Different phosphorylation states of the anaphase promoting complex in response to antimitotic drugs: A quantitative proteomic analysis

Steen, Judith A.; Steen, Hanno; Georgi, Ann; Parker, Kenneth C.; Springer, Michael; Kirchner, Marc; Hamprecht, Fred A.; Kirschner, Marc W.

doi:10.1073/pnas.0709807104

Cited by 82 publications

(100 citation statements)

References 26 publications

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“…Nevertheless, the phosphopeptide signal was higher in all of the R light than the dark samples, and peptide-level quantification from other peptides indicated that the overall PIF3 levels were lower in the R light samples, suggesting that this site may also be induced by R light. A second method of calculating phosphorylation changes employed ratios between detected signal intensities of phosphorylated and unphosphorylated peptides (bottom panel in Supplemental Figure 1C online) (Steen et al, 2008). The results from this analysis are also presented in Supplemental Data Set 1 and Supplemental Table 2 online and generally agreed with those determined using the percentage phosphopeptide signals.…”

Section: Light Induces Multisite Phosphorylation Of Pif3 In Vivosupporting

confidence: 70%

“…This value is compared directly for dark control and R light-treated seedlings at each site in Figure 1D. In the second method, a value termed the relative phospho ratio was defined as the ratio of two values, each of which are themselves ratios (Steen et al, 2008). In this procedure, the ratio of the abundance of phosphorylated to nonphosphorylated residues, detected at a given site, is calculated separately for dark control and R-treated seedlings, and then the ratio of these two derived values is expressed as the ratio of R to dark values (see Supplemental Figure 1C, bottom panel, Supplemental Data Set 1, and Supplemental Table 2 online).…”

Section: Identification Of Phosphorylation Sites and Quantificationmentioning

confidence: 99%

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Multisite Light-Induced Phosphorylation of the Transcription Factor PIF3 Is Necessary for Both Its Rapid Degradation and Concomitant Negative Feedback Modulation of Photoreceptor phyB Levels in Arabidopsis

et al. 2013

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Plants constantly monitor informational light signals using sensory photoreceptors, which include the phytochrome (phy) family (phyA to phyE), and adjust their growth and development accordingly. Following light-induced nuclear translocation, photoactivated phy molecules bind to and induce rapid phosphorylation and degradation of phy-interacting basic Helix Loop Helix (bHLH) transcription factors (PIFs), such as PIF3, thereby regulating the expression of target genes. However, the mechanisms underlying the signal-relay process are still not fully understood. Here, using mass spectrometry, we identify multiple, in vivo, light-induced Ser/Thr phosphorylation sites in PIF3. Using transgenic expression of site-directed mutants of PIF3, we provide evidence that a set of these phosphorylation events acts collectively to trigger rapid degradation of the PIF3 protein in response to initial exposure of dark-grown seedlings to light. In addition, we show that phyB-induced PIF3 phosphorylation is also required for the known negative feedback modulation of phyB levels in prolonged light, potentially through codegradation of phyB and PIF3. This mutually regulatory intermolecular transaction thus provides a mechanism with the dual capacity to promote early, graded, or threshold regulation of the primary, PIF3-controlled transcriptional network in response to initial light exposure, and later, to attenuate global sensitivity to the light signal through reductions in photoreceptor levels upon prolonged exposure.

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Section: Light Induces Multisite Phosphorylation Of Pif3 In Vivosupporting

confidence: 70%

Section: Identification Of Phosphorylation Sites and Quantificationmentioning

confidence: 99%

Multisite Light-Induced Phosphorylation of the Transcription Factor PIF3 Is Necessary for Both Its Rapid Degradation and Concomitant Negative Feedback Modulation of Photoreceptor phyB Levels in Arabidopsis

et al. 2013

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“…Protein/peptide identification, the phosphorylation site assignment, as well as information on the relative stoichiometry were obtained from two LC-MS/MS experiments with nonphosphorylated (control) and phosphorylated (sample) preparations (40,41). Briefly, two samples were prepared as described under the CYP3A4 phos-phorylation assay (11).…”

Section: Methodsmentioning

confidence: 99%

Multisite Phosphorylation of Human Liver Cytochrome P450 3A4 Enhances Its gp78- and CHIP-mediated Ubiquitination

Wang

Guan²,

Acharya

et al. 2012

Molecular & Cellular Proteomics

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CYP3A4, an integral endoplasmic reticulum (ER)-anchored protein, is the major human liver cytochrome P450 enzyme responsible for the disposition of over 50% of clinically relevant drugs. Alterations of its protein turnover can influence drug metabolism, drug-drug interactions, and the bioavailability of chemotherapeutic drugs. Such CYP3A4 turnover occurs via a classical ER-associated degradation (ERAD) process involving ubiquitination by both UBC7/gp78 and UbcH5a/CHIP E2-E3 complexes for 26 S proteasomal targeting. These E3 ligases act sequentially and cooperatively in CYP3A4 ERAD because RNA interference knockdown of each in cultured hepatocytes results in the stabilization of a functionally active enzyme. We have documented that UBC7/gp78-mediated CYP3A4 ubiquitination requires protein phosphorylation by protein kinase (PK) A and PKC and identified three residues (Ser-478, Thr-264, and Ser-420) whose phosphorylation is required for intracellular CYP3A4 ERAD. We document herein that of these, Ser-478 plays a pivotal role in UBC7/ gp78-mediated CYP3A4 ubiquitination, which is accelerated and enhanced on its mutation to the phosphomimetic Asp residue but attenuated on its Ala mutation. Intriguingly, CYP3A5, a polymorphically expressed human liver CYP3A4 isoform (containing Asp-478) is ubiquitinated but not degraded to a greater extent than CYP3A4 in HepG2 cells. This suggests that although Ser-478 phosphorylation is essential for UBC7/gp78-mediated CYP3A4 ubiquitination, it is not sufficient for its ERAD. Additionally, we now report that CYP3A4 protein phosphorylation by PKA and/or PKC at sites other than Ser-478, Thr-264, and Ser-420 also enhances UbcH5a/CHIP-mediated ubiquitination. Through proteomic analyses, we identify (i) 12 additional phosphorylation sites that may be involved in CHIP-CYP3A4 interactions and (ii) 8 previously unidentified CYP3A4 ubiquitination sites within spatially associated clusters of Asp/Glu and phosphorylatable Ser/Thr residues that may serve to engage each E2-E3 complex. Collectively, our findings underscore the interplay between protein phosphorylation and ubiquitination in ERAD and, to our knowledge, provide the very first example of gp78 substrate recognition via protein phosphorylation. Molecular & Cellular Proteomics 11: 10.1074/mcp.M111.010132, 1-17, 2012. Hepatic cytochromes P450 (P450s)1 are endoplasmic reticulum (ER)-anchored hemoproteins involved in the metabolism of numerous endo-and xenobiotics. Of these, CYP3A4 is particularly noteworthy because it comprises 30% of the human liver microsomal P450 complement and is responsible for the metabolism of Ͼ50% clinically relevant drugs, as well as hepatotoxins such as aflatoxin B1 (1). In common with other ER-integral P450s, it is a monotopic protein, N-terminally anchored to the ER membrane, with the bulk of its catalytic domain in the cytosol. We have documented that CYP3A4, in common with its CYP3A orthologs, incurs ubiquitin (Ub)-dependent proteasomal degradation (UPD) in a classical ER-associated degradation (ERAD) process...

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“…Protein/peptide identification, the phosphorylation site assignment, as well as information on the relative stoichiometry can be obtained from two LC-MS/MS experiments with unphosphorylated (control) and phosphorylated (sample) preparations (65,66).…”

Section: ␥-S-[mentioning

confidence: 99%

Ubiquitin-dependent Proteasomal Degradation of Human Liver Cytochrome P450 2E1

Wang

Guan²,

Acharya

et al. 2011

Journal of Biological Chemistry

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Human liver CYP2E1 is a monotopic, endoplasmic reticulumanchored cytochrome P450 responsible for the biotransformation of clinically relevant drugs, low molecular weight xenobiotics, carcinogens, and endogenous ketones. CYP2E1 substrate complexation converts it into a stable slow-turnover species degraded largely via autophagic lysosomal degradation. Substrate decomplexation/withdrawal results in a fast turnover CYP2E1 species, putatively generated through its futile oxidative cycling, that incurs endoplasmic reticulum-associated ubiquitin-dependent proteasomal degradation (UPD). CYP2E1 thus exhibits biphasic turnover in the mammalian liver. We now show upon heterologous expression of human CYP2E1 in Saccharomyces cerevisiae that its autophagic lysosomal degradation and UPD pathways are evolutionarily conserved, even though its potential for futile catalytic cycling is low due to its sluggish catalytic activity in yeast. This suggested that other factors (i.e. post-translational modifications or "degrons") contribute to its UPD. Indeed, in cultured human hepatocytes, CYP2E1 is detectably ubiquitinated, and this is enhanced on its mechanismbased inactivation. Studies in Ubc7p and Ubc5p genetically deficient yeast strains versus corresponding isogenic wild types identified these ubiquitin-conjugating E2 enzymes as relevant to CYP2E1 UPD. Consistent with this, in vitro functional reconstitution analyses revealed that mammalian UBC7/gp78 and UbcH5a/CHIP E2-E3 ubiquitin ligases were capable of ubiquitinating CYP2E1, a process enhanced by protein kinase (PK) A and/or PKC inclusion. Inhibition of PKA or PKC blocked intracellular CYP2E1 ubiquitination and turnover. Here, through mass spectrometric analyses, we identify some CYP2E1 phosphorylation/ubiquitination sites in spatially associated clusters. We propose that these CYP2E1 phosphorylation clusters may serve to engage each E2-E3 ubiquitination complex in vitro and intracellularly.Hepatic cytochromes P450 (P450s) 2 are endoplasmic reticulum (ER)-anchored hemoproteins involved in the metabolism of numerous endo-and xenobiotics. These substrates can modulate P450 content, diversity, and/or function (see Refs. 1, 2 and references therein) through induction via either increased synthesis or protein stabilization, i.e. half-life prolongation (3-9). By contrast, "suicide" substrate/inactivators accelerate the degradation of certain P450s and dramatically curtail their halflives (10 -23). Such substrate-mediated P450 induction and/or enhanced turnover can influence the severity and the time course of certain pharmacokinetic/pharmacodynamic drugdrug interactions and is an important therapeutic consideration (24 -27).P450 turnover has been proposed to involve various proteolytic mechanisms (6 -9, 28 -38). However, it is now increasingly evident that in common with other type I monotopic ER proteins, P450s such as CYPs 3A (both native and structurally inactivated) undergo ER-associated degradation (ERAD) involving the ubiquitin (Ub)-dependent 26 S proteasomal system (UPS) (6 ...

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Different phosphorylation states of the anaphase promoting complex in response to antimitotic drugs: A quantitative proteomic analysis

Cited by 82 publications

References 26 publications

Multisite Light-Induced Phosphorylation of the Transcription Factor PIF3 Is Necessary for Both Its Rapid Degradation and Concomitant Negative Feedback Modulation of Photoreceptor phyB Levels in Arabidopsis

Multisite Light-Induced Phosphorylation of the Transcription Factor PIF3 Is Necessary for Both Its Rapid Degradation and Concomitant Negative Feedback Modulation of Photoreceptor phyB Levels in Arabidopsis

Multisite Phosphorylation of Human Liver Cytochrome P450 3A4 Enhances Its gp78- and CHIP-mediated Ubiquitination

Ubiquitin-dependent Proteasomal Degradation of Human Liver Cytochrome P450 2E1

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