DARPP-32 and Phosphatase Inhibitor-1, Two Structurally Related Inhibitors of Protein Phosphatase-1, Are Both Present in Striatonigral Neurons

DARPP-32 (dopamine- and cyclic AMP-regulated phosphoprotein of Mr = 32,000) and phosphatase inhibitor-1, two previously characterized inhibitors of protein phosphatase-1, were identified in both the neostriatum and the substantia nigra. Phosphatase inhibitor-1 was partially purified from bovine caudate nucleus and found to be distinct from DARPP-32 in some of its biochemical properties. The neuronal localization of DARPP-32 and phosphatase inhibitor-1 within the rat neostriatum and substantia nigra was investigated by studying the effects of kainic acid. Injection into the neostriatum of kainic acid, which destroys striatonigral neurons and striatonigral fibers, decreased the amounts of DARPP-32 and phosphatase inhibitor-1 to the same extent, both in the lesioned neostriatum and in the ipsilateral substantia nigra. The specific activity of protein phosphatase-1 in the neostriatum was unaffected by kainic acid. The results indicate that, in rat brain, DARPP-32 and phosphatase inhibitor-1 are both present in striatal neurons and in striatonigral fibers, and that they probably coexist in at least a subpopulation of striatonigral neurons. In contrast, protein phosphatase-1 does not appear to be enriched in any specific neuronal subpopulation in the neostriatum.     

Na+,K(+)-ATPase phosphorylation in the choroid plexus: synergistic regulation by serotonin/protein kinase C and isoproterenol/cAMP-PK/PP-1 pathways.

BACKGROUND: The ion pump Na+,K(+)-ATPase is responsible for the secretion of cerebrospinal fluid from the choroid plexus. In this tissue, the activity of Na+,K(+)-ATPase is inhibited by serotonin via stimulation of protein kinase C-catalyzed phosphorylation. The choroid plexus is highly enriched in two phosphoproteins which act as regulators of protein phosphatase-1 activity, DARPP-32 and inhibitor-1. Phosphorylation catalyzed by cAMP-dependent protein kinase on a single threonyl residue converts DARPP-32 and inhibitor-1 into potent inhibitors of protein phosphatase-1. Previous work has shown that in the choroid plexus, phosphorylation of DARPP-32 and I-1 is enhanced by isoproterenol and other agents that activate cAMP-PK. We have now examined the possible involvement of the cAMP-PK/protein phosphatase-1 pathway in the regulation of Na+,K(+)-ATPase. MATERIALS AND METHODS: The state of phosphorylation of Na+,K(+)-ATPase was measured by determining the amount of radioactivity incorporated into the ion pump following immunoprecipitation from 32P-prelabeled choroid plexuses incubated with various drugs (see below). Two-dimensional phosphopeptide mapping was employed to identify the protein kinase involved in the phosphorylation of Na+,K(+)-ATPase. RESULTS: The serotonin-mediated increase in Na+,K(+)-ATPase phosphorylation is potentiated by okadaic acid, an inhibitor of protein phosphatases-1 and -2A, as well as by forskolin or the beta-adrenergic agonist, isoproterenol, activators of cAMP-dependent protein kinase. Two-dimensional phosphopeptide maps suggest that this potentiating action occurs at the level of a protein kinase C phosphorylation site. Forskolin and isoproterenol also stimulate the phosphorylation of DARPP-32 and protein phosphatase inhibitor-1, which in their phosphorylated form are potent inhibitors of protein phosphatase-1. CONCLUSIONS: The results presented here support a model in which okadaic acid, forskolin, and isoproterenol achieve their synergistic effects with serotonin through phosphorylation of DARPP-32 and inhibitor-1, inhibition of protein phosphatase-1, and a reduction of dephosphorylation of Na+,K(+)-ATPase at a protein kinase C phosphorylation site.     

Phosphorylation of DARPP-32, a dopamine- and cAMP-regulated phosphoprotein, by casein kinase II

DARPP-32 (dopamine- and cAMP-regulated phosphorprotein, Mr = 32,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) is an inhibitor of protein phosphatase-1 and is enriched in dopaminoceptive neurons possessing the D1 dopamine receptor. Purified bovine DARPP-32 was phosphorylated in vitro by casein kinase II to a stoichiometry greater than 2 mol of phosphate/mol of protein whereas two structurally and functionally related proteins, protein phosphatase inhibitor-1 and G-substrate, were poor substrates for this enzyme. Sequencing of chymotryptic and thermolytic phosphopeptides from bovine DARPP-32 phosphorylated by casein kinase II suggested that the main phosphorylated residues were Ser45 and Ser102. In the case of rat DARPP-32, the identification of these phosphorylation sites was confirmed by manual Edman degradation. The phosphorylated residues are located NH2-terminal to acidic amino acid residues, a characteristic of casein kinase II phosphorylation sites. Casein kinase II phosphorylated DARPP-32 with an apparent Km value of 3.4 microM and a kcat value of 0.32 s-1. The kcat value for phosphorylation of Ser102 was 5-6 times greater than that for Ser45. Studies employing synthetic peptides encompassing each phosphorylation site confirmed this difference between the kcat values for phosphorylation of the two sites. In slices of rat caudate-putamen prelabeled with [32P]phosphate, DARPP-32 was phosphorylated on seryl residues under basal conditions. Comparison of thermolytic phosphopeptide maps and determination of the phosphorylated residue by manual Edman degradation identified the main phosphorylation site in intact cells as Ser102. In vitro, DARPP-32 phosphorylated by casein kinase II was dephosphorylated by protein phosphatases-1 and -2A. Phosphorylation by casein kinase II did not affect the potency of DARPP-32 as an inhibitor of protein phosphatase-1, which depended only on phosphorylation of Thr34 by cAMP-dependent protein kinase. However, phosphorylation of DARPP-32 by casein kinase II facilitated phosphorylation of Thr34 by cAMP-dependent protein kinase with a 2.2-fold increase in the Vmax and a 1.4-fold increase in the apparent Km. Phosphorylation of DARPP-32 by casein kinase II in intact cells may therefore modulate its phosphorylation in response to increased levels of cAMP.     

Synthetic peptide analogs of DARPP-32 (Mr 32,000 dopamine- and cAMP-regulated phosphoprotein), an inhibitor of protein phosphatase-1. Phosphorylation, dephosphorylation, and inhibitory activity

Synthetic peptides based on the threonine phosphorylation site and proposed inhibitory site of DARPP-32 (dopamine- and cAMP-regulated phosphoprotein, Mr = 32,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) were prepared and analyzed as substrates for cAMP-dependent protein kinase and protein phosphatases-1c, -2Ac (the catalytic subunits of protein phosphatase-1 and 2A, respectively) and -2B, and as inhibitors of protein phosphatase-1c. Studies of the kinetics of phosphorylation of the peptides by cAMP-dependent protein kinase indicated an important role in facilitating phosphorylation for the region COOH-terminal to the phosphorylatable threonyl residue. Studies of the dephosphorylation of the phosphopeptides demonstrated that they were effectively dephosphorylated by protein phosphatase-2A and -2B and poorly dephosphorylated by protein phosphatase-1. The active inhibitory region of phospho-DARPP-32 was analyzed by determining the effects of synthetic phosphopeptides on the activity of protein phosphatase-1c. Phospho-D32-(8-48) and phospho-D32-(8-38) inhibited protein phosphatase-1c with IC50 values of 2 x 10(-8) and 4 x 10(-8) M, respectively, compared with an IC50 of 8 x 10(-9) M for intact phospho-DARPP-32. Phospho-D32-(9-38) was equipotent with phospho-D32-(8-38); however, further NH2-terminal deletions resulted in marked reductions in IC50 values. An analog of an active DARPP-32 phosphopeptide containing a phosphoseryl residue in place of the phosphothreonyl residue also exhibited a much reduced IC50. These data identify the essential inhibitory region of phospho-DARPP-32 as residues 9-38, which contains the phosphorylation site (Thr34). This region exhibits extensive amino acid sequence identity with phosphatase inhibitor-1, a distinct inhibitor of protein phosphatase-1. Kinetic studies of the inhibition of protein phosphatase-1c by phospho-D32-(9-38), a potent inhibitor, as well as by phospho-D32-(10-38), a weak inhibitor, indicated a mixed competitive/noncompetitive mechanism of inhibition, as has been previously found for both intact phospho-DARPP-32 and intact phospho-inhibitor-1. These findings support the hypothesis that a 30-amino acid domain in the NH2-terminal region of phospho-DARPP-32 is sufficient for the inhibition of protein phosphatase-1.     

Distribution of Protein Phosphatase Inhibitor-1 in Brain and Peripheral Tissues of Various Species: Comparison with DARPP-32

The distribution of inhibitor-1, a cyclic AMP-regulated inhibitor of protein phosphatase-1, was analyzed in various brain regions and peripheral tissues of various species by immunolabeling of sodium dodecyl sulfate-poly-acrylamide gel transfers using specific antibodies. The distribution of inhibitor-1 was directly compared to that of DARPP-32, a structurally related cyclic AMP-regulated inhibitor of protein phosphatase-1. In rat CNS, a single immunoreactive protein of M(r) 30,000, identified as inhibitor-1, was widely distributed. In contrast, DARPP-32 was highly concentrated in the basal ganglia. Inhibitor-1 was detected in brain tissue from frog (M(r) 27,000), turtle (M(r) 29,000/33,000), canary (M(r) 26,000), pigeon (M(r) 28,000), mouse (M(r) 30,500), rabbit (M(r) 26,500), cow (M(r) 27,000), and monkey (M(r) 27,500), but not from goldfish. Inhibitor-1 was detected at various levels in most peripheral tissues of the species studied; however, it was not detectable in certain tissues of particular species (e.g., rat and cow liver). DARPP-32 was detected in brain tissue of all the species tested except frog and goldfish, but was not detectable in most peripheral tissues. Both inhibitor-1 and DARPP-32 were concentrated in the cytosol and synaptosomal cytosol of rat striatum. The developmental expressions of inhibitor-1 and DARPP-32 in rat striatum differed: the level of inhibitor-1 peaked in the first postnatal week and then declined by the third postnatal week, whereas the level of DARPP-32 increased to a peak level by the third postnatal week and remained elevated thereafter.(ABSTRACT TRUNCATED AT 250 WORDS)     

DARPP-32, a dopamine- and cyclic AMP-regulated neuronal phosphoprotein. Primary structure and homology with protein phosphatase inhibitor-1

The complete amino acid sequence of bovine brain DARPP-32, a dopamine- and cyclic AMP-regulated neuronal phosphoprotein, which is a potent and specific inhibitor of the catalytic subunit of protein phosphatase-1, has been determined. The S-14C-carboxymethylated protein was subjected to enzymatic cleavage by endoproteinase Lys-C, endoproteinase Arg-C, trypsin, chymotrypsin, and Staphylococcus aureus V8 protease, and to chemical cleavage by cyanogen bromide. The overlapping sets of peptides were purified by high performance liquid chromatography and subjected to amino acid sequencing by automated Edman degradation to deduce the complete sequence. The protein consists of a single NH2-terminal blocked polypeptide chain of 202 residues, with a calculated molecular mass of 22,591 daltons, excluding the unidentified NH2-terminal blocking group. This molecular mass is significantly lower than earlier estimates based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis or hydrodynamic measurements. The threonine residue that is phosphorylated by cyclic AMP-dependent protein kinase (Hemmings, H. C., Jr., Williams, K. R., Konigsberg, W. H., and Greengard, P. (1984) J. Biol. Chem. 259, 14486-14490), and that must be phosphorylated for the expression of inhibitory activity, is located at position 34. The molecule contains only 1 cysteine residue and 1 tryptophan residue, at positions 72 and 161, respectively. DARPP-32 is very hydrophilic, and contains a stretch of 16 consecutive acidic residues from position 119 to 134. The predicted secondary structure suggests the presence of 47% alpha-helix, 7% beta-sheet, and 46% random coil, with 11 beta-turns. Comparison of the complete amino acid sequence of bovine DARPP-32 with that of rabbit skeletal muscle protein phosphatase inhibitor-1 revealed a significant amount of sequence identity in the NH2-terminal regions of these two proteins. The active region of inhibitor-1 has been localized to an NH2-terminal fragment (Aitken, A., and Cohen, P. (1982) FEBS Lett. 147, 54-58), the part of the molecule that is most similar to DARPP-32. These data suggest that these two protein phosphatase inhibitors may share a common structural basis for their inhibitory activity and may be related by a common ancestral gene.     

DARPP-32, a dopamine- and adenosine 3':5'-monophosphate-regulated neuronal phosphoprotein. I. Amino acid sequence around the phosphorylated threonine

DARPP-32 (dopamine- and cyclic AMP-regulated phosphoprotein, Mr = 32,000) is a major endogenous cytosolic substrate for dopamine- and cyclic AMP-stimulated protein phosphorylation in neurons of the basal ganglia of mammalian brain. It shares many properties with phosphatase inhibitor 1, a substrate for cyclic AMP-dependent protein kinase, and with G-substrate, a substrate for cyclic GMP-dependent protein kinase. We have, therefore, undertaken an analysis of the amino acid sequence around the site at which purified DARPP-32 is phosphorylated by the catalytic subunit of cyclic AMP-dependent protein kinase. The results indicate that DARPP-32 is phosphorylated at a single threonine residue contained in the sequence Arg-Arg-Arg-Pro-Thr(P)-Pro-Ala-Met-Leu-Phe-Arg. This sequence was obtained by automated solid phase sequencing of two overlapping tryptic phosphopeptides and one overlapping chymotryptic phosphopeptide which were purified by reverse-phase high-performance liquid chromatography. A 9-amino acid sequence containing the phosphorylatable threonine residue in DARPP-32 shares 8 identical residues with a sequence containing the phosphorylatable threonine residue in phosphatase inhibitor 1, and shares 5 identical residues with the two identical sequences surrounding the 2 phosphorylatable threonine residues in G-substrate. These observations support the view that DARPP-32, inhibitor 1, and G-substrate are members of a family of regulatory proteins which are involved in the control of protein phosphatase activity by both cyclic AMP and cyclic GMP, but which differ in their cellular and tissue distributions.     

Role of calcineurin and protein phosphatase-2A in the regulation of DARPP-32 dephosphorylation in neostriatal neurons

DARPP-32, a dopamine- and cyclic AMP-regulated phosphoprotein of Mr 32 kDa, is phosphorylated on Thr34 by cyclic AMP-dependent protein kinase, resulting in its conversion to a potent inhibitor of protein phosphatase-1 (PP-1). Conversely, Thr34-phosphorylated DARPP-32 is dephosphorylated and inactivated in vitro by calcineurin and protein phosphatase-2A (PP-2A). We have investigated the relative contributions of these protein phosphatases to the regulation of DARPP-32 dephosphorylation in mouse neostriatal slices. Cyclosporin A (5 microM), a calcineurin inhibitor, maximally increased the level of phosphorylated DARPP-32 by 17+/-2-fold. Okadaic acid (1 microM), an inhibitor of PP-1 and PP-2A, had a smaller effect, increasing phospho-DARPP-32 by 5.1+/-1.3-fold. The effect of okadaic acid on DARPP-32 phosphorylation was shown to be due to inhibition of PP-2A activity. Incubation of slices in the presence of cyclosporin A plus either okadaic acid or calyculin A, another PP-1/PP-2A inhibitor, caused a synergistic increase in the level of phosphorylated DARPP-32. The use of Ca2(+)-free/EGTA medium mimicked the effects of cyclosporin A on DARPP-32 phosphorylation, supporting the conclusion that the action of cyclosporin on DARPP-32 phosphorylation was attributable to blockade of the Ca2(+)-dependent activation of calcineurin. The results indicate that calcineurin and PP-2A, but not PP-1, act synergistically to maintain a low level of phosphorylated DARPP-32 in neostriatal slices.     

Adipose tissue protein phosphatase inhibitor-2

Rat fat cells contain three species of spontaneously active inhibitor proteins of protein phosphatase 1, as resolved by SDS-PAGE, with apparent molecular masses of 40 kDa, and 28 kDa respectively. The 33-kDa, thermostable inhibitor was highly purified from bovine adipose tissue and shown to be very similar to inhibitor-2 of skeletal muscle. It was phosphorylated, on threonine only, by glycogen synthase kinase 3. It formed an inactivated complex with protein phosphatase 1, that was reactivated by incubation with ATP-Mg and glycogen synthase kinase 3. By gel filtration it had a Stokes radius of 3.4 nm. Peptide and phosphopeptide maps, generated by Staphylococcus aureus V8 proteinase, trypsin or thermolysin, of the inhibitor and of the skeletal muscle inhibitor-2 were similar. The 40-kDa inhibitor, which was denatured by boiling, represents a novel protein phosphatase inhibitor protein or an undegraded precursor of inhibitor-2. The total activity of inhibitor-2-like material (thermostable and macromolecular) in an adipocyte cytosol extract corresponded to an intracellular concentration of 0.3 microM inhibitor-2.     

Inhibitor-1 is not required for the activation of glycogen synthase by insulin in skeletal muscle.

Glycogen synthase is an excellent in vitro substrate for protein phosphatase-1 (PP1), which is potently inhibited by the phosphorylated forms of DARPP-32 (dopamine- and cAMP-regulated phosphoprotein, M(r) = 32,000) and Inhibitor-1. To test the hypothesis that the activation of glycogen synthase by insulin is due to a decrease in the inhibition of PP1 by the phosphatase inhibitors, we have investigated the effects of insulin on glycogen synthesis in skeletal muscles from wild-type mice and mice lacking Inhibitor-1 and DARPP-32 as a result of targeted disruption of the genes encoding the two proteins. Insulin increased glycogen synthase activity and the synthesis of glycogen to the same extent in wild-type and knockout mice, indicating that neither Inhibitor-1 nor DARPP-32 is required for the full stimulatory effects of insulin on glycogen synthase and glycogen synthesis in skeletal muscle.     

Phosphorylation of protein phosphatase inhibitor-1 by Cdk5

Protein phosphatase inhibitor-1 is a prototypical mediator of cross-talk between protein kinases and protein phosphatases. Activation of cAMP-dependent protein kinase results in phosphorylation of inhibitor-1 at Thr-35, converting it into a potent inhibitor of protein phosphatase-1. Here we report that inhibitor-1 is phosphorylated in vitro at Ser-67 by the proline-directed kinases, Cdk1, Cdk5, and mitogen-activated protein kinase. By using phosphorylation state-specific antibodies and selective protein kinase inhibitors, Cdk5 was found to be the only kinase that phosphorylates inhibitor-1 at Ser-67 in intact striatal brain tissue. In vitro and in vivo studies indicated that phospho-Ser-67 inhibitor-1 was dephosphorylated by protein phosphatases-2A and -2B. The state of phosphorylation of inhibitor-1 at Ser-67 was dynamically regulated in striatal tissue by glutamate-dependent regulation of N-methyl-d-aspartic acid-type channels. Phosphorylation of Ser-67 did not convert inhibitor-1 into an inhibitor of protein phosphatase-1. However, inhibitor-1 phosphorylated at Ser-67 was a less efficient substrate for cAMP-dependent protein kinase. These results demonstrate regulation of a Cdk5-dependent phosphorylation site in inhibitor-1 and suggest a role for this site in modulating the amplitude of signal transduction events that involve cAMP-dependent protein kinase activation.     

Regulation of DARPP-32 dephosphorylation at PKA- and Cdk5-sites by NMDA and AMPA receptors: distinct roles of calcineurin and protein phosphatase-2A

Glutamatergic inputs from corticostriatal and thalamostriatal pathways have been shown to modulate dopaminergic signaling in neostriatal neurons. DARPP-32 (dopamine- and cAMP-regulated phosphoprotein of M (r) 32 kDa) is a signal transduction molecule that regulates the efficacy of dopamine signaling in neostriatal neurons. Dopamine signaling is mediated in part through phosphorylation of DARPP-32 at Thr34 by cAMP-dependent protein kinase, and antagonized by phosphorylation of DARPP-32 at Thr75 by cyclin-dependent protein kinase 5. We have now investigated the effects of the ionotropic glutamate NMDA and AMPA receptors on DARPP-32 phosphorylation in neostriatal slices. Activation of NMDA and AMPA receptors decreased the state of phosphorylation of DARPP-32 at Thr34 and Thr75. The decrease in Thr34 phosphorylation was mediated through Ca(2+) -dependent activation of the Ca(2+) -/calmodulin-dependent phosphatase, calcineurin. In contrast, the decrease in Thr75 phosphorylation was mediated through Ca(2+) -dependent activation of dephosphorylation by protein phosphatase-2A. The results provide support for a complex effect of glutamate on dopaminergic signaling through the regulation of dephosphorylation of different sites of DARPP-32 by different protein phosphatases.     

Neurotensin regulates DARPP-32 thr34 phosphorylation in neostriatal neurons by activation of dopamine D1-type receptors

Neurotensin modulates dopaminergic transmission in the nigrostriatal system. DARPP-32, a dopamine- and cAMP-regulated phosphoprotein of Mr 32 kDa, is phosphorylated on Thr34 by cAMP-dependent protein kinase, resulting in its conversion into a potent inhibitor of protein phosphatase-1 (PP 1). Here, we examined the effect of neurotensin on DARPP-32 Thr34 phosphorylation using mouse neostriatal slices. Neurotensin stimulated DARPP-32 Thr34 phosphorylation by 4-7-fold with a K(0.5) of approximately 50 nM. The effect of neurotensin was antagonized by a combined neurotensin receptor type-1 (NTR1)/type-2 (NTR2) antagonist, SR142948. It was not antagonized by a NTR1 antagonist, SR48692 or by a NTR2 antagonist, levocabastine; neither was it antagonized by the two combined. Pretreatment with TTX or cobalt abolished the effect of neurotensin. The effect of neurotensin was antagonized by a dopamine D1 antagonist, SCH23390, and by ionotropic glutamate receptor antagonists, MK801 and CNQX. These results indicate that neurotensin stimulates the release of dopamine from nigrostriatal presynaptic terminals in an NMDA receptor- and AMPA receptor-dependent manner, leading to the increase in DARPP-32 Thr34 phosphorylation. Neurotensin stimulated the phosphorylation of Ser845 of the AMPA receptor GluR1 subunit in wild-type mice but not in DARPP-32 knockout mice. Thus, neurotensin, by stimulating the release of dopamine, activates the dopamine D1-receptor/cAMP/PKA/DARPP-32/PP 1 cascade.     

Hormonal regulation of protein dephosphorylation. Identification and hormonal regulation of protein phosphatase inhibitor-1 in rat adipose tissue

An inhibitor (inhibitor-1) of phosphorylase a phosphatase has been identified in rat epididymal fat pads. This heat-stable, acid-soluble protein only exhibits phosphatase inhibitory activity when it itself is phosphorylated. Inhibitor-1 in rat adipose tissue migrates at 32,000 Da on sodium dodecyl sulfate-polyacrylamide gels, and at 64,000 Da on gel filtration. Exposure of fat pads to insulin (1 milliunit/ml) resulted in a 50% decrease in inhibitor-1 activity, compared to control (p less than 0.001). Isoproterenol (10(-6) M) caused a 25% increase in inhibitor-1 activity (p less than 0.05). Electrophoresis of heat-stable proteins prepared from hormone-treated 32P-labeled fat cells showed that insulin caused a dephosphorylation of the 32,000 Da phosphoprotein by 30% (p less than 0.01), whereas isoproterenol stimulated 32P incorporation in this protein by 35% compared to control (p less than 0.05). Thus, insulin appears to dephosphorylate and inactivate inhibitor-1, and might thereby result in an increase of protein phosphatase activity. Insulin regulation of inhibitor-1 is a mechanism which may underlie other of insulin's effects in adipose tissue, such as the activation of glycogen synthase.     

The protein phosphatases involved in cellular regulation. Purification and characterisation of the glycogen-bound form of protein phosphatase-1 from rabbit skeletal muscle

A type-1 protein phosphatase (protein phosphatase-1G) was purified to homogeneity from the glycogen-protein particle of rabbit skeletal muscle. Approximately 3 mg of enzyme were isolated within 4 days from 5000 g of muscle. Protein phosphatase-1G had a molecular mass of 137 kDa and was composed of two subunits G (103 kDa) and C (37 kDa) in a 1:1 molar ratio. The subunits could be dissociated by incubation in the presence of 2 M NaCl, separated by gel-filtration on Sephadex G-100, and recombined at low ionic strength. The C component was the catalytic subunit, and was identical to the 37-kDa type-1 protein phosphatase catalytic subunit (protein phosphatase-1C) isolated from ethanol-treated muscle extracts, as judged by peptide mapping. The G component was the glycogen-binding subunit. It was very asymmetric, extremely sensitive to proteolytic degradation, and failed to silver stain on SDS/polyacrylamide gels. Protein phosphatase-1G was inhibited by inhibitor-1 and inhibitor-2, but unlike protein phosphatase-1C, the rate of inactivation was critically dependent on the ionic strength, temperature and time of preincubation with the inhibitor protein. At near physiological temperature and ionic strength, protein phosphatase-1G was inactivated very rapidly by inhibitor-1. Protein phosphatase-1G interacted with inhibitor-2 (I-2) to form an inactive species, with the structure GCI-2. This form could be activated by preincubation with Mg-ATP and glycogen synthase kinase-3. The G subunit could be phosphorylated on a serine residue(s) by cyclic-AMP-dependent protein kinase, but not by phosphorylase kinase or glycogen synthase kinase-3. Phosphorylation was rapid and stoichiometric, and increased the rate of inactivation of protein phosphatase-1G by inhibitor-1. The relationship of the G subunit to the 'deinhibitor protein' is discussed.     

Progesterone and cAMP-dependent protein kinase regulate in vivo the level of phosphorylation of two proteins (Mr 20,000 and Mr 32,000) in Xenopus oocytes

The [32P]phosphoproteins and [35S]thiophosphoproteins were analyzed by electrophoresis and autoradiography after microinjection of [gamma-32P]ATP or of [35S]ATP-gamma-S into living Xenopus oocytes. The level of 32P incorporation into a 20-kDA protein was decreased following progesterone treatment (between 1 and 2 hr). This 20-kDa protein was partially thiophosphorylated in vivo by [35S]ATP-gamma-S. Furthermore it was found that this phosphoprotein was partially purified by TCA (1%) extraction and heat treatment. Microinjection of the C-subunit of cAMP-dependent protein kinase (0.6 to 1.2 pmole) inhibited maturation and provoked an increase in the level of phosphorylation of the 20-kDa protein and of a 32-kDa protein, indicating that both proteins were in vivo substrates (directly or indirectly) for cAMP-dependent protein kinase. When inhibitor-1 of protein phosphatase-1 was microinjected (5 to 10 pmole per oocyte) meiotic maturation was inhibited and the level of phosphorylation of the 32-kDa protein was increased; the same result was obtained following ATP-gamma-S (1 mM) microinjection. Altogether these results suggest that a 20-kDa phosphoprotein, whose level of phosphorylation is decreased by progesterone, could be involved in the regulation of maturation by lowering the level phosphorylation of a 32-kDa phosphoprotein. An attractive hypothesis would be that the 20-kDa phosphoprotein is an inhibitor of protein phosphatase-1.     

Identification of protein phosphatases-1 and 2A and inhibitor-2 in oocytes of the starfish Asterias rubens and Marthasterias glacialis

Protein phosphatases present in the particulate and soluble fractions of oocytes of the starfish Asterias rubens and Marthasterias glacialis have been classified according to the criteria used for these enzymes from mammalian cells. The major protein phosphatase activity in the particulate fraction had very similar properties to protein phosphatase-1 from mammalian tissues, including preferential dephosphorylation of the beta subunit of phosphorylase kinase, sensitivity to inhibitor-1 and inhibitor-2, inhibition of phosphorylase phosphatase activity by protamine and heparin, and retention by heparin-Sepharose. The major protein phosphatase in the soluble fraction had very similar properties to mammalian protein phosphatase-2A, including preferential dephosphorylation of the alpha subunit of phosphorylase kinase, insensitivity to inhibitors-1 and 2, activation by protamine and heparin, and exclusion from heparin-Sepharose. An acid-stable and heat-stable protein was detected in the soluble fraction of starfish oocytes, whose properties were indistinguishable from those of inhibitor-2 from mammalian tissues. It inhibited protein phosphatase-1 specifically, and its apparent molecular mass on SDS polyacrylamide gels was 31 kDa. Furthermore, an inactive hybrid formed between the starfish oocyte inhibitor and the catalytic subunit of mammalian protein phosphatase-1 could be reactivated by preincubation with MgATP and mammalian glycogen synthase kinase-3. The remarkable similarities between starfish oocyte protein phosphatases and their mammalian counterparts are indicative of strict phylogenetic conservation of these enzymes. The results will facilitate further analysis of the role of protein phosphorylation in the control of starfish oocyte maturation by the hormone 1-methyladenine.     

Phosphorylation of DARPP-32 Is Regulated by GABA in Rat Striatum and Substantia Nigra

Abstract: In the medium-sized spiny neurons of the striatonigral pathway, a cascade of events involving the activation of dopamine D₁ receptors, an increase in cyclic AMP, and activation of cyclic AMP-dependent protein kinase causes the phosphorylation of DARPP-32 on Thr³⁴, converting DARPP-32 into a powerful inhibitor of protein phosphatase-1. In the present study, the incubation of striatal or substantia nigra slices with GABA also increased the phosphorylation of DARPP-32 on Thr³⁴. GABA did not significantly increase cyclic AMP levels in slices. The phosphorylation of DARPP-32 by GABA was blocked in both brain regions by pretreatment of slices with the GABA_A receptor antagonist, bicuculline, but not with the GABA_B receptor antagonist, phaclofen. Moreover, the threonine phosphorylation of DARPP-32 produced by maximally effective doses of either forskolin (in striatum) or l -3,4-dihydroxyphenylalanine (in substantia nigra) was increased further by GABA. The data are consistent with a model in which GABA increases the phosphorylation state of DARPP-32 by inhibiting dephosphorylation of the protein by the calcium/calmodulin-dependent protein phosphatase, calcineurin.     

DARPP—32的结构,功能及其调节机制

DARPP-32是一种多巴胺（DA）和cAMP调节的磷蛋白，存在于所有接受DA能投射的神经元中，在中枢神经系统的分布与DAD1受体的分布非常一致。DA通过D1受体使DARPP-32第34位苏氨酸磷酸化，磷酸化DARPP-32成为蛋白磷酸酶1（PP-1）的强效抑制剂，在两个不同位点与PP-1相互作用，从而抑制PP-1活性。DARPP-32／PP-1级联反应在调节，如钙通道、电压依赖性钠通道、Na＋,K＋-ATPase和NMDANR1受体的功能等神经元兴奋性过程中起重要作用。DA对DARPP—32的磷酸化状态有双向调节作用，其他许多神经递质亦可调节其磷酸化状态。     

Specificity of the heat-stable protein inhibitor of the branched-chain alpha-keto acid dehydrogenase phosphatase

A potent, heat-stable protein inhibitor of branched-chain alpha-keto acid dehydrogenase (BCKDH) phosphatase has been identified and purified to near homogeneity from bovine kidney mitochondria (Damuni, Z., Humphreys, J. S., and Reed, L. J., Proc. Natl. Acad. Sci. U.S.A., in press). This protein is a noncompetitive inhibitor of BCKDH phosphatase, with a Ki about 0.13 nM. By contrast, this protein inhibitor did not affect the activity of the cytosolic protein phosphatase-1 and phosphatase-2A or the mitochondrial pyruvate dehydrogenase (PDH) phosphatase at concentrations up to 10 nM. The cytosolic protein phosphatase inhibitor-1 and inhibitor-2 had no effect on the activity of BCKDH phosphatase or PDH phosphatase at concentrations up to 50 and 300 nM respectively. These results, together with previous evidence, demonstrate that BCKDH phosphatase and its inhibitor protein are distinct from the cytosolic protein phosphatase-1 and phosphatase-2A and from protein phosphatase inhibitor-1 and inhibitor-2, respectively.