Adenosine-3': 5'-Monophosphate-Binding Proteins from Bovine Kidney. Isolation by Affinity Chromatography and Limited Proteolysis of the Regulatory Subunit of Protein Kinase II

Abstract

Affinity chromatography on cyclic AMP columns allowed a two-step isolation of the cyclic-AMP-binding proteins from bovine kidney cytosol. An AMP-binding protein (apparent molecular weight zz 60000) and large amounts of a low affinity binding protein ('P35'; apparent subunit size z 35000) were obtained in practically pure form besides the high affinity binding proteins of the R type. Among the R proteins the dimer RZ of the regulatory subunit of protein kinase I1 (apparent subunit size z 54000) represented the bulk material. Small amounts of monomer, of higher aggregates, and of a protein 'P49' (subunit size ~4 9 0 0 0 ) presumably identical with the regulatory subunit of protein kinase I were also detected.The R protein fraction of kidney also contained a high affinity binding protein of smaller size (designated as R'; molecular weight z 37000) which appeared to be derived from protein Rz of protein kinase I1 by limited proteolysis. At all stages of purification, R protein and its aggregates could be quantitatively transformed into R' protein (or a closely related polypeptide) by several proteases including the relatively unspecific proteinase K. The degradation product exhibited unchanged cyclic-AMP-binding capacities but had largely lost the ability to inhibit the catalytic subunit C of protein kinase, to be phosphorylated by C, and to form a dimer. Preliminary experiments indicate that protein R' may be a natural component of kidney tissue.Mammalian tissues contain cyclic-AMP-dependent protein kinases which are composed of catalytic (C) and regulatory (R) subunits. Dissociation of the holoenzymes occurs by binding of cyclic AMP to the regulatory R proteins (for a recent review see [l]). Two types of protein kinases (type I and type 11) have been found in many tissues [2] which appear to possess identical catalytic subunits, but differ in their regulatory subunits (cf. [l]). The detection of additional cyclic-AMP-binding proteins in mammalian tissues ~~

Degradative inactivation of cyclic AMP-dependent protein kinase by a membranal proteinase is restricted to the free catalytic subunit in its native conformation.

“…Alternatively, the specific degradation of C might be a means of transferring a unique physiological stimulus if, for example, one or more of the degradation products will be found to possess an important function (catalysis, inhibition, activation, etc). In any case, the fact that the membranal enzyme exhibits such a specific, restricted, and limited action on C, the fact that the biodegradative inactivation takes place when C is in its free form but not in its R2C2 (inactive or "stored") form, the fact that it degrades C only when it is in its native conformation, and the fact that the proteinase is found in a membrane whose function (fluid and electrolyte secretion) is known to be t The observation that free R is susceptible to degradation by a variety of proteinases is in agreement with reports from other laboratories (23,24). …”

A new cAMP affinity matrix for the rapid purification of protein kinase regulatory subunits

“…Residual R protein may be desorbed by prolonged elution with 30 mM CAMP, or with 6 M urea/l 2 mM methylamine in TG buffer (10 mM TrisHCl, 1 mM EDTA, 6 mM fl-mercaptoethanol, 10% glycerol (pH 7.4, cf. [4]). Excess CAMP in the fractions was removed by dialysis or, especially when rapid determination of CAMP binding capacity was indicated, by double charcoal treatment (see below).…”

“…1 mM CAMP. In some tissues, this procedure separated out contaminating low affinity binding proteins [4]. The regulatory subunit of protein kinase II was eluted by slow passage of 30 mM CAMP at room temperature.…”

“…Isolation of its regulatory subunit R II by affinity chromatography requires prior separation of other high affinity CAMP-binding proteins like R I or the products of limited proteolysis (cf. [4,14]. A clear separation of protein kinase II from type I holoenzyme was achieved by fractionation on DEAE cellulose.…”

A constitutively active holoenzyme form of the cAMP-dependent protein kinase.

“…Weber and Hilz (18,19) demonstrated that an RII fragment generated by chymotryptic cleavage at the hinge region, which still contained the arginine residues, was capable of inhibiting the C subunit and undergoing autophosphorylation but that a similar fragment lacking the arginine residues obtained after tryptic digestion lost the ability to undergo autophosphorylation and to inhibit the C subunit. By using a monoclonal antibody to detect the stable R-C complex, Weldon and Taylor (20) showed that removal of the first 90 residues by chymotryptic digestion did not affect the ability of the remaining RII fragment to form a stable complex with the C subunit, whereas an RII fragment, in which aminoterminal residues 1-93 were removed by thermolysin digestion, lost the ability to form the complex.…”