title: The activity of CK2 in the extracts of COS-7 cells transfected with wild type and mutant subunits of protein kinase CK2.
abstract: Protein kinase CK2 is ubiquitous in eukaryotes and is known to phosphorylate many protein substrates. The enzyme is normally a heterotetramer composed of catalytic (alpha and alpha') and regulatory (beta) subunits. The physiological regulation of the enzyme is still unknown but one of the factors that may play an important role in this regulation is the ratio of the catalytic and regulatory subunits present in cells. The possible existence of 'free' CK2 subunits, not forming part of the holoenzyme, may be relevant to the physiological function of the enzyme in substrate selection or in the interaction of the subunits with other partners. The objective of this work was to study in COS-7 cells the effects of transient expression of CK2 subunits and mutants of the catalytic subunit on the CK2 phosphorylating activity of the extracts of these cells. Using pCEFL vectors that introduce hemagglutinin (HA) or a heptapeptide (AU5) tags in the expressed proteins, COS-7 cells were transfected with alpha and beta subunits of Xenopus CK2, with the alpha' subunit of D. rerio, and with Xl CK2alphaA156, which although inactive can bind tightly to CK2beta, and with Xl CK2alphaE75E76, which is resistant to heparin and polyanion inhibition. The efficiency of transient transfection was of 10-20% of treated cells. Expression of CK2alpha or CK2alphaE75E76 in COS-7 cells caused an increase of 5-7-fold of the CK2 activity in the soluble cell extracts. If these catalytic subunits were cotransfected with CK2beta, the activity increased further to 15-20-fold of the controls. Transfection of CK2beta alone also increase the activity of the extracts about 2-fold. Transfection with the inactive CK2alphaA156 yielded extracts with CK2 activities not significantly different from those transfected with the empty vectors. However, co-transfection of CK2alpha or CK2alphaE75E76 with CK2alphaA156 caused a 60-70% decrease in the CK2 activity as compared to those of cells transfected with only the active CK2alpha subunits. These results can be interpreted as meaning that CK2alphaA156 is a dominant negative mutant that can compete with the other catalytic subunits for the CK2beta subunit. Addition of recombinant CK2beta to the assay system of extracts of cells transfected with catalytic subunits causes a very significant increase in their CK2 activity, demonstrating that CK2beta subunit is limiting in the extracts and that an excess of free CK2alpha has been produced in the transfected cells. Transfection of cells with CK2alphaE75E76 results in a CK2 activity of extracts that is 90% resistant to heparin demonstrating that a very large proportion of the CK2 activity is derived from the expression of the exogenous mutant. In both the in vivo and in vitro systems, the sensitivity of CK2alphaE75E76 to heparin increases considerably when it forms part of the holoenzyme CK2alpha2beta2.
authors.affiliation: Programa de Biología Celular y Molecular, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Santiago.
authors.forename: I
authors.initials: I
authors.lastname: Korn
authors.forename: G
authors.initials: G
authors.lastname: Jacob
authors.forename: C C
authors.initials: CC
authors.lastname: Allende
authors.forename: J E
authors.initials: JE
authors.lastname: Allende
mesh_terms: D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D047390:Casein Kinase II; D020134:Catalytic Domain; D018076:DNA, Complementary / Q000378:metabolism; D005799:Genes, Dominant; D006388:Hemagglutinins / Q000737:chemistry; D006493:Heparin / Q000378:metabolism; D009154:Mutation; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D013997:Time Factors; D014162:Transfection; D014981:Xenopus; D015027:Zebrafish
chemical_list: D018076:DNA, Complementary; D006388:Hemagglutinins; D011994:Recombinant Proteins; D006493:Heparin; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases