Substrate-effected release of surface-located protein kinase from intact cells.

Kübler, Dieter; Pyerin, Walter; Burow, E; Kinzel, V.

doi:10.1073/pnas.80.13.4021

Cited by 61 publications

(50 citation statements)

References 26 publications

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“…The results for the Western Blot were negative. The results for the cytosolic enzyme assay, which indicate that incubation with phosvitin did not cause any measurable increase in the very low levels of activity of the dehydrogenase, agree with those reported by Kübler et al (2).…”

Section: Control Of Cell Lysissupporting

confidence: 82%

“…Specifically, there are several reports that an ectokinase has the characteristics of protein kinase casein kinase 2 (CK2) (2)(3)(4). A CK2-like ectokinase activity has been reported to be responsible for the phosphorylation of vitronectin, an extracellular matrix protein (5,6), and of the external domain of the ␤-amyloid precursor protein (7) and T lymphocyte surface proteins (8).…”

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confidence: 99%

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Protein kinase casein kinase 2 holoenzyme produced ectopically in human cells can be exported to the external side of the cellular membrane

Rodrı́guez

Allende

2005

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

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Ectokinases can phosphorylate extracellular proteins and external domains of membrane proteins influencing cell adhesion, movement, and cellular interactions. An ectokinase with the properties of casein kinase 2 (CK2) has been previously described, but little is known about the structural characteristics that allow this enzyme to be exported from the cell. Transfection of human embryonic kidney-293 cells with cDNAs coding for the catalytic (CK2␣ or CK2␣ ) and regulatory (CK2␤) subunits with hemaglutinin tags allowed us to study the export of ectopically synthesized enzyme. When the catalytic (CK2␣ or CK2␣ ) and the CK2␤ regulatory subunits are cotransfected, the tetrameric enzyme composed of both subunits (holoenzyme) is detected outside the cell. This observation has been confirmed by assaying protein kinase activity in immunoprecipitates obtained with antihemaglutinin antibody by using a CK2-specific peptide substrate and by Western blots as well as by immunofluorescence of nonpermeabilized cells. Transfection with cDNA of catalytic or regulatory subunit alone does not result in export of these subunits. A study of the kinetics of appearance of the ectopically synthesized protein at different times after transfection indicates that a 5-to 7-h delay after the synthesis of the protein before it appears in the extracellular compartment. Using mutations of CK2␣ that eliminate phosphorylating activity [CK2␣(Asp-156-Ala)] or that make it less sensitive to heparin inhibition [CK2␣(Lys-75-Glu,Lys-76-Glu)] demonstrated that these mutations do not prevent the holoenzyme to be exported from the cells.ectokinase ͉ protein phosphorylation T here are a number of reports that protein kinases are present on the external side of the cellular membrane and that these ectokinases are responsible for phosphorylating proteins of the extracellular matrix and extracellular domains of proteins that are attached to cells (1). Specifically, there are several reports that an ectokinase has the characteristics of protein kinase casein kinase 2 (CK2) (2-4). A CK2-like ectokinase activity has been reported to be responsible for the phosphorylation of vitronectin, an extracellular matrix protein (5, 6), and of the external domain of the ␤-amyloid precursor protein (7) and T lymphocyte surface proteins (8). However, there is no information about the structural features that are responsible for the export of CK2 or other ectokinases.Protein kinase CK2 is ubiquitous in eukaryotes and is responsible for the phosphorylation of hundreds of proteins (9-11). There is strong evidence that CK2 is involved in the control of cell proliferation, apoptosis, and circadian rhythms (12)(13)(14). In addition, CK2 may play a role in controlling the activity of a number of protein kinases through a positive feedback loop with the Cdc37 protein that acts as a chaperone that activates these kinases (15,16).The CK2 holoenzyme is a heterotetramer composed of catalytic subunits (␣ and ␣Ј) and regulatory ␤ subunits conforming ␣ 2 ␤ 2 , ␣␣Ј␤ 2 and ␣Ј 2 ␤ 2 combination...

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Section: Control Of Cell Lysissupporting

confidence: 82%

mentioning

confidence: 99%

Protein kinase casein kinase 2 holoenzyme produced ectopically in human cells can be exported to the external side of the cellular membrane

Rodrı́guez

Allende

2005

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

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“…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.''…”

mentioning

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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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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“…The substrate for cell .,urface protein kinases (ecto-PK) and nucleotidases is M2+ATI~-) z-, and the Km for the enzymatic reaction is in ~he micromolar range [24][25][26]. (iii) Ecto-PK was found in all ,:ells examined, and it mediates the phosphorylation of mem~rane and extracellular proteins [24,[27][28][29], and alters the res~onse of the cell to extracellular ATP [26].…”

Section: Discussionmentioning

confidence: 99%

Combined effects of ATP and its analogs on the membrane permeability in transformed mouse fibroblasts

Arav

Friedberg

1996

FEBS Letters

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Extracellular ATP (0.6 mM) induces a marked decrease in the membrane potential, followed by an increase in cell membrane permeability in transformed mouse fibroblasts. The effects of the ATP analogs, pICHaIppA and p[NH]ppA (0.6 m VI), on the membrane potential and permeability are much less pronounced. ATP at 0.05 mM has no effect by itself, but markedly increases the analog-induced membrane potential di~ipation and permeability. The data suggest that ATP-induced membrane permeation is composed of two processes: One is common to ATP and its analogs and appears to be a receptormediated process. The second is unique for ATP, effective even at low concentration (0.05 mM), and might be mediated by cell surface enzymes, for which ATP, but not its analogs, serves as a substrate.

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Substrate-effected release of surface-located protein kinase from intact cells.

Cited by 61 publications

References 26 publications

Protein kinase casein kinase 2 holoenzyme produced ectopically in human cells can be exported to the external side of the cellular membrane

Protein kinase casein kinase 2 holoenzyme produced ectopically in human cells can be exported to the external side of the cellular membrane

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

Combined effects of ATP and its analogs on the membrane permeability in transformed mouse fibroblasts

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