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.

Tight binding between a pool of the heterodimeric α/β tubulin and a protein kinase CK2 inTrypanosoma cruziepimastigotes

Parasitology ◽  
2005 ◽  
Vol 132 (4) ◽  
pp. 511-523 ◽  
Author(s):  
A. R. DE LIMA ◽  
R. MEDINA ◽  
G. L. UZCANGA ◽  
K. NORIS SUÁREZ ◽  
V. T. CONTRERAS ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinase Ck2 ◽  
Divalent Cations ◽  
Polyacrylamide Gel Electrophoresis ◽  
Tight Binding ◽  
Α Subunit ◽  
Native Polyacrylamide Gel Electrophoresis ◽  
Tubulin Antibodies ◽  
Kinase Ck2 ◽  
Β Tubulin

Tubulin is the predominant phosphoprotein inTrypanosoma cruziepimastigotes and is phosphorylated by a protein kinase CK2. Interestingly, the presence or absence of divalent cations affected the solubilization of a pool of the parasite tubulin and the CK2 responsible for its phosphorylation. This fraction of tubulin and its kinase co-eluted using phosphocellulose, DEAE-Sepharose and Sephacryl S-300 chromatographies. Anti-α tubulin antibodies co-immunoprecipitated both tubulin and the CK2 responsible for its phosphorylation, and anti-CK2 α-subunit antibodies immunoprecipitated radioactively labelled α and β tubulin from phosphorylated epimastigote homogenates. Additionally, native polyacrylamide gel electrophoresis of the purified and radioactively labelled fraction containing tubulin and its kinase demonstrated the phosphorylation of a unique band that reacted with both anti-CK2 α-subunit and anti-tubulin antibodies. Together, these results establish a strong interaction between a pool of the heterodimeric α/β tubulin and a CK2 in this parasite. Hydrodynamic measurements indicated that theT. cruzitubulin-CK2 complex is globular with an estimated size of 145·4–147·5 kDa.

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

scholarly journals The 47-kD fragment of talin is a substrate for protein kinase P

Blood ◽  
1993 ◽  
Vol 82 (11) ◽  
pp. 3343-3349 ◽  
Author(s):  
PC Simons ◽  
L Elias
Keyword(s):  
Protein Kinase ◽  
Ion Exchange ◽  
Sequence Data ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Exchange Chromatography ◽  
Myelogenous Leukemia ◽  
Blue Staining ◽  
Major Protein

Abstract This laboratory has been characterizing protein serine/threonine kinase reactions of hematopoietic tissues, whose most distinguishing characteristics in vitro are stimulation with vesicular phosphatidyl glycerol, and the ability to function using Mn2+ as the sole divalent cation. The major protein substrates are a 73-kD protein and a protein migrating near ovalbumin on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The 47-kD protein was partially purified from cells harvested by leukapheresis from a patient with acute myelogenous leukemia, using ammonium sulfate precipitation and ion exchange chromatography. This partially purified ion-exchange fraction contained an endogenous kinase activity with characteristics similar to those we previously described of protein kinase P (protein kinase, phospholipid- stimulable: PK-P), but not typical of any form of protein kinase C (PK- C). With longer phosphorylation, the 47-kD band showed increasingly lower mobility demonstrable both by Coomassie blue staining and autoradiography, suggesting both that it was multiply phosphorylated, and that the excisable band was pure. The protein was thus eluted from preparative gel slices and digested with endoproteinase lys C. Sequence data from the fragments identified the protein as the 47-kD calpain fragment of talin, a protein found in focal adhesion plaques and some cell-cell contacts. PK-C phosphorylated the 47-kD protein, as has been reported previously, and phosphopeptide mapping disclosed a similar pattern of phosphorylation using either PK-C or the endogenous activity. The 47-kD protein labeled with the endogenous kinase contained predominantly phosphoserine, with some phosphothreonine and a trace of phosphotyrosine. Intact, purified talin was also phosphorylated by PK-P in a phospholipid-stimulable manner, but at 1/20 the rate of the 47-kD fragment.

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 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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