scholarly journals Phosphorylation by Protein Kinase CK2 Changes the DNA Binding Properties of the Human Chromatin Protein DEK

2004 ◽  
Vol 24 (13) ◽  
pp. 6011-6020 ◽  
Author(s):  
Ferdinand Kappes ◽  
Catalina Damoc ◽  
Rolf Knippers ◽  
Michael Przybylski ◽  
Lorenzo A. Pinna ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Protein Kinase ◽  
Protein Kinase Ck2 ◽  
Ion Trap ◽  
Quadrupole Ion Trap ◽  
Chromatin Protein ◽  
Binding Assays ◽  
Kinase Ck2

ABSTRACT We have examined the posttranslational modification of the human chromatin protein DEK and found that DEK is phosphorylated by the protein kinase CK2 in vitro and in vivo. Phosphorylation sites were mapped by quadrupole ion trap mass spectrometry and found to be clustered in the C-terminal region of the DEK protein. Phosphorylation fluctuates during the cell cycle with a moderate peak during G1 phase. Filter binding assays, as well as Southwestern analysis, demonstrate that phosphorylation weakens the binding of DEK to DNA. In vivo, however, phosphorylated DEK remains on chromatin. We present evidence that phosphorylated DEK is tethered to chromatin throughout the cell cycle by the un- or underphosphorylated form of DEK.

Protein Kinase CK2 Regulates Leukocyte-Endothelial Cell Interactions during Ischemia and Reperfusion in Striated Skin Muscle

2016 ◽  
Vol 57 (1-2) ◽  
pp. 111-124 ◽  
Author(s):  
Emmanuel Ampofo ◽  
Daniela Widmaier ◽  
Mathias Montenarh ◽  
Michael D. Menger ◽  
Matthias W. Laschke
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Protein Kinase ◽  
Protein Kinase Ck2 ◽  
Tissue Injury ◽  
Cell Interactions ◽  
Ischemia And Reperfusion ◽  
Kinase Ck2

Background: Ischemia and reperfusion (I/R) causes tissue injury by inflammatory processes. This involves the upregulation of endothelial surface proteins by phospho-regulated signaling pathways, resulting in enhanced interactions of leukocytes with endothelial cells. Recently, we found that protein kinase CK2 is a crucial regulator of leukocyte-mediated inflammation. Therefore, in this study we investigated the involvement of CK2 in leukocyte-endothelial cell interactions during I/R injury. Methods: We first analyzed the inhibitory action of (E)-3-(2,3,4,5-tetrabromophenyl)acrylic acid (TBCA) and CX-4945 on CK2 kinase activity and the viability of human dermal microvascular endothelial cells (HDMEC). To mimic I/R conditions in vitro, HDMEC were exposed to hypoxia and reoxygenation and the expression of adhesion molecules was analyzed by flow cytometry. Moreover, we analyzed in vivo the effect of CK2 inhibition on leukocyte-endothelial cell interactions in the dorsal skinfold chamber model of I/R injury by means of repetitive intravital fluorescence microscopy and immunohistochemistry. Results: We found that TBCA and CX-4945 suppressed the activity of CK2 in HDMEC without affecting cell viability. This was associated with a significant downregulation of E-selectin and intercellular adhesion molecule (ICAM)-1 after in vitro hypoxia and reoxygenation. In vivo, CX-4945 treatment significantly decreased the numbers of adherent and transmigrated leukocytes in striated muscle tissue exposed to I/R. Conclusion: Our findings indicate that CK2 is involved in the regulation of leukocyte-endothelial cell interactions during I/R by mediating the expression of E-selectin and ICAM-1.

scholarly journals Effect of etimizole structural analogues on protein kinase CK2, protein phosphorylation and transcription of chromatin in rat brain cortex and hippocampus

2020 ◽  
Vol 66 (2) ◽  
pp. 130-137
Author(s):  
B.A. Reikhardt ◽  
P.D. Shabanov
Keyword(s):  
Protein Kinase ◽  
Rat Brain ◽  
Protein Kinase Ck2 ◽  
Long Term Memory ◽  
Term Memory ◽  
Structural Analogues ◽  
Kinase Ck2

Protein kinase CK2 is an important enzyme in the nervous system. The nuclear forms of CK2 regulate chromatin structure and gene expression, the key processes for long-term memory formation. Memory modulators, the Structural Analogues of Etimizole (SAE), were able to increase or decrease the activity of chromatin-associated CK in the cortex and hippocampus of rat brain in vitro. In vivo memory enhancers from SAE-group (3 mg/kg) stimulated CK2 activity and the transcriptional ability of chromatin in the cortex and hippocampus, starting from 30 min with a peak for 60 min and a duration up to 180 min. At these periods the memory inhibitor from the SAE-group reduced CK2 activity and chromatin transcription. It is assumed that the modulating effect of SAE on CK2 activity and transcription underlies the effects of these compounds on long-term memory.

scholarly journals Dynamic Localization of the Swe1 Regulator Hsl7 During theSaccharomyces cerevisiae Cell Cycle

2001 ◽  
Vol 12 (6) ◽  
pp. 1645-1669 ◽  
Author(s):  
Victor J. Cid ◽  
Mark J. Shulewitz ◽  
Kent L. McDonald ◽  
Jeremy Thorner
Keyword(s):  
Cell Cycle ◽  
Protein Kinase ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Fine Tuning ◽  
Dependent Manner ◽  
Binding Assays ◽  
Bud Neck

In Saccharomyces cerevisiae, entry into mitosis requires activation of the cyclin-dependent kinase Cdc28 in its cyclin B (Clb)-associated form. Clb-bound Cdc28 is susceptible to inhibitory tyrosine phosphorylation by Swe1 protein kinase. Swe1 is itself negatively regulated by Hsl1, a Nim1-related protein kinase, and by Hsl7, a presumptive protein-arginine methyltransferase. In vivo all three proteins localize to the bud neck in a septin-dependent manner, consistent with our previous proposal that formation of Hsl1-Hsl7-Swe1 complexes constitutes a checkpoint that monitors septin assembly. We show here that Hsl7 is phosphorylated by Hsl1 in immune-complex kinase assays and can physically associate in vitro with either Hsl1 or Swe1 in the absence of any other yeast proteins. With the use of both the two-hybrid method and in vitro binding assays, we found that Hsl7 contains distinct binding sites for Hsl1 and Swe1. A differential interaction trap approach was used to isolate four single-site substitution mutations in Hsl7, which cluster within a discrete region of its N-terminal domain, that are specifically defective in binding Hsl1. When expressed in hsl7Δ cells, each of these Hsl7 point mutants is unable to localize at the bud neck and cannot mediate down-regulation of Swe1, but retains other functions of Hsl7, including oligomerization and association with Swe1. GFP-fusions of these Hsl1-binding defective Hsl7 proteins localize as a bright perinuclear dot, but never localize to the bud neck; likewise, inhsl1Δ cells, a GFP-fusion to wild-type Hsl7 or native Hsl7 localizes to this dot. Cell synchronization studies showed that, normally, Hsl7 localizes to the dot, but only in cells in the G1 phase of the cell cycle. Immunofluorescence analysis and immunoelectron microscopy established that the dot corresponds to the outer plaque of the spindle pole body (SPB). These data demonstrate that association between Hsl1 and Hsl7 at the bud neck is required to alleviate Swe1-imposed G2-M delay. Hsl7 localization at the SPB during G1 may play some additional role in fine-tuning the coordination between nuclear and cortical events before mitosis.

scholarly journals Protein kinase CK2 phosphorylates Hsp105alpha at Ser509 and modulates its function

2003 ◽  
Vol 371 (3) ◽  
pp. 917-925 ◽  
Author(s):  
Keiichi ISHIHARA ◽  
Nobuyuki YAMAGISHI ◽  
Takumi HATAYAMA
Keyword(s):  
Heat Shock ◽  
Protein Kinase ◽  
Mammalian Cells ◽  
Protein Kinase Ck2 ◽  
Peptide Mapping ◽  
Stress Protein ◽  
Cellular Events ◽  
Kinase Ck2

The 105 kDa heat-shock protein (Hsp) Hsp105α is a mammalian stress protein that belongs to the HSP105/HSP110 family. We have shown previously that Hsp105α exists as non-phosphorylated and phosphorylated forms in vivo, and is phosphorylated by protein kinase CK2 (CK2) in vitro. In this study, to elucidate the role of phosphorylation of Hsp105α, we first analysed the site of phosphorylation of Hsp105α by CK2. Peptide mapping analysis of Hsp105α phosphorylated by CK2 and in vitro phosphorylation experiments using various deletion and substitution mutants of Hsp105α revealed that Hsp105α is phosphorylated at Ser509 in the β-sheet domain. Furthermore, Ser509 in Hsp105α was also phosphorylated in mammalian COS-7 cells, although other sites were phosphorylated as well. Next, we examined the effects of phosphorylation of Hsp105α on its functions using CK2-phosphorylated Hsp105α. Interestingly, Hsp105α suppressed 70 kDa heat-shock cognate protein (Hsc70)-mediated protein folding, whereas the phosphorylation of Hsp105α at Ser509 abolished the inhibitory activity of Hsp105α in vitro. In accordance with these findings, wild-type Hsp105α, which was thought to be phosphorylated in vivo, had no effect on Hsp70-mediated refolding of heat-denatured luciferase, whereas a non-phosphorylatable mutant of Hsp105α suppressed the Hsp70-mediated refolding of heat-denatured luciferase in mammalian cells. Thus it was suggested that CK2 phosphorylates Hsp105α at Ser509 and modulates the function of Hsp105α. The regulation of Hsp105α function by phosphorylation may play an important role in a variety of cellular events.

Assembly of protein kinase CK2: investigation of complex formation between catalytic and regulatory subunits using a zinc-finger-deficient mutant of CK2β

2001 ◽  
Vol 358 (1) ◽  
pp. 87-94 ◽  
Author(s):  
David A. CANTON ◽  
Cunjie ZHANG ◽  
David W. LITCHFIELD
Keyword(s):  
Protein Kinase ◽  
Zinc Finger ◽  
Protein Kinase Ck2 ◽  
Deficient Mutant ◽  
Binding Assays ◽  
Catalytic Subunits ◽  
Regulatory Subunits ◽  
Regulatory Ck2β Subunit ◽  
Kinase Ck2

Protein kinase CK2 is a tetrameric enzyme comprised of two regulatory subunits (CK2β) and two catalytic subunits (CK2α and/or CK2α′). The crystal structure of dimeric CK2β demonstrated that a zinc finger mediates CK2β dimerization, therefore we constructed a mutant in which cysteine residues 109 and 114 were mutated to serine. Our objectives were to examine the effects of disrupting the zinc finger of the regulatory CK2β subunit on CK2 tetramer assembly. Examination of this zinc-finger-deficient mutant of CK2β using a yeast two-hybrid assay demonstrates that the mutant fails to form CK2β homodimers. In order to extend these studies, we co-transfected COS-7 cells with epitope-tagged constructs and performed co-immunoprecipitation assays. The results from these studies demonstrate that the mutant fails to form CK2β homodimers and fails to interact with catalytic CK2 subunits. Furthermore, we demonstrate that the mutant CK2β is not appreciably phosphorylated in cells. Using in vitro binding assays, we demonstrated that the mutant CK2β protein fails to interact with glutathione S-transferase–CK2α′. Finally, we demonstrate that the mutant is translated at an equivalent rate to wild-type CK2β, but is degraded much more rapidly. Overall, our results are consistent with the model that β–β dimerization precedes incorporation of catalytic subunits into tetrameric CK2 complexes, and that β–β dimerization is a prerequisite for the stable incorporation of catalytic subunits into CK2 complexes.

In Vitro and In Vivo Assays of Protein Kinase CK2 Activity

2010 ◽  
pp. 597-610 ◽  
Author(s):  
Renaud Prudent ◽  
Céline F. Sautel ◽  
Virginie Moucadel ◽  
Béatrice Laudet ◽  
Odile Filhol ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinase Ck2 ◽  
In Vivo Assays ◽  
Kinase Ck2

scholarly journals Inhibition of Protein Kinase CK2 by Condensed Polyphenolic Derivatives. An in Vitro and in Vivo Study†

Biochemistry ◽  
2004 ◽  
Vol 43 (40) ◽  
pp. 12931-12936 ◽  
Author(s):  
Flavio Meggio ◽  
Mario A. Pagano ◽  
Stefano Moro ◽  
Giuseppe Zagotto ◽  
Maria Ruzzene ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinase Ck2 ◽  
In Vivo Study ◽  
Kinase Ck2

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 Protein Kinase C Signaling Mediates a Program of Cell Cycle Withdrawal in the Intestinal Epithelium

2000 ◽  
Vol 151 (4) ◽  
pp. 763-778 ◽  
Author(s):  
Mark R. Frey ◽  
Jennifer A. Clark ◽  
Olga Leontieva ◽  
Joshua M. Uronis ◽  
Adrian R. Black ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Intestinal Epithelium ◽  
Molecular Mechanisms ◽  
Model Systems ◽  
Intestinal Epithelial ◽  
Kinase C

Members of the protein kinase C (PKC) family of signal transduction molecules have been widely implicated in regulation of cell growth and differentiation, although the underlying molecular mechanisms involved remain poorly defined. Using combined in vitro and in vivo intestinal epithelial model systems, we demonstrate that PKC signaling can trigger a coordinated program of molecular events leading to cell cycle withdrawal into G0. PKC activation in the IEC-18 intestinal crypt cell line resulted in rapid downregulation of D-type cyclins and differential induction of p21waf1/cip1 and p27kip1, thus targeting all of the major G1/S cyclin-dependent kinase complexes. These events were associated with coordinated alterations in expression and phosphorylation of the pocket proteins p107, pRb, and p130 that drive cells to exit the cell cycle into G0 as indicated by concomitant downregulation of the DNA licensing factor cdc6. Manipulation of PKC isozyme levels in IEC-18 cells demonstrated that PKCα alone can trigger hallmark events of cell cycle withdrawal in intestinal epithelial cells. Notably, analysis of the developmental control of cell cycle regulatory molecules along the crypt–villus axis revealed that PKCα activation is appropriately positioned within intestinal crypts to trigger this program of cell cycle exit–specific events in situ. Together, these data point to PKCα as a key regulator of cell cycle withdrawal in the intestinal epithelium.

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