Protein Phosphatase 2Ac Research Articles

Expression of protein phosphatase 2AC in the brain of mice and Alzheimer's patients

Objective: To investigate the biological functions of protein phosphatase 2AC(PP2AC) in the brain, and to detect its spatio-temporal expression and its involvement in neurological disorders in the brains of mice and Alzheimer's patients. Methods: Western blot was used to evaluate the expression level of PP2AC in different organs. Immunohistochemical staining was performed to detect the in situ expression levels of PP2AC in the brains of mice and patients, and the pathological changes were confirmed in the brains of patients with Alzheimer's disease. Results: Among all the tested organs in adult mouse, the expression of PP2AC protein was the highest in the brain. From embryonic day 18.5 to postnatal 2-year-old mice, PP2AC exhibited spatio-temporal specific expression in the brains. Furthermore, an age-dependent increased expression in the cerebral cortex at both protein and RNA levels was observed. Compared to control group, PP2AC protein expression was lower in the frontal cortex of Alzheimer's patients. Conclusions: The spatio-temporal specific expression profiles of PP2AC in mouse brain implicate its biological significance. Its diminished expression in the frontal cortex of Alzheimer's disease patients implies that PP2AC plays a potential role in the pathogenesis of Alzheimer's disease.

Protein phosphatase 2A activation mechanism contributes to JS-K induced caspase-dependent apoptosis in human hepatocellular carcinoma cells

BackgroundJS-K is a nitric oxide (NO) donor and could generate intracellularly high levels of NO. The study explores PP2A as a tumor suppressor is a major determinant mediating JS-K-caused apoptosis in human hepatocellular carcinoma (HCC) cells.MethodsThe human HCC cell lines (PLC5, Huh-7, Bel-7402, SMMC-7721 and HepG2) were used to assess effects of JS-K on cell viability, apoptosis induction and PP2A activation. Effects of JS-K on cell morphology, mitochondrial membrane potential, apoptosis and NO levels were determined in HCC cells expressing PP2A. Simultaneously, the expression of PP2A family including PP2A-A(α/β), PP2A-B55, PP2A-C(α/β) and the substrates of PP2A, such as β-catenin, c-Myc and p-Bcl-2 (Ser70) were detected in sensitive HCC cells. Furthermore, the role of NO in mediating the expression of PP2A was further validated with Z-VAD-FMK (a caspase inhibitor), Carboxy-PTIO (a NO scavenger), okadaic acid (OA, a PP2A inhibitor) and FTY720 (a PP2A agonist) in JS-K treated cells. In addition, the genetic manuplation of PP2A including overexpression and knockdown have been also performed in JS-K treated cells. Moreover, the rat model of primary hepatic carcinoma was established with diethylnitrosamine for 16 weeks to verify the anti-tumor effects of JS-K in vivo. Immunohistochemical and Western blot analysis were used to determine the expression of proteins in rat primary hepatic carcinoma tissues.ResultsJS-K significantly inhibited cell proliferation, increased apoptosis rate and activated PP2A activity in five HCC cells viability, especially SMMC7721 and HepG2 cells. It was characterized by loss of mitochondrial membrane potential, significant externalization of phosphatidylserine, nuclear morphological changes. Moreover, JS-K enhanced Bax-to-Bcl-2 ratio, released cytochrome c (Cyt c) from mitochondria, activated cleaved-caspase-9/3 and the cleavage of PARP, and decreased the expression of X-linked inhibitor of apoptosis protein (XIAP). Both Z-VAD-FMK and Carboxy-PTIO suppressed the activation of cleaved-caspase-9/3 and of cleaved-PARP in JS-K-treated sensitive HCC cells. Simultaneously, JS-K treatment could lead to the activation of protein phosphatase 2A-C (PP2A-C) but not PP2A-A and PP2A-B55, which subsequently inactivated and dephosphorylated the PP2A substrates including β-catenin, c-Myc, and p-Bcl-2 (Ser70). However, silencing PP2A-C could abolish both the activation of PP2A-C and down-regulation of β-catenin, c-Myc and p-Bcl-2 (Ser70) in sensitive HCC cells. Conversely, PP2A overexpression could enhance the effects of JS-K on activation of PP2A and down-regulation of β-catenin, c-Myc and p-Bcl-2 (Ser70). In addition, adding okadaic acid (OA), a PP2A inhibitor, abolished the effects of JS-K on apoptosis induction, PP2A activation and the substrates of PP2A dephosphorylation; FTY720, a PP2A agonist, enhanced the effects of JS-K including apoptosis induction, PP2A activation and the substrates of PP2A dephosphorylation. The mice exhibited a lower number and smaller tumor nodules in response to JS-K-treated group. A marked increase in the number of hepatocytes with PCNA-positive nuclei (proliferating cells) was evident in DEN group and tended to decrease with JS-K treatment. Furthermore, JS-K treatment could induce PP2A activation and the substrates of PP2A inactivation such as β-catenin, c-Myc and p-Bcl-2(Ser70) in DEN-induced hepatocarcinogenesis.ConclusionsHigh levels of NO released from JS-K induces a caspase-dependent apoptosis through PP2A activation.

Protein phosphatase 2A mediates JS-K-induced apoptosis by affecting Bcl-2 family proteins in human hepatocellular carcinoma HepG2 cells.

Protein phosphatase 2A (PP2A) is an important enzyme within various signal transduction pathways. The present study was investigated PP2A mediates JS-K-induced apoptosis by affecting Bcl-2 family protein. JS-K showed diverse inhibitory effects in five HCC cell lines, especially HepG2 cells. JS-K caused a dose- and time-dependent reduction in cell viability and increased in levels of LDH release. Meanwhile, JS-K- induced apoptosis was characterized by mitochondrial membrane potential reduction, Hoechst 33342+ /PI+ dual staining, release of cytochrome c (Cyt c), and activation of cleaved caspase-9/3. Moreover, JS-K-treatment could lead to the activation of protein phosphatase 2A-C (PP2A-C), decrease of anti-apoptotic Bcl-2 family-protein expression including p-Bcl-2 (Ser70), Bcl-2, Bcl-xL, and Mcl-1 as well as the increase of pro-apoptosis Bcl-2 family-protein including Bim, Bad, Bax, and Bak. Furthermore, JS-K caused a marked increase of intracellular NO levels while pre-treatment with Carboxy-PTIO (a NO scavenger) reduced the cytotoxicity effects and the apoptosis rate. Meanwhile, pre-treatment with Carboxy-PTIO attenuated the JS-K-induced up-regulation of PP2A, Cyt c, and cleaved-caspase-9/3 activation. The silencing PP2A-C by siRNA could abolish the activation of PP2A-C, down-regulation of anti-apoptotic Bcl-2 family-protein (p-Bcl-2, Bcl-2, Bcl-xL, and Mcl-1), increase of pro-apoptosis Bcl-2 family-protein (Bim, Bad, Bax, and Bak) and apoptotic-related protein (Cyt c, cleaved caspase-9/3) that were caused by JS-K in HepG2 cells. In addition, pre-treatment with OA (a PP2A inhibitor) also attenuated the above effects induced by JS-K. In summary, NO release from JS-K induces apoptosis through PP2A activation, which contributed to the regulation of Bcl-2 family proteins.

Mid1/Mid2 expression in craniofacial development and a literature review of X-linked opitz syndrome.

Opitz syndrome (OS) is a genetic disorder that affects mainly the development of midline structures, including the craniofacial region, embryonic heart, and urogenital system. The manifestations of X-linked OS are believed to be results of a malfunctioned gene, MID1, whose product has been shown to have ubiquitin E3 ligase activity and regulate the turnover of microtubular protein phosphatase 2Ac. MID2, a homolog of MID1, shares high structural and functional similarities with MID1. Identification of a missense mutation in MID2 in an Indian family causing overlapping phenotypes with OS provided the first evidence that MID2 might be involved in similar pathogenesis. The clinic features and the genetic findings of all reported X-linked OS were collectively summarized in this research. Real-time RT-PCR and in situ hybridization were used in the expression studies of Mid1/Mid2 in mouse embryos. Up-to-date, 88 different mutations have been identified in MID1 and most mutations occurred on the conserved amino acids of MID1 and MID2. Expression studies using real-time RT-PCR implicated a tendency of a mutually repressive expression pattern between Mid1 and Mid2 in mouse embryos. Further investigations using in situ hybridization revealed strong expressions of Mid1 and Mid2 in the epithelium of approaching facial prominences and downregulated expressions after fusion in mouse embryos. Our results support the hypothesis of functional redundancy of Mid1/Mid2 and their potential roles in regulating tissue remodelling in early development.

Effect of Protein Phosphatase 2Ac and Norcantharidin on Smad3 Linker Region Phosphorylation in HK-2 Cells

Effect of Protein Phosphatase 2Ac and Norcantharidin on Smad3 Linker Region Phosphorylation in HK-2 Cells

TNF-related apoptosis-inducing ligand (TRAIL) regulates midline-1, thymic stromal lymphopoietin, inflammation, and remodeling in experimental eosinophilic esophagitis

Eosinophilic esophagitis (EoE) is an inflammatory disorder of the esophagus defined by eosinophil infiltration and tissue remodeling with resulting symptoms of esophageal dysfunction. TNF-related apoptosis-inducing ligand (TRAIL) promotes inflammation through upregulation of the E3 ubiquitin-ligase midline-1 (MID1), which binds to and deactivates the catalytic subunit of protein phosphatase 2Ac, resulting in increased nuclear factor κB activation. We sought to elucidate the role of TRAIL in EoE. We used Aspergillus fumigatus to induce EoE in TRAIL-sufficient (wild-type) and TRAIL-deficient (TRAIL(-/-)) mice and targeted MID1 in the esophagus with small interfering RNA. We also treated mice with recombinant thymic stromal lymphopoietin (TSLP) and TRAIL. TRAIL deficiency and MID1 silencing with small interfering RNA reduced esophageal eosinophil and mast cell numbers and protected against esophageal circumference enlargement, muscularis externa thickening, and collagen deposition. MID1 expression and nuclear factor κB activation were reduced in TRAIL(-/-) mice, whereas protein phosphatase 2Ac levels were increased compared with those seen in wild-type control mice. This was associated with reduced expression of CCL24, CCL11, CCL20, IL-5, IL-13, IL-25, TGFB, and TSLP. Treatment with TSLP reconstituted hallmark features of EoE in TRAIL(-/-) mice and recombinant TRAIL induced esophageal TSLP expression in vivo in the absence of allergen. Post hoc analysis of gene array data demonstrated significant upregulation of TRAIL and MID1 in a cohort of children with EoE compared with that seen in controls. TRAIL regulates MID1 and TSLP, inflammation, fibrosis, smooth muscle hypertrophy, and expression of inflammatory effector chemokines and cytokines in experimental EoE.

Role of p53–fibrinolytic system cross-talk in the regulation of quartz-induced lung injury

Silica is the major component of airborne dust generated by wind, manufacturing and/or demolition. Chronic occupational inhalation of silica dust containing crystalline quartz is by far the predominant form of silicosis in humans. Silicosis is a progressive lung disease that typically arises after a very long latency and is a major occupational concern with no known effective treatment. The mechanism of silicosis is not clearly understood. However, silicosis is associated with increased cell death, expression of redox enzymes and pro-fibrotic cytokines and chemokines. Since alveolar epithelial cell (AEC) death and disruption of alveolar fibrinolysis is often associated with both acute and chronic lung injuries, we explored whether p53-mediated changes in the urokinase-type plasminogen activator (uPA) system contributes to silica-induced lung injury. We further sought to determine whether caveolin-1 scaffolding domain peptide (CSP), which inhibits p53 expression, mitigates lung injury associated with exposure to silica. Lung tissues and AECs isolated from wild-type (WT) mice exposed to silica exhibit increased apoptosis, p53 and PAI-1, and suppression of uPA expression. Treatment of WT mice with CSP inhibits PAI-1, restores uPA expression and prevents AEC apoptosis by suppressing p53, which is otherwise induced in mice exposed to silica. The process involves CSP-mediated inhibition of serine-15 phosphorylation of p53 by inhibition of protein phosphatase 2A-C (PP2A-C) interaction with silica-induced caveolin-1 in AECs. These observations suggest that changes in the p53–uPA fibrinolytic system cross-talk contribute to lung injury caused by inhalation of silica dust containing crystalline quartz and is protected by CSP by targeting this pathway.

Silibinin suppresses the maintenance of colorectal cancer stem‐like cells by inhibiting PP2A/AKT/mTOR pathways

Silibinin, an effective chemo-preventive agent in various cancer types, suppresses cancer cell growth, but its effects on cancer stem-like cells (CSLCs) remain unclear. This study aimed to examine whether silibinin inhibited the development of CSLCs and disclose the underlying signaling. The colorectal cancer spheroid culture system was used for enriching CSLCs. The effects of silibinin on CSLCs were evaluated by counting sphere numbers, and calculating the percentage of CD133+ cells by flow cytometry and immunofluorescence both in the absence and presence of different concentrations of silibinin. The results showed the sphere number of CCS was 36 ± 9.6 after 15 days of CSLC enrichment in spheroid culture, and the percentage of CD133+ cells increased to 18 ± 6.4% compared to 3 ± 0.8% before enrichment. Treatment with silibinin reduced the sphere formation to 5 ± 3.3 and decreased the CD133+ percentage to 8 ± 2.3%. Interestingly, treatment of silibinin suppressed the activation of the AKT Ser473/mTOR pathway in spheroid culture through suppressing the activity of protein phosphatase 2Ac subunit (PP2Ac). In a xenograft tumor model, treatment with silibinin also inhibited tumor formation rate and tumor growth. Silibinin, which inhibits colon CSLCs self-renewal and sphere formation by suppressing the PP2Ac/AKT Ser473/mTOR pathway, may be a compound for developing new strategies in modulating CSLCs in cancer therapy.

MRNA expression of protein phosphatase 2A/C (PP2A/C) in erlotinib-treated non-small cell lung cancer (NSCLC) patients (p) with EGFR mutations.

e18037 Background: EGFR mutations can influence double-strand break (DSB) repair due to impairment of DNA-PKcs/Ku complexes. PP2A enhances Ku and DNA-PK activities, accelerating DSB repair. In addi...

ADP ribosylation factor like 2 (Arl2) protein influences microtubule dynamics in breast cancer cells

ADP ribosylation factor like 2 (Arl2) protein is involved in the folding of tubulin peptides. Variants of the human adenocarcinoma line MCF7 cells with increased or reduced content of Arl2 protein were produced and characterized. Western blot analysis performed after separation of the different fractions of tubulins showed that the content in polymerizable soluble heterodimers was significantly increased in cells with the highest Arl2 expression level (MA+) and reduced in cells with the lowest Arl2 expression level (MA−) in comparison to control cells (MP). Microtubule dynamic instability, measured after microinjection of rhodamine-labelled tubulin in living cells, was significantly enhanced in MA+ cells and reduced in MA− cells. These alterations involved modifications of the microtubule growth and shortening rates, duration of attenuation phases, percentage of time spent in each phase (growth, shortening and attenuation) and catastrophe frequency. We also observed modifications in the expression level of the tumor suppressor protein phosphatase 2Ac, which has been shown to form a complex with Arl2. Finally, cell cycle progression was modified in these cells, particularly in regard to duration of telophase. In summary, alterations in Arl2 protein content were found to be associated with modifications in tubulin pools, microtubule dynamics as well as cell cycle progression.

Upregulation of protein phosphatase 2Ac by hepatitis C virus modulates NS3 helicase activity through inhibition of protein arginine methyltransferase 1.

Hepatitis C virus (HCV) is a major cause of chronic liver disease, cirrhosis, and hepatocellular carcinoma worldwide. HCV has a positive-strand RNA genome of about 9.4 kb in size, which serves as a template for replication and for translation of a polyprotein of about 3,000 amino acids. The polyprotein is cleaved co- and posttranslationally by cellular and viral proteases into at least 10 different mature proteins. One of these proteins, nonstructural protein 3 (NS3), has serine protease and NTPase/RNA helicase activity. Arginine 467 in the helicase domain of NS3 (arginine 1493 in the polyprotein) can be methylated by protein arginine methyltransferase 1 (PRMT1). Here we report that the methylation of NS3 inhibits the enzymatic activity of the helicase. Furthermore, we found that PRMT1 activity itself is regulated by protein phosphatase 2A (PP2A). PP2A inhibits PRMT1 enzymatic activity and therefore increases the helicase activity of NS3. This is important, because we found an increased expression of PP2A in cell lines with inducible HCV protein expression, in transgenic mice expressing HCV proteins in hepatocytes, and in liver biopsy samples from patients with chronic hepatitis C. Interestingly, up-regulation of PP2A not only modulates the enzymatic activity of an important viral protein, NS3 helicase, but also interferes with the cellular defense against viruses by inhibiting interferon-induced signaling through signal transducer and activator of transcription 1 (STAT1). We conclude that up-regulation of PP2A might be crucial for the efficient replication of HCV and propose PP2A as a potential target for anti-HCV treatment strategies.

Antiapoptotic Activity of Akt Is Down-regulated by Ca2+ in Myocardiac H9c2 Cells: EVIDENCE OF Ca2+-DEPENDENT REGULATION OF PROTEIN PHOSPHATASE 2Ac

Cell survival signaling of the Akt/protein kinase B pathway was influenced by a change in the cytoplasmic free calcium concentration ([Ca2+]i) for over 2 h via the regulation of a Ser/Thr phosphatase, protein phosphatase 2Ac (PP2Ac), in rat myocardiac H9c2 cells. Akt was down-regulated when [Ca2+]i was elevated by thapsigargin, an inhibitor of the endoplasmic reticulum Ca(2+)-ATPase, but was up-regulated when it was suppressed by 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl)ester (BAPTA-AM), a cell permeable Ca2+ chelator. The inactivation of Akt was well correlated with the susceptibility to oxidant-induced apoptosis in H9c2 cells. To investigate the mechanism of the Ca(2+)-dependent regulation of Akt via the regulation of PP2A, we examined the transcriptional regulation of PP2Acalpha in H9c2 cells with Ca2+ modulators. Transcription of the PP2Acalpha gene was increased by thapsigargin but decreased by BAPTA-AM. The promoter activity was examined and the cAMP response element (CRE) was found responsible for the Ca(2+)-dependent regulation of PP2Acalpha. Furthermore, phosphorylation of CRE-binding protein increased with thapsigargin but decreased with BAPTA-AM. A long term change of [Ca2+]i regulates PP2Acalpha gene transcription via CRE, resulting in a change in the activation status of Akt leading to an altered susceptibility to apoptosis.

Dephosphorylation of PKCδ by protein phosphatase 2Ac and its inhibition by nucleotides

The protein phosphatases PP1 c, PP2A c and PP2Cα are shown to dephosphorylate protein kinase Cδ (PKCδ) in vitro; of these PP2A c displayed the highest specific activity towards PKCδ. The role of PP2A c in the dephosphorylation of PKCδ in cells was supported by the demonstration that these proteins could be co-immunoprecipitated from NIH3T3 cells. However the observation that binding of Mg-ATP to PKCδ could protect the enzyme from dephosphorylation by PP2A c in vitro indicates that an additional input/factor is required for dephosphorylation in vivo.

Characterization and partial purification of a novel neutrophil membrane-associated kinase capable of phosphorylating the respiratory burst component p47phox

The phosphorylation of p47phox is widely viewed as an important step in the activation of the neutrophil respiratory burst oxidase system. The exact nature of the kinase(s) responsible remains to be elucidated. We show here that such a kinase was detected on neutrophil membranes activated by either PMA or formyl-methionyl-leucyl-phenylalanine. This enzyme is not intrinsic to the neutrophil membrane and could be eluted with 0.5 M NaCl. The kinase activity was partially purified and was found not to be due to the presence of previously suggested kinases, including protein kinase C isotypes, mitogen-activated protein kinase and protein kinase B. Gel filtration and renaturation in substrate gels suggest a molecular mass of between 45 and 51 kDa. The kinase activity was independent of calcium and lipids but was potently inhibited by staurosporine. Treatment with protein phosphatase 2Ac suggested that the kinase was activated by serine/threonine phosphorylation. Phosphopeptide maps indicated that the kinase phosphorylated p47phox on similar sites to those found in vivo. These results indicate that activation of neutrophils by PMA results in the activation of a membrane-associated kinase that may play a part in the regulation of neutrophil NADPH oxidase through its ability to phosphorylate p47phox.

Identification and characterization of an auto-activating MEK kinase from bovine brain: Phosphorylation of serine-298 in the proline-rich domain of the mammalian MEKs

Mitogen-activated protein kinase kinases (MKKs or MEKs) are dual specificity tyrosine/threonine protein kinases that are activated by phosphorylation at two closely spaced serine residues (serines-218 and -222) by the c-mos and raf proto-oncogenes. This double phosphorylation is both necessary and sufficient for MEKs to activate the MAP kinase enzymes in vitro. The specificity or regulation of in vivo signaling to the mammalian MEKs (MEK1 and MEK2) was recently reported also to involve the differential phosphorylation of a proline-rich peptide located between the MEK kinase-subdomains IX and X. Here we report the purification and characterization of an auto-activating protein kinase from bovine brain that phosphorylates serine-298 of the MEK1 and MEK2 proline-rich insert peptides. The auto-activation of the MEK-S298 peptide kinase is the result of an intermolecular phosphorylation event that can be prevented by the peptide substrates. The inactive kinase migrates on gel filtration as a 90 kDa protein, and after activation as a 43 kDa phosphoprotein. Incorporation of 32P[phosphate] into 40–42 kDa proteins on SDS-PAGE parallels the activation of the enzyme, and dephosphorylation by protein phosphatase 2Ac reverses the activation. SDS-PAGE renaturation assays show that the 40 kDa protein has the capacity to autophosphorylate, and exhibits kinase activity towards myelin basic protein after activation. Phosphorylation of purified bovine brain MEK or recombinant MEK1 by the auto-activated kinase does not activate the enzyme, and does not interfere with the in vitro raf-mediated MEK activation. We conclude that still unknown kinases may control the MAP kinase pathway by targeting MEK.

Correlation between high temperature dependence of smooth muscle myosin light chain phosphatase activity and muscle relaxation rate.

Q10 values of the protein phosphatases that can dephosphorylate the regulatory light chain of smooth muscle myosin were determined. Six phosphatases were examined, i.e. skeletal muscle protein phosphatase 1c; protein phosphatase 2Ac; smooth muscle phosphatases (SMP) I, II, and IV; and myosin-associated protein phosphatase (MAP phosphatase). Among them, SMP-IV and MAP phosphatase, which can dephosphorylate intact smooth muscle myosin, showed extremely high Q10 values (5.3 and 5.2, respectively). On the other hand, the Q10 values of other tested phosphatases were within the range of the normal enzyme reaction (Q10 = 2.0). The rate of dephosphorylation of the myosin light chain in alpha-toxin-skinned strips was measured at different temperatures. The results provided a Q10 of 5.1, which was quite similar to those values obtained for SMP-IV and MAP phosphatase. These results suggest that the physiological myosin light chain phosphatases are SMP-IV and/or MAP phosphatase, i.e. type 1 protein phosphatases. The temperature dependence of maximum force, the steady-state extent of myosin light chain phosphorylation, and the relaxation rate of alpha-toxin-permeabilized rabbit portal vein smooth muscle strips were measured. Both maximum force and the extent of myosin light chain phosphorylation were significantly higher at lower temperature (15 degrees C) than at higher temperature (25 degrees C) under all pCa conditions tested, i.e. > 8, 6.3, and 5. The temperature dependence of the relaxation rate was much steeper (decreased 4 times by lowering the temperature from 25 to 15 degrees C) than that of the initial rate of increase in force development (decreased 1.4 times by lowering the temperature from 25 to 15 degrees C). These results are consistent with the Q10 values of myosin light chain phosphatases (Q10 = 5) and myosin light chain kinase (Q10 = 1.7) and further show that the smooth muscle type 1 phosphatases are responsible for the dephosphorylation of smooth muscle myosin in situ.

The protein phosphatases involved in cellular regulation. Influence of polyamines on the activities of protein phosphatase-1 and protein phosphatase-2A.

The effects of polyamines on the oligomeric forms of protein phosphatase-1 (1G), protein phosphatase-2A (2A0, 2A1 and 2A2) and their free catalytic subunits (1C and 2AC) has been studied using homogeneous enzymes isolated from rabbit skeletal muscle. Spermine increased the activity of protein phosphatase-2A towards eight of nine substrates tested. Half-maximal activation was observed at 0.2 mM with optimal effects at 1-2 mM. Above 2 mM, spermine became inhibitory. The most impressive activation of protein phosphatase-2A was obtained with glycogen synthase, especially when phosphorylated at sites-3 (8-15-fold with protein phosphatase-2A1) and phenylalanine hydroxylase (6-7-fold with protein phosphatase-2A1) as substrates. Activation of protein phosphatases 2A0, 2A1 and 2A2 was greater than that observed with 2AC. Spermine was a more potent activator than spermidine, while putrescine had only a small effect. Qualitatively similar results were obtained with five other substrates, although maximal activation was much less (1.3-3-fold with protein phosphatase-2A1). The rate of dephosphorylation of glycogen phosphorylase was decreased by spermine, inhibition being more pronounced with protein phosphatase-2AC than with 2A0, 2A1 and 2A2. Spermine (I50 = 0.1 mM with protein phosphatase-2AC) was a more potent inhibitor than spermidine (I50 = 0.9 mM) or putrescine (I50 = 8 mM). Partially purified preparations of protein phosphatases-2A0, 2A1 and 2A2 from from rat liver were affected by spermine in a similar manner to the homogeneous enzymes from rabbit skeletal muscle. Spermine did not activate protein phosphatase-1 to the same extent as protein phosphatase-2A. Greatest stimulation (2.5-fold) was again observed with glycogen synthase labelled in sites-3, with half-maximal activation at 0.2 mM and optimal effects at 1-2 mM spermine. Spermine was a much more effective stimulator than spermidine, while putrescine was ineffective. Very similar results were obtained with protein phosphatases 1G and 1C. With four other substrates maximal activation by spermine was less than 1.5-fold, while the dephosphorylation of glycogen synthase (labelled in site-2), phosphorylase kinase, pyruvate kinase and glycogen phosphorylase were inhibited. Spermine (I50 = 0.04 mM) was a more potent inhibitor of the dephosphorylation of glycogen phosphorylase than spermidine (I50 = 0.9 mM) or putrescine (I50 = 9 mM).(ABSTRACT TRUNCATED AT 400 WORDS)