Crystal structure of the complex between calyculin A and the catalytic subunit of protein phosphatase 1

The crystal structure of the catalytic subunit of the protein phosphatase 1 (PP1), PP1 gamma, in complex with a marine toxin, calyculin A, was determined at 2.0 A resolution. The metal binding site contains the phosphate group of calyculin A and forms a tight network via the hydrophilic interactions between PP1 and calyculin A. Calyculin A is located in two of the three grooves, namely, in the hydrophobic groove and the acidic groove on the molecular surface. This is the first observation to note that the inhibitor adopts not a pseudocyclic conformation but an extended conformation in order to form a complex with the protein. The amino acid terminus of calyculin A contributes, in a limited manner, to the binding to PP1 gamma, which is consistent with findings from the studies of dose-inhibition analysis.     

Phosphorylation of the catalytic subunit of type-1 protein phosphatase by the v-abl tyrosine kinase.

The catalytic subunit of type-1 protein phosphatase (PP1) was phosphorylated by the tyrosine kinase v-abl as follows: (i) cytosolic PP1 was phosphorylated more (0.73 mol/mol) than PP1 obtained from the glycogen particles (0.076 mol/mol), while free catalytic subunit isolated in the active or inactive form from cytosolic PP1 was phosphorylated even less and catalytic subunit complexed with inhibitor-2 was not phosphorylated; (ii) phosphorylation stoichiometry was dependent on the concentration of PP1 and 3 h incubation at 30 degrees C was required for maximal phosphorylation; (iii) phosphorylation was on a tyrosine residue located in the C-terminal region of PP1 which is lost during proteolysis; (iv) phosphorylation did not affect enzyme activity but allowed conversion from the active to the inactive form upon incubation with inhibitor-2 of a PP1 form that in its dephospho-form did not convert.     

Expression of the catalytic subunit of phosphorylase phosphatase (protein phosphatase-1) in Escherichia coli.

The catalytic subunit of rabbit skeletal muscle protein phosphatase-1 was expressed in Escherichia coli. Expression of phosphatase-1 in the pET3a vector, which is based on the use of the T7 promoter, resulted in the expression of the enzyme as an insoluble aggregate. The insoluble enzyme could be renatured by high dilutions of the urea-solubilized protein in buffers containing dithiothreitol, Mn2+, and high NaCl concentrations. However, under all conditions tested, only partial (less than 5%) renaturation was achieved. A second attempt was made using a vector with the trp-lac hybrid promoter. In this case it was possible to express the enzyme as a soluble protein at levels of 3-4% of the soluble E. coli protein. The recombinant enzyme was purified by DEAE-Sepharose and heparin-Sepharose chromatography. Approximately 20 mg of purified enzyme was reproducibly obtained from the cells derived from 2 liters of culture. The purified enzyme had a specific activity toward phosphorylase alpha comparable to that reported for the authentic protein and had an Mr of 37,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The recombinant enzyme displayed similar sensitivities to inhibition by inhibitor-2, okadaic acid, and microcystin-LR as for the protein isolated from rabbit muscle. At all stages of purification the recombinant phosphatase behaved as an essentially inactive enzyme that required the presence of microM Mn2+ for full expression of its activity.     

Protein phosphatase type 1 catalytic subunit forms nondissociable dimers

Protein phosphatase type 1 is the major enzyme in skeletal muscle and liver for the dephosphorylation of Ser(P) and Thr(P) phosphoproteins. The cDNA for the catalytic subunit encodes a polypeptide of Mr 35,400 kDa, consistent with the Mr of 36,000-38,000 of the active protein purified in various laboratories. However, several investigators have found a Mr 70,000 protein for phosphatase type 1. In this report proteins of Mr 38,000 and 70,000 were resolved by Mono Q chromatography after extensive copurification from rabbit skeletal muscle. Antibodies affinity-purified against a type 1 phosphatase catalytic fragment reacted with both proteins in Western immunoblotting. Fractions from each peak were cleaved with cyanogen bromide and the major peptides were the same size by electrophoresis in gradient polyacrylamide gels. Cyanogen bromide peptides of the individual bands also were mapped by reversed-phase high-performance liquid chromatography. The purified Mr 38,000 and 70,000 proteins had identical HPLC peptide maps and also gave the same amino acid compositions after acid hydrolysis. Purified Mr 38,000 phosphatase catalytic subunit spontaneously formed a Mr 70,000 dimer that resisted usual dissociation conditions, i.e., boiling dodecyl sulfate plus 2-mercaptoethanol, but could be cleaved to about half size by various proteases, indicating that monomers were bound together near their amino or carboxy termini. Physiological changes in protein phosphatase type 1 are reflected in the amount of nondissociable dimers detected in tissue extracts.     

Isolation and sequence analysis of a cDNA clone encoding a type-1 protein phosphatase catalytic subunit: homology with protein phosphatase 2A

A 1.5 kb clone containing the full-length coding sequence of a type-1 protein phosphatase catalytic subunit has been isolated from a rabbit skeletal muscle cDNA library constructed in lambda gt10. The protein sequence deduced from the cDNA contains 311 residues and has a molecular mass of 35.4 kDa. A single mRNA species at 1.6 kb was visualized by Northern blotting. The type-1 protein phosphatase was strikingly homologous to protein phosphatase 2A, 49% of the amino acids between residues 11 and 280 being identical. The first 10 and last 31 residues were dissimilar. Residues 1-101 of the type-1 protein phosphatase also showed 21% sequence identity with a region of mammalian alkaline phosphatases.     

Structural basis for the recognition of regulatory subunits by the catalytic subunit of protein phosphatase 1.

The diverse forms of protein phosphatase 1 in vivo result from the association of its catalytic subunit (PP1c) with different regulatory subunits, one of which is the G-subunit (G(M)) that targets PP1c to glycogen particles in muscle. Here we report the structure, at 3.0 A resolution, of PP1c in complex with a 13 residue peptide (G(M[63-75])) of G(M). The residues in G(M[63-75]) that interact with PP1c are those in the Arg/Lys-Val/Ile-Xaa-Phe motif that is present in almost every other identified mammalian PP1-binding subunit. Disrupting this motif in the G(M[63-75]) peptide and the M(110[1-38]) peptide (which mimics the myofibrillar targeting M110 subunit in stimulating the dephosphorylation of myosin) prevents these peptides from interacting with PP1. A short peptide from the PP1-binding protein p53BP2 that contains the RVXF motif also interacts with PP1c. These findings identify a recognition site on PP1c, invariant from yeast to humans, for a critical structural motif on regulatory subunits. This explains why the binding of PP1 to its regulatory subunits is mutually exclusive, and suggests a novel approach for identifying the functions of PP1-binding proteins whose roles are unknown.     

Identification of a novel protein phosphatase catalytic subunit by cDNA cloning

A cDNA encoding a novel protein phosphatase catalytic subunit (protein phosphatase X) has been isolated from a rabbit liver library. It codes for a protein having 45% and 65% amino acid sequence identity, respectively, to the catalytic subunits of protein phosphatase 1 and protein phosphatase 2A from skeletal muscle. The enzyme is neither the hepatic form of protein phosphatase 1 or 2A, nor is it protein phosphatase 2B or 2C. The possible identity of protein phosphatase X is discussed.     

Modulation of myosin phosphatase targeting subunit and protein phosphatase 1 in the heart

Myosin light chain 2 (LC2) phosphorylation is of both physiological and pathological importance to myocardial function. The phosphatase that directly dephosphorylates LC2 is a type 1 protein phosphatase (PP1) that contains a catalytic subunit that complexes with a myosin-binding phosphatase targeting subunit (MYPT). The goal of the present study was to examine the role of MYPT in the regulation of PP1 in ventricular myocytes. In the first part of the study, regional distribution of MYPT expression and phosphorylation were determined in unstimulated hearts. The pattern of MYPT phosphorylation was inversely related to the LC2 phosphorylation spatial gradient as described by Epstein and colleagues (Davis JS, Hassanzadeh S, Winitsky S, Lin H, Satorius C, Vemuri R, Aletras AH, Wen H, and Epstein ND. Cell 107: 631-641, 2001). In the second part of the study, adult rat isolated ventricular myocytes were exposed to an alpha-adrenergic receptor agonist, and properties of MYPT, PP1, and LC2 were studied. We found MYPT associates with cardiac myofilaments, and this association increases upon alpha-adrenergic receptor stimulation. Activation of alpha-adrenergic receptors also led to a decrease in the PP1-myofilament association. Furthermore, alpha-adrenergic receptor stimulation results in phosphorylation of MYPT and LC2 and an increase in myocyte Ca(2+) sensitivity of tension that all depend on Rho kinase activation. These data support the hypothesis that alpha-adrenergic receptor activation works through Rho kinase to phosphorylate MYPT, and phosphorylated MYPT dissociates from PP1 so that PP1 is no longer physically associated with LC2. Hence, we propose a pathway for the dynamic modulation of LC2 phosphorylation through receptor-dependent phosphorylation of MYPT, and a spatial gradient of LC2 phosphorylation under basal conditions that occurs due to varied levels of phosphorylation of MYPT in ventricles.     

Overexpression of the catalytic subunit of protein phosphatase 2A impairs cardiac function

Reversible protein phosphorylation is an essential regulatory mechanism in many cellular functions. In contrast to protein kinases, the role and regulation of protein phosphatases has remained ambiguous. To address this issue, we generated transgenic mice that overexpress the catalytic subunit alpha of protein phosphatase 2A (PP2A) (PP2Acalpha) in the heart driven by the alpha-myosin heavy chain promoter. Overexpression of the PP2Acalpha gene in the heart led to increased levels of the transgene both at RNA and protein levels. This was accompanied by a significant increase of PP2A enzyme activity in the myocardium. Morphological analysis revealed isles of necrosis and fibrosis. The phosphorylation state of phospholamban, troponin inhibitor, and eukaryotic elongation factor 2 was reduced significantly. The expression of junctional (calsequestrin) and free SR proteins (SERCA and phospholamban) was not altered. Whereas no increase in morbidity or mortality was noted, transgenic mice developed cardiac hypertrophy and reduced contractility of the heart, as well as cardiac dilatation as shown by biplane echocardiography. Taken together, these findings are indicative of the fundamental role of PP2A in cardiac function and imply that disturbances in protein phosphatases expression and activity may cause or aggravate the course of cardiac diseases.     

Phoslactomycin targets cysteine-269 of the protein phosphatase 2A catalytic subunit in cells

According to the chemical genetic approach, small molecules that bind directly to proteins are used to analyze protein function, thereby enabling the elucidation of complex mechanisms in mammal cells. Thus, it is very important to identify the molecular targets of compounds that induce a unique phenotype in a target cell. Phoslactomycin A (PLMA) is known to be a potent inhibitor of protein Ser/Thr phosphatase 2A (PP2A); however, the inhibitory mechanism of PP2A by PLMA has not yet been elucidated. Here, we demonstrated that PLMA directly binds to the PP2A catalytic subunit (PP2Ac) in cells by using biotinylated PLMA, and the PLMA-binding site was identified as the Cys-269 residue of PP2Ac. Moreover, we revealed that the Cys-269 contributes to the potent inhibition of PP2Ac activity by PLMA. These results suggest that PLMA is a PP2A-selective inhibitor and is therefore expected to be useful for future investigation of PP2A function in cells.     

The modulator protein dissociates the catalytic subunit of hepatic protein phosphatase G from glycogen.

1. The phosphorylase phosphatase and glycogen-synthase phosphatase activities associated with the glycogen particles from rat liver were progressively inhibited by incubation with modulator protein. However, the phosphorylase phosphatase activity of the catalytic subunit was entirely recovered after destruction of the modulator and the regulatory subunit(s) by trypsin. 2. Inhibition of protein phosphatase G by modulator was associated with a translocation of the phosphorylase phosphatase activity (measured after incubation with trypsin) from glycogen to the soluble fraction. The degree of inhibition of phosphatase G corresponded closely to the extent to which the phosphorylase phosphatase activity was released from the glycogen particles. Incubation of glycogen-free protein phosphatase G with modulator did not change the affinity of the enzyme for added glycogen, but decreased the amount of phosphatase that could be bound to glycogen. 3. The phosphorylase phosphatase activity that was released from the glycogen particles by modulator migrated on gel filtration as a complex (Mr 106,000) of the catalytic subunit with modulator. Phosphorylase phosphatase activity could be transferred from glycogen-bound protein phosphatase G to modulator that was covalently bound to Sepharose. After elution from the column, the enzyme was identified as the free catalytic subunit (Mr 37,000).     

The catalytic subunit of protein phosphatase 2A associates with the translation termination factor eRF1.

By a number of criteria, we have demonstrated that the translation termination factor eRF1 (eukaryotic release factor 1) associates with protein phosphatase 2A (PP2A). Trimeric PP2A1 was purified from rabbit skeletal muscle using an affinity purification step. In addition to the 36 kDa catalytic subunit (PP2Ac) and established regulatory subunits of 65 kDa (PR65) and 55 kDa (PR55), purified preparations contained two proteins with apparent Mrs of 54 and 55 kDa. Protein microsequencing revealed that the 55 kDa component is a novel protein, whereas the 54 kDa protein was identified as eRF1, a protein that functions in translational termination as a polypeptide chain release factor. Using the yeast two-hybrid system, human eRF1 was shown to interact specifically with PP2Ac, but not with the PR65 or PR55 subunits. By deletion analysis, the binding domains were found to be located within the 50 N-terminal amino acids of PP2Ac, and between amino acid residues 338 and 381 in the C-terminal part of human eRF1. This association also occurs in vivo, since PP2A can be co-immunoprecipitated with eRF1 from mammalian cells. We observed a significant increase in the amount of PP2A associated with the polysomes when eRF1 was transiently expressed in COS1 cells, and eRF1 immunoprecipitated from those fractions contained associated PP2A. Since we did not observe any dramatic effects of PP2A on the polypeptide chain release activity of eRF1 (or vice versa), we postulate that eRF1 also functions to recruit PP2A into polysomes, thus bringing the phosphatase into contact with putative targets among the components of the translational apparatus.     

Brain actin-associated protein phosphatase 1 holoenzymes containing spinophilin, neurabin, and selected catalytic subunit isoforms

We previously characterized PP1bp134 and PP1bp175, two neuronal proteins that bind the protein phosphatase 1 catalytic subunit (PP1). Here we purify from rat brain actin-cytoskeletal extracts PP1(A) holoenzymes selectively enriched in PP1gamma(1) over PP1beta isoforms and also containing PP1bp134 and PP1bp175. PP1bp134 and PP1bp175 were identified as the synapse-localized F-actin-binding proteins spinophilin (Allen, P. B., Ouimet, C. C., and Greengard, P. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 9956-9561; Satoh, A., Nakanishi, H., Obaishi, H., Wada, M., Takahashi, K., Satoh, K., Hirao, K., Nishioka, H., Hata, Y., Mizoguchi, A., and Takai, Y. (1998) J. Biol. Chem. 273, 3470-3475) and neurabin (Nakanishi, H., Obaishi, H., Satoh, A., Wada, M., Mandai, K., Satoh, K., Nishioka, H. , Matsuura, Y., Mizoguchi, A., and Takai, Y. (1997) J. Cell Biol. 139, 951-961), respectively. Recombinant spinophilin and neurabin interacted with endogenous PP1 and also with each other when co-expressed in HEK293 cells. Spinophilin residues 427-470, or homologous neurabin residues 436-479, were sufficient to bind PP1 in gel overlay assays, and selectively bound PP1gamma(1) from a mixture of brain protein phosphatase catalytic subunits; additional N- and C-terminal sequences were required for potent inhibition of PP1. Immunoprecipitation of spinophilin or neurabin from crude brain extracts selectively coprecipitated PP1gamma(1) over PP1beta. Moreover, immunoprecipitation of PP1gamma(1) from brain extracts efficiently coprecipitated spinophilin and neurabin, whereas PP1beta immunoprecipitation did not. Thus, PP1(A) holoenzymes containing spinophilin and/or neurabin target specific neuronal PP1 isoforms, facilitating efficient regulation of synaptic phosphoproteins.     

The MyD116 African swine fever virus homologue interacts with the catalytic subunit of protein phosphatase 1 and activates its phosphatase activity

The DP71L protein of African swine fever virus (ASFV) shares sequence similarity with the herpes simplex virus ICP34.5 protein over a C-terminal domain. We showed that the catalytic subunit of protein phosphatase 1 (PP1) interacts specifically with the ASFV DP71L protein in a yeast two-hybrid screen. The chimeric full-length DP71L protein, from ASFV strain Badajoz 71 (BA71V), fused to glutathione S-transferase (DP71L-GST) was expressed in Escherichia coli and shown to bind specifically to the PP1-alpha catalytic subunit expressed as a histidine fusion protein (6xHis-PP1alpha) in E. coli. The functional effects of this interaction were investigated by measuring the levels of PP1 and PP2A in ASFV-infected Vero cells. This showed that infection with wild-type ASFV strain BA71V activated PP1 between two- and threefold over that of mock-infected cells. This activation did not occur in cells infected with the BA71V isolate in which the DP71L gene had been deleted, suggesting that expression of DP71L leads to PP1 activation. In contrast, no effect was observed on the activity of PP2A following ASFV infection. We showed that infection of cells with wild-type BA71V virus resulted in decreased phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF-2alpha). ICP34.5 recruits PP1 to dephosphorylate the alpha subunit of eukaryotic translational initiation factor 2 (also known as eIF-2alpha); possibly the ASFV DP71L protein has a similar function.     

Purification and characterization of the polycation-stimulated protein phosphatase catalytic subunit from porcine renal cortex

The predominant form of phosphorylase phosphatase activity in porcine renal cortical extracts was a polycation-stimulated protein phosphatase. This activity was present in extracts in a high-molecular-weight form which could be converted to a free catalytic subunit by treatment with ethanol, urea, or freezing and thawing in the presence of beta-mercaptoethanol. The catalytic subunit of the polycation-stimulated phosphatase was purified by chromatography on DEAE-Sephacel, heparin-Sepharose, and Sephadex G-75. The phosphatase appeared to be homogeneous on SDS-polyacrylamide gel electrophoresis. The enzyme had an apparent Mr of 35 000 on gel filtration and SDS-polyacrylamide gel electrophoresis. The purified phosphatase could be stimulated by histone H1, protamine, poly(D-lysine), poly(L-lysine) or polybrene utilizing phosphorylase a as the substrate. It preferentially dephosphorylated the alpha-subunit of phosphorylase kinase. The phosphatase was highly sensitive to inhibition by ATP. These results suggest that the renal polycation-stimulated phosphatase catalytic subunit is very similar to or identical with the skeletal muscle phosphatase form which has been previously designated phosphatase-2Ac.     

Cellular mechanisms regulating protein phosphatase-1. A key functional interaction between inhibitor-2 and the type 1 protein phosphatase catalytic subunit

Inhibitor-1 (I-1) and inhibitor-2 (I-2) selectively inhibit type 1 protein serine/threonine phosphatases (PP1). To define the molecular basis for PP1 inhibition by I-1 and I-2 charged-to-alanine substitutions in the Saccharomyces cerevisiae, PP1 catalytic subunit (GLC7), were analyzed. Two PP1 mutants, E53A/E55A and K165A/E166A/K167A, showed reduced sensitivity to I-2 when compared with wild-type PP1. Both mutants were effectively inhibited by I-1. Two-hybrid analysis and coprecipitation or pull-down assays established that wild-type and mutant PP1 catalytic subunits bound I-2 in an identical manner and suggested a role for the mutated amino acids in enzyme inhibition. Inhibition of wild-type and mutant PP1 enzymes by full-length I-2(1-204), I-2(1-114), and I-2(36-204) indicated that the mutant enzymes were impaired in their interaction with the N-terminal 35 amino acids of I-2. Site-directed mutagenesis of amino acids near the N terminus of I-2 and competition for PP1 binding by a synthetic peptide encompassing an I-2 N-terminal sequence suggested that a PP1 domain composed of amino acids Glu-53, Glu-55, Asp-165, Glu-166, and Lys-167 interacts with the N terminus of I-2. This defined a novel regulatory interaction between I-2 and PP1 that determines I-2 potency and perhaps selectivity as a PP1 inhibitor.     

Interaction of p130 with, and consequent inhibition of, the catalytic subunit of protein phosphatase 1alpha

The protein p130 was originally isolated from rat brain as an inositol 1,4,5-trisphosphate-binding protein with a domain organization similar to that of phospholipase C-delta1 but which lacks phospholipase C activity. Yeast two-hybrid screening of a human brain cDNA library for clones that encode proteins that interact with p130 has now led to the identification of the catalytic subunit of protein phosphatase 1alpha (PP1calpha) as a p130-binding protein. The association between p130 and PP1calpha was also confirmed in vitro by an overlay assay, a "pull-down" assay, and surface plasmon resonance analysis. The interaction of p130 with PP1calpha resulted in inhibition of the catalytic activity of the latter in a p130 concentration-dependent manner. Immunoprecipitation and immunoblot analysis of COS-1 cells that stably express p130 and of mouse brain extract with antibodies to p130 and to PP1calpha also detected the presence of a complex of p130 and PP1calpha. The activity of glycogen phosphorylase, which is negatively regulated by dephosphorylation by PP1calpha, was higher in COS-1 cells that stably express p130 than in control COS-1 cells. These results suggest that, in addition to its role in inositol 1,4,5-trisphosphate and Ca(2+) signaling, p130 might also contribute to regulation of protein dephosphorylation through its interaction with PP1calpha.     

Characterization of cyclic AMP-requiring yeast mutants altered in the catalytic subunit of protein kinase

The cyr2 mutant of yeast, Saccharomyces cerevisiae, required cAMP for growth at 35 degrees C. The cyr2 mutation was suppressed by the bcy1 mutation which resulted in deficiency of the regulatory subunit of cAMP-dependent protein kinase. The DEAE-Sephacel elution profile of cyr2 cAMP-dependent protein kinase was markedly different from that observed for the wild-type enzyme. With histone as substrate, the cAMP-dependent protein kinase activity of cyr2 cells showed 100-fold greater Ka value for activation by cAMP at 35 degrees C than that of the wild-type cells, while the Kd value for cAMP of the mutant enzyme was not altered. The electrophoretic character, molecular weight, and pI value of the regulatory subunit of the mutant enzyme were the same as those of the wild-type enzyme. When histone, trehalase, and glutamate dehydrogenase were used as substrate, the free catalytic subunit of the mutant enzyme showed a markedly decreased affinity for ATP and was more thermolabile compared to that of the wild-type enzyme. The results indicated that the cyr2 phenotype was produced by a structural mutation in the cyr2 gene coding for the catalytic subunit of cAMP-dependent protein kinase in yeast.