Subunit structure and autophosphorylation mechanism of casein kinase-TS (type-2) from rat liver cytosol.
Type-2 casein kinase-TS (Ck-TS) purified to homogeneity from rat liver cytosol exhibits a molecular mass of 130000 daltons in non-denaturating media and a subunit composition consistent with an alpha 2 beta 2 heterotetramer. The quaternary structure of Ck-TS is not compromised by limited proteolysis with trypsin which converts the 38-kDa alpha subunit into 36-kDa (alpha') and 34-kDa (alpha") derivatives, inducing a parallel decrease of enzymatic activity. Since the 25-kDa beta subunit is unaffected under comparable conditions, the catalytic activity seemingly resides in the alpha subunits. The beta subunit, on the other hand, undergoes a very rapid phosphorylation upon incubation of Ck-TS with ATP/Mg2+: 0.8-1.5 mol P/mol Ck-TS are incorporated within 30 s. Such a fast autophosphorylation is neither prevented nor slowed down by the addition of a large excess of phosphorylatable substrates and takes place through an intra-molecular rather than inter-molecular process. This conclusion is supported by the following data. (a) The autophosphorylation rate is linearly proportional to the concentration of Ck-TS. (b) Thermally inactivated Ck-TS is not phosphorylated by catalytic amounts of active enzyme. (c) Basic polypeptides like protamine and polylysine stimulate the activity of Ck-TS toward phosphorylatable substrates while preventing the autophosphorylation reaction. Since the effectors that inhibit autophosphorylation also induce a remarkable decrease of the Km values for the protein substrates, the possibility is discussed that autophosphorylation might represent a regulatory device by which Ck-TS could be converted into a partially inactivated form exhibiting reduced affinity toward its endogenous targets.
D000818:Animals; D047388:Casein Kinases; D002499:Centrifugation, Density Gradient; D055598:Chemical Phenomena; D002621:Chemistry; D003600:Cytosol / Q000201:enzymology; D006868:Hydrolysis; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D010446:Peptide Fragments / Q000032:analysis; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D051381:Rats
D010446:Peptide Fragments; D011494:Protein Kinases; D047388:Casein Kinases