Sds22p is a subunit of a stable isolatable form of protein phosphatase 1 (Glc7p) from Saccharomyces cerevisiae.

Protein phosphatase 1 (PP1) is one of the major protein phosphatases in eukaryotic cells. PP1 activity is believed to be controlled by the interaction of PP1 catalytic subunit with various regulatory subunits. The essential gene GLC7 encodes the PP1 catalytic subunit in Saccharomyces cerevisiae. In this study, full-length GLC7(1-312), C-terminal deletion mutants, and C-terminally poly-his tagged mutants were constructed and expressed in a GLC7 knockout strain of S. cerevisiae. Viability studies of the GLC7 knockout strains carrying the plasmids expressing GLC7 C-terminal deletion mutants and their tagged forms showed that the mutants 1-295 and 1-304 were functional, whereas the mutant 1-245 was not. The C-terminally poly-his tagged Glc7p with and without an N-terminal hemagglutinin (HA) tag was partially purified by immobilized Ni(2+) affinity chromatography and further analyzed by gel filtration and ion exchange chromatography. Phosphatase activity assays, SDS-PAGE, and Western blot analyses of the chromatographic fractions suggested that the Glc7p associated with regulatory subunits in vivo. A 40-kDa protein was copurified with tagged Glc7p through several chromatographic procedures. Monoclonal antibody against the HA tag coimmunoprecipitated the tagged Glc7p and the 40-kDa protein. This protein was further purified by a reverse phase HPLC column. Analysis by CNBr digestion, peptide sequencing, and electrospray mass spectrometry showed that this 40-kDa protein is Sds22p, one of the proteins proposed to be a regulatory subunit of Glc7. These results demonstrate that Sds22p forms a complex with Glc7p and that Sds22p:Glc7p is a stable isolatable form of yeast PP1.

Cardiac myosin-binding protein C (MyBP-C): identification of protein kinase A and protein kinase C phosphorylation sites.

Myosin binding protein C (MyBP-C) is a major myofibril-associated protein in cardiac muscle which is subject to reversible phosphorylation. Cardiac MyBP-C is a substrate in vivo and in vitro for cAMP-dependent protein kinase (PKA) and calcium/phospholipid-dependent protein kinase (PKC). Chicken cardiac MyBP-C was phosphorylated by PKA to 3.0 mol phosphate/mol and by PKC to 2.0 mol phosphate/mol. Tryptic phosphopeptides from MyBP-C were purified by successive iron iminodiacetate column chromatography and reversed-phase high-performance liquid chromatography. Three phosphopeptides purified from PKA-phosphorylated MyBP-C contained phosphoserine [T1, (RTS[P]LAGGGR) and T2, (KRDS[P]FLR)] or phosphothreonine (CT3, MT[P]SAFL). PKC phosphorylated two of the same sites (T1 and T2) as PKA and an additional site [T2a (TGTTYKPPS[P]YK)]. PKA phosphorylation sites corresponding to peptides T1, T2, and T3 were identified in the N-terminus of the cDNA deduced amino acid sequence (S265, S300, and T274, respectively). The PKC-specific site in peptide T2a was at position S1169. cDNA clones encoding rat cardiac MyBP-C were isolated, and the segment corresponding to PKA and major PKC phosphorylation sites was sequenced. Chicken cardiac MyBP-C has a threonine at position 274 (CT3), whereas rat cardiac MyBP-C has a serine at the corresponding position. Only chicken cardiac MyBP-C had a phosphorylatable residue at the position corresponding to S1169. All of the cardiac MyBP-C phosphorylation sites are absent in known sequences of skeletal muscle MyBP-C isoforms.

Purification and characterization of type 1 protein phosphatase from Saccharomyces cerevisiae: effect of the R73C mutation.

Type 1 protein phosphatase encoded by the GLC7 gene was purified from Saccharomyces cerevisiae as a 1:1 complex with mammalian inhibitor 2 fused to glutathione S-transferase. The complex was inactive and required treatment with Co2+ and trypsin for maximal activity. The specific activity toward phosphorylase a was about 1.8 units/mg of Glc7p, and IC50's for inhibitor 2, okadaic acid, and microcystin-LR were 7.3, 81, and 0.30 nM, respectively. The complex could be activated by glycogen synthase kinase-3 in the presence of Mg2+ and ATP to 20% of the activity seen with Co2+ and trypsin. Thus, the catalytic properties of the yeast type 1 phosphatase are similar to those of the mammalian protein phosphatase 1. The R73C mutant phosphatase from the glycogen-deficient strain, glc7-1, purified as a 1:1 complex with the inhibitor 2 fusion, had a specific activity toward phosphorylase a of 0.9 unit/mg of Glc7p, and IC50's for inhibitor 2, okadaic acid, and microcystin-LR were 13. 1, 113, and 0.37 nM, respectively. The R73C mutation slightly decreases the specific activity and sensitivity to inhibitors, suggesting that changes in biochemical properties may affect glycogen levels. However, the modest changes are consistent with our previous proposal (E. M. Reimann et al., 1993, Adv. Protein Phosphatases 7,173-182) and with the results of Stuart et al. (1994, Mol. Cell. Biol. 14, 896-905) that the mutation may selectively alter the interaction of Glc7p with regulatory proteins.

Effect of activation of protein phosphatase 1 on sulfhydryl reactivity.

Myofibril protein phosphatase 1 (PP1) from bovine heart, identified as PP1alpha, was purified in a latent form which was dependent on Co2+ or Mn2+ for activity (Y. Chu, S. E. Wilson, and K. K. Schlender (1994) Biochim. Biophys. Acta 1208, 45-54). This was also true for recombinant PP1 alpha expressed in Escherichia coli (Z. Zhang, G. Bai, S. Deans-Zirattu, M. F. Browner, and E. Y. C. Lee (1992) J. Biol. Chem. 267, 1484-1490). Here we report on the change in the sulfhydryl reactivity during the cation activation process. The activation of myofibrillar PP1 by Co2+ was prevented by 10 mM dithiothreitol (DTT) and incubation of the Co2+-activated enzyme with 50 mM DTT reversed the activation. Activation of recombinant PP1alpha was associated with 57Co2+ incorporation into PP1. DTT reversal of Co2+-activated PP1 was accompanied by release of Co2+ from the enzyme. The latent PP1 modified with 2-nitro-5-thiocyanobenzoic acid (NTCB) or N-ethylmaleimide (NEM) did not bind Co2+ and could not be activated by Co2+. Conversely, the Co2+-activated PP1 was resistant to inactivation with NTCB and less sensitive to NEM. Similarly, PP1 pretreated with NTCB was not activated by Mn2+ and the Mn2+-activated enzyme was also resistant to NTCB inhibition. The number of sulfhydryls of nondenatured PP1, reactive with 5, 5'-dithiobis[2-nitrobenzoic acid] (DTNB), was reduced from approximately 8 to 2-3 mol/mol when the enzyme was activated with Co2+ or Mn2+. After denaturation with guanidine-HCl, the number of reactive sulfhydryls of nonactivated PP1 and Co2+-activated PP1 was approximately 10 mol/mol enzyme. These results suggest that when PP1 is activated by Co2+ or Mn2+, the enzyme undergoes a conformational change resulting in some of the cysteine sulfhydryls no longer being accessible to chemical modification.

Gap-junction disassembly and connexin 43 dephosphorylation induced by 18 beta-glycyrrhetinic acid.

Gap-junction channels connect the interiors of adjacent cells and can be arranged into aggregates or plaques consisting of hundreds to thousands of channel particles. The mechanism of channel aggregation into plaques and whether plaques can disaggregate are not known. Many carcinogenic and tumor-promoting chemicals have been identified that inhibit cell-cell gap-junctional coupling. Here, we provide morphological evidence that 18 beta-glycyrrhetinic acid (18 beta-GA), a saponin isolated from licorice root that is an inhibitor of gap-junctional communication, caused the disassembly of gap-junction plaques in WB-F344 rat liver epithelial cells. This effect was dose (5-40 microM) and time dependent (1-4 h treatment). Gap-junction channels in WB-F344 cells are comprised of connexin 43 (Cx43), and the protein is phosphorylated to a species known as Cx43-P2 coincident with the assembly of channels into plaques. Consistent with this, the disassembly of plaques induced by 18 beta-GA was correlated with decreases in Cx43-P2 levels and increases in nonphosphorylated Cx43. Biochemical evidence indicated that these changes in the P2 and NP forms of Cx43 represented 18 beta-GA-induced dephosphorylation of Cx43-P2 and not its degradation or the inhibition of Cx43-NP phosphorylation. Okadaic acid and calyculin A, which are inhibitors of type 1 and type 2A protein phosphatases, prevented the dephosphorylation of Cx43, suggesting that one or both of these phosphatases were involved in Cx43 dephosphorylation. These data indicate that 18 beta-GA causes type 1 or type 2A protein phosphatase-mediated Cx43 dephosphorylation coincident with the disassembly of gap-junction plaques.

Activation of protein phosphatase 1. Formation of a metalloenzyme.

The recombinant catalytic subunit of protein phosphatase 1 is produced as an inactive enzyme which can be activated by Mn2+ (Zhang, Z., Bai, G., Deans-Zirattu, S., Browner, M. F., and Lee, E. Y. C. (1992) J. Biol. Chem. 267, 1484-1490). In this report, we have investigated the effects of divalent cations on the activity of recombinant catalytic subunit of protein phosphatase 1. Latent phosphatase 1 can be activated by Co2+ or Mn2+, whereas other metal ions tested including Fe2+, Zn2+, Mg2+, Ca2+, Cu2+, or Ni2+ were not effective or were only weakly effective in activating the enzyme. The Mn(2+)-stimulated activity was susceptible to inactivation by EDTA; however, the Co(2+)-activated phosphatase was stable after dilution and chelation of the Co2+ with excess EDTA. After stable activation of phosphatase 1 using 57Co2+, a stoichiometric amount of 57Co2+ was shown to be tightly bound to phosphatase 1. These findings demonstrate for the first time the generation of a stable metalloenzyme form of phosphatase 1. Fe2+ reversibly deactivated the Co(2+)-stimulated activity, but did not displace the bound Co2+. Interestingly, treatment of the enzyme with a combination of Fe2+ and Zn2+ (but not the individual metal ions) significantly activated phosphatase 1. These results suggest that at least two metal binding sites exist on the enzyme and that protein phosphatase 1 may be an iron/zinc metalloprotein in vivo.

Phosphorylation and inactivation of rat heart glycogen synthase by cAMP-dependent and cAMP-independent protein kinases.

The regulation of cardiac muscle glycogen metabolism is not well understood. Previous studies have indicated that heart glycogen synthase is heavily phosphorylated in vivo on multiple sites. Using purified enzymes, we have investigated the effect of phosphorylation of different sites on the activity of rat heart glycogen synthase. A convenient procedure was developed for the purification of rat heart glycogen synthase. The enzyme was phosphorylated by selected kinases, and glycogen synthase activity, extent of phosphorylation, and phosphopeptide maps were analyzed. Rat heart glycogen synthase, purified to apparent homogeneity (M(r) 87,000 on SDS-PAGE), had a specific activity of 18 U/mg protein and had an activity ratio of 0.74 (activity in the absence divided by the activity in the presence of glucose 6-P). cAMP-dependent protein kinase, glycogen synthase kinase 3, Ca2+/calmodulin-dependent protein kinase II, protein kinase C, and phosphorylase kinase phosphorylated the enzyme with a concomitant decrease in the activity ratio to values ranging from 0.1 to 0.4. Casein kinase II phosphorylated but did not inactivate glycogen synthase. Six tryptic phosphopeptides, obtained from heart glycogen synthase phosphorylated by the various kinases, were separated by reverse-phase chromatography. The phosphopeptide(s) obtained with each kinase eluted at the same position(s) as corresponding phosphopeptides obtained from rat skeletal muscle glycogen synthase. The study shows that the pattern of phosphorylation and effects on activity are very similar for cardiac and skeletal muscle glycogen synthase. It is suggested that the well known differences in heart and glycogen metabolism may be due to the interplay of kinases and phosphatases which could lead to different phosphorylation and activity states of glycogen synthase.

A metal-dependent form of protein phosphatase 2A.

Highly purified bovine heart protein phosphatase 2A catalytic subunit lost virtually all of its activity during storage at -70 degrees. When the enzyme was preincubated with Co2+, over 35% of the original activity was restored. Freshly prepared protein phosphatase 2A purified from bovine heart was stimulated at least 3 to 4-fold by pretreatment with Co2+ or Mn2+. Activation by Co2+ appeared to be irreversible whereas activation by Mn2+ was partially reversed after the cation was chelated with excess EDTA/EGTA. The sensitivity of Co2(+)-stimulated protein phosphatase 2A to okadaic acid or inhibitor-2 was similar to that of spontaneously active protein phosphatase 2A. The enzyme was converted to a latent form by treatment with phosphate or pyrophosphate. The latent form was completely reactivated by preincubation with Co2+. These results demonstrate that protein phosphatase 2A, like phosphatase 1, can exist in a metal ion-dependent form and may represent a new mechanism for the regulation of protein phosphatase 2A activity.

A latent form of protein phosphatase 1 alpha associated with bovine heart myofibrils.

The catalytic subunit of the major protein phosphatase associated with bovine cardiac myofibrils was purified to homogeneity. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the enzyme revealed only one band with an apparent molecular weight of 37,000. On gel filtration chromatography, the phosphatase activity and the protein co-eluted as a single peak with an apparent molecular weight of 37,000. The purified enzyme was identified as the catalytic subunit of protein phosphatase 1, as determined by sensitivity to inhibitor 1, inhibitor 2, okadaic acid and by specific immunostaining. Evidence obtained with specific antipeptide antibodies demonstrated that this myofibril protein phosphatase was predominantly the alpha isoform of protein phosphatase 1. The purified catalytic subunit was completely inactive. It was activated by pretreatment with Co2+/trypsin in the presence of high ionic strength. Treatment with trypsin alone did not activate the latent enzyme. The enzyme was also activated by Co2+ or Mn2+ alone but not by Ca2+, Mg2+, Ni2+, Cu2+ or Zn2+. Activation of the enzyme was not reversed by removal of Co2+, but Mn(2+)-activated phosphatase activity was partially reversed when Mn2+ was removed. The catalytic subunit could form a 1:1 complex with inhibitor 2 in vitro. The resulting holoenzyme was also activated by pretreatment with Co2+. Since phosphatase 1 alpha is the major phosphatase associated with cardiac myofibril, it is suggested that it is responsible for the dephosphorylation of myosin and other myofibril phosphoproteins.

Protein phosphatases 1 and 2A dephosphorylate B-50 in presynaptic plasma membranes from rat brain.

The protein B-50 is dephosphorylated in rat cortical synaptic plasma membranes (SPM) by protein phosphatase type 1 and 2A (PP-1 and PP-2A)-like activities. The present studies further demonstrate that B-50 is dephosphorylated not only by a spontaneously active PP-1-like enzyme, but also by a latent form after pretreatment of SPM with 0.2 mM cobalt/20 micrograms of trypsin/ml. The activity revealed by cobalt/trypsin was inhibited by inhibitor-2 and by high concentrations (microM) of okadaic acid, identifying it as a latent form of PP-1. In the presence of inhibitor-2 to block PP-1, histone H1 (16-64 micrograms/ml) and spermine (2 mM) increased B-50 dephosphorylation. This sensitivity to polycations and the reversal of their effects on B-50 dephosphorylation by 2 nM okadaic acid are indicative of PP-2A-like activity. PP-1- and PP-2A-like activities from SPM were further displayed by using exogenous phosphorylase alpha and histone H1 as substrates. Both PP-1 and PP-2A in rat SPM were immunologically identified with monospecific antibodies against the C-termini of catalytic subunits of rabbit skeletal muscle PP-1 and PP-2A. Okadaic acid-induced alteration of B-50 phosphorylation, consistent with inhibition of protein phosphatase activity, was demonstrated in rat cortical synaptosomes after immunoprecipitation with affinity-purified anti-B-50 immunoglobulin G. These results provide further evidence that SPM-bound PP-1 and PP-2A-like enzymes that share considerable similarities with their cytosolic counterparts may act as physiologically important phosphatases for B-50.

Antibodies directed against synthetic peptides distinguish between the catalytic subunits of protein phosphatases 1 and 2A.

Two antipeptide antibodies (designated type 1 antibody and type 2A antibody) were raised against synthetic peptides, Cys-Thr-Pro-Pro-Arg-Asn-Ser-Ala-Lys-Ala-Lys-Lys and Cys-Val-Thr-Arg-Arg-Thr-Pro-Asp-Try-Phe-Leu, corresponding to the carboxyl termini of the catalytic subunits of protein phosphatase 1 and phosphatase 2A (Cys was added for specific coupling to carrier protein). These antipeptide antibodies were highly specific and were useful in discriminating between protein phosphatase 1 and phosphatase 2A in crude extracts or purified preparations. Type 2A antibody reacted with both native and denatured protein phosphatase 2A whereas under the same condition type 1 antibody reacted only with denatured protein phosphatase 1.

The yeast GLC7 gene required for glycogen accumulation encodes a type 1 protein phosphatase.

The glc7 mutant of the yeast Saccharomyces cerevisiae does not accumulate glycogen due to a defect in glycogen synthase activation (Peng, Z., Trumbly, R. J., and Reimann, E.M. (1990) J. Biol. Chem. 265, 13871-13877) whereas wild-type strains accumulate glycogen as the cell cultures approach stationary phase. We isolated the GLC7 gene by complementation of the defect in glycogen accumulation and found that the GLC7 gene is the same as the DIS2S1 gene (Ohkura, H., Kinoshita, N., Miyatani, S., Toda, T., and Yanagida, M. (1989) Cell 57, 997-1007). The protein product predicted by the GLC7 DNA sequence has a sequence that is 81% identical with rabbit protein phosphatase 1 catalytic subunit. Protein phosphatase 1 activity was greatly diminished in extracts from glc7 mutant cells. Two forms of protein phosphatase 1 were identified after chromatography of extracts on DEAE-cellulose. Both forms were diminished in the glc7 mutant and were partly restored by transformation with a plasmid carrying the GLC7 gene. Southern blots indicate the presence of a single copy of GLC7 in S. cerevisiae, and gene disruption experiments showed that the GLC7 gene is essential for cell viability. The GLC7 mRNA was identified as a 1.4-kilobase RNA that increases 4-fold at the end of exponential growth in wild-type cells, suggesting that activation of glycogen synthase is mediated by increased expression of protein phosphatase 1 as cells reach stationary phase.

Deficiency in phosphorylase phosphatase activity despite elevated protein phosphatase type-1 catalytic subunit in skeletal muscle from insulin-resistant subjects.

Glycogen synthase is activated by protein phosphatase type-1 (PP-1). The spontaneous PP-1 activity accounts for only a small fraction of total PP-1 activity, which can be exposed by trypsin digestion of inhibitor proteins in the presence of Mn2+. We determined total PP-1 activity in muscle biopsies from insulin-sensitive and -resistant nondiabetic Pima Indians. Inhibitor-2 sensitive PP-1 represented 90% of total phosphatase activity. Spontaneous and total PP-1 activities were reduced in insulin resistant subjects (P less than 0.05-0.01), suggesting that the reduced PP-1 activity is not the result of inhibition by trypsin-labile phosphatase regulatory subunits. This difference was further investigated by Western blots using two different antibodies. An antibody raised against the rabbit muscle PP-1 catalytic subunit was used to analyze muscle extracts concentrated by DEAE-Sepharose adsorption. An antibody raised against a peptide derived from the COOH-terminal end of the PP-1 catalytic subunit was used to analyze crude muscle extracts. Both antibodies recognized a PP-1 catalytic subunit of approximately 33 kD, which unexpectedly was more abundant in insulin-resistant subjects (P less than 0.05-0.01). The increase in the tissue PP-1 protein content may be a response to compensate for the impairment in the enzyme activity.

Identification of a glycogen synthase phosphatase from yeast Saccharomyces cerevisiae as protein phosphatase 2A.

A glycogen synthase phosphatase was purified from the yeast Saccharomyces cerevisiae. The purified yeast phosphatase displayed one major protein band which coincided with phosphatase activity on nondenaturing polyacrylamide gel electrophoresis. This phosphatase had a molecular mass of about 160,000 Da determined by gel filtration and was comprised of three subunits, termed A, B, and C. The subunit molecular weights estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were 60,000 (A), 53,000 (B), and 37,000 (C), indicating that this yeast glycogen synthase phosphatase is a heterotrimer. On ethanol treatment, the enzyme was dissociated to an active species with a molecular weight of 37,000 estimated by gel filtration. The yeast phosphatase dephosphorylated yeast glycogen synthase, rabbit muscle glycogen phosphorylase, casein, and the alpha subunit of rabbit muscle phosphorylase kinase, was not sensitive to heat-stable protein phosphatase inhibitor 2, and was inhibited 90% by 1 nM okadaic acid. Dephosphorylation of glycogen synthase, phosphorylase, and phosphorylase kinase by this yeast enzyme could be stimulated by histone H1 and polylysines. Divalent cations (Mg2+ and Ca2+) and chelators (EDTA and EGTA) had no effect on dephosphorylation of glycogen synthase or phosphorylase while Mn2+ stimulated enzyme activity by approximately 50%. The specific activity and kinetics for phosphorylase resembled those of mammalian phosphatase 2A. An antibody against a synthetic peptide corresponding to the carboxyl terminus of the catalytic subunit of rabbit skeletal muscle protein phosphatase 2A reacted with subunit C of purified yeast phosphatase on immunoblots, whereas the analogous peptide antibody against phosphatase 1 did not. These data show that this yeast glycogen synthase phosphatase has structural and catalytic similarity to protein phosphatase 2A found in mammalian tissues.

Phosphorylation of chicken cardiac C-protein by calcium/calmodulin-dependent protein kinase II.

Chicken cardiac C-protein was readily phosphorylated by purified calcium/calmodulin-dependent protein kinase II (CaM-kinase II). Maximum incorporation was about 4 mol of 32P/mol of C-protein subunit. Peptide mapping indicated that some of the sites phosphorylated by CaM-kinase II were located on the same phosphopeptides obtained when C-protein was phosphorylated by the cAMP-dependent protein kinase (peptides T1, T2, and T3). There was a fourth peptide (T3a) which was unique to CaM-kinase II phosphorylation. 32P-Amino acid analysis showed that essentially all of the 32P of peptides T1, T2, and T3a was in phosphoserine. cAMP-dependent protein kinase incorporated 32P only into threonine of peptide T3. Threonine was the preferred site of phosphorylation by CaM-kinase II, but there was significant phosphorylation of a serine in peptide T3. Partially purified C-protein preparations contained an associated calcium/calmodulin-dependent protein kinase. Peptide maps obtained from C-protein phosphorylated by the endogenous kinase were similar to those obtained from C-protein phosphorylated by CaM-kinase II. However, the ratio of phosphothreonine to phosphoserine in peptide T3 was lower. This was due to a contaminating phosphatase in the partially purified C-protein which preferentially dephosphorylated the phosphothreonine of peptide T3. It is suggested that the calcium/calmodulin-dependent protein kinase associated with C-protein is similar or identical to CaM-kinase II and that CaM-kinase II may play a role in the phosphorylation of C-protein in the heart.

The grid-blot: a procedure for screening large numbers of monoclonal antibodies for specificity to native and denatured proteins.

This report describes a procedure referred to as a grid-blot for simultaneously testing up to 30 monoclonal antibodies for specificity with an equivalent number of different proteins on a single sheet of nitrocellulose paper. Only 150 microliters of hybridoma culture supernatant is required for the screening and the entire procedure can be completed in less than five hours. This assay was developed to quickly identify those hybridoma cultures producing antibodies that preferentially recognize the native form of a protein and those that also recognize the SDS denatured form and were optimal for use in Western blots. Monoclonal antibodies raised against two distinct proteins, myofibril C-protein (120 antibodies) and the catalytic subunit of cyclic-AMP dependent protein kinase (240 antibodies) were tested. The grid-blot results indicated that 85 of the C-protein antibodies and 55 of the catalytic subunit antibodies were monospecific. Only 4 of the C-protein and 9 catalytic subunit antibodies showed a preferential staining for the appropriate native protein. The antibodies that stained the denatured protein most intensely in the grid-blot corresponded with those that produced the best immunostain in the Western blot. Finally, a version of the grid-blot was found to be an efficient means of determining antibody isotypes.

Inhibitory effect of polycations on phosphorylation of glycogen synthase by glycogen synthase kinase 3.

Several polycations were tested for their abilities to inhibit the activity of glycogen synthase kinase 3 (GSK-3). L-Polylysine was the most powerful inhibitor of GSK-3 with half-maximal inhibition of glycogen synthase phosphorylation occurring at approx. 100 nM. D-Polylysine and histone H1 were also inhibitory, but the concentration dependence was complex, and DL-polylysine was the least effective inhibitor. Spermine caused about 50% inhibition of GSK-3 at 0.7 mM and 70% inhibition at 4 mM. Inhibition of GSK-3 by L-polylysine could be blocked or reversed by heparin. A heat-stable polycation antagonist isolated from swine kidney cortex also blocked the inhibitory effect of L-polylysine on GSK-3 and blocked histone H1 stimulation of protein phosphatase 2A activity. Under the conditions tested, L-polylysine also inhibited GSK-3 catalyzed phosphorylation of type II regulatory subunit of cAMP-dependent protein kinase and a 63 kDa brain protein, but only slightly inhibited phosphorylation of inhibitor 2 or proteolytic fragments of glycogen synthase that contain site 3 (a + b + c). L-Polylysine at a concentration (200 nM) that caused nearly complete inhibition of GSK-3 stimulated casein kinase I and casein kinase II, but had virtually no effect on the catalytic subunit of cAMP-dependent protein kinase. These results suggest that polycations can be useful in controlling GSK-3 activity. Polycations have the potential to decrease the phosphorylation state of glycogen synthase at site 3, both by inhibiting GKS-3 as shown in this study and by stimulating the phosphatase reaction as shown previously (Pelech, S. and Cohen, P. (1985) Eur. J. Biochem. 148, 245-251).

Evidence for a latent form of protein phosphatase 1 associated with cardiac myofibrils.

Detergent-purified myofibrils from bovine heart contained very little spontaneously active protein phosphatase 1 activity. Phosphatase 1, extracted from the myofibrils by freeze-thawing in the presence of 500 mM KCl, was markedly activated by cobalt/trypsin treatment. Myofibril phosphatase 1 was separated from phosphatase 2A by chromatography on heparin-Sepharose. The phosphatase 1 was isolated in a latent form. Pretreatment with trypsin released free catalytic subunit and increased activity about 25-fold. Addition of cobalt with the trypsin increased activity another 2-fold. The latent myofibril phosphatase 1 did not appear to be the same as previously characterized forms of protein phosphatase 1. We suggest that cardiac myofibril phosphatase 1 contains a unique inhibitory subunit which directs the enzyme to the myofibril and regulates the dephosphorylation of myofibril phosphoproteins.

Modulation of glycogen synthase kinase activity of skeletal and smooth muscle casein kinase I by spermine.

Casein kinase I (CK-I) from skeletal muscle was stimulated 2-3 fold by 0.25-1 mM spermine. The polyamine also stimulated the phosphorylation of glycogen synthase by another casein kinase purified from aortic smooth muscle [DiSalvo et al. (1986) Biochem. Biophys. Res. Commun. 136, 789-796]. Phosphopeptide maps and phosphoamino acid analysis of [32P]glycogen synthase revealed that smooth muscle casein kinase phosphorylated glycogen synthase in the same sites that undergo phosphorylation by CK-I. The stimulatory effect of spermine on glycogen synthase kinase activity of CK-I was accompanied by increased phosphorylation of all peptide sites of glycogen synthase. Increased phosphorylation was observed in both seryl and threonyl residues. Higher concentrations (4 mM) of spermine inhibited CK-I activity by about 50%. These results indicate that aortic smooth muscle casein kinase is a CK-I enzyme and that skeletal and smooth muscle CK-I can be modulated by spermine.

Histone H1 phosphorylated by protein kinase C is a selective substrate for the assay of protein phosphatase 2A in the presence of phosphatase 1.

A protein phosphatase assay, selective for protein phosphatase 2A, has been developed. Bovine histone H1 phosphorylated by protein kinase C and [gamma-32P]ATP, designated H1(C), was tested as the substrate for various preparations of protein phosphatases 1 and 2A. The phosphatase 2A preparations were 10-60-times more active with H1(C) as the substrate when compared to phosphorylase a. The phosphatase 1 enzymes showed very little dephosphorylation of the H1(C) substrate, the activity being less than 5% of the phosphorylase phosphatase activity. This preference and selectivity was demonstrated for purified phosphatase preparations in addition to fresh tissue extracts. The assay provides a rapid, simple assay for the routine analysis of phosphatase 2A in the presence of phosphatase 1, without the use of heat-stable inhibitor proteins.