Phosphorylation of murine double minute clone 2 (MDM2) protein at serine-267 by protein kinase CK2 in vitro and in cultured cells

2001 ◽  
Vol 355 (2) ◽  
pp. 347-356 ◽  
Author(s):  
Majbrit HJERRILD ◽  
Diane MILNE ◽  
Nicolas DUMAZ ◽  
Trevor HAY ◽  
Olaf-Georg ISSINGER ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinase Ck2 ◽  
Cultured Cells ◽  
Specific Inhibitor ◽  
Mdm2 Protein ◽  
Acidic Domain ◽  
Double Minute ◽  
Murine Double Minute ◽  
Kinase Ck2

Murine double minute clone 2 oncoprotein (MDM2) is a key component in the regulation of the tumour suppressor p53. MDM2 mediates the ubiqutination of p53 in the capacity of an E3 ligase and targets p53 for rapid degradation by the proteosome. Stress signals which impinge on p53, leading to its activation, promote disruption of the p53-MDM2 complex, as in the case of ionizing radiation, or block MDM2 synthesis and thereby reduce cellular MDM2 levels, as in the case of UV radiation. It is therefore likely that MDM2, which is known to be modified by ubiquitination, SUMOylation and multi-site phosphorylation, may itself be a target for stress signalling (SUMO is small ubiquitin-related modifier-1). In the present study we show that, like p53, the MDM2 protein is a substrate for phosphorylation by the protein kinase CK2 (CK2) in vitro. CK2 phosphorylates a single major site, Ser267, which lies within the central acidic domain of MDM2. Fractionation of cellular extracts revealed the presence of a single Ser267 protein kinase which co-purified with CK2 on ion-exchange chromatography and, like CK2, was subject to inhibition by micromolar concentrations of the CK2-specific inhibitor 5,6-dichlororibofuranosylbenzimidazole. Radiolabelling of cells expressing tagged recombinant wild-type MDM2 or a S267A (Ser267 → Ala) mutant, followed by phosphopeptide analysis, confirmed that Ser267 is a cellular target for phosphorylation. Ser267 mutants are still able to direct the degradation of p53, but in a slightly reduced capacity. These data highlight a potential route by which one of several physiological modifications occurring within the central acidic domain of the MDM2 protein can occur.

scholarly journals Phosphorylation of murine double minute clone 2 (MDM2) protein at serine-267 by protein kinase CK2 in vitro and in cultured cells

2001 ◽  
Vol 355 (2) ◽  
pp. 347 ◽  
Author(s):  
Majbrit HJERRILD ◽  
Diane MILNE ◽  
Nicolas DUMAZ ◽  
Trevor HAY ◽  
Olaf-Georg ISSINGER ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinase Ck2 ◽  
Cultured Cells ◽  
Mdm2 Protein ◽  
Double Minute ◽  
Murine Double Minute ◽  
Kinase Ck2

scholarly journals Protein kinase CK2 inhibitor 4,5,6,7-tetrabromobenzotriazole (TBB) induces apoptosis and caspase-dependent degradation of haematopoietic lineage cell-specific protein 1 (HS1) in Jurkat cells

2002 ◽  
Vol 364 (1) ◽  
pp. 41-47 ◽  
Author(s):  
Maria RUZZENE ◽  
Daniele PENZO ◽  
Lorenzo A. PINNA
Keyword(s):  
Protein Kinase ◽  
Protein Kinase Ck2 ◽  
Specific Inhibitor ◽  
Specific Protein ◽  
Jurkat Cells ◽  
Similar Data ◽  
Caspase Cleavage ◽  
Protein Substrates ◽  
Kinase Ck2

Incubation of Jurkat cells with 4,5,6,7-tetrabromobenzotriazole (TBB), a specific inhibitor of protein kinase CK2, induces dose-and time-dependent apoptosis as judged by several criteria. TBB-promoted apoptosis is preceded by inhibition of Ser/Thr phosphorylation of haematopoietic lineage cell-specific protein 1 (HS1) and is accompanied by caspase-dependent fragmentation of the same protein. Both effects are also observable if apoptosis is promoted by anti-Fas antibodies and by etoposide. Moreover, in vitro experiments show that HS1, once phosphorylated by CK2, becomes refractory to cleavage by caspase-3. These findings, in conjunction with similar data in the literature concerning two other CK2 protein substrates, Bid and Max, suggest that CK2 may play a general anti-apoptotic role through the generation of phosphorylated sites conferring resistance to caspase cleavage.

scholarly journals The catalytic subunit of protein kinase CK2 phosphorylates in vitro the movement protein of Tomato mosaic virus

2003 ◽  
Vol 84 (2) ◽  
pp. 497-505 ◽  
Author(s):  
Yasuhiko Matsushita ◽  
Mayumi Ohshima ◽  
Kuniaki Yoshioka ◽  
Masamichi Nishiguchi ◽  
Hiroshi Nyunoya
Keyword(s):  
Protein Kinase ◽  
Mosaic Virus ◽  
Protein Kinase Ck2 ◽  
Movement Protein ◽  
Catalytic Subunit ◽  
Tomato Mosaic Virus ◽  
Kinase Ck2

Yeast surviving factor Svf1 as a new interacting partner, regulator and in vitro substrate of protein kinase CK2

2008 ◽  
Vol 312 (1-2) ◽  
pp. 61-69 ◽  
Author(s):  
Maciej Masłyk ◽  
Elżbieta Kochanowicz ◽  
Rafał Zieliński ◽  
Konrad Kubiński ◽  
Ulf Hellman ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinase Ck2 ◽  
Kinase Ck2

Interaction of tubulin and protein kinase CK2 in Trypanosoma equiperdum

2017 ◽  
Vol 72 (11-12) ◽  
pp. 459-465 ◽  
Author(s):  
Beatriz E. Boscán ◽  
Graciela L. Uzcanga ◽  
Maritza Calabokis ◽  
Rocío Camargo ◽  
Frank Aponte ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinase Ck2 ◽  
Polyacrylamide Gel Electrophoresis ◽  
Direct Interaction ◽  
Exchange Chromatography ◽  
Parasite Protein ◽  
Α Subunit ◽  
Trypanosoma Equiperdum ◽  
Kinase Ck2

AbstractA polypeptide band with an apparent molecular weight of 55,000 was phosphorylated in vitro in whole-cell lysates ofTrypanosoma equiperdum. This band corresponds to tubulin as demonstrated by immunoprecipitation of the phosphorylated polypeptide fromT. equiperdumextracts when anti-α and anti-β tubulin monoclonal antibodies were employed. A parasite protein kinase CK2 was in charge of modifying tubulin given that common mammalian CK2 inhibitors such as emodin and GTP, hindered the phosphorylation of tubulin and exogenously added casein. Interestingly, a divalent cation-dependent translocation of theT. equiperdumtubulin and the CK2 responsible for its phosphorylation was noticed, suggesting a direct interaction between these two proteins. Additionally, this fraction of tubulin and its kinase coeluted using separations based on parameters as different as charge (DEAE-Sepharose anion-exchange chromatography) and size (Sephacryl S-300 gel filtration chromatography). Analyses by non-denaturing polyacrylamide gel electrophoresis and immunoblot of the purified and radioactively labeled fraction containing both tubulin and the CK2 enzyme, established the phosphorylation of a single band that was recognized by anti-CK2 α-subunit and anti-tubulin antibodies. All these findings revealed a physical association between a pool of tubulin and a CK2 inT. equiperdum.

Phosphorylation of the acidic domain of Mdm2 by protein kinase CK2

2005 ◽  
Vol 274 (1-2) ◽  
pp. 85-90 ◽  
Author(s):  
Nerea Allende-Vega ◽  
Sylvia Dias ◽  
Diane Milne ◽  
David Meek
Keyword(s):  
Protein Kinase ◽  
Protein Kinase Ck2 ◽  
Acidic Domain ◽  
Kinase Ck2

scholarly journals Protein Kinase CK2 Subunits Differentially Perturb the Adhesion and Migration of GN11 Cells: A Model of Immature Migrating Neurons

2019 ◽  
Vol 20 (23) ◽  
pp. 5951 ◽  
Author(s):  
Antonella Lettieri ◽  
Christian Borgo ◽  
Luca Zanieri ◽  
Claudio D’Amore ◽  
Roberto Oleari ◽  
...  
Keyword(s):  
Cell Migration ◽  
Protein Kinase ◽  
Protein Kinase Ck2 ◽  
Primary Role ◽  
Functional Requirements ◽  
Adhesion Properties ◽  
And Migration ◽  
The Individual ◽  
Kinase Ck2

Protein kinase CK2 (CK2) is a highly conserved and ubiquitous kinase is involved in crucial biological processes, including proliferation, migration, and differentiation. CK2 holoenzyme is a tetramer composed by two catalytically active (α/α’) and two regulatory (β) subunits and exerts its function on a broad range of targets. In the brain, it regulates different steps of neurodevelopment, such as neural differentiation, neuritogenesis, and synaptic plasticity. Interestingly, CK2 mutations have been recently linked to neurodevelopmental disorders; however, the functional requirements of the individual CK2 subunits in neurodevelopment have not been yet investigated. Here, we disclose the role of CK2 on the migration and adhesion properties of GN11 cells, an established model of mouse immortalized neurons, by different in vitro experimental approaches. Specifically, the cellular requirement of this kinase has been assessed pharmacologically and genetically by exploiting CK2 inhibitors and by generating subunit-specific CK2 knockout GN11 cells (with a CRISPR/Cas9-based approach). We show that CK2α’ subunit has a primary role in increasing cell adhesion and reducing migration properties of GN11 cells by activating the Akt-GSK3β axis, whereas CK2α subunit is dispensable. Further, the knockout of the CK2β regulatory subunits counteracts cell migration, inducing dramatic alterations in the cytoskeleton not observed in CK2α’ knockout cells. Collectively taken, our data support the view that the individual subunits of CK2 play different roles in cell migration and adhesion properties of GN11 cells, supporting independent roles of the different subunits in these processes.

scholarly journals Hepatitis C Virus NS2 Protein Is Phosphorylated by the Protein Kinase CK2 and Targeted for Degradation to the Proteasome

2005 ◽  
Vol 79 (5) ◽  
pp. 2700-2708 ◽  
Author(s):  
Nathalie Franck ◽  
Jacques Le Seyec ◽  
Christiane Guguen-Guillouzo ◽  
Lars Erdtmann
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Sequence Analysis ◽  
Protein Kinase ◽  
Protein Turnover ◽  
Protein Kinase Ck2 ◽  
Kinase Assay ◽  
Serine Residue ◽  
Kinase Ck2

ABSTRACT 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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