Protein phosphorylation is now recognized to be the major general mechanism by which intracellular events in mammalian tissues are controlled by external physiological stimuli However, only recently has the idea that different cellular functions are controlled by common protein kinases and protein phosphatases started to gain widespread acceptance Thus there is an integrated network of regulatory pathways, mediated by phosphorylation-dephosphorylation, that allows diverse cellular events to be coordinated by neural and hormonal stimuli The evidence that supports this concept is reviewed, with emphasis on the role of protein phosphorylation in enzyme regulation

The role of protein phosphorylation in neural and hormonal control of cellular activity

Publisher Summary This chapter discusses glycogen synthase kinase-3 from rabbit skeletal muscle. Glycogen synthase kinase-3 is one of the five glycogen synthase kinases that are identified in skeletal muscle, and is of major importance in determining the kinetic properties of glycogen synthase in vivo. It catalyzes the phosphorylation of three serine residues on glycogen synthase, converting the enzyme from a form that is almost fully active in the absence of glucose-6P, to one that is largely dependent on this allosteric activator. Glycogen synthase kinase-3 also has a second activity that is not shared by any other protein kinase—namely, the ability to activate an enzyme termed the MgATP-dependent protein phosphatase. Glycogen synthase may also contain traces of a modified form of phosphorylase kinase that has lost its sensitivity to regulation by calcium ions, and is therefore, no longer inhibited by ethylene glycol tetraacetic acid (EGTA). This is largely removed by passing glycogen synthase through phosphocellulose.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1980.tb06059.x

Glycogen Synthase Kinase-3 from Rabbit Skeletal Muscle

Protein phosphorylation is a principal regulatory mechanism in the control of almost all cellular processes. The nature of the protein phosphatases that participate in these reactions has been a subject of controversy. Four enzymes, termed protein phosphatases 1, 2A, 2B, and 2C, account for virtually all of the phosphatase activity toward phosphoproteins involved in controlling glycogen metabolism, glycolysis, gluconeogenesis, fatty acid synthesis, cholesterol synthesis, and protein synthesis. The properties, physiological roles, and mechanisms for regulating the four protein phosphatases are reviewed.

https://science.sciencemag.org/content/sci/221/4608/331.full.pdf

Protein phosphatases: properties and role in cellular regulation

One of the important metabolic effects of insulin is its ability to stimulate the synthesis of glycogen in skeletal muscle within minutes; much research has been directed toward understanding the molecular basis of this effect. Almost 25 years ago, Joseph Larner and co-workers showed that glycogen synthase, the rate-limiting enzyme in this pathway, could exist in at least two forms. One possessed little activity in the absence of glucose 6-phosphate (G6P), while the other was almost fully active in the absence of G6P.(1) Incubation of rat hemidiaphragms with insulin decreased the proportion of glycogen synthase in the G6P-dependent form which paralleled the increased rate of glycogen production.(1–3) Furthermore, the conversion of glycogen synthase to a G6Pindependent form was still observed when glucose was omitted from the perfusion medium,(2,3) indicating that it was not a consequence of the increased rate of transport of glucose into muscle, which is also stimulated by insulin. Conversely, incubation of the hemidiaphragms with epinephrine increased the proportion of the G6P-dependent form and decreased the rate of glycogen synthesis.(2,3)

The molecular mechanism by which insulin stimulates glycogen synthesis in mammalian skeletal muscle

The effects of polyamines on the oligomeric forms of protein phosphatase-1 (1G), protein phosphatase-2A (2A0, 2A1 and 2A2) and their free catalytic subunits (1C and 2AC) has been studied using homogeneous enzymes isolated from rabbit skeletal muscle. Spermine increased the activity of protein phosphatase-2A towards eight of nine substrates tested. Half-maximal activation was observed at 0.2 mM with optimal effects at 1–2 mM. Above 2 mM, spermine became inhibitory. The most impressive activation of protein phosphatase-2A was obtained with glycogen synthase, especially when phosphorylated at sites-3 (8–15-fold with protein phosphatase-2A1) and phenylalanine hydroxylase (6–7-fold with protein phosphatase-2A1) as substrates. Activation of protein phosphatases 2A0, 2A1 and 2A2 was greater than that observed with 2AC. Spermine was a more potent activator than spermidine, while putrescine had only a small effect. Qualitatively similar results were obtained with five other substrates, although maximal activation was much less (1.3–3-fold with protein phosphatase-2A1). The rate of dephosphorylation of glycogen phosphorylase was decreased by spermine, inhibition being more pronounced with protein phosphatase-2AC than with 2A0, 2A1 and 2A2. Spermine (I50= 0.1 mM with protein phosphatase-2AC) was a more potent inhibitor than spermidine (I50= 0.9 mM) or putrescine (I50= 8 mM). Partially purified preparations of protein phosphatases-2A0, 2A1 and 2A2 from from rat liver were affected by spermine in a similar manner to the homogeneous enzymes from rabbit skeletal muscle.



Spermine did not activate protein phosphatase-1 to the same extent as protein phosphatase-2A. Greatest stimulation (2.5-fold) was again observed with glycogen synthase labelled in sites-3, with half-maximal activation at 0.2 mM and optimal effects at 1–2 mM spermine. Spermine was a much more effective stimulator than spermidine, while putrescine was ineffective. Very similar results were obtained with protein phosphatases 1G and 1C. With four other substrates maximal activation by spermine was < 1.5-fold, while the dephosphorylation of glycogen synthase (labelled in site-2), phosphorylase kinase, pyruvate kinase and glycogen phosphorylase were inhibited. Spermine (I50= 0.04 mM) was a more potent inhibitor of the dephosphorylation of glycogen phosphorylase than spermidine (I50= 0.9 mM) or putrescine (I50= 9 mM).



There was some resemblance between the effects of Mn2+ and spermine on the activity of protein phosphatase-2A, although with the substrates inhibitor-1 and phenylalanine hydroxylase, activation by Mn2+ was greater than that observed with spermine, while with glycogen synthase the converse was true. Mn2+ could not mimic the activating effect of spermine on dephosphorylation of glycogen synthase (labelled in sites-3) by protein phosphatase 1. By contrast, spermine hardly affected the dephosphorylation of inhibitor-1 by phosphatase-1, a reaction that was almost completely dependent on Mn2+. Physiological concentrations of Mg2+ (1.0 mM) neither mimicked, nor influenced, the response to spermine with either protein phosphatase-1 or 2A.



The effects of spermine appear to result from interaction of the polyamine with both the protein phosphatases and their substrates. The possibility that activation of protein phosphatase-1 and/or 2A by spermine underlies the relatively specific dephosphorylation of sites-3 of glycogen synthase by insulin is discussed.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1985.tb08832.x

The protein phosphatases involved in cellular regulation

Muscle extracts were subjected to fractionation with ethanol, chromatography on DEAE-cellulose, precipitation with (NH4)2SO4 and gel filtration on Sephadex G-200. These fractions were assayed for protein phosphatase activities by using the following seven phosphoprotein substrates: phosphorylase a, glycogen synthase b1, glycogen synthase b2, phosphorylase kinase (phosphorylated in either the alpha-subunit or the beta-subunit), histone H1 and histone H2B. Three protein phosphatases with distinctive specificities were resolved by the final gel-filtration step and were termed I, II and III. Protein phosphatase-I, apparent mol.wt. 300000, was an active histone phosphatase, but it accounted for only 10-15% of the glycogen synthase phosphatase-1 and glycogen synthase phosphatase-2 activities and 2-3% of the phosphorylase kinase phosphatase and phosphorylase phosphatase activity recovered from the Sephadex G-200 column. Protein phosphatase-II, apparent mol.wt. 170000, possessed histone phosphatase activity similar to that of protein phosphatase-I. It possessed more than 95% of the activity towards the alpha-subunit of phosphorylase kinase that was recovered from Sephadex G-200. It accounted for 10-15% of the glycogen synthase phosphatase-1 and glycogen synthase phosphatase-2 activity, but less than 5% of the activity against the beta-subunit of phosphorylase kinase and 1-2% of the phosphorylase phosphatase activity recovered from Sephadex G-200. Protein phosphatase-III was the most active histone phosphatase. It possessed 95% of the phosphorylase phosphatase and beta-phosphorylase kinase phosphatase activities, and 75% of the glycogen synthase phosphatase-1 and glycogen synthase phosphatase-2 activities recovered from Sephadex G-200. It accounted for less than 5% of the alpha-phosphorylase kinase phosphatase activity. Protein phosphatase-III was sometimes eluted from Sephadex-G-200 as a species of apparent mol.wt. 75000(termed IIIA), sometimes as a species of mol.wt. 46000(termed IIIB) and sometimes as a mixture of both components. The substrate specificities of protein phosphatases-IIA and -IIB were identical. These findings, taken with the observation that phosphorylase phosphatase, beta-phosphorylase kinase phosphatase, glycogen synthase phosphatase-1 and glycogen synthase phosphatase-2 activities co-purified up to the Sephadex G-200 step, suggest that a single protein phosphatase (protein phosphatase-III) catalyses each of the dephosphorylation reactions that inhibit glycogenolysis or stimulate glycogen synthesis. This contention is further supported by results presented in the following paper [Cohen, P., Nimmo, G.A. & Antoniw, J.F. (1977) Biochem. J. 1628 435-444] which describes a heat-stable protein that is a specific inhibitor of protein phosphatase-III.

/pdf/comparison-of-the-substrate-specificities-of-protein-54ectb6a1v.pdf

Comparison of the substrate specificities of protein phosphatases involved in the regulation of glycogen metabolism in rabbit skeletal muscle.

Cyclic-AMP-dependent protein kinase catalyses the activation of phosphorylase kinase and the phosphorylation of two serine residues on the α subunit and β subunit of phosphorylase kinase [Cohen, P., Watson, D. C. and Dixon, G. H. (1975)].



The dephosphorylation of phosphorylase kinase has been shown to be catalysed by two distinct enzymes, termed α-phosphorylase kinase phosphatase and β-phosphorylase kinase phosphatase. These two enzymes show essentially absolute specificity towards the α and β subunits respectively. The two phosphatases copurified through ethanol fractionation, DEAE-cellulose chromaiograpity and ammonium sulphate precipitation, but were separated from each other by gel filtration on Sephadex G,200. α-Phosphorylase kinase phosphatase was purified 500-fold from the ethanol precipitation step, and β-phosphorylase kinase phosphatase 320-fold. The molecular weights estimated by gel filtration were 170-180000 for α-phosphorylase kinase phosphatase and 75-80000 for β- phosphorylase kinase phosphatase.



Since the activity of phosphorylase kinase correlates with the state of phosphorylation of the β subunit (Cohen, P. (1974)), β-phosphorylase kinase phosphatase is the enzyme which reverses the activation of phosphorylase kinase. α-Phosphorylase kinase phosphatase is an enzyme activity that has not been recognised previously. Since the role of the a-subunit phosphorylation is to stimulate the rate-of dephosphorylation of the β subunit (Cohen, P. (1974)), α-phosphorylase kinase phosphatase can be regarded as the enzyme which inhibits the reversal of the activation of phosphorylase kinase. The implications of these findings for the hormonal control of phosphorylase kinase activity by multisite phosphorylation are discussed.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1976.tb10763.x

Separation of two phosphorylase kinase phosphatases from rabbit skeletal muscle.

A hear-stable protein, which is a specific inhibitor of protein phosphatase-III, was purified 700-fold from skeletal muscle by a procedure that involved heat-treatment at 95 degrees C, chromatography on DEAE-cellulose and gel filtration on Sephadex G-100. The final step completely resolved the protein phosphatase inhibitor from the protein inhibitor of cyclic AMP-dependent protein kinase. The phosphorylase phosphatase, beta-phosphorylase kinase phosphatase, glycogen synthase phosphatase-1 and glycogen synthase phosphatase-2 activities of protein phosphatase-III [Antoniw, J. F., Nimmo, H. G., Yeaman, S. J. & Cohen, P.(1977) Biochem.J. 162, 423-433] were inhibited in a very similar manner by the protein phosphatase inhibitor and at least 95% inhibition was observed at high concentrations of inhibitor. The two forms of protein phosphatase-III, termed IIIA and IIIB, were equally susceptible to the protein phosphatase inhibitor. The protein phosphatase inhibitor was at least 200 times less effective in inhibiting the activity of protein phosphatase-I and protein phosphatase-II. The high degree of specificity of the inhibitor for protein phosphatase-III was used to show that 90% of the phosphorylase phosphatase and glycogen synthase phosphatase activities measured in muscle extracts are catalysed by protein phosphatase-III. Protein phosphatase-III was tightly associated with the protein-glycogen complex that can be isolated from skeletal muscle, whereas the protein phosphatase inhibitor and protein phosphatase-II were not. The results provide further evidence that the enzyme that catalyses the dephosphorylation of the alpha-subunit of phosphorylase kinase (protein phosphatase-II) and the enzyme that catalyses the dephosphorylation of the beta-subunit of phosphorylase kinase (protein phosphatase-III) are distinct. The results suggest that the protein phosphatase inhibitor may be a useful probe for differentiating different classes of protein phosphatases in mammalian cells.

/pdf/specificity-of-a-protein-phosphatase-inhibitor-from-rabbit-25yevawpnf.pdf

Specificity of a protein phosphatase inhibitor from rabbit skeletal muscle.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1016/0014-5793%2873%2980699-4

The control of phosphorylase kinase phosphatase by "second site phosphorylation"; a new form of enzyme regulation.

Although the scheme hormone leads to raised cyclic AMP levels leads to activated protein kinase leads to phosphorylated protein leads to physiological response may represent an outline for the action of several hormones, in the best understood example, namely regulation of glucogen metabolism in mammalian muscle, the picture is more complex. Modification of phosphorylase kinase by cyclic AMP-dependent protein kinase, after stimulation by adrenaline, leads to phosphorylation of the enzyme at two sites. Activation is associated exclusively with the phosphorylation of the primary site, but the secondary phosphorylation indirectly antagonizes the primary phosphorylation in that it is necessary to render the primary site susceptible to dephosphorylation. The recent separation of two distinct phosphorylase kinase phosphatases specific for the two sites shows that reversal of the hormonal stimulation is controlled by the relative activities of two enzymes with opposing functions. Glycogen synthetase, which is phosphorylated and inactivated by cyclic AMP-dependent protein kinase, is also under the control of insulin. Although insulin appears to stimulate glycogen synthetase by reversal of the inactivation catalysed by the cyclic AMP-dependent protein kinase, tissue cyclic AMP concentrations do not alter. The recent identification of a second glycogen synthetase kinase, unaffected by cyclic AMP, therefore raises the possibility that insulin action may also be mediated through phosphorylation-dephosphorylation mechanisms, which antagonize those mediated through cyclic AMP-dependent protein kinase.

John F. Antoniw

Papers

Comparison of the substrate specificities of protein phosphatases involved in the regulation of glycogen metabolism in rabbit skeletal muscle.

Separation of two phosphorylase kinase phosphatases from rabbit skeletal muscle.

Specificity of a protein phosphatase inhibitor from rabbit skeletal muscle.

The control of phosphorylase kinase phosphatase by "second site phosphorylation"; a new form of enzyme regulation.

Protein phosphorylation and hormone action.