I. Korn et al., The activity of CK2 in the extracts of COS-7 cells transfected with wild type and mutant subunits of protein kinase CK2, MOL C BIOCH, 227(1-2), 2001, pp. 37-44
Protein kinase CK2 is ubiquitous in eukaryotes and is known to phosphorylat
e many protein substrates. The enzyme is normally a heterotetramer composed
of catalytic (alpha and alpha') and regulatory (beta) subunits. The physio
logical regulation of the enzyme is still unknown but one of the factors th
at may play an important role in this regulation is the ratio of the cataly
tic and regulatory subunits present in cells. The possible existence of 'fr
ee' CK2 subunits, not forming part of the holoenzyme, may be relevant to th
e physiological function of the enzyme in substrate selection or in the int
eraction of the subunits with other partners. The objective of this work wa
s to study in COS-7 cells the effects of transient expression of CK2 subuni
ts and mutants of the catalytic subunit on the CK2 phosphorylating activity
of the extracts of these cells. Using pCEFL vectors that introduce hemaggu
tinin (HA) or a heptapeptide (AU5) tags in the expressed proteins, COS-7 ce
lls 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 ina
ctive 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 transie
nt transfection was of 10-20% of treated cells.
Expression of CK2 alpha or CK2 alphaE(75)E(76) in COS-7 cells caused an inc
rease of 5-7-fold of the CK2 activity in the soluble cell extracts. If thes
e catalytic subunits were cotransfected with CK2 beta, the activity increas
ed further to 15-20-fold of the controls. Transfection of CK2 beta alone al
so increase the activity of the extracts about 2-fold. Transfection with th
e inactive CK2 alphaA(156) yielded extracts with CK2 activities not signifi
cantly different from those transfected with the empty vectors. However, co
transfection of CK2 alpha or CK2 alphaE(75)E(76) with CK2 alphaA(156) cause
d a 60-70% decrease in the CK2 activity as compared to those of cells trans
fected with only the active CK2 alpha subunits. These results can be interp
reted as meaning that CK2 alphaA(156) is a dominant negative mutant that ca
n compete with the other catalytic subunits for the CK2 beta subunit. Addit
ion of recombinant CK2 beta to the assay system of extracts of cells transf
ected with catalytic subunits causes a very significant increase in their C
K2 activity, demonstrating that CK2 beta subunit is limiting in the extract
s 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 act
ivity of extracts that is 90% resistant to heparin demonstrating that a ver
y large proportion of the CK2 activity is derived from the expression of th
e exogenous mutant. In both the in vivo and in vitro systems, the sensitivi
ty of CK2 alphaE(75)E(76) to heparin increases considerably when it forms p
art of the holoenzyme CK2 alpha (2)beta (2).