The activity of CK2 in the extracts of COS-7 cells transfected with wild type and mutant subunits of protein kinase CK2

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 hemaggutinin (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 CK2 alphaA(156), which although inactive can bind tightly to CK2 beta, and with Xl CK2 alphaE(75)E(76), which is resistant to heparin and polyanion inhibition. The efficiency of transient transfection was of 10-20% of treated cells. Expression of CK2 alpha or CK2 alphaE(75)E(76) 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 CK2 beta, the activity increased further to 15-20-fold of the controls. Transfection of CK2 beta alone also increase the activity of the extracts about 2-fold. Transfection with the inactive CK2 alphaA(156) yielded extracts with CK2 activities not significantly different from those transfected with the empty vectors. However, cotransfection of CK2 alpha or CK2 alphaE(75)E(76) with CK2 alphaA(156) caused a 60-70% decrease in the CK2 activity as compared to those of cells transfected with only the active CK2 alpha subunits. These results can be interpreted as meaning that CK2 alphaA(156) is a dominant negative mutant that can compete with the other catalytic subunits for the CK2 beta subunit. Addition of recombinant CK2 beta to the assay system of extracts of cells transfected with catalytic subunits causes a very significant increase in their CK2 activity, demonstrating that CK2 beta subunit is limiting in the extracts and that an excess of free CK2 alpha has been produced in the transfected cells. Transfection of cells with CK2 alphaE(75)E(76) 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 CK2 alphaE(75)E(76) to heparin increases considerably when it forms part of the holoenzyme CK2 alpha (2)beta (2).