.Plant cell division is completed by the highly dynamic process of de novo cell plate construction leading to the separation of two daughter cells. Formation of this unique cytokinetic organelle involves at least three key membrane fusion steps: (a) fusion of secretory vesicles across the division plane to form a membranous tubular-vesicular network, (b) consolidation of the tubular-vesicular network, and (c) fusion of the cell plate leading-edge with the original parental plasma membrane to complete division (Samuels et al., 1995). These distinct stages of cell plate biogenesis likely involve both heterotypic and homotypic membrane fusion events. In addition to the cell plate, the division plane contains an extensive endoplasmic reticulum (ER) network that has been suggested to function in the formation of the cell plate (Hepler, 1982). Assembly of cell plateassociated ER is likely to involve homotypic fusion of ER membrane within the division plane.Two homologous classes of ATPases associated with various cellular activities (AAA) proteins (Frö hlich, 2001), Sec18p N-ethylmaleimide-sensitive fusion protein (NSF) and Cdc48p/p97 (p97 is also known as VCP), regulate a variety of secretory membrane fusion processes (Acharya et al., 1995;Latterich et al., 1995;Rabouille et al., 1995). Monomers of Sec18p/NSF and Cdc48p/p97 consist of two Mg 2ϩ -dependent ATPase domains and an N-terminal substrate/adapter domain that assemble into biologically active ring-shaped oligohexamers (Peters et al., 1990; Hanson et al., 1997). Of these two AAA complexes, the biochemical function of Sec18p/NSF, which along with its cofactor, soluble NSF-attachment protein (␣-SNAP) regulates hetero-and homotypic membrane fusion, has...

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“…VCP is required for the disassembly of the mitotic spindle proteins preceding cell division (83), and VCP function is crucial during homotypic fusion of transient vesicles into membranes that reassemble into the ER and Golgi apparatus following cell division (73,78,84,85). VCP also participates in cell cycle (86) and transcription factor regulation (87), and it even plays a role in apoptosis (69) and DNA repair (88,89). Consequently, in situations of extreme cellular stress, such as occurs during Hsp90 and proteasome inhibition, when VCP becomes relocalized from its constitutive sites of action into insoluble aggresomes, all of the functions of VCP would be compromised.…”

Section: Discussionmentioning

confidence: 99%

Endoplasmic Reticulum Vacuolization and Valosin-Containing Protein Relocalization Result from Simultaneous Hsp90 Inhibition by Geldanamycin and Proteasome Inhibition by Velcade

Mimnaugh

Vos

et al. 2006

Molecular Cancer Research

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Geldanamycin and Velcade, new anticancer drugs with novel mechanisms of action, are currently undergoing extensive clinical trials. Geldanamycin interrupts Hsp90 chaperone activity and causes down-regulation of its many client proteins by the ubiquitin-proteasome pathway; Velcade is a specific proteasome inhibitor. Misfolded Hsp90 clients within the endoplasmic reticulum (ER) lumen are cleared by ER-associated protein degradation, a sequential process requiring valosin-containing protein (VCP) -dependent retrotranslocation followed by ubiquitination and proteasomal proteolysis. Cotreatment of cells with geldanamycin and Velcade prevents destruction of destabilized, ubiquitinated Hsp90 client proteins, causing them to accumulate. Here, we report that misfolded protein accumulation within the ER resulting from geldanamycin and Velcade exposure overwhelms the ability of the VCP-centered machine to maintain the ER secretory pathway, causing the ER to distend into conspicuous vacuoles. Overexpression of dominantnegative VCP or the ''small VCP-interacting protein'' exactly recapitulated the vacuolated phenotype provoked by the drugs, associating loss of VCP function with ER vacuolization. In cells transfected with a VCP-enhanced yellow fluorescent protein fluorescent construct, geldanamycin plus Velcade treatment redistributed VCP-enhanced yellow fluorescent protein from the cytoplasm and ER into perinuclear aggresomes. In further support of the view that compromise of VCP function is responsible for ER vacuolization, small interfering RNA interference of VCP expression induced ER vacuolization that was markedly increased by Velcade. VCP knockdown by small interfering RNA eventually deconstructed both the ER and Golgi and interdicted protein trafficking through the secretory pathway to the plasma membrane. Thus, simultaneous geldanamycin and Velcade treatment has far-reaching secondary cytotoxic consequences that likely contribute to the cytotoxic activity of this anticancer drug combination. (Mol Cancer Res 2006;4(9):667 -81)

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Phosphorylation of p97(VCP) and p47 in vitro by p34cdc2 kinase

Cited by 19 publications

References 59 publications

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

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

Characterization of AtCDC48. Evidence for Multiple Membrane Fusion Mechanisms at the Plane of Cell Division in Plants

Endoplasmic Reticulum Vacuolization and Valosin-Containing Protein Relocalization Result from Simultaneous Hsp90 Inhibition by Geldanamycin and Proteasome Inhibition by Velcade

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