Regulation of PTP-1 and insulin receptor kinase by fractions from cinnamon: implications for cinnamon regulation of insulin signalling.

Bioactive compound(s) extracted from cinnamon potentiate insulin activity, as measured by glucose oxidation in the rat epididymal fat cell assay. Wortmannin, a potent PI 3'-kinase inhibitor, decreases the biological response to insulin and bioactive compound(s) from cinnamon similarly, indicating that cinnamon is affecting an element(s) upstream of PI 3'-kinase. Enzyme studies done in vitro show that the bioactive compound(s) can stimulate autophosphorylation of a truncated form of the insulin receptor and can inhibit PTP-1, a rat homolog of a tyrosine phosphatase (PTP-1B) that inactivates the insulin receptor. No inhibition was found with alkaline phosphate or calcineurin suggesting that the active material is not a general phosphatase inhibitor. It is suggested, then, that a cinnamon compound(s), like insulin, affects protein phosphorylation-dephosphorylation reactions in the intact adipocyte. Bioactive cinnamon compounds may find further use in studies of insulin resistance in adult-onset diabetes.

Conversion of protein phosphatase 1 catalytic subunit to a Mn(2+)-dependent enzyme impairs its regulation by inhibitor 1.

The phosphorylase phosphatase activity of protein phosphatase 1 (PP1) catalytic subunit from freshly purified rabbit skeletal muscle was inhibited by MnCl2. Prolonged storage or inhibition by nonspecific phosphatase inhibitors ATP, sodium pyrophosphate, and NaF converted the muscle PP1 to a form that required Mn2+ for enzyme activity. Recombinant PP1 catalytic subunit expressed in Escherichia coli was also a Mn2+-dependent enzyme. While native PP1 was inhibited by the phosphoprotein inhibitor I (I-1), with an IC50 of 1 nM, 40-50-fold higher concentrations of I-1 were required to inhibit the Mn2+-dependent PP1 enzymes. Conversion to the Mn2+-dependent state was accompanied by a 20-fold increase in PP1's ability to dephosphorylate and inactivate I-1. Inhibition by thiophosphorylated I-1 established that dephosphorylation does not play a significant role in I-1's reduced potency as an inhibitor of Mn2+-dependent PP1. The Mn2+-dependent PP1 enzymes were poorly inhibited by N-terminal phosphopeptides of I-1, indicating their impaired interaction with the I-1 functional domain. Mutation of a residue conserved in I-1 and DARPP-32, a structurally related PP1 inhibitor, preferentially attenuated I-1's activity as an inhibitor of Mn2+-dependent PP1. These data showed that, in addition to changes in its catalytic properties, Mn2+-dependent PP1 was modified in its interaction with I-1 at a site that was distinct from its catalytic domain. Our studies suggest that conversion to a Mn2+-dependent state alters multiple structural elements in PP1 catalytic subunit that together define its regulation by I-1.

Thiophosphorylated RCM-lysozyme, an active site-directed protein tyrosine phosphatase inhibitor, inhibits G2/M transition during mitotic cell cycle and uncouples MPF activation from G2/M transition.

Microinjection of thiophosphotyrosylated RCM-lysozyme (TRCML), a potent and specific inhibitor of protein tyrosine phosphatases (PTPs) into sea urchin (Lytechinus pictus) eggs prior to fertilization inhibited the first zygotic cell division in a concentration-dependent fashion. Microinjection of TRCML at varying times after fertilization indicated that at least one site of action is late in the first cell cycle near the G2/M boundary. In order to further study the mechanism for the TRCML effect, a cell-free cell cycling system prepared from electrically activated Xenopus eggs was used. The addition of TRCML to cycling extracts delayed the entrance and progression of extracts through mitosis, as indicated by the inhibition of chromatin condensation, nuclear envelope breakdown, M-phase-promoting factor (MPF) inactivation, and cyclin degradation. Surprisingly, TRCML did not inhibit MPF activation. These results suggest that (1) the target(s) of TRCML lies in late G2- or early M-phase before the onset of metaphase, (2) TRCML uncouples MPF activation from progression through M-phase, and (3) there is a potential involvement of a novel PTP(s) in the control of the cell cycle which may act either downstream of the MPF activation or alternatively in an additional but essential mitotic pathway that is parallel to the MPF activation pathway.

Casein kinase II stimulates Xenopus laevis DNA topoisomerase I by physical association.

A Xenopus laevis casein kinase II-like activity copurified with X. laevis DNA topoisomerase I activity during chromatography on DEAE-cellulose, phosphocellulose, and hydroxylapatite, but the two activities were resolved by chromatography on DNA-agarose [Kaiserman, H. B., Ingebritsen, T. S., & Benbow, R. M. (1988) Biochemistry 27, 3216-3222]. Phosphorylation of the catalytic polypeptides of dephosphorylated X. laevis DNA topoisomerase I by the endogenous X. laevis casein kinase II-like activity apparently resulted in a severalfold increase in catalytic activity. In this study, we show that incubation of purified X. laevis DNA topoisomerase I with electrophoretically homogeneous bovine brain casein kinase II and ATP strongly stimulated catalytic activity. Surprisingly, purified bovine casein kinase II stimulated X. laevis DNA topoisomerase I activity by more than an order of magnitude in the absence of ATP, although ATP resulted in additional stimulation. Other basic proteins, such as histone H1 and HMG proteins, also stimulated X. laevis DNA topoisomerase I catalytic activity 2-3-fold in the absence of ATP. Modulation of catalytic activity by direct physical association (protein-protein interactions) must, therefore, be considered in addition to phosphorylation in assessing the physiological role of casein kinase II and other basic proteins during regulation of X. laevis DNA topoisomerase I activity in vivo.

Thiophosphorylated substrate analogs are potent active site-directed inhibitors of protein-tyrosine phosphatases.

Thiophosphotyrosyl protein and peptide substrate analogs were found to be potent and specific protein-tyrosine phosphatase inhibitors with IC50s in the range of 0.2-30 microM. The analogs were based on highly reactive substrates and included thiophosphotyrosyl forms of reduced carboxamidomethylated and maleylated lysozyme and peptides based on tyrosine phosphorylation sites of lysozyme, alpha s2-casein, and platelet-derived growth factor receptor. These analogs inhibited protein-tyrosine phosphatases from both the intracellular and transmembrane classes and from a variety of species ranging from a prokaryote (Yersinia enterolitica) to man. The extent of inhibition of phosphatase activity by a given analog varied with the phosphatase species. In contrast, protein kinases and protein-serine/threonine phosphatases were not significantly affected by these analogs. The mechanism of inhibition was investigated using rat brain protein-tyrosine phosphatase-1 as a prototype. These studies indicated that the inhibition was rapid and reversible and was competitive in nature. The Ki for inhibition by various thiophosphotyrosyl analogs was generally proportional to the apparent Km for the corresponding phosphorylated substrates. Unphosphorylated substrate molecules were generally much weaker inhibitors than the corresponding thiophosphotyrosyl substrate analogs. Taken together these results point to an active site-directed mechanism for inhibition. These specific inhibitory probes could be used to study substrate binding mechanisms as well as physiological roles of various protein-tyrosine phosphatases.

Acidic residues are involved in substrate recognition by two soluble protein tyrosine phosphatases, PTP-5 and rrbPTP-1.

The mechanisms for substrate recognition by two cytoplasmic protein tyrosine phosphatases, PTP-5 and rrbPTP-1, were investigated. Phosphorylation sites on tyrosine-phosphorylated casein, a model PTP substrate, were characterized. Two peptides based on casein phosphorylation sites and one peptide based on the tyrosine phosphorylation site of reduced, carboxamidomethylated and maleylated (RCM) lysozyme were tested as PTP substrates. The three peptides were dephosphorylated by PTP-5 and rrbPTP-1 at rates comparable to those of the corresponding sites on the intact proteins. This indicates that peptides based on the two model PTP substrates, casein and RCM-lysozyme, contained all or most of the structural information necessary for PTP-5 and rrbPTP-1 substrate recognition. Structural elements required for substrate recognition by PTP-5 and rrbPTP-1 were also investigated. Km values for dephosphorylation of three simple aromatic phosphate esters (phosphotyrosine, p-nitrophenyl phosphate, and phenyl phosphate) by rrbPTP-1 were about 5000-fold higher than those obtained for the peptide and protein substrates. This indicates that recognition of protein and peptide substrates involves structural elements in addition to the phosphate group and the aromatic tyrosine ring of phosphotyrosine. Analysis of the effects of truncations and Ala for polar substitutions on the reactivity with PTP-5 and rrbPTP-1 of peptides based on casein, RCM-lysozyme, and angiotensin II indicated that Asp or Glu within the first five residues on the N-terminal side of phosphotyrosine increased peptide reactivity with both PTP's. Asn residues were unable or only weakly able to substitute for Asp residues.(ABSTRACT TRUNCATED AT 250 WORDS)

Structurally different members of the okadaic acid class selectively inhibit protein serine/threonine but not tyrosine phosphatase activity.

The relative potencies of four main types of okadaic acid class compounds as inhibitors of the catalytic subunits of protein serine/threonine phosphatases 1 and 2A and the protein tyrosine phosphatase 1 were determined. These four types of compounds are okadaic acid, calyculin A, microcystin-LR, and tautomycin, which are isolated from different natural sources, a black sponge Halichondria okadai, a marine sponge Discodermia calyx, a blue-green alga Microcystis aeruginosa, and Streptomyces spirover ticillatus, respectively. While okadaic acid was a more effective inhibitor of protein phosphatase 2A (IC50, 0.07 nM) than protein phosphatase 1 (IC50, 3.4 nM), other compounds of the okadaic acid class were equally effective against the two protein serine/threonine phosphatases. The order of potency was microcystin greater than calyculin A greater than tautomycin, and the IC50S ranged from 0.1 to 0.7 nM. None of the okadaic acid class compounds inhibited protein tyrosine phosphatase 1 activity at concentrations up to 0.01 mM. These results indicate that the compounds of the okadaic acid class are selective inhibitors of protein serine/threonine but not tyrosine phosphatases.

Protein tyrosine phosphatase stimulates Ca(2+)-dependent amylase secretion from pancreatic acini.

Eukaryotic cells respond to various stimuli by an increase or decrease in levels of phosphoproteins. Phosphotyrosine levels on eukaryotic cellular proteins are tightly regulated by the opposing actions of protein-tyrosine kinases and protein-tyrosine phosphatases (PTPases, EC 3.1.3.48). Studies on permeabilized mast cells suggest that the enabling reaction for exocytosis might involve protein dephosphorylation. In the present studies, a recombinant form of rat brain PTPase (rrbPTP-1) has been used to examine the potential role of PTPases in Ca(2+)-dependent amylase secretion from permeabilized rat pancreatic acini. Additionally, the concentrations and subcellular distributions of endogenous PTPase activity in rat pancreas were determined. The results from these experiments indicate that addition of exogenous PTPase stimulated Ca(2+)-dependent amylase secretion from pancreatic acinar cells and that endogenous PTPase activity was associated with the postgranule supernatant, zymogen granules, and in particular zymogen granule membranes. Our data suggest that protein tyrosine dephosphorylation is potentially involved in regulated secretion at a site(s) distal to receptor-mediated elevation of intracellular second messengers.

pp54 microtubule-associated protein-2 kinase requires both tyrosine and serine/threonine phosphorylation for activity.

pp54 microtubule-associated protein-2 (MAP-2) kinase, a recently discovered protein serine/threonine kinase (Kyriakis, J., and Avruch, J. (1990) J. Biol. Chem. 265, 17355-17363), is shown to contain immunoreactive phosphotyrosine residues. Treatment with recombinant rat brain protein tyrosine phosphatase-1 deactivates pp54 MAP-2 kinase, concomitant with the removal of phosphotyrosine residues. Protein (serine/threonine) phosphatase-1 also deactivates pp54 MAP-2 kinase in a specific fashion. pp54 MAP-2 kinase joins pp42 MAP-2 kinase and cdc2/maturation-promoting factor as one of only three serine/threonine protein kinases known to be regulated by phosphorylation at both tyrosine and, independently, at serine/threonine residues. In view of these shared regulatory properties, a role for pp54 MAP-2 kinase in the control of cell division is likely.

Regulation of rhodopsin dephosphorylation by arrestin.

We have characterized the opsin phosphatase activities in extracts of rod outer segments and determined their relationship to known protein phosphatases. The opsin phosphatase activity in the extracts was not due to protein phosphatases 1, 2B, or 2C because it was neither stimulated by Mg2+ or Ca2+/calmodulin nor inhibited by protein phosphatase inhibitors-1 or -2. Opsin phosphatase activity in rod outer segment extracts was potently inhibited by okadaic acid (IC50 approximately 10 nM), a preferential inhibitor of protein phosphatase 2A. Moreover, during chromatography on DEAE-Sepharose, the opsin phosphatase activity co-eluted with three peaks of protein phosphatase 2A activity, termed protein phosphatases 2A0, 2A1, and 2A2. The opsin phosphatase activity of each peak was stimulated by polylysine, a known activator of protein phosphatase 2A. Finally, treatment of rod outer segment extracts with 80% ethanol at room temperature converted the activity from a high molecular weight form characteristic of the protein phosphatase 2A0, 2A1, and 2A2 species to a low molecular weight form characteristic of the protein phosphatase 2A catalytic subunit. We conclude that protein phosphatase 2A is likely to be the physiologically relevant rhodopsin phosphatase. The 48-kDa rod outer segment protein arrestin (S-antigen) was found to inhibit the dephosphorylation of freshly photolyzed rhodopsin by protein phosphatase 2A but did not inhibit the dephosphorylation of unbleached rhodopsin. Arrestin has no effect on the dephosphorylation of phorphorylase a, indicating that the effect was substrate-directed. It appears that dephosphorylation of the photoreceptor protein phosphorhodopsin occurs only after decay of the photoactivated protein and that this may be regulated in vivo by arrestin. The binding of arrestin to photolyzed phosphorylated rhodopsin, i.e. the binding of a regulatory protein to a protein phosphatase substrate to form a complex resistant to dephosphorylation represents a novel mechanism for the regulation of protein phosphatase 2A.

Immobilized inhibitor-1 binds and inhibits protein phosphatase 1.

Inhibitor-1 is a potent and specific inhibitor of protein phosphatase 1. Phosphorylation by cAMP-dependent protein kinase is required for expression of its inhibitor activity. In the present study, we have used immobilized inhibitor-1 preparations to study the mechanism underlying protein phosphatase 1 inhibition. Protein phosphatase 1 bound to phosphorylated inhibitor-1 covalently coupled to Sepharose or Affi-Gel beads but did not bind to immobilized preparations of dephosphorylated inhibitor-1 or bovine serum albumin. Phosphorylated inhibitor-1 coupled to Sepharose or Affi-Gel beads retained its ability to inhibit protein phosphatase 1, although the apparent IC50 was decreased about 500-fold. The extent of protein phosphatase 1 binding to immobilized phosphorylated inhibitor-1 was comparable to the degree of protein phosphatase inhibition when the inhibitor protein was present at a concentration near the IC50. The efficiency of protein phosphatase 1 binding to immobilized phosphorylated inhibitor-1 was dependent on the inhibitor concentration on the matrix. Taken together these data indicate that the inhibition of protein phosphatase 1 by phosphorylated inhibitor-1 is a consequence of the binding of the inhibitor protein to one or more sites on protein phosphatase 1.

Phosphotyrosyl-protein phosphatases. I. Separation of multiple forms from bovine brain and purification of the major form to near homogeneity.

Seven Tyr-protein phosphatase activities were isolated from bovine brain using phosphotyrosyl-casein as a model substrate. The activities were resolved from the cytosolic fraction by a three-step procedure employing successive DEAE-cellulose, phosphocellulose, and gel permeation chromatography steps. The seven activities accounted for 70% of the Tyr-protein phosphatase activity in bovine brain extracts and were distinct from type 1 and type 2 Ser/Thr-protein phosphatases and from the major alkaline phosphatase activities. Apparent molecular weights of the activities by gel permeation chromatography were: phosphotyrosyl-protein phosphatase (PTP)-1A (Mr 86,000), PTP-1B (Mr 24,000), PTP-2 (Mr 88,000), PTP-3 (Mr 90,000), PTP-4 (Mr 80,000), PTP-5 (Mr 48,000), and PTP-6 (Mr 104,000). PTP-5 was the major activity accounting for 26% of total while the remaining activity was divided rather evenly among the other six activities. PTP-5 was further purified to near homogeneity by additional chromatographies on Affi-Gel Blue, heparin-agarose, and Mono S giving an overall purification of 50,000-fold and a yield of 5.8%. One of two major polypeptides (Mr 46,000) in the preparation was identified as PTP-5 since it alone expressed protein phosphatase activity when protein-staining bands were eluted from sodium dodecyl sulfate-polyacrylamide gels and renatured. PTP-5 had a neutral pH optimum, and using phosphotyrosyl-casein as substrate it had a Km of 130 nM and a Vmax of 10 mumol Pi released.min-1.mg protein-1. These kinetic parameters are well within the range of values obtained for other pure protein phosphatases. PTP-5 also dephosphorylated pp60v-src (autophosphorylated at Tyr-416) at 10% of the rate observed with phosphotyrosyl-casein. Additionally the ratio of phosphotyrosyl-casein/pp60v-src phosphatase activity was relatively constant throughout the PTP-5 purification procedure. These results indicate that PTP-5 is able to bind and efficiently dephosphorylate phosphotyrosyl-proteins and suggest that it is a physiologically relevant Tyr-protein phosphatase.

Phosphotyrosyl-protein phosphatases. II. Identification and characterization of two heat-stable protein inhibitors.

Two Tyr-protein phosphatase inhibitors, termed inhibitor H (Mr greater than 500,000) and inhibitor L (Mr 38,000), have been detected in bovine brain extracts. The inhibitors were partially purified by chromatography on DEAE-cellulose and Sephacryl S-300. Both inhibitors are proteins, as judged by their inactivation by proteinase K, and they exhibited remarkable stability during incubation at 95 degrees C. Of seven Tyr-protein phosphatase activities that we have isolated from bovine brain, PTP-4 and PTP-5 were most sensitive to the inhibitor proteins. Inhibition of the other five Tyr-protein phosphatases was only observed at very high inhibitor concentrations. The IC50 values for the inhibition of PTP-4 by inhibitor H and inhibitor L were 2- and 10-fold higher than those for the inhibition of PTP-5. Inhibition of PTP-5 by either inhibitor was rapid (maximum effect in less than 1 min) and readily reversed upon removal of the inhibitors by dilution. Inhibitor H and inhibitor L are distinct from the three heat-stable protein inhibitors of Ser/Thr-protein phosphatase 1. The ability of inhibitor H and inhibitor L to preferentially inhibit PTP-4 and PTP-5 provides an important new criterion that can be used to distinguish these enzymes from other Tyr-protein phosphatases. The two inhibitor proteins may be involved in regulating the activity of PTP-4 and PTP-5.

The catalytic subunit of phosphatase 2A dephosphorylates phosphoopsin.

Rod cell outer segments were found to contain a protein phosphatase activity toward phosphoopsin with properties very similar to those of protein phosphatase 1 or 2A. The opsin phosphatase activity was stable to ethanol precipitation, had a Mr of 35,000-38,000 as determined by gel filtration, and was not dependent on divalent cations for activity. The chromatographic properties on DEAE-cellulose of the rod outer segment protein phosphatase were also similar to those reported for protein phosphatase 1 or 2A. In order to distinguish between these two protein phosphatases, we tested homogeneous preparations of protein phosphatases 1 and 2A from skeletal muscle for activity toward phosphoopsin. Protein phosphatase 2A dephosphorylated phosphoopsin at approximately 10% of its rate toward phosphorylase a, whereas protein phosphatase 1 had no activity toward phosphoopsin. We conclude that protein phosphatase 2A is present in the rod cell outer segment and that it is a likely candidate to perform the in vivo dephosphorylation of rhodopsin in the visual cycle.

Regulation of Xenopus laevis DNA topoisomerase I activity by phosphorylation in vitro.

DNA topoisomerase I has been purified to electrophoretic homogeneity from ovaries of the frog Xenopus laevis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the most purified fraction revealed a single major band at 110 kDa and less abundant minor bands centered at 62 kDa. Incubation of the most purified fraction with immobilized calf intestinal alkaline phosphatase abolished all DNA topoisomerase enzymatic activity in a time-dependent reaction. Treatment of the dephosphorylated X. laevis DNA topoisomerase I with a X. laevis casein kinase type II activity and ATP restored DNA topoisomerase activity to a level higher than that observed in the most purified fraction. In vitro labeling experiments which employed the most purified DNA topoisomerase I fraction, [gamma-32P]ATP, and the casein kinase type II enzyme showed that both the 110- and 62-kDa bands became phosphorylated in approximately molar proportions. Phosphoamino acid analysis showed that only serine residues became phosphorylated. Phosphorylation was accompanied by an increase in DNA topoisomerase activity in vitro. Dephosphorylation of DNA topoisomerase I appears to block formation of the initial enzyme-substrate complex on the basis of the failure of the dephosphorylated enzyme to nick DNA in the presence of camptothecin. We conclude that X. laevis DNA topoisomerase I is partially phosphorylated as isolated and that this phosphorylation is essential for expression of enzymatic activity in vitro. On the basis of the ability of the casein kinase type II activity to reactivate dephosphorylated DNA topoisomerase I, we speculate that this kinase may contribute to the physiological regulation of DNA topoisomerase I activity.

Effects of phosphorylation of protein phosphatase 1 by pp60v-src on the interaction of the enzyme with substrates and inhibitor proteins.

Phosphorylation of protein phosphatase 1 by pp60v-src decreased its activity towards phosphorylase kinase and glycogen synthase as well as towards phosphorylase a. Kinetic experiments indicated that the primary effect of phosphorylation was to increase the Km for each of the substrate proteins. There was little or no change in the Vmax for the reactions. The possibility that phosphorylation of protein phosphatase 1 altered its regulation by inhibitors-1 and -2 was also examined. Phosphorylation of protein phosphatase 1 did not prevent the reversible inhibition of the enzyme by inhibitor-1 or inhibitor-2 nor did it prevent the association of inhibitor-2 with protein phosphatase 1 to form the MgATP-dependent protein phosphatase. Protein phosphatase 1 is not a substrate for pp60v-src when it is complexed with inhibitor-2 to form the inactive MgATP-dependent protein phosphatase. Here we have shown that protein phosphatase 1 is also not phosphorylated by pp60v-src following activation of the MgATP-dependent protein phosphatase with glycogen synthase kinase-3 and MgATP. This indicates that the inability of pp60v-src to phosphorylate protein phosphatase 1 is not due to the change in protein phosphatase 1 conformation which accompanies the inactivation of the MgATP-dependent protein phosphatase. Rather, it appears to be the result of steric hindrance by inhibitor-2. This suggests that the pp60v-src phosphorylation site is closely associated with the inhibitor-2 binding site involved in the formation of the MgATP dependent protein phosphatase. The pp60v-src phosphorylation site was previously localized to a small (Mr less than or equal to 4000) domain which can be selectively degraded by chymotrypsin. Here we have shown that chymotryptic digestion increased the Km of unphosphorylated protein phosphatase 1 for each of the three phosphoprotein substrates used in this study. This effect was similar to that observed after phosphorylation of protein phosphatase 1. These results indicate that the pp60v-src phosphorylation site is in a region of protein phosphatase 1 which influences substrate binding and which may be near the active site.

Apparent activation of the MgATP-dependent protein phosphatase by pp60v-src. Identification of an activity like that of glycogen synthase kinase 3 in immunoaffinity purified pp60v-src preparations.

Immunoaffinity purified pp60v-src was found to activate the MgATP-dependent protein phosphatase in the presence of MgATP. Although preliminary evidence suggested that phosphorylation of the inhibitor-2 subunit on tyrosine residues was responsible for the activation, preincubation of the pp60v-src preparation at 41 degrees C resulted in a rapid loss of its protein kinase activities towards both casein and inhibitor-2 while its ability to activate the protein phosphatase complex was relatively insensitive to this treatment. This result demonstrated that pp60v-src was not responsible for activation of the MgATP-dependent protein phosphatase. A protein kinase activity which phosphorylated glycogen synthase on serine residues was detected in the pp60v-src preparation. The protein kinase was active in the presence of inhibitors of phosphorylase kinase, glycogen synthase kinase 5/casein kinase II, and cAMP-dependent protein kinase. It is, therefore, likely that activation of the MgATP-dependent protein phosphatase resulted from the presence of a glycogen synthase kinase 3 like activity in the pp60v-src preparation. Our results illustrate the importance of applying multiple criteria to link the phosphorylation of a protein with an observed change in its activity.

Phosphorylation and inactivation of protein phosphatase 1 by pp60v-src.

Protein phosphatase 1, one of four major protein phosphatases involved in cellular regulation, was phosphorylated in vitro by pp60v-src, the transforming gene product of Rous sarcoma virus. Phosphorylation was accompanied by a loss of protein phosphatase activity. The inactivation of protein phosphatase 1 was time-dependent and the extent of inactivation correlated closely with the stoichiometry of phosphorylation. Under optimal conditions, 0.34 +/- 0.01 mol of phosphate were incorporated per mol of protein phosphatase and the activity of the enzyme was decreased by 39 +/- 2%. The inactivation required the presence of both MgATP and pp60v-src. There was no loss of activity when adenosine 5'-[beta gamma-imido]triphosphate was used in place of ATP. Phosphorylation of protein phosphatase 1 occurred exclusively on tyrosine residues and was blocked by specific antibodies to pp60v-src. During preincubation of pp60v-src at 41 degrees C, its protein kinase activity towards casein was lost rapidly. The ability of pp60v-src to phosphorylate and inactivate protein phosphatase 1 declined in parallel with the loss of casein kinase activity. Limited chymotryptic digestion of 32P-labeled protein phosphatase 1 (Mr 37,000) resulted in its quantitative conversion to a Mr 33,000 species. Conversion to this species was accompanied by the loss of 32P-labeling and by reactivation of the protein phosphatase. When various concentrations of chymotrypsin were used in the digestion, there was a close correlation between conversion to the Mr 33,000 species and the restoration of protein phosphatase activity. pp60v-src was unable to phosphorylate or inactivate a partially proteolyzed species of protein phosphatase 1 (Mr 33,000/34,000).

Identification of protein phosphatase 1 in synaptic junctions: dephosphorylation of endogenous calmodulin-dependent kinase II and synapse-enriched phosphoproteins.

A calcium/calmodulin-dependent protein kinase termed CaM-kinase II is a major component of synaptic junctions from forebrain and constitutes approximately 12% of total synaptic junction protein. CaM-kinase II phosphorylates at least seven polypeptides that are enriched in synaptic junctions, of which two represent the 50- and 60-kilodalton subunits of the protein kinase. In this report the nature of endogenous protein phosphatases which dephosphorylate each of the seven synaptic junction phosphoproteins was examined. Assays of synaptic junctions and other subcellular fractions from rat forebrain for type-1 and type-2 protein phosphatases revealed that protein phosphatase 1 (PrP-1) was specifically enriched in synaptic junctions with respect to cytosolic fractions. The activity of type-2 protein phosphatases was very low in synaptic junctions. Homogeneous PrP-1 from rabbit skeletal muscle was found to dephosphorylate each of the seven phosphoproteins in synaptic junctions. Inhibitors-1 and -2 were found to inhibit endogenous protein phosphatase activity by 70 to 80%. Since inhibitors-1 and -2 are specific inhibitors of PrP-1, these results indicate that this enzyme accounts for the majority of endogenous protein phosphatase activity in synaptic junctions. Approximately 15% of the protein phosphatase activity in synaptic junctions was type 2A, whereas PrP-2B and PrP-2C accounted for little, if any, of the activity toward endogenous or exogenous phosphoproteins. These results indicate that PrP-1 may be important in controlling the state of phosphorylation of synaptic junction proteins.