Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.25.1 (proteasome)
28,817 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Protein kinase CK2 is a ubiquitous serine/threonine kinase involved in many biological processes. It is overexpressed in many malignancies including rodent and human breast cancer, and is up-regulated in Wnt-transfected mammary epithelial cells, where it can be found in a complex with dishevelled and beta-catenin. beta-Catenin is a substrate for CK2 and inhibition of CK2 reduces levels of beta-catenin and dishevelled. Here we report that inhibition of CK2 using pharmacologic agents or expression of kinase inactive subunits reduces beta-catenin-dependent transcription and protein levels in a proteasome-dependent fashion. The major region of phosphorylation of beta-catenin by CK2 is the central armadillo repeat domain, where carrier proteins like axin and the adenomatous polyposis coli gene product APC interact with beta-catenin. The major CK2 phosphorylation site in this domain is Thr393, a solvent-accessible residue in a key hinge region of the molecule. Mutation of this single amino acid reduces beta-catenin phosphorylation, cotranscriptional activity, and stability. Thus, CK2 is a positive regulator of Wnt signaling through phosphorylation of beta-catenin at Thr393, leading to proteasome resistance and increased protein and co-transcriptional activity.
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PMID:CK2 phosphorylation of the armadillo repeat region of beta-catenin potentiates Wnt signaling. 1270 Feb 39

In animal cells there are several regulatory complexes which interact with 20S proteasomes and give rise to functionally distinct proteasome complexes. gamma-Interferon upregulates three immuno beta catalytic subunits of the 20S proteasome and the PA28 regulator, and decreases the level of 26S proteasomes. It also decreases the level of phosphorylation of two proteasome alpha subunits, C8 (alpha7) and C9 (alpha3). In the present study we have investigated the role of phosphorylation of C8 by protein kinase CK2 in the formation and stability of 26S proteasomes. An epitope-tagged C8 subunit expressed in mammalian cells was efficiently incorporated into both 20S proteasomes and 26S proteasomes. Investigation of mutants of C8 at the two known CK2 phosphorylation sites demonstrated that these are the two phosphorylation sites of C8 in animal cells. Although phosphorylation of C8 was not absolutely essential for the formation of 26S proteasomes, it did have a substantial effect on their stability. Also, when cells were treated with gamma-interferon, there was a marked decrease in phosphorylation of C8, a decrease in the level of 26S proteasomes, and an increase in immunoproteasomes and PA28 complexes. These results suggest that the down-regulation of 26S proteasomes after gamma-interferon treatment results from the destabilization that occurs after dephosphorylation of the C8 subunit.
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PMID:Phosphorylation of 20S proteasome alpha subunit C8 (alpha7) stabilizes the 26S proteasome and plays a role in the regulation of proteasome complexes by gamma-interferon. 1458 91

Recently, evidence is accumulating pointing to a function of the COP9 signalosome (CSN) in regulation of ubiquitination by specific ubiquitin ligases. Here, we demonstrate by mammalian two-hybrid analysis that the transcriptional regulators and substrates of the ubiquitin system Id1 and Id3, but not Id2 and Id4, bind to the CSN subunit CSN5. Pull-down experiments revealed that Id3 physically interacts with the CSN complex. Additional far Western and pull-down studies with Id3 support our two-hybrid data and show that the transcription regulator can bind to CSN5 and CSN7. Recombinant Id3 is not phosphorylated by the CSN-associated kinases CK2 and PKD. However, it inhibits c-Jun and CSN2 phosphorylation by the isolated CSN complex and by the recombinant CK2. The inhibitors of CSN associated kinases, curcumin and emodin, significantly induce ubiquitination and proteasome-dependent degradation of transiently expressed Id3 in HeLa cells. Proteasome-dependent degradation of endogenous Id1 in HeLa cells is also stimulated by treatment with curcumin or emodin. Ubiquitination of Id3 is shown directly by cotransfection of HeLa cells with Id3 and His-ubiquitin cDNA. Curcumin increased Id3-ubiquitin conjugate formation, as shown by Western blotting and His-pull-downs. In addition, overexpression of CSN2 leads to stabilization of Id3 protein. On the basis of these data, it is speculated that CSN-mediated phosphorylation inhibits ubiquitination of Id1 and Id3.
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PMID:Ubiquitin-dependent degradation of Id1 and Id3 is mediated by the COP9 signalosome. 1545 66

The 26S proteasome complex, which consists of a 20S proteasome and a pair of 19S regulatory particles, plays important roles in the degradation of ubiquitinated proteins in eukaryotic cells. The alpha7 subunit of the budding yeast 20S proteasome is a major phosphorylatable subunit; serine residue(s) in its C-terminal region are phosphorylated in vitro by CKII. However, the exact in vivo phosphorylation sites have not been identified. In this study, using electrospray ionization quadrupole time-of-flight mass spectrometry analysis, we detected a mixture of singly, doubly, and triply phosphorylated C-terminal peptides isolated from a His-tagged construct of the alpha7 subunit by nickel-immobilized metal affinity chromatography. In addition, we identified three phosphorylation sites in the C-terminal region using MS/MS analysis and site-directed mutagenesis: Ser258, Ser263, and Ser264 residues. The MS/MS analysis of singly phosphorylated peptides showed that phosphorylation at these sites did not occur successively.
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PMID:Identification of three phosphorylation sites in the alpha7 subunit of the yeast 20S proteasome in vivo using mass spectrometry. 1546 21

Hepatitis C virus (HCV) nonstructural 2 (NS2) protein is a hydrophobic transmembrane protein, described to be involved in different functions, such as apoptosis inhibition and gene transcription modulation. We investigated here NS2 protein turnover and found that NS2 was rapidly degraded by the proteasome in different cell lines, as in primary human hepatocytes. Since posttranslational modifications can influence protein turnover, we looked for potential phosphoacceptor sites in NS2. Computational sequence analysis in combination with screening of NS2 point mutants revealed that serine residue 168 was critical for degradation. In the quest of a protein kinase for NS2, we identified by sequence analysis that the serine residue 168 was part of a consensus casein kinase 2 (CK2) recognition site (S/TXXE). This motif was highly conserved since it could be found in the NS2 primary consensus sequences from all HCV genotypes. To verify whether CK2 is involved in NS2 phosphorylation, we showed by an in vitro kinase assay that CK2 phosphorylated NS2, as far as this CK2 motif was conserved. Interestingly, NS2 became resistant to protein degradation when the CK2 motif was modified by a single point mutation. Furthermore, inhibition of CK2 activity by curcumin decreased NS2 phosphorylation in vitro and stabilized NS2 expression in HepG2 cells. Finally, we showed in Huh-7.5 replicon cells that NS2, expressed in the context of the HCV polyprotein, was also sensitive to both proteasome-mediated degradation and CK2 inhibitor treatment. We suggest that NS2 is a short-lived protein whose degradation by the proteasome is regulated in a phosphorylation-dependent manner through the protein kinase CK2.
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PMID:Hepatitis C virus NS2 protein is phosphorylated by the protein kinase CK2 and targeted for degradation to the proteasome. 1570 89

At the onset of M phase, the activity of somatic Wee1 (Wee1A), the inhibitory kinase for cyclin-dependent kinase (CDK), is down-regulated primarily through proteasome-dependent degradation after ubiquitination by the E3 ubiquitin ligase SCF(beta-TrCP). The F-box protein beta-TrCP (beta-transducin repeat-containing protein), the substrate recognition component of the ubiquitin ligase, binds to its substrates through a conserved binding motif (phosphodegron) containing two phosphoserines, DpSGXXpS. Although Wee1A lacks this motif, phosphorylation of serines 53 and 123 (S53 and S123) of Wee1A by polo-like kinase 1 (Plk1) and CDK, respectively, are required for binding to beta-TrCP. The sequence surrounding phosphorylated S53 (DpSAFQE) is similar to the conserved beta-TrCP-binding motif; however, the role of S123 phosphorylation (EEGFGSSpSPVK) in beta-TrCP binding was not elucidated. In the present study, we show that phosphorylation of S123 (pS123) by CDK promoted the binding of Wee1A to beta-TrCP through three independent mechanisms. The pS123 not only directly interacted with basic residues in the WD40 repeat domain of beta-TrCP but also primed phosphorylation by two independent protein kinases, Plk1 and CK2 (formerly casein kinase 2), to create two phosphodegrons on Wee1A. In the case of Plk1, S123 phosphorylation created a polo box domain-binding motif (SpSP) on Wee1A to accelerate phosphorylation of S53 by Plk1. CK2 could phosphorylate S121, but only if S123 was phosphorylated first, thereby generating the second beta-TrCP-binding site (EEGFGpS121). Using a specific inhibitor of CK2, we showed that the phosphorylation-dependent degradation of Wee1A is important for the proper onset of mitosis.
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PMID:Cyclin-dependent kinase (CDK) phosphorylation destabilizes somatic Wee1 via multiple pathways. 1608 15

Diminished expression of NKX3.1 is associated with prostate cancer progression in humans, and in mice, loss of nkx3.1 leads to epithelial cell proliferation and altered gene expression patterns. The NKX3.1 amino acid sequence includes multiple potential phosphoacceptor sites for protein kinase CK2. To investigate posttranslational regulation of NKX3.1, phosphorylation of NKX3.1 by CK2 was studied. In vitro kinase assays followed by mass spectrometric analyses demonstrated that CK2 phosphorylated recombinant NKX3.1 on Thr89 and Thr93. Blocking CK2 activity in LNCaP cells with apigenin or 5,6-dichlorobenzimidazole riboside led to a rapid decrease in NKX3.1 accumulation that was rescued by proteasome inhibition. Replacing Thr89 and Thr93 with alanines decreased NKX3.1 stability in vivo. Small interfering RNA knockdown of CK2alpha' but not CK2alpha also led to a decrease in NKX3.1 steady-state level. In-gel kinase assays and Western blot analyses using fractionated extracts of LNCaP cells demonstrated that free CK2alpha' could phosphorylate recombinant human and mouse NKX3.1, whereas CK2alpha' liberated from the holoenzyme could not. These data establish CK2 as a regulator of NKX3.1 in prostate tumor cells and provide evidence for functionally distinct pools of CK2alpha' in LNCaP cells.
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PMID:NKX3.1 is regulated by protein kinase CK2 in prostate tumor cells. 1658 76

Most organisms have evolved an internal timing mechanism, the circadian clock, that is able to generate and maintain 24 h rhythmic oscillation in molecular, biochemical and metabolic activities. In Arabidopsis, the clock-dependent synchronization of physiology with the environment is essential for successful growth and development. The mechanisms of the Arabidopsis clockwork have been described as transcriptional feedback loops at the core of the oscillator. However, an increasing body of evidence points towards a key role of post-translational regulation of clock components as an essential mechanism of circadian function. Here, we identify CKB4, a CK2 regulatory subunit, as a component of the Arabidopsis circadian system. We demonstrate that the nuclear-localized CKB4 protein exists in vivo as different isoforms, resulting from phosphorylation on serine residues. Our findings show that the phosphorylated isoforms are the preferred substrate for ubiquitination and degradation by the proteasome pathway. We provide evidence of the involvement of the biological clock in the circadian regulation of CKB4 protein abundance, which itself is important for an accurate control of circadian period by the clock. Overexpression of CKB4 results in elevated CK2 overall activity and period-shortening of clock-controlled genes peaking at different phase angles. Restriction of CKB4 protein phosphorylation and/or degradation to specific phases within the circadian cycle might provide the cell with a fine-tuning mechanism to selectively regulate the CK2 phosphorylation activity on specific substrates.
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PMID:The proteasome-dependent degradation of CKB4 is regulated by the Arabidopsis biological clock. 1670 99

Sensitive to Apoptosis Gene (SAG), a RING component of SCF E3 ubiquitin ligase, was shown to be phosphorylated by protein kinase CK2 at the Thr10 residue. It is, however, unknown whether this phosphorylation is stress-responsive or whether the phosphorylation changes its E3 ubiquitin ligase activity. To address these, we made a specific antibody against the phosphor-SAG(Thr10). Transient transfection experiment showed that SAG was phosphorylated at Thr10 which can be significantly inhibited by TBB, a relatively specific inhibitor of protein kinase CK2. To determine whether this SAG phosphorylation is stress-responsive, we defined a chemical-hypoxia condition in which SAG and CK2 were both induced. Under this condition, we failed to detect SAG phosphorylation at Thr10, which was readily detected, however, in the presence of MG132, a proteasome inhibitor, suggesting that the phosphorylated SAG has undergone a rapid degradation. To further define this, we made two SAG mutants, SAG-T10A which abolishes the SAG phosphorylation and SAG-T10E, which mimics the constitutive SAG phosphorylation. The half-life study revealed that indeed, SAG-T10E has a much shorter protein half-life (2 h), as compared to wild-type SAG (10 h). Again, rapid degradation of SAG-T10E in cells can be blocked by MG132. Thus, it appears that CK2-induced SAG phosphorylation at Thr10 regulates its stability through a proteasome-dependent pathway. Immunocytochemistry study showed that SAG as well as its phosphorylation mutants, was mainly localized in nucleus and lightly in cytoplasm. Hypoxia condition did not change their sub-cellular localization. Finally, an in vitro ubiqutination assay showed that SAG mutation at Thr10 did not change its E3 ligase activity when complexed with cullin-1. These studies suggested that CK2 might regulate SAG-SCF E3 ligase activity through modulating SAG's stability, rather than its enzymatic activity directly.
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PMID:CK2 phosphorylation of SAG at Thr10 regulates SAG stability, but not its E3 ligase activity. 1687 60

A number of cancers are characterized by elevated expression of CK2 (formerly casein kinase II), which has been implicated as a key component in cell proliferation and transformation. Two lines of evidence, (a) deregulated expression of CK2 and (b) CK2beta ubiquitination and degradation of these in a proteasome-dependent manner prompted further investigation of the regulation of CK2beta protein stability. We demonstrate that mutating six surface-exposed lysine residues to arginine (6KR) to interfere with ubiquitin attachment can stabilize CK2beta. Examination of 6KR expression in cells revealed increased stability over time and increased its steady-state expression level compared with CK2beta. In cells, 6KR was no longer sensitive to proteasome inhibition but maintained an elevated expression level. In our studies, 6KR functioned as a normal CK2 regulatory subunit, because it participated in CK2beta dimerization, associated with catalytic subunits, was autophosphorylated, and formed active, stable CK2 tetramers. The physiological role of CK2beta stabilization was investigated in cell proliferation assays, which showed a significant decrease in proliferation in cells expressing 6KR compared with CK2beta. Overall, our results indicate that a stabilized form of CK2beta can be used to inhibit cell proliferation.
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PMID:Development of a stabilized form of the regulatory CK2beta subunit that inhibits cell proliferation. 1768 43


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