Subunit structure and autophosphorylation mechanism of casein kinase-TS (type-2) from rat liver cytosol

Abstract

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 α₂β₂ heterotetramer. The quaternary structure of Ck‐TS is not compromised by limited proteolysis with trypsin which converts the 38‐kDa α subunit into 36‐kDa (α′) and 34‐kDa (α”) derivatives, inducing a parallel decrease of enzymatic activity. Since the 25‐kDa β subunit is unaffected under comparable conditions, the catalytic activity seemingly resides in the α subunits. The β 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 K_m 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.