Shiaw-Der Yang

A simplified procedure for the purification of the protein phosphatase modulator (inhibitor-2) from rabbit skeletal-muscle

Two heat-stable proteins have been identified as specific inhibitors of the phosphatase activity [l-l 11. Inhibitor-l requires phosphorylation by the cyclic AMPdependent protein kinase for its activity [3,4] and has been clearly implicated in the hormonal regulation of glycogen metabolism [12-l 41. The inhibitor-2 protein has been purified up to homogeneity from rabbit muscle [lo] and liver [7] and its specific role as modulator of the protein phosphatase activity has been described [ 15 ,161. ma1 amount of FA required for full activation of Fc under these conditions. The phosphatase activity unit is defined as the amount of enzyme which releases 1 nM [3zP]phosphate/min at 30°C from 32P-labeled phosphorylase a (2 mg/ml). The assay time is 10 min. Heat-stable phosphatase inhibitor-2 was purified to homogeneity from rabbit muscle according to [lo] or by an alternative method, described in section 3. One unit of inhibitor-2 decreased the activity of 20 munits ATP-Mg-dependent phosphatase by 50% when included in the 10 min preincubation of FC (20 000 U/mg) [ 171 with FA and ATP-Mg. A highM, multisubstrate protein phosphatase has been extensively purified from rabbit muscle and shown to interconvert to the inactive ATP-Mg-dependent enzyme form (F,) [ 171. The activation of purified FCenzyme [ 181 is brought about by a preincubation with ATP-Mg and FA, a bifunctional protein also displaying synthase kinase activity [ 191. This reversible ATP-Mgdependent activation was consequently shown to depend upon the presence of inhibitor-2 which we therefore called phosphatase modulator [15,16]. 3. Results

Regulation of ATP—Mg-Dependent Protein Phosphatase

“ATP,Mg-dependent protein phosphatase” designates a family of enzyme forms whose catalytic subunit can interconvert between an active and an inactive conformation through the specific actions of the heat-stable modulator protein (inactivation) and protein kinase FA (activation). The catalytic subunit of these enzymes has a wide substrate specificity (1, 2) so that the ATP, Mg-dependent phosphatase activity can account for the major dephosphorylation of the key enzymes that regulate glycogen metabolism, i.e., glycogen phosphorylase, glycogen synthase, and phosphorylase kinase (β subunit).

ATP-Mg-dependent phosphorylase phosphatase in mammalian tissues

In [ 1 ] we described in dog liver a phosphorylase phosphatase which depends on the interaction of two protein fractions and ATP-Mg for activity. These two protein fractions (called F, and Fc) can be separated by DEAE-cellulose chromatography, and recombination of both fractions in the presence of ATP-Mg results in an active phosphatase enzyme. We have now extended these observations to other tissues and animals: fresh high-speed supernatant fractions of both rat and rabbit tissues (liver, heart and skeletal muscle) were shown to contain a similar ATP-Mg requiring phosphatase system. It was observed that this phosphatase constitutes an important fraction of the total phosphorylase phosphatase activity present in these high-speed supernatant fractions.

Conversion of active protein phosphatase to the ATP-Mg-dependent enzyme form by inhibitor-2

Brandt et al. [ 1,2] made the original observation that liver and muscle extracts contain heat-stable, trypsin-labile proteins which inhibit phosphorylase phosphatase. The regulatory role of these heat-stable proteins in the control of phosphatase activity was accentuated when two heat-stable inhibitors, termed inhibitor-l and -2 were discovered and the inhibitory capacity of inhibitor-l demonstrated to depend upon its phosphorylation by the cyclic AMP-dependent protein kinase [3,4]. Heat-stable phosphatase inhibitors have since been identified in a variety of animals and tissues [S-l 0] and purified to homogeneity from rabbit muscle (inhibitor-l , -2) [8 ,101 and rabbit liver [7]. The implication of inhibitor-l in the hormonal regulation of glycogen metabolism has been clearly demonstrated [ll-131. We have reported the purification and characterisation of an ATP-Mgdependent protein phosphatase system from rabbit muscle [14-191: an inactive multifunctional protein phosphatase (FC) was shown to be activated by another protein factor (FA) in the presence of ATP-Mg ions (without phosphorylation). This report provides evidence that purified inhibitor-2 preparations [lo] have the capacity to reverse the F, and ATP-Mg-mediated activation.

On the mechanism of activation of rabbit skeletal-muscle atp-mg-dependent protein phosphatase

An inactive form of protein phosphatase (F,) has been purified to near homogeneity from rabbit skeletal muscle [ 11. The enzyme can be activated by another protein factor (FA) in the presence of ATP and Mg2+ without demonstrable incorporation Of Pi into the protein [ 1,2]. The activated phosphatase has a broad substrate specificity [3,4]. The two protein factors (FA,FC) which constitute the ATP-Mg-dependent phosphatase system have been demonstrated in a variety of mammalian tissues [5,6] and can be used interchangeably from the different sources. It has been suggested that F, may represent the inactive form of the physiologically active protein phosphatase, responsible for the major dephosphorylation reactions of the enzymes involved in the hormonal control of glycogen metabolism [3]. The mechanism of activation of the ATP-Mg-dependent phosphatase has now been studied using different nucleotides and metal ions.

The heat labile phosphatase modulator (inhibitor-2) complex from rabbit skeletal muscle

The heat stable phosphatase modulator protein (inhibitor-2) has been shown to play a crucial role in the reversible ATP, Mg-dependent activation of a multisubstrate protein phosphatase. The modulator activity is acid and heat stable and resides in a small asymmetrical protein which, after boiling migrates in sucrose density gradient centrifugation with a molecular weight of 17K. The present report shows that in unboiled rabbit skeletal muscle preparations all the modulator activity is found associated with a heat labile protein component, which imposes an important regulatory feature on the heat stable activity. The heat labile complex migrates in sucrose density gradient centrifugation as a Mr = 70K protein.

Role of the modulator protein in the interconversion of rabbit skeletal muscle protein phosphatase

The major active protein phosphatase present in a rabbit skeletal muscle extract is associated with the glycogen particle and migrates in sucrose density gradient centrifugation as a Mr = 70,000 protein and contains modulator activity. Addition of extra modulator protein causes a time- and concentration-dependent conversion of the enzyme to an inactive FA-ATP, Mg-dependent form. The intrinsic modulator in the active phosphatase is destroyed by limited proteolysis without an appreciable change in the phosphatase activity. The proteolyzed active enzyme has a lower molecular weight (Mr = 40,000) and it reassociates with the modulator producing a FA-ATP, Mg-dependent enzyme form (Mr = 60,000). The modulator protein is used stoichiometrically in the activation of the ATP, Mg-dependent phosphatase. This is in agreement with the presence of one unit of modulator activity per unit of native spontaneously active phosphatase.

Purification of a latent protein phosphatase from rabbit skeletal muscle

A high molecular weight protein phosphatase (Mr = 260K) has been isolated from rabbit skeletal muscle. The enzyme has a very low activity towards phosphorylase a isolated from the same tissue, but its activity towards this substrate is stimulated several fold after dissociation by 2-mercaptoethanol treatment. The purified phosphatase shows one major protein staining band on non denaturing polyacrylamide gel electrophoresis, and contains four subunits with molecular weights of 95K, 75K, 65K and 38K. The catalytic activity resides in the Mr = 38K subunit and is not sensitive to inhibition by the heat stable protein phosphatase inhibitor-1 or modulator protein. Polyamines stimulate the holoenzyme in a dose dependent, biphasic manner, but inhibit the activity of the dissociated Mr = 38K catalytic subunit.

Control of the ATP,Mg-dependent protein phosphatase by heat-stable regulatory proteins.

Protein Phosphatases Involved in Glycogen Metabolism. Covalent modifications are involved in the control of almost every type of cellular regulation, and the phosphorylation–dephosphorylation cycle appears to be the most prevalent among them. This type of regulation has assumed increasing importance not only in exploring enzyme regulation but also in the study of the signal transfer by receptors, muscular contraction, membrane transport systems, and by the observation that gene products of certain oncogenic viruses display protein kinase activity. The mode of action and regulation of a substantial number of protein kinases is well documented while development in the field of protein phosphatases, which has been reviewed in detail (1–4), is much more recent. This is probably so, because their activity and specificity is basically the result of the interaction of several regulatory proteins and because of the problems involved in the use of phospho-proteins in screening studies and early purification procedures. Glycogen metabolism has always been an outstanding area in metabolic research resulting in remarkable contributions to the development of enzymology. Protein phosphatases have also been investigated most thoroughly in the regulation of the pathways of glycogen synthesis and glycogenolysis. Dephosphorylation by the ATP, Mg-dependent protein phosphatase of all the phosphoproteins known to be relevant in the regulation of glycogen metabolism (Fig. 1) and the activation of this phosphatase by kinase FA have been reviewed recently (3, 4). A Ca2+-calmodulin-dependent protein phosphatase that appears to be identical with calcineurin (5, 6) is present at high concentrations in brain and skeletal muscle but its concentration in liver (7) and vascular smooth muscle (E. Waelkens, J. Goris, and W. Merlevede, unpublished observations) is low. The Ca2+-calmodulin-dependent phosphatase could promote glycogen synthesis through dephosphorylation of inhibitor-1 although this proposal is not easy to reconcile with the role of Ca2+ in muscular contraction.

Protein phosphatase interconversions

This report illustrates the complex enzymology of the multisubstrate protein phosphate that reverses most of the cyclic AMP-mediated protein phosphorylation reactions that regulate glycogen metabolism. The activity of the protein phosphatase is controlled in a dual way: it interconverts between an active and an inactive form, while the expression of its activity can furthermore be prevented by a heat-stable protein (inhibitor-1). The interconversion of the mutisubstrate protein phosphatase is made possible by the presence of a modulator protein, which constitutes the enzymes regulatory subunit, and by the action of an activating protein, the kinase FA, which is responsible for the transition of the enzymes catalytic subunit into its active conformation.