A futher theoretical treatment of the turnover of protein bound phospate in the presence of both protein kinase and phosphatase activities.

Many subcellular fractions contain both protein kinase and phosphatase enzymes which can act on endogenous proteins in the fractions.When studying the phosphorylation of the endogenous protein it is necessary to take into account the presence of both enzymes.In a previous paper (Weller, 1974) an equation was derived which described the time-course of phosphorylation of a protein in the presence of both kinase and phosphatase activities. To derive this equation the assumption was made that the activity of the kinase was very much greater than that of the phosphatase. This assumption may not be valid in all cases and the present paper describes the derivation of a similar equation without making any assumptions about the relative rates of kinase and phosphatase activities.The time-course of protein phosphorylation predicted by the new equation is compared to that predicted by the previous equation with varying rates of protein phosphatase activity. It is found that the new equation should be used to describe the time-course of protein phosphorylation in the presence of protein kinase and phosphatase activities if the activity of the phosphatase is more than about 7% of that of the kinase.     

Endogenous phosphorylation of proteins and phosphatidylinositol in the plasma membranes of a human astrocytoma

Incubation of plasma membrane preparations from several tissues with [gamma-32P]ATP resulted in the phosphorylation of phosphatidylinositol as well as of proteins. The presence of an active phosphatidylinositol kinase in these membranes was indicated by equal or greater incorporation of 32P into phosphatidylinositol phosphate than into proteins. Phosphorylation of endogenous protein and lipid substrates by protein and phosphatidylinositol kinases in the plasma membranes of a human astrocytoma was investigated in detail. Maximal protein phosphorylation required the presence of Nonidet-P40 and phosphatase inhibitors (vanadate or fluoride). The rate of protein phosphorylation was greater with Mg2+ than with Mn2+, and phosphoserine accounted for 60% of the radioactivity incorporated into proteins. In the presence of Mn2+, phosphorylation of tyrosine was increased and was equal to that of serine phosphorylation (40%). With one exception, the overall pattern of phosphorylated proteins was similar with either Mg2+ or Mn2+. Maximal phosphatidylinositol phosphorylation of the astrocytoma plasma membranes also required detergent and phosphatase inhibitors. However, the enzymatic characteristics of lipid phosphorylation differed from those of protein phosphorylation with respect to divalent cation activation, ATP dependence, and sensitivity to inhibition by p-chloromercuriphenyl sulfonate, quercetin, and nucleoside derivatives. These results suggest that phosphorylation of plasma membrane proteins and phosphatidylinositol is catalyzed by different enzymes. The fact that membrane preparations exhibited phosphatidylinositol kinase activity almost 100,000 times greater than that exhibited by the purified tyrosine kinase of ros gene would exclude this and similar oncogene proteins from making a significant contribution to the overall phosphatidylinositol phosphorylation of cell membranes.     

Defective sarcolemmal phosphorylation associated with noninsulin-dependent diabetes

Noninsulin-dependent diabetes is associated with a decrease in the activity of sarcolemmal phosphatase 1, but no change in the activities of phosphatase 2A, 2B, or 2C. Also unaffected by diabetes were the activities of protein kinase C, cAMP-dependent protein kinase and calcium-calmodulin protein kinase. Because of the decrease in phosphatase 1 activity, 32P incorporation into sarcolemmal phosphoproteins catalyzed by either intrinsic protein kinases or extrinsic cAMP-dependent protein kinase was elevated in the diabetic. Among the proteins whose phosphorylation was elevated in diabetes was the phospholamban-like protein, which has been implicated in the regulation of ATP-dependent calcium transport. The phosphate-linked increase could be prevented by exposing the membranes to a phosphatase inhibitor and either extrinsic cAMP-dependent protein kinase or alamethicin. In addition to the phosphatase-linked effects, analysis of individual sarcolemmal phosphoproteins by SDS-polyacrylamide gel electrophoresis indicated that diabetes caused a specific elevation in membrane phosphorylation of some proteins (43 kDa and 78 kDa), but a decrease in the phosphorylation state of other phosphoproteins (31 kDa and 49 kDa). The data indicate that membrane phosphorylation is dramatically altered by diabetes. The possibility that this contributes to altered myocardial function is discussed.     

Distribution of protein kinase activities in subcellular fractions of rat brain

The subcellular distribution of histone and phosvitin kinase activities in brain has been studied and the ability of the various fractions to catalyse the phosphorylation of their endogenous proteins (intrinsic protein kinase activity) also examined. Synaptosome membrane fragments have little or no histone or phosvitin kinase activity but contain the highest concentration of cyclic AMP-stimulated intrinsic protein kinase activity. Homogenisation of the membrane fragments in Triton X-100 increased the histone kinase activity but on centrifugation it was all recovered in the supernatant, while the insoluble material contained all the intrinsic protein kinase activity. These results indicate that the intrinsic protein kinase activity of cerebral membrane fragments is due to the presence of a kinase enzyme which is specific to certain membrane proteins. The intrinsic protein kinase activity of synaptosome membrane fragments is a rather slow reaction which takes several minutes to saturate all the acceptor proteins.     

Distribution of protein kinase activities in subcellular fractions of rat brain.

The subcellular distribution of histone and phosvitin kinase activities in brain has been studied and the ability of the various fractions to catalyse the phosphorylation of their endogenous proteins (intrinsic protein kinase activity) also examined. Synaptosome membrane fragments have little or no histone or phosvitin kinase activity but contain the highest concentration of cyclic AMP-stimulated intrinsic protein kinase activity. Homogenisation of the membrane fragments in Triton X-100 increased the histone kinase activity but on centrifugation it was all recovered in the supernatant, while the insoluble material contained all the intrinsic protein kinase activity. These results indicate that the intrinsic protein kinase activity of cerebral membrane fragments is due to the presence of a kinase enzyme which is specific to certain membrane proteins. The intrinsic protein kinase activity of synaptosome membrane fragments is a rather slow reaction which takes several minutes to saturate all the acceptor proteins.     

Cyclic adenosine monophosphate-dependent and -independent protein kinase activity of renal brush border membranes.

Kinase(s) in brush border membranes, isolated from rabbit renal proximal tubules, phosphorylated proteins intrinsic to the membrane and exogenous proteins. cAMP stimulated phosphorylation of histone; phosphorylation of protamine was cAMP independent. cAMP-dependent increases in phosphorylation of endogenous membrane protein were small, but highly reproducible. Most of the ³²P incorporated into membranes represented phosphorylation of serine residues, with phosphorylthreonine comprising a minor component. cAMP did not alter the electrophoretic pattern of ³²P-labeled membrane polypeptides. The small cAMP-dependent phosphorylation of brush border membrane proteins was not due to membrane phosphodiesterase or adenylate cyclase activities. Considerable cAMP was found “endogenously” bound to the membranes as prepared. However, this did not result in preactivation of the kinase since activity was not inhibited by a heat-stable protein inhibitor of cAMP-dependent protein kinases. With intrinsic membrane protein as phosphate acceptor, the relationship between rate of phosphorylation and ATP concentration appeared to follow Michaelis-Menton kinetics. With histone the relationship was complex. cAMP did not affect the apparent K_m for histone. One-half maximal stimulation of the rate of histone phosphorylation was obtained with 7 × 10^?8m cAMP. The K_a values for dibutyryl cAMP, cIMP, and cGMP were one to two orders of magnitude greater. Treatment of brush border membranes with detergent greatly increased the dependency of histone phosphorylation on cAMP. Phosphorylations of intrinsic membrane protein and histone were nonlinear with time, due in part to the lability of the protein kinase, the hydrolysis of ATP, and minimally to the presence of phosphoprotein phosphatase in the border membrane. The membrane phosphoprotein phosphatase was unaffected by cyclic nucleotides. Protein kinase activity was also found in cytosolic and crude particulate fractions of the renal cortex. Activity was enriched in the brush border membrane relative to that in the crude membrane preparation. The kinase activities in the different loci were distinct both in relative activities toward different substrates and in responsiveness to cAMP.     

Phosphorylation and dephosphorylation of spectrin

The phosphorylation of spectrin polypeptide 2 is thought to be involved in the metabolically dependent regulation of red cell shape and deformability. Spectrin phosphorylation is not affected by cAMP. The reaction in isolated membranes resembles the cAMP-independent, salt-stimulated phosphorylation of an exogenous substrate, casein, by enzyme(s) present both in isolated membranes and cytoplasmic extracts. Spectrin kinase is selectively eluted from membranes by 0.5 M NaCl and co-fractionates with eluted casein kinase. Phosphorylation of band 3 in the membrane is inhibited by salt, but the band 3 kinase is otherwise indistinguishable operationally from spectrin kinase. The membrane-bound casein (spectrin) kinase is not eluted efficiently with spectrin at low ionic strength; about 80% of the activity is apparently bound at sites (perhaps on or near band 3) other than spectrin. Partitioning of casein kinase between cytoplasm and membrane is metabolically dependent; the proportion of casein kinase on the membrane can range from 25% to 75%, but for fresh cells is normally about 40%. Dephosphorylation of phosphorylated spectrin has not been studied intensively. Slow release of ³²Pi from [³²P] spectrin on the membrane can be demonstrated, but phosphatase activity measured against solubilized [³²P] spectrin is concentrated in the cytoplasm. The crude cytoplasmic phosphospectrin phosphatase is inhibited by various anions – notably, ATP and 2,3-DPG at physiological concentrations. Regulation of spectrin phosphorylation in intact cells has not been studied. We speculate that spectrin phosphorylation state may be regulated (1) by metabolic intermediates and other internal chemical signals that modulate kinase and phosphatase activities per se or determine their intracellular localization and (2) by membrane deformation that alters enzyme–spectrin interaction locally. Progress in the isolation and characterization of spectrin kinase and phosphospectrin phosphatase should lead to the resolution of major questions raised by previous work: the relationships between membrane-bound and cytoplasmic forms of the enzymes, the nature of their physical interactions with the membrane, and the regulation of their activities in defined cell-free systems.     

Complex I and the cAMP cascade in human physiopathology

A cAMP-dependent protein kinase (PKA) is localized in mammalian mitochondria with the catalytic site at the matrix side of the membrane where it phosphorylates a number of proteins. One of these is the 18 kDa(IP) subunit of the mammalian complex I of the respiratory chain, encoded by the nuclear NDUFS4 gene. Mitochondria have a Ca²⁺-inhibited phosphatase, which dephosphorylates the 18 kDa phosphoprotein of complex I. In fibroblast and myoblast cultures cAMP-dependent phosphorylation of the 18 kDa protein is associated with stimulation of complex I and overall respiratory activity with NAD-linked substrates. Mutations in the human NDUFS4 gene have been found, which in the homozygous state are associated with deficiency of complex I and fatal neurological syndrome.     

erbB1 functions as a sensor of airway epithelial integrity by regulation of protein phosphatase 2A activity

Two enzymes, protein phosphatase 2A and atypical protein kinase C, are associated with the tight junction and regulate its function. For example, phosphorylation of the tight junction protein occludin is required for its incorporation into the junction. The association of a kinase and phosphatase with the tight junction suggests that a balance between their activities exists and is required for normal tight junction function. This hypothesis predicts that loss of epithelial integrity may disrupt this balance in such a way as to facilitate restoration of epithelial integrity. Our previous data have shown that apically localized growth factors segregate from their basolaterally localized erbB receptors. Loss of epithelial integrity allows ligand access to the basolateral membrane where it immediately binds to and activates erbB receptors. We found that activation of erbB1 leads to phosphorylation of protein phosphatase 2A, inhibiting its activity. Importantly, this phosphorylation event was dependent on factors in the overlying airway surface liquid; washing away this liquid prevented phosphorylation. erbB1-mediated inhibition of phosphatase activity would shift the balance in favor of the kinase such that tight junction proteins would regain their phosphorylation, allowing for their incorporation into the junction complex. This mechanism provides a rapid means of sensing the loss of epithelial integrity and subsequently restoring barrier function.     

Selective modulation of the two antagonistic activities of protein kinase FA (the activator of ATP.Mg-dependent protein phosphatase)

The ATP.Mg-dependent protein phosphatase activating factor (FA) has been identified as a protein kinase. The results are unexpected since factor FA possesses two activities which are antagonistic. As a kinase, factor FA catalyzes protein phosphorylation, while as a phosphatase activator, it catalyzes protein dephosphorylation. In this report, we found that the two opposing activities of factor FA could be selectively modulated. For instance, heparin at concentrations of 0.1-0.3 mg/ml could stimulate FA to work preferentially as a kinase towards phosphorylation of proteins but simultaneously inhibit it to work as a phosphatase activator towards dephosphorylation of the same proteins. In a similar manner, alkaline pH could stimulate FA to work as a kinase but block it to work as a phosphatase activator. This is the first report providing initial evidence that the two opposing activities of factor FA can be selectively modulated in a reciprocal manner by various triggers, suggesting that a simultaneous coordinate control mechanism may well be involved in regulating the activities of factor FA in the cell.     

Phosphorylation and dephosphorylation of human platelet surface proteins by an ecto-protein kinase/phosphatase system.

We have characterized a novel ecto-protein kinase activity and a novel ecto-protein phosphatase activity on the membrane surface of human platelets. Washed intact platelets, when incubated with [gamma-32P]ATP in Tyrode's buffer, showed the phosphorylation of a membrane surface protein migrating with an apparent molecular mass of 42 kDa on 5-15% SDS polyacrylamide gradient gels. The 42 kDa protein could be further resolved on 15% SDS gels into two proteins of 39 kDa and 42 kDa. In this gel system, it was found that the 39 kDa protein became rapidly phosphorylated and dephosphorylated, whereas the 42 kDa protein was phosphorylated and dephosphorylated at a much slower rate. NaF inhibited the dephosphorylation of these proteins indicating the involvement of an ecto-protein phosphatase. The platelet membrane ecto-protein kinase responsible for the phosphorylation of both of these proteins was identified as a serine kinase and showed dependency on divalent cations Mg2+ or Mn2+ ions. Ca2+ ions potentiated the Mg(2+)-dependent ecto-protein kinase activity. The ecto-protein kinase rapidly phosphorylated histone and casein added exogenously to the extracellular medium of intact platelets. Following activation of platelets by alpha-thrombin, the incorporation of [32P]phosphate from exogenously added [gamma-32P]ATP by endogenous protein substrates was reduced by 90%, suggesting a role of the ecto-protein kinase system in the regulation of platelet function. The results presented here demonstrate that both protein kinase and protein phosphatase activities reside on the membrane surface of human platelets. These activities are capable of rapidly phosphorylating and dephosphorylating specific surface platelet membrane proteins which may play important roles in early events of platelet activation and secretion.     

Protein modification enzymes associated with the protein-synthesizing complex from rabbit reticulocytes. Protein kinase, phosphoprotein phosphatase, and acetyltransferase.

A number of protein modification activities are present in the protein-synthesizing complex isolated from rabbit reticulocytes. These enzymes are solubilized by sedimentation of the ribosomes through buffered sucrose containing 0.5 M KCl, and have been partially purified from the high salt wash fraction by chromatography on DEAE-cellulose and phosphocellulose. The ribosomal-associated enzymatic activities include cyclic AMP-regulated and cyclic nucloetide-independent protein kinase, phosphoprotein phosphatase, and acetyltransferase activities. These enzymatic activities have been shown to modify specific ribosomal and ribosomal-associated proteins. The cycli c AMP-regulated protein kinase phosphorylate the 40 S ribosomal subunit from rabbit reticulocytes. One of the cyclic nucleotide-independent protein kinase catalyzes the phosphorylation of two different factors involved in the initiation of hemoglobin synthesis. A single phosphoprotein phosphatase activity is shown to remove phosphate from 40 S ribosomal subunits. The major acetyltransferase activity associated with ribosomes acetylates a 60 S ribosomal protein.     

Thyroxine-stimulated synthesis of microvillus membrane glycoproteins during culture of chick embryonic duodenum

The effect of thyroxine on biosynthesis of microvillus membrane glycoproteins has been investigated in organ culture of 18-day-old chick embryonic duodenum. Explants incorporate [³H]leucine and [³H]glucosamine continuously, and overall incorporation is enhanced by 10 nM thyroxine during 48 h of labeling; this increase in radioactivity is associated with vesicles released from the microvilli. Light microscope autoradiography, pulse labeling of brush border fragments, and pulse chase experiments reveal that [³H]glucosamine is incorporated into brush border at an increasing rate during culture, and that newly synthesized glycoproteins are discharged into the medium along with brush border enzymes (alkaline phosphatase and maltase). These results suggest that thyroxine stimulates biosynthesis of microvillus membrane glycoproteins, in addition to stimulating vesiculation of the membrane. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of ³H-labeled vesicles and brush border fragments show that [³H]leucine and [³H]glucosamine are incorporated into proteins of high molecular weight. Two protein bands are identified as alkaline phosphatase and maltase. Thyroxine stimulates glycosylation of these enzymes, but does not change protein patterns. Radioactivity assay of alkaline phosphatase- and maltase-active gel slices suggests that thyroxine stimulation of these enzyme activities during culture is not correlated with de novo synthesis of these proteins.     

The protein phosphatases involved in cellular regulation. 1. Classification and substrate specificities

The protein phosphatase activities involved in regulating the major pathways of intermediary metabolism can be explained by only four enzymes which can be conveniently divided into two classes, type-1 and type-2. Type-1 protein phosphatases dephosphorylate the beta-subunit of phosphorylase kinase and are potently inhibited by two thermostable proteins termed inhibitor-1 and inhibitor-2, whereas type-2 protein phosphatases preferentially dephosphorylate the alpha-subunit of phosphorylase kinase and are insensitive to inhibitor-1 and inhibitor-2. The substrate specificities of the four enzymes, namely protein phosphatase-1 (type-1) and protein phosphatases 2A, 2B and 2C (type-2) have been investigated. Eight different protein kinases were used to phosphorylate 13 different substrate proteins on a minimum of 20 different serine and threonine residues. These substrates include proteins involved in the regulation of glycogen metabolism, glycolysis, fatty acid synthesis, cholesterol synthesis, protein synthesis and muscle contraction. The studies demonstrate that protein phosphatase-1 and protein phosphatase 2A have very broad substrate specificities. The major differences, apart from the site specificity for phosphorylase kinase, are the much higher myosin light chain phosphatase and ATP-citrate lyase phosphatase activities of protein phosphatase-2A. Protein phosphatase-2C (an Mg2+-dependent enzyme) also has a broad specificity, but can be distinguished from protein phosphatase-2A by its extremely low phosphorylase phosphatase and histone H1 phosphatase activities, and its slow dephosphorylation of sites (3a + 3b + 3c) on glycogen synthase relative to site-2 of glycogen synthase. It has extremely high hydroxymethylglutaryl-CoA (HMG-CoA) reductase phosphatase and HMG-CoA reductase kinase phosphatase activity. Protein phosphatase-2B (a Ca2+-calmodulin-dependent enzyme) is the most specific phosphatase and only dephosphorylated three of the substrates (the alpha-subunit of phosphorylase kinase, inhibitor-1 and myosin light chains) at a significant rate. It is specifically inhibited by the phenathiazine drug, trifluoperazine. Examination of the amino acid sequences around each phosphorylation site does not support the idea that protein phosphatase specificity is determined by the primary structure in the immediate vicinity of the phosphorylation site.     

The MgATP-dependent protein phosphatase and protein phosphatase 1 have identical substrate specificities

The MgATP-dependent phosphorylase phosphatase was found to have a broad substrate specificity. Its activity against all phosphoproteins tested was dependent upon preincubation with the activating factor FA and MgATP. The enzyme dephosphorylated and inactivated phosphorylase kinase and inhibitor 1, and dephosphorylated and activated glycogen synthase and acetyl-CoA carboxylase. Glycogen synthase was dephosphorylated at similar rates whether it had been phosphorylated by cyclic-AMP-dependent protein kinase, phosphorylase kinase or glycogen synthase kinase 3. The enzyme also catalysed the dephosphorylation of ATP citrate lyase, initiation factor eIF-2, and troponin I. The properties of the MgATP-dependent protein phosphatase from either dog liver or rabbit skeletal muscle showed a remarkable similarity to highly purified preparations of protein phosphatase 1 from rabbit skeletal muscle. The relative activities of the two enzymes against all phosphoproteins tested was very similar. Both enzymes dephosphorylated the beta-subunit of phosphorylase kinase 40-fold faster than the alpha-subunit, and both enzymes were inhibited by identical concentrations of the two proteins termed inhibitor 1 and inhibitor 2, which inhibit protein phosphatase 1 specifically. These results demonstrate that the MgATP-dependent protein phosphatase is a type-1 protein phosphatase, and is distinct from type-2 protein phosphatases which dephosphorylate the alpha-subunit of phosphorylase kinase and are unaffected by inhibitor 1 and inhibitor 2. The possibility that the MgATP-dependent protein phosphatase is an inactive form of protein phosphatase 1 and that both proteins share the same catalytic subunit is discussed.     

Specific association of calmodulin-dependent protein kinase and related substrates with the junctional sarcoplasmic reticulum of skeletal muscle

A systematic study of protein kinase activity and phosphorylation of membrane proteins by ATP was carried out with vesicular fragments of longitudinal tubules (light SR) and junctional terminal cisternae (JTC) derived from skeletal muscle sarcoplasmic reticulum (SR). Following incubation of JTC with ATP, a 170,000-Da glycoprotein, a 97,500-Da protein (glycogen phosphorylase), and a 55,000-60,000-Da doublet (containing calmodulin-dependent protein kinase subunit) underwent phosphorylation. Addition of calmodulin in the presence of Ca2+ (with no added protein kinase) produced a 10-fold increase of phosphorylation involving numerous JTC proteins, including the large (approximately 450,000 Da) ryanodine receptor protein. Calmodulin-dependent phosphorylation of the ryanodine receptor protein was unambiguously demonstrated by Western blot analysis. The specificity of these findings was demonstrated by much lower levels of calmodulin-dependent phosphorylation in light SR as compared to JTC, and by much lower cyclic AMP dependent kinase activity in both JTC and light SR. These observations indicate that the purified JTC contain membrane-bound calmodulin-dependent protein kinase that undergoes autophosphorylation and catalyzes phosphorylation of various membrane proteins. Protein dephosphorylation was very slow in the absence of added phosphatases, but was accelerated by the addition of phosphatase 1 and 2A (catalytic subunit) in the absence of Ca2+, and calcineurin in the presence of Ca2+. Therefore, in the muscle fiber, dephosphorylation of SR proteins relies on cytoplasmic phosphatases. No significant effect of protein phosphorylation was detected on the Ca2(+)-induced Ca2+ release exhibited by isolated JTC vesicles. However, the selective and prominent association of calmodulin-dependent protein kinase and related substrates with junctional membranes, its Ca2+ sensitivity, and its close proximity to the ryanodine and dihydropyridine receptor Ca2+ channels suggest that this phosphorylation system is involved in regulation of functions linked to these structures.     

Effects of cyclic adenosine 3': 5'-monophosphate on phosphoprotein kinase and phosphatase fractions prepared from rat liver nuclei.

A soluble rat liver nuclear extract containing total RNA polymerase activities also exhibits appreciable amounts of protein kinase activity. This unfractionated protein kinase catalyzes the phosphorylation of both endogenous proteins and exogenous lysine-rich histone in the presence of [γ-³²P]ATP and Mg²⁺. The optimal concentration of Mg²⁺ is 5 mm for histone phosphorylation and 25 mm for the phosphorylation of endogenous proteins. Cyclic AMP has no effect on the phosphorylation of lysine-rich histone by this unfractionated nuclear protein kinase. However, addition of cyclic AMP causes a reduction in the ³²P-labeling of an endogenous protein (CAI) which can be characterized by its mobility during SDS-acrylamide gel electrophoresis and elution in the unbound fraction of a DEAESephadex column. If CAI is first labeled with ³²P and then incubated with 10^?6m cyclic AMP under conditions where protein kinase activity is inhibited, the presence of the cyclic nucleotide causes a loss of the ³²P-labeling of this protein, implying the activation of a substrate-specific protein phosphatase. When rat liver RNA polymerases are purified by DEAE-Sephadex chromatography, protein kinase activity is found in the unbound fraction and in those column fractions containing RNA polymerase I and II. The fractionated protein kinases exhibit different responses to cyclic AMP, the unbound protein kinase being stimulated and the RNA polymerase-associated protein kinases being dramatically inhibited. A second protein (CAII) whose phosphorylated state is modified by cyclic AMP is found within the DEAE-Sephadex column fractions containing RNA polymerase II. The cyclic nucleotide in this case appears to reduce labeling of CAII by inhibition of the protein kinase activity which co-chromatographs with both CAII and RNA polymerase II. Based on molecular weight estimates, neither CAI nor CAII appears to be an RNA polymerase subunit. The identity of CAI as a protein factor whose phosphorylated state influences nuclear RNA synthesis is suggested by the fact that addition of fractions containing CAI to purified RNA polymerase II inhibits the activity of this enzyme, but only if CAI has been previously incubated in the presence of cyclic AMP.     

Separation of two phosphorylase kinase phosphatases from rabbit skeletal muscle.

Cyclic-AMP-dependent protein kinase catalyses the activation of phosphorylase kinase and the phosphorylation of two serine residues on the alpha subunit and beta 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 alpha-phosphorylase kinase phosphatase and beta-phosphorylase kinase phosphatase. These two enzymes show essentially absolute specificity towards the alpha and beta subunits respectively. The two phosphatases copurified through ethanol fractionation, DEAE-cellulose chromatography and ammonium sulphate precipitation, but were separated from each other by a gel filtration on Sephadex G-200. alpha-Phosphorylase kinase phosphatase was purified 500-fold from the ethanol precipitation step, and beta-phosphorylase kinase phosphatase 320-fold. The molecular weights estimated by gel filtration were 170--180 000 for alpha-phosphorylase kinase phosphatase and 75--80 000 for beta-phosphorylase kinase phosphatase. Since the activity of phosphorylase kinase correlates with the state of phosphorylation of the beta subunit (Cohen, P. (1974)), beta-phosphorylase kinase phosphatase is the enzyme which reverses the activation of phosphorylase kinase. alpha-Phosphorylase kinase phosphatase is an enzyme activity that has not been recognised previously. Since the role of the alpha-subunit phosphorylation is to stimulate the rate of dephosphorylation of the beta subunit (Cohen, P. (1974)), alpha-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.     

Dephosphorylation of the beta 2-adrenergic receptor and rhodopsin by latent phosphatase 2

Recent evidence suggests that the function of receptors coupled to guanine nucleotide regulatory proteins may be controlled by highly specific protein kinases, e.g. rhodopsin kinase and the beta-adrenergic receptor kinase. In order to investigate the nature of the phosphatases which might be involved in controlling the state of receptor phosphorylation we studied the ability of four highly purified well characterized protein phosphatases to dephosphorylate preparations of rhodopsin or beta 2-adrenergic receptor which had been highly phosphorylated by beta-adrenergic receptor kinase. These included: type 1 phosphatase, calcineurin phosphatase, type 2A phosphatase, and the high molecular weight latent phosphatase 2. Under conditions in which all the phosphatases could dephosphorylate such common substrates as [32P]phosphorylase a and [32P]myelin basic protein at similar rates only the latent phosphatase 2 was active on the phosphorylated receptors. Moreover, a latent phosphatase activity was found predominantly in a sequestered membrane fraction of frog erythrocytes. This parallels the distribution of a beta-adrenergic receptor phosphatase activity recently described in these cells (Sibley, D. R., Strasser, R. H., Benovic, J. L., Daniel, K., and Lefkowitz, R. J. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 9408-9412). These data suggest a potential role for the latent phosphatase 2 as a specific receptor phosphatase.