Activity of the E75E76 mutant of the α subunit of casein kinase II from Xenopus laevis

Gatica, Marta; Jedlicki, Ana; Allende, Catherine C.; Allende, Jorge E.

doi:10.1016/0014-5793(94)80392-7

Cited by 23 publications

(22 citation statements)

References 15 publications

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“…The effect of this mutation is actually comparable to that induced by the mutation of just K74 and K75 inside the 74-77 stretch [17], although a precise comparison is hampered by the different substrate (casein) used in that study.…”

Section: Resultsmentioning

confidence: 81%

“…With one of these, RRKDLHDDEEDEEMSETADGE, lacking the crucial acidic determinant at position +3, but still having the one at position + 1, no stimulation occurs while a slight inhibition is observable reaching about 40% with 120 j~g/ml heparin. The notion that the 74-80 basic stretch unique to CK2 is implicated in heparin inhibition was already provided by double mutations of K74/K75 [16] and K75/K76 [17] giving rise to enzymes defective in this property. Interestingly however these mutations caused a 70-and 13-fold increase of ICs0 values, respectively, but they did not suppress heparin inhibition as it is observed with the quadruple mutated K74-77A mutant used here.…”

Section: Resultsmentioning

confidence: 99%

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Mapping the residues of protein kinase CK2 α subunit responsible for responsiveness to polyanionic inhibitors

et al. 1996

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The quadruple mutation of the whole basic cluster, K74KKK 77 conserved in the catalytic subunits of protein kinase CK2 and implicated in substrate recognition, not only abolishes inhibition by heparin but even induces with some peptide substrates an up to 5-fold stimulation by heparin in the 0.5-5/tg/ml concentration range. Two other mutants defective in substrate recognition, R191,195K198A and K79R80K83A, display either a 100-fold reduction or no alteration at all in heparin inhibition, respectively. In contrast sensitivity to heparin inhibition is increased 30-fold by a single mutation affecting Arg-228 while it is not altered by a triple mutation in the small insert of subdomain XI (mutant R278K279R280A). The effect of the same mutations on inhibition by pseudosubstrate EEEEEYEEEEEEE is different, the mutant displaying the most reduced sensitivity being R191,195K198A, followed by K74-77A and K79R80K83A; the other mutants are almost indistinguishable from CK2 wild type. Substantial reduction of inhibition by poly(Glu,Tyr)4:l is only observable with mutant R191,195K198A, whereas R228A is significantly more sensitive to inhibition. These data show that the mode of inhibition of CK2 by polyanionic compounds occurs through substantially different mechanisms involving residues that are variably concerned with substrate recognition.

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Section: Resultsmentioning

confidence: 81%

Section: Resultsmentioning

confidence: 99%

Mapping the residues of protein kinase CK2 α subunit responsible for responsiveness to polyanionic inhibitors

et al. 1996

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“…The CK2 activity of ectopically expressed CK2 subunits is assayed in the lysates (A) and extracellular liquid (B) of cells transfected with HA-tagged subunits. In this experiment, transfection of the mutant CK2␣(Lys-75-Glu,Lys-76-Glu) (shown as HA-EE), which is resistant in vitro to heparin inhibition (25), was included either alone or in combination with CK2␤. The activity was measured in the presence of heparin (1 g͞ml) or in its absence as indicated.…”

Section: Discussionmentioning

confidence: 99%

“…A second mutant, CK2␣(Lys-75-Glu,Lys-76-Glu), which was described by our laboratory several years ago (25), has Lys-75 and Lys-76 changed to Glu. This mutation affects a basic region of CK2␣ that is important for binding heparin and other polyanion inhibitors, making it more resistant to heparin inhibition.…”

Section: Discussionmentioning

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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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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“…The changes produced neither a significant increase in the K m of the CK2 subunit for the casein and model peptide substrates nor changes in the affinity of the mutated CK2 subunit for the CK2 subunit during assembling a fully competent CK2 holoenzyme. The same mutations, however, had a significant effect on the affinity of CK2 for heparin and for other polyanionic inhibitors (Hu & Rubin, 1990;Gatica at al., 1994). Complete suppression of heparin inhibition was observed with the quadruple mutated K74-77A CK2 used by Vaglio and collaborators (1996).…”

Section: Mutation Of Ck2α In the Basic Regionsmentioning

confidence: 99%

Site-Directed Mutagenesis in the Research of Protein Kinases - The Case of Protein Kinase CK2

Sajnaga¹,

Szyszka²,

Kubiński³

2013

Genetic Manipulation of DNA and Protein - Examples From Current Research

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Activity of the E⁷⁵E⁷⁶ mutant of the α subunit of casein kinase II from Xenopus laevis

Cited by 23 publications

References 15 publications

Mapping the residues of protein kinase CK2 α subunit responsible for responsiveness to polyanionic inhibitors

Mapping the residues of protein kinase CK2 α subunit responsible for responsiveness to polyanionic inhibitors

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

Site-Directed Mutagenesis in the Research of Protein Kinases - The Case of Protein Kinase CK2

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