Synthetic peptides as model substrates for the study of the specificity of the polycation-stimulated protein phosphatases

The substrate specificity of the different forms of the polycation-stimulated (PCS, type 2A) protein phosphatases and of the active catalytic subunit of the ATP, Mg-dependent (type 1) phosphatase (AMDC) was investigated, using synthetic peptides phosphorylated by either cyclic-AMP-dependent protein kinase or by casein kinase-2. The PCS phosphatases are very efficient toward the Thr(P) peptides RRAT(P)VA and RRREEET(P)EEE when compared with the Ser(P) analogues RRAS(P)VA and RRREEES(P)EEEAA. Despite their distinct sequence, both Thr(P) peptides are excellent substrates for the PCSM and PCSH1 phosphatases, being dephosphorylated faster than phosphorylase a. The slow dephosphorylation of RRAS(P)VA by the PCS phosphatases could be increased substantially by the insertion of N-terminal (Arg) basic residues. In contrast with the latter, the AMDC phosphatase shows very poor activity toward all the phosphopeptides tested, without preference for either Ser(P) or Thr(P) peptides. However, N-terminal basic residues also favor the dephosphorylation of otherwise almost inert substrates by the AMDC phosphatase. Hence, while the dephosphorylation of Thr(P) substrates by the PCS phosphatases is highly favored by the nature of the phosphorylated amino acid, phosphatase activity toward Ser(P)-containing peptides may require specific determinants in the primary structure of the phosphorylation site.     

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.     

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.     

Identification of the ATP + Mg-dependent and polycation-stimulated protein phosphatases in the germinal vesicle of the Xenopus oocyte.

Two protein phosphatase activities were characterized in the germinal vesicle of Xenopus laevis oocytes after manual dissection of the nucleus. One enzyme can be classified as an active form of the ATP + Mg-dependent (AMD) phosphatase, the other as a polycation-stimulated (PCS) phosphatase. The activity of the PCS phosphatase is localized exclusively in the soluble compartment of the nucleus (nucleoplasm). The catalytic subunit of the AMD phosphatase activity is associated either with the nuclear particulate fraction or with an inhibitory subunit in the nucleoplasm.     

Purification and properties of polycation-stimulated phosphorylase phosphatases from rabbit skeletal muscle

Four types of polycation-stimulated (PCS) phosphorylase phosphatases have been isolated from rabbit skeletal muscle. They are called PCSH (390 kDa), PCSM (250 kDa), and PCSL (200 kDa) phosphatase according to the apparent molecular weight of the native enzymes in gel filtration. Two forms of PCSH phosphatase could be separated by Mono Q fast protein liquid chromatography: PCSH1 and PCSH2. In the absence of polycations, the specific activities of the PCSH1, PCSH2, PCSM, and PCSL phosphatase were 400, 680, 600, and 3000 units/mg, respectively, using phosphorylase a as a substrate. They all contain a 62-65- and a 35-kDa subunit, the latter being the catalytic subunit. In addition PCSH1 phosphatase contains a 55-kDa subunit and the PCSM phosphatase a 72-75-kDa subunit in a substoichiometric ratio. All the PCS phosphatases are insensitive to Ca2+ calmodulin, inhibitor-1, and modulator protein. They display a high specificity for the alpha-subunit of phosphorylase kinase and a broad substrate specificity. The PCSH1 and PCSH2 phosphatases, but not the catalytic subunit (PCSC phosphatase), show a high degree of specificity for the deinhibitor protein. During the purification the phosphorylase to inhibitor-1 phosphatase activity ratio (10:1) remained constant for the PCSH and PCSL enzymes but decreased for the PCSM phosphatase. The stimulation observed with low concentrations of polycations is enzyme directed. The different enzyme forms show a characteristic concentration optimum and degree of stimulation. At higher concentrations, polycations become inhibitory and a time-dependent deactivation of the phosphatases is observed.     

Purification and characterization of three distinct types of protein phosphatase catalytic subunits in bovine platelets.

The catalytic subunits of bovine platelet protein phosphatases were separated into three distinct forms by chromatography on heparin-Sepharose. Each phosphatase was further purified to apparent homogeneity as judged in sodium dodecyl sulfate-polyacrylamide gel yielding single protein bands of 37, 41, and 36 kDa. The 37-kDa phosphatase was excluded from heparin-Sepharose and preferentially dephosphorylated the alpha-subunit of phosphorylase kinase. It was stimulated by polycations (polybrene or histone H1) and was inhibited by okadaic acid (IC50 = 0.3 nM), but its activity was not influenced by inhibitor-2 or heparin. The 41-kDa phosphatase was eluted from heparin-Sepharose by 0.20-0.25 M NaCl and preferentially dephosphorylated the beta-subunit of phosphorylase kinase. It was stimulated by polycations and inhibited by okadaic acid (IC50 = 2 nM), but its activity was not affected by inhibitor-2 or heparin. The 36-kDa phosphatase was eluted from heparin-Sepharose by 0.45-0.50 M NaCl and preferentially dephosphorylated the beta-subunit of phosphorylase kinase. It was inhibited by inhibitor-2, heparin, histone H1, and okadaic acid (IC50 = 70 nM). The 37- and 36-kDa phosphatases can be classified as type-2A and type-1 enzymes, respectively. The 41-kDa phosphatase does not precisely fit the criteria of either type, showing only partial similarities to both type-1 and type-2A enzymes and it may represent a novel type of protein phosphatase in bovine platelets.     

Three different genes in S. cerevisiae encode the catalytic subunits of the cAMP-dependent protein kinase

We have isolated three genes (TPK1, TPK2, and TPK3) from the yeast S. cerevisiae that encode the catalytic subunits of the cAMP-dependent protein kinase. Gene disruption experiments demonstrated that no two of the three genes are essential by themselves but at least one TPK gene is required for a cell to grow normally. Comparison of the predicted amino acid sequences of the TPK genes indicates conserved and variable domains. The carboxy-terminal 320 amino acid residues have more than 75% homology to each other and more than 50% homology to the bovine catalytic subunit. The amino-terminal regions show no homology to each other and are heterogeneous in length. The TPK1 gene carried on a multicopy plasmid can suppress both a temperature-sensitive ras2 gene and adenylate cyclase gene.     

The catalytic mechanism of protein tyrosine phosphatases revisited.

Experimental and theoretical studies of the catalytic mechanism in protein tyrosine phosphatases and dual specific phosphatases are reviewed. The structural properties of these enzymes contributing to the efficient rate enhancement of phosphate monoester hydrolysis have been established during the last decade. There are, however, uncertainties in the interpretation of available experimental data that make the commonly assumed reaction mechanism somewhat doubtful. Theoretical calculations as well as analysis of crystal structures point towards an alternative interpretation of the ionisation state in the reactive complex.     

Modulation of catalytic activity in multi-domain protein tyrosine phosphatases

Signaling mechanisms involving protein tyrosine phosphatases govern several cellular and developmental processes. These enzymes are regulated by several mechanisms which include variation in the catalytic turnover rate based on redox stimuli, subcellular localization or protein-protein interactions. In the case of Receptor Protein Tyrosine Phosphatases (RPTPs) containing two PTP domains, phosphatase activity is localized in their membrane-proximal (D1) domains, while the membrane-distal (D2) domain is believed to play a modulatory role. Here we report our analysis of the influence of the D2 domain on the catalytic activity and substrate specificity of the D1 domain using two Drosophila melanogaster RPTPs as a model system. Biochemical studies reveal contrasting roles for the D2 domain of Drosophila Leukocyte antigen Related (DLAR) and Protein Tyrosine Phosphatase on Drosophila chromosome band 99A (PTP99A). While D2 lowers the catalytic activity of the D1 domain in DLAR, the D2 domain of PTP99A leads to an increase in the catalytic activity of its D1 domain. Substrate specificity, on the other hand, is cumulative, whereby the individual specificities of the D1 and D2 domains contribute to the substrate specificity of these two-domain enzymes. Molecular dynamics simulations on structural models of DLAR and PTP99A reveal a conformational rationale for the experimental observations. These studies reveal that concerted structural changes mediate inter-domain communication resulting in either inhibitory or activating effects of the membrane distal PTP domain on the catalytic activity of the membrane proximal PTP domain.     

Isolation of the catalytic subunits of cyclic AMP-dependent protein kinases from different mammalian tissues on the basis of charge differences of their subunits.

Both, the experimental data and a literature survey presented reveal common charge differences in the subunit composition of cAMP-dependent protein kinases from a variety of mammalian tissues. In general, the holoenzymes (type I and II) focus at pH 4.5-5.5, the cAMP-binding regulatory subunit below pH 4, whereas catalytic subunits are found at pH 6.7-9.1. For the purification of the catalytic subunits, the anionic holoenzymes of a variety of rat and rabbit organs as well as mouse and human-derived tissue culture cells were adsorbed to DEAE-cellulose. The catalytic subunit from the particular isoenzyme were selectively eluted at an appropriate ionic strength depending on the isoenzyme elution pattern by small amounts of cAMP. Extraction of tissues with Triton X-100 increased enzyme yield. Improved elution of the enzyme was accomplished by 8-(4-aminobutyl)amino-cAMP instead of cyclic AMP. Carboxymethyl-cellulose chromatography leads to both, concentration and, if necessary, to further purification of the enzyme.     

Characterization of major protein phosphatases from selected species of Kluyveromyces. Comparison with protein phosphatases from Yarrowia lipolytica.

Casein phosphatase activities have been identified in five yeast strains grown on Pi-deficient medium. Maximal endocellular activities appeared in the exponential phase. Exocellular phosphatases were significantly produced from Yarrowia lipolytica W-29 and Kluyveromyces marxianus, in the early stationary phase. Major phosphatases from K. marxianus were one heavy acid phosphatase composed of 64-67 kDa subunits, which could be secreted in the medium, and one type 2A protein phosphatase with an apparent molecular mass of 147 kDa and a 52 kDa catalytic subunit dissociated by 80% ethanol treatment. The characteristics of phosphatases purified from K. marxianus were compared with those previously purified from Y. lipolytica.     

Characterization of the major phosphofructokinase-dephosphorylating protein phosphatases from Ascaris suum muscle.

In contrast to the mammalian enzyme, PFK from the nematode Ascaris suum is activated following phosphorylation (Daum et al. (1986) Biochem. Biophys. Res. Commun. 139, 215-221) catalyzed by a cAMP-dependent protein kinase (Thalhofer et al. (1988) J. Biol. Chem. 263, 952-957). In the present report, we describe the characterization of the major PFK dephosphorylating phosphatases from Ascaris muscle. Two of these phosphatases exhibit apparent M(r) values of 174,000 and 126,000, respectively, and are dissociated to active 33 kDa proteins by ethanol precipitation. Denaturing electrophoresis of each of the enzyme preparations showed two bands of M(r) 33,000 and 63,000. The enzymes are classified as type 2A phosphatases according to their inhibition by subnanomolar concentrations of okadaic acid, the lack of inhibition by heat-stable phosphatase inhibitors 1 and 2, and their preference for the alpha- rather than for the beta-subunit of phosphorylase kinase. Like other type 2A phosphatases, they exhibit broad substrate specificities, are activated by divalent cations and polycations, and inhibited by fluoride, inorganic phosphate and adenine nucleotides. In addition, we have found that PFK is also dephosphorylated by an unusual protein phosphatase. This exhibits kinetic properties similar to type 2A protein phosphatases, but has a distinctly lower sensitivity towards inhibition by okadaic acid (IC50 approx. 20 nM). Partial purification of the enzyme provided evidence that it is composed of a 30 kDa catalytic subunit and probably two other subunits (molecular masses 66 and 72 kDa). The dephosphorylation of PFK by protein phosphatases is strongly inhibited by heparin. This effect, however, is substrate-specific and does not occur with Ascaris phosphorylase a.     

Dephosphorylation of phosphoproteins and synthetic phosphopeptides. Study of the specificity of the polycation-stimulated and MgATP-dependent phosphorylase phosphatases

The substrate specificity of different forms of polycation-stimulated (PCSH, PCSL, and PCSC) phosphorylase phosphatases and of the catalytic subunit of the MgATP-dependent protein phosphatase from rabbit skeletal muscle was investigated. This was done, with phosphorylase a as the reference substrate, using the synthetic phosphopeptides patterned after the phosphorylated sites of pyruvate kinase (type L) (Arg2-Ala-Ser(32P)-Val-Ala (S2), and its Thr(32P) substitute (T4)), inhibitor-1 (Arg4-Pro-Thr(32P)-Pro-Ala (T5), Arg2-Pro-Thr(32P)-Pro-Ala (T1), and its Ser(32P) substitute (S1)), and some modified phosphopeptides (Arg2-Ala-Thr(32P)-Pro-Ala (T2) and Arg2-Pro-Thr(32P)-Val-Ala (T3)), all phosphorylated by cyclic AMP-dependent protein kinase. In addition, casein(Thr-32P), phosphorylated by casein kinase-2, was also tested. The PCS phosphatases show a striking preference for the T4 configuration, PCSC being the least efficient. The catalytic subunit of the MgATP-dependent phosphatase was almost completely inactive toward all these substrates. As shown for the PCSH phosphatase, and comparing with T4, the two proline residues flanking the Thr(P) in T1 and T5, just as in inhibitor-1, drastically imparied the dephosphorylation by lowering the Vmax and not by affecting the apparent Km. The C-terminal proline (as in T2) by itself represents a highly unfavorable factor in the dephosphorylation. The critical effect of the sequence X-Thr(P)-Pro or Pro-Thr(P)-Pro (T1, T2, T5, and inhibitor-1) can be overcome by manganese ions. The additional finding that this is not the case with the Pro-Ser(P)-Pro sequence (S1) suggests that the effect of Mn2+ is highly substrate specific. These observations show the considerable importance of the primary structure of the substrate in determining the specificity of the protein phosphatases.     

Preparation of catalytic subunits of cAMP-dependent protein kinase from cattle myocardium

A method is suggested for obtaining a catalytic subunit of cAMP-dependent protein kinase from the cattle myocardium. The specific activity of protein kinase is 0.5 mumol 32P per 1 mg of the enzyme. The method is based on the difference of protein kinase in the subunit and choloenzyme charges, it embraces the stages of homogenization, ultracentrifugation and biospecific elution on anion exchanger of DEAE-Sepharose-CL-6B using 10(-4) M cAMP in the stationary variant.     

Developmental expression patterns of protein kinase A catalytic subunits in zebrafish

Protein kinase A (PKA), also known as cAMP dependent protein kinase, is an essential component of many signaling pathways, many of which regulate key developmental processes. Inactive PKA is a tetrameric holoenzyme, comprised of two catalytic (PRKAC), and two regulatory subunits. Upon cAMP binding, the catalytic subunits are released and thereby activated. There are multiple isoforms of PKA catalytic subunits, but their individual roles are not well understood. In order to begin studying their roles in zebrafish development, it is first necessary to identify the spatial and temporal expression profiles for each prkac subunit. Here we evaluate the expression profiles for the four zebrafish prkacs: prkacαa, αb, βa, and βb, at key developmental time points: 24, 48 and 72 h post fertilization. We show that zebrafish prkacs are expressed throughout the developing nervous system, each showing unique expression patterns. This body of work will inform future functional studies into the roles of PKA during development.     

The complement of protein phosphatase catalytic subunits encoded in the genome of Arabidopsis

Reversible protein phosphorylation is critically important in the modulation of a wide variety of cellular functions. Several families of protein phosphatases remove phosphate groups placed on key cellular proteins by protein kinases. The complete genomic sequence of the model plant Arabidopsis permits a comprehensive survey of the phosphatases encoded by this organism. Several errors in the sequencing project gene models were found via analysis of predicted phosphatase coding sequences. Structural sequence probes from aligned and unaligned sequence models, and all-against-all BLAST searches, were used to identify 112 phosphatase catalytic subunit sequences, distributed among the serine (Ser)/threonine (Thr) phosphatases (STs) of the protein phosphatase P (PPP) family, STs of the protein phosphatase M (PPM) family (protein phosphatases 2C [PP2Cs] subfamily), protein tyrosine (Tyr) phosphatases (PTPs), low-M(r) protein Tyr phosphatases, and dual-specificity (Tyr and Ser/Thr) phosphatases (DSPs). The Arabidopsis genome contains an abundance of PP2Cs (69) and a dearth of PTPs (one). Eight sequences were identified as new protein phosphatase candidates: five dual-specificity phosphatases and three PP2Cs. We used phylogenetic analyses to infer clustering patterns reflecting sequence similarity and evolutionary ancestry. These clusters, particularly for the largely unexplored PP2C set, will be a rich source of material for plant biologists, allowing the systematic sampling of protein function by genetic and biochemical means.     

Specific binding of protein kinase CK2 catalytic subunits to tubulin

Protein kinase CK2 is composed of two regulatory beta-subunits and two catalytic alpha- or alpha'-subunits. To analyse these subunits individually we generated antibodies against unique peptides derived from the alpha-, alpha'- and beta-subunit. Immunofluorescence studies with these antibodies revealed the presence of all three CK2 subunits in the cytoplasm and weakly in the nucleus with strong signals around the nuclear membrane. Double staining experiments revealed a co-localisation of all three subunits with tubulin. A direct association between the CK2 alpha- and the alpha'-subunit and tubulin was confirmed by co-immunoprecipitation experiments as well as by Far Western analysis. There was no binding of the CK2 beta-subunit to tubulin. Thus, with tubulin we have identified a new binding partner specific for the catalytic subunits of CK2.     

Receptor-like protein tyrosine phosphatases: alike and yet so different

Reversible phosphorylation on tyrosine residues is an extremely rapid and powerful posttranslational modification that is used in signalling pathways for the regulation of cell growth and differentiation. Over the past several years an impressive number of receptor-like protein tyrosine phosphatase (RPTPase) family members have been identified by molecular cloning, and undoubtedly many more will follow. This review provides an overview of the molecular data that are available for the currently identified RPTPases and discusses their possible biological implications. This revised version was published online in June 2006 with corrections to the Cover Date.     

Discrimination between the activity of protein kinase CK2 holoenzyme and its catalytic subunits

The acronym CK2 denotes a highly pleiotropic Ser/Thr protein kinase whose over-expression correlates with neoplastic growth. A vexed question about the enigmatic regulation of CK2 concerns the actual existence in living cells of the catalytic (alpha and/or alpha') and regulatory beta-subunits of CK2 not assembled into the regular heterotetrameric holoenzyme. Here we take advantage of novel reagents, namely a peptide substrate and an inhibitor which discriminate between the holoenzyme and the catalytic subunits, to show that CK2 activity in CHO cells is entirely accounted for by the holoenzyme. Transfection with individual subunits moreover does not give rise to holoenzyme formation unless the catalytic and regulatory subunits are co-transfected together, arguing against the existence of free subunits in CHO cells.