Dephosphorylation of the deinhibitor protein by the PCSH protein phosphatase

The deinhibitor protein, responsible for the decreased sensitivity of the ATP,Mg-dependent protein phosphatase to inhibitor-1 and the modulator protein, is inactivated by cyclic AMP-dependent protein kinase and reactivated by dephosphorylation. The specificity of this reaction was tested with the ATP,Mg-dependent phosphatase in its activated or spontaneously active form, four different forms of polycation-stimulated phosphatases (PCSH, PCSM, PCSL and PCSC) and calcineurin. Only the high -Mr polycation-stimulated protein phosphatase (PCSH), but not its catalytic subunit (PCSC), shows a high degree of specificity for the deinhibitor protein. Deinhibitor phosphatase activity of PCSH is affected neither by polycations nor by Mn ions.     

Stimulation of Ca(2+)/calmodulin-dependent protein kinase phosphatase by polycations

Ca(2+)/calmodulin-dependent protein kinase phosphatase (CaMKPase) dephosphorylates and regulates multifunctional Ca(2+)/calmodulin-dependent protein kinases (CaMKs). One of the prominent features of CaMKPase is stimulation of phosphatase activity by polycations such as poly-L-lysine (poly(Lys)). Using various polycations, basicity and molecular weight of the polymer proved to be important for the stimulation. Surface plasmon resonance (SPR) analysis showed that CaMKIV(T196D), which mimics CaMKPase substrate, and CaMKPase could form tight complexes with poly(Lys). Pull-down binding experiments suggested that the formation of a tightly associated ternary complex consisting of CaMKPase, poly(Lys), and phosphorylated CaMKIV is essential for stimulation. Dilution experiments also supported this contention. Poly(Lys) failed to stimulate a CaMKPase mutant in which a Glu cluster corresponding to residues 101-109 in the N-terminal domain was deleted, and the mutant could not interact with poly(Lys) in the presence of Mn(2+). Thus, the Glu cluster appeared to be the binding site for polycations and to play a pivotal role in the polycation stimulation of CaMKPase activity.     

Evidence for the coordinate control of activity of liver glycogen synthase and phosphorylase by a single protein phosphatase.

Homogeneous rabbit liver phosphorylase phosphatase (Brandt, H., Capulong, Z. L., and Lee, E. Y. C. (1975) J. Biol. Chem. 250, 8038-8044) also dephosphorylates glycogen synthase b. During purification, phosphorylase phosphatase and glycogen synthase phosphatase co-purified with a constant ratio of activities. The two activities co-migrated on disc gel electrophoresis. Both substrates competed with each other for the phosphatase, and both phosphatase activities were inhibited by lysine ethyl ester. It is concluded that liver phosphorylase phosphatase and glycogen synthase phosphatase have a common identity and that coordinate regulation of the phosphatase-catalyzed activation of glycogen synthase and inactivation of phosphorylase occurs in vivo. This provides a parallel and opposing mechanism to that mediated by adenosine 3':5'-monophosphate-dependent protein kinase, which coordinately inactivates glycogen synthase and, via phosphorylase kinase, activates phosphorylase. Maximal glycogen synthase phosphatase activity was observed near neutrality. Mg2+ and glucose-6-P activated the glycogen synthase phosphatase reaction and this activation was pH-dependent. The Km for glycogen synthase b was 0.12 muM.     

The deinhibitor protein: regulation by phosphorylation-dephosphorylation

The deinhibitor protein, which protects the multisubstrate protein phosphatase from inhibition by inhibitor-1 and the modulator protein, stabilizes the enzyme in its active conformation preventing its conversion to the ATP,Mg-dependent enzyme form and controls the dephosphorylation of inhibitor-1, was shown to exist under active and inactive forms. It can be inactivated by the catalytic unit of the cyclic AMP-dependent protein kinase and reactivated by an inhibitor-1 phosphatase, also described as histone-H1 ("latent") stimulated protein phosphatase.     

Identification of the phosphatase deinhibitor protein phosphatases in rabbit skeletal muscle.

In rabbit skeletal muscle the polycation-stimulated (PCS) protein phosphatases [Merlevede (1985) Adv. Protein Phosphatases 1, 1-18] are the only phosphatases displaying significant activity toward the deinhibitor protein. Among them, the PCSH protein phosphatase represents more than 80% of the measurable deinhibitor phosphatase activity associated with the PCS phosphatases. The deinhibitor phosphatase activity co-purifies with the PCSH phosphatase to apparent homogeneity. In the last purification step two forms of PCSH phosphatase were separated (PCSH1, containing 62, 55 and 34 kDa subunits, and PCSH2, containing 62 and 35 kDa subunits), both showing the same deinhibitor/phosphorylase phosphatase activity ratio. The activity of the PCSH phosphatase toward the deinhibitor is not stimulated by polycations such as protamine, histone H1 or polylysine, unlike the stimulation observed with phosphorylase as the substrate. The phosphorylase phosphatase activity of PCSH phosphatase is inhibited by ATP, PPi and Pi, whereas the deinhibitor phosphatase activity of the enzyme is much less sensitive to these agents.     

Role of the deinhibitor protein in the interconversion of the ATP,Mg-dependent protein phosphatase

The small molecular weight (± 9,000) heat stable deinhibitor protein, isolated from dog liver, not only protects the multisubstrate protein phosphatase from inhibition by inhibitor-1 and the modulator protein. It prevents the conversion of the active enzyme to the ATP,Mg-dependent enzyme form brought about by the modulator protein, and also affects the activation of the ATP,Mg-dependent protein phosphatase, probably by stabilizing the enzyme in its active conformation during the reversible activation by protein kinase F_A. Therefore the deinhibitor protein could be an important factor in the process of glycogen synthesis, which requires glycogen synthase and phosphorylase as dephosphorylated enzymes.     

The ATPase activity of phosphorylase kinase is regulated in parallel with its protein kinase activity.

Phosphorylase kinase from rabbit skeletal muscle has been found to have an intrinsic ATPase activity that occurs at a rate approximately 0.2% of that of its phosphorylase conversion activity and about three times that of its autophosphorylation activity. The characteristics of this ATPase activity were in all aspects tested essentially the same as the kinase's phosphorylase conversion activity. The ATPase requires Mg2+ and is dramatically stimulated by Ca2+ ions. At neutral pH there is a pronounced lag in the rate of product formation that is not present at alkaline pH, a condition that greatly stimulates both the phosphorylase conversion and ATPase activities. ATP is preferentially hydrolyzed over GTP and the Km for MgATP determined in the ATPase assay is 0.14 mM. ADP, an allosteric activator of phosphorylase conversion, also stimulates the ATPase activity, whereas beta-glycerophosphate, an inhibitor of phosphorylase conversion, is an inhibitor of the ATPase activity. Phosphorylation or partial proteolysis of the kinase, which are known to activate phosphorylase conversion, also activate the ATPase activity. Because the phosphorylase conversion and ATPase activities are regulated in parallel, we conclude that activation of the two catalytic activities must share a common underlying basis, namely an enhanced phosphotransferase activity that is independent of the phosphoryl acceptor.     

The phosphorylase kinase activity of hearts from phosphorylase kinase-deficient mice

In an assay measuring radioactive incorporation from gamma--P32P]ATP into phosphorylase b, cardiac muscle extracts from mice with the phosphorylase kinase deficiency mutation showed significant, calcium-dependent phosphorylase kinase activity that was 10 to 15% of that of Swiss mice, the control strain. Isoproterenol stimulated significant phosphorylase a accumulation in both isolated atria and right ventricular strips of phosphorylase kinase-deficient mice, and the drug-stimulated increases in phosphorylase a activity the the contractile responses of right ventricular strips were similar in Swiss and phosphorylase kinase/deficient mice.     

Protein inhibitors of phosphorylase phosphatase and cyclic AMP-dependent protein kinase from rabbit skeletal muscle

Summary A heat- and acid-stable proten inhibitor of phosphorylase phosphatase is present in a highly purified preparation of protein inhibitor of cyclic AMP-dependent protein kinase from rabbit skeletal muscle. Although these two inhibitors have strikingly similar properties to each other, such as sensitivity to trypsin and behavior on gel permeation chromatography, they can be separated by polyacrylamide disc gel electrophoresis. This indicates that the phosphatase-inhibitory and kinase-inhibitory activities reside with different protein species. The inhibition of both the enzymes is not altered by incubating the inhibitor preparation with a general phosphoprotein phosphatase, with phosvitin kinase, or with cyclic AMP-dependent protein kinase. Inhibition of phosphorylase phosphatase is of a non-competitive type supporting the idea that the phosphatase inhibitor is not an alternative substrate for the enzyme. Inhibition of phosphatase activity is selective in that it does not occur when phosphorylated histone or phosphorylated protamine are used as substrates.     

Inhibition of mitogen-activated protein kinase phosphatase 3 activity by interdomain binding

Mitogen-activated protein (MAP) kinase phosphatase 3 (MKP3) is a cytoplasmic dual specificity phosphatase that functions to attenuate signaling via dephosphorylation and subsequent deactivation of its substrate and allosteric regulator, extracellular signal-regulated protein kinase 2 (ERK2). Expression of MKP3 has been shown to be under the control of ERK2, thus providing an elegant feedback mechanism for regulating the rate and duration of proliferative signals. Previously published studies suggest that MKP3 might serve as a tumor suppressor; however, significantly elevated, rather than reduced, levels of this protein have been reported in early lesions. Because overexpression of this phosphatase is counterintuitive to a proposed tumor suppressor function, the observed cellular tolerance suggested a self-inactivation mechanism. Using surface plasmon resonance, we have provided direct evidence of physical interaction between the N- and C-terminal domains. Kinetic analysis using dimethyl sulfoxide to activate the C-terminal fragment in the absence of ERK2 showed that the isolated C-terminal domain had higher catalytic efficiency than the similarly activated full-length protein. Furthermore, when the isolated N-terminal domain was added to the activated C-terminal domain, a dose-dependant inhibition of catalytic activity was observed. The similarity between the K(I) and K(D) values obtained indicate that interdomain binding stabilizes the inactive conformation of the catalytic site and implies that the N-terminal domain functions as an allosteric inhibitor of phosphatase activity. Finally, we have provided evidence for oligomerization of MKP3 in pancreatic cancer cells expressing elevated levels of this phosphatase.     

Thiophosphate-activated phosphorylase kinase as a probe in the regulation of phosphorylase phosphatase.

Rabbit muscle nonactivated phosphorylase kinase (EC 2.7.1.38) is converted to thiophosphate-activated phosphorylase kinase by cyclic AMP dependent protein kinase, Mg2+ and ATP-gamma-S/adenosine-5'-O-(s-thiotriphosphate)/. The formation of thiophosphate-activated phosphorylase kinase wal also observed in the protein-glycogen complex from skeletal muscle. This new form of kinase is resistant to the action of phosphatase and behaves as a competitive inhibitor in the dephosphorylation of phosphorylase alpha by phosphorylase phosphatase (Ki = 0.04 mg per ml). The fact that the inhibitory effect of thiophosphate-activated phosphorylase kinase is 3 times higher than in the case of nonactivated kinase, may explain the transient inhibition of phosphorylase phosphatase in the protein-glycogen complex. The use of activated (phosphorylated) phosphorylase kinase supports this assumption since it causes a delay in the dephosphorylation of phosphorylase alpha, i.e. the conversion of phosphorylase alpha into beta could start only after the dephosphorylation of activated phosphorylase kinase.     

Activation of glycogen synthetase and inactivation of phosphorylase kinase by the same phosphoprotein phosphatase

Purified rabbit muscle glycogen synthetase D phosphatase inactivates phosphorylase kinase. The inactivation is reversed by cyclic AMP-dependent protein kinase. It is postulated that the synthetase D phosphatase is a general phosphoprotein phosphatase which dephosphorylates proteins that are phosphorylated $\text{in}$$\text{vivo}$ by the cyclic AMP-dependent kinase.