Mass Spectrometry Analysis of a Protein Kinase CK2β Subunit Interactome Isolated from Mouse Brain by Affinity Chromatography

Arrigoni, Giorgio; Pagano, Mario A.; Sarno, Stefania; Cesaro, Luca; James, Peter; Pinna, Lorenzo A.

doi:10.1021/pr070500s

Cited by 34 publications

(32 citation statements)

References 100 publications

(173 reference statements)

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“…As seen in Fig. 1B, mutant Myc-CK2␤ F21F22-A contains F 21 and F 22 mutated to alanine, and mutant Myc-CK2␤ D26E27D28-A has D 26 , E 27 , and D 28 mutated to alanine. These mutants were transfected into cells ( Fig.…”

Section: Resultsmentioning

confidence: 87%

“…It is pertinent therefore to ask whether these other kinases can also be carried out of cells by this regulatory subunit and by this mechanism they may function as exo or ectokinases. CK2␤ has also been shown to interact with a large number of other proteins, many of which are substrates of CK2 (21). It is possible that these proteins may also be exported through the binding of CK2␤.…”

Section: Resultsmentioning

confidence: 99%

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Protein kinase CK2 as an ectokinase: The role of the regulatory CK2β subunit

Rodrı́guez

Contreras

Bolaños-García

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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Protein kinase CK2 (also known as casein kinase 2) is present in the cytoplasm, nuclei, and several other organelles. In addition, this enzyme has been found bound to the external side of the cell membrane where it acts as an ectokinase phosphorylating several extracellular proteins. Previous experiments with transfection of HEK-293T cells demonstrated that expression of both subunits, CK2␣ (catalytic) and CK2␤ (regulatory), was necessary for the appearance of the ectopic enzyme as an ectokinase. In this work, using deletion and point mutations of CK2␤, it was possible to demonstrate that the region between amino acids 20 and 33 was necessary for the export of the enzyme as an ectokinase. Phenylalanines 21 and 22 and acidic residues in positions 26 -28 are involved in the structural aspects that are required for export. However, the region encompassing amino acids 20 -33 of CK2␤ is not sufficient to make the carboxyl half of this subunit functional in bringing CK2 to the ectokinase locus. In cells transfected with only CK2␤, it was demonstrated that 3-4% of the subunit is exported to the cell medium, but the subunit is not bound to the external membrane.casein kinase 2 ͉ export of proteins ͉ shedding I t has been known for a number of years that protein kinase CK2 (formerly called casein kinase 2) is present in the cytoplasm, nuclei, and several other cell organelles. It has also been found on the external side of the cellular membrane where, acting as an ectokinase, it can phosphorylate extracellular proteins and external domains of proteins. Kubler et al. (1) originally discovered that incubation of cells with CK2 substrates such as casein or phosvitin resulted in the liberation of the enzyme activity into the medium. This substrate-mediated release from the cell membrane was called ''shedding.'' Several extracellular proteins have been identified as substrates of the CK2 ectokinase activity. Vitronectin has been shown to be phosphorylated extracellularly by CK2, and evidence has been presented to demonstrate that its phosphorylation regulates the adhesion of cells to the extracellular matrix (2, 3). More recently, it has been shown that CK2 phosphorylates the C9 complement and it has been suggested that this phosphorylation might control cell lysis caused by this complement protein (4). Another recent example of an extracellular domain phosphorylated by ecto CK2 is the collagen XVII receptor. This phosphorylation may inhibit its degradation catalyzed by metaloproteases (5).In our previous work (6), we explored the conditions and requirements necessary to observe the presence of ectopically expressed CK2 holoenzyme that could be obtained after shedding of human cells in culture that had been transfected with the cDNAs coding for the two CK2 subunits. This work demonstrated that transfection with both catalytic (CK2␣) and regulatory (CK2␤) subunits was necessary to detect ectopically expressed CK2 bound externally to the cellular membrane. It was further shown that the export outside the cell did not require c...

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Section: Resultsmentioning

confidence: 87%

Section: Resultsmentioning

confidence: 99%

Protein kinase CK2 as an ectokinase: The role of the regulatory CK2β subunit

Rodrı́guez

Contreras

Bolaños-García

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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“…BN-PAGE gel bands containing the monomeric and dimeric forms of ATP synthase isolated from the different yeast strains were digested with trypsin as described in [24]. Briefly, gel bands were cut into small pieces, and treated for 1 h at 56°C with 10 mM dithiothreitol (Fluka) in 50 mM NH 4 HCO 3 .…”

Section: Protein Digestion and Mass Spectrometry Analysismentioning

confidence: 99%

High-Conductance Channel Formation in Yeast Mitochondria is Mediated by F-ATP Synthase e and g Subunits

Carraro

Checchetto

Sartori

et al. 2018

Cell Physiol Biochem

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Background/Aims: The permeability transition pore (PTP) is an unselective, Ca²⁺-dependent high conductance channel of the inner mitochondrial membrane whose molecular identity has long remained a mystery. The most recent hypothesis is that pore formation involves the F-ATP synthase, which consistently generates Ca²⁺-activated channels. Available structures do not display obvious features that can accommodate a channel; thus, how the pore can form and whether its activity can be entirely assigned to F-ATP synthase is the matter of debate. In this study, we investigated the role of F-ATP synthase subunits e, g and b in PTP formation. Methods: Yeast null mutants for e, g and the first transmembrane (TM) α-helix of subunit b were generated and evaluated for mitochondrial morphology (electron microscopy), membrane potential (Rhodamine123 fluorescence) and respiration (Clark electrode). Homoplasmic C23S mutant of subunit a was generated by in vitro mutagenesis followed by biolistic transformation. F-ATP synthase assembly was evaluated by BN-PAGE analysis. Cu²⁺ treatment was used to induce the formation of F-ATP synthase dimers in the absence of e and g subunits. The electrophysiological properties of F-ATP synthase were assessed in planar lipid bilayers. Results: Null mutants for the subunits e and g display dimer formation upon Cu²⁺ treatment and show PTP-dependent mitochondrial Ca²⁺ release but not swelling. Cu²⁺ treatment causes formation of disulfide bridges between Cys23 of subunits a that stabilize dimers in absence of e and g subunits and favors the open state of wild-type F-ATP synthase channels. Absence of e and g subunits decreases conductance of the F-ATP synthase channel about tenfold. Ablation of the first TM of subunit b, which creates a distinct lateral domain with e and g, further affected channel activity. Conclusion: F-ATP synthase e, g and b subunits create a domain within the membrane that is critical for the generation of the high-conductance channel, thus is a prime candidate for PTP formation. Subunits e and g are only present in eukaryotes and may have evolved to confer this novel function to F-ATP synthase.

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“…Temporal changes in the local concentration or in the relative affinities of the partners could be instrumental in dictating the segregation of individual CK2 subunits to different places. CK2b, in particular is a promiscuous protein interacting with a myriad of intracellular proteins, further implying that this subunit can exist independently of CK2a [76]. Indeed, localization and translocation are often mediated by modular proteins, and as such CK2b can generate multiple CK2-containing complexes.…”

Section: Functional Consequences Of Ck2 Dynamicsmentioning

confidence: 99%

Protein Kinase CK2 in Health and Disease

Filhol

Cochet

2009

Cell. Mol. Life Sci.

114

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Protein kinase CK2 targets a vast array of substrates located in a number of cellular compartments, making the challenge of discriminating among these substrates a daunting task. However, as a signaling protein, CK2 could be targeted to different cellular compartments in response to various stress stimuli such as heat shock, UV irradiation, hypoxia, DNA damage and viral infections. This review will be focused on the evidence that the dynamic association of CK2 subunits and the substrate-dependent subcellular targeting of the enzyme are a likely point of regulation in response to a variety of signaling events. We propose that in addition to enzymatic substrate recognition, regulated CK2 localization to specific compartments should help to provide the exquisite specificity required for robust signal transduction.

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Mass Spectrometry Analysis of a Protein Kinase CK2β Subunit Interactome Isolated from Mouse Brain by Affinity Chromatography

Cited by 34 publications

References 100 publications

Protein kinase CK2 as an ectokinase: The role of the regulatory CK2β subunit

Protein kinase CK2 as an ectokinase: The role of the regulatory CK2β subunit

High-Conductance Channel Formation in Yeast Mitochondria is Mediated by F-ATP Synthase e and g Subunits

Protein Kinase CK2 in Health and Disease

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