protein phosphatase protocols

1 Prokaryotic Protein-SerineiThreonine Phosphatases Peter J. Kennelly 1. Introduction 1.1. Prokaryotic Protein-Serine/Threonine Phosphatases: A Brief Review 1.1.1. Why Study Protein Phosphorylation Events in Prokaryotes? As this chapter deals with the protein-serine/threonine phosphatases of prokaryotic organisms, some comments on the role of prokaryotes in the study of these important enzymes would appear to be in order. Prokaryottc organ- isms dominate the living world. They represent by the largest source of biomass on the planet, forming the indtspensable foundation of the food cham upon which all other living organisms depend. They are the exclusive agents for carrying out biological nitrogen fixation, and are responsible for the majority of the photosynthetic activtty that generates the oxygen we breath. In absolute numbers, in number of species, in range of habitat, and in the spectrum of their metabolic activities, the prokaryotes far outpace their eukaryotic brethren. More immediately, in humans prokaryotes perform essential functions in the digestion and asstmilation of nutrients, whereas infection by bacterial patho- gens can lead to illness or death. The intrinsic biological importance of prokaryotic organisms m the bio- sphere renders them important and interesting objects of study (1). Be that as it may, the question remams as to why protein phosphorylation in prokaryotes should be of interest to “mainstream” signal transduction researchers whose attention has long been fixed on humans and other higher eukaryotes. At least part of the answer lies in the recent realization that prokaryotes and eukaryotes employ many of the same molecular themes for the construction and operatton of their protein phosphorylation networks (2,3). Virtually every major family From Methods IR Molecular Biology, Vol 93 Protern Phosphatase Protocols Edlted by J W Ludlow 0 Humana Press Inc , Totowa, NJ 1 2 Kennel/y of protem kinases or protein phosphatases identified in eukaryotlc organisms possesses a prokaryotic homolog(s), and vice versa. Consequently, the prokary- otes represent a volummous library of fundamentally important, universally applicable information concerning the structure, function, origins, and evolu- tion of protein kmases, protein phosphatases, and their target phosphoprotems. In addition, prokaryotes offer significant advantages as venues for the study of protem kmases and protein phosphatases, particularly with regard to dlssectmg thetr physiological functions and the factors that Influence them. Prokaryotes carry out their life functions and the regulation thereof utlhzmg a many-fold smaller suite of genes and gene products than does the typical eukaryote. Although they employ molecular mechanisms as subtle and sophlstlcated as any found in “higher” orgamsms, the fewer “moving parts” in the prokaryotes materially faclhtates the design, execution, and analysis of molecular genetic experiments. In addition, their robustness m the face of a wide range of nutn- tional and environmental challenges greatly facilitates the ldentlficatlon and analysis of resulting phenotypes. The prokaryotes thus represent a rich and presently underutilized tool for understanding the fundamental prmclples gov- erning the form and function of protein phosphorylatlon networks. 1.1.2. Not All Prokaryotes Are Created Equal: A Brief Outline of Phylogeny Most readers of this chapter were taught that all living organisms could be grouped mto two phylogenetlc domains whose names were often given as the eukaryotes and the prokaryotes (4), However, these latter terms actually refer to a morphological classlficatlon, not a genetic/hereditary one (5). The term eukaryote describes those organisms whose cells manifest internal compart- mentation, more precisely the presence of a nuclear membrane. The prokary- otes include all organisms lacking such mtracellular orgamzatlon, m other words everythmg that 1s not a eukaryote. Early studies of phylogeny based on the first protein sequences, the gross structural and functional characteristics of key macromolecules, the architecture of common metabolic pathways, and so forth, suggested that this morphological classificatron of hvmg organisms paralleled their hereditary relationships. However, as researchers gamed facll- ity with the isolation, sequencmg, and analysis of DNA, a truly genetic out- line of phylogeny has emerged, one that groups living organisms mto three distinct phylogenetlc domains-the Eucarya, Bacteria, and Archaea (Archae- bacteria) (6). Whereas the prior supposition that the eukaryote morphological phenotype characterized members of a coherent phylogenetic domain-the Eucarya- proved correct, molecular genetic analysis revealed that the prokaryotes segre- gated into two distinct and very different domains: the Bacteria and the Prokaryotlc Phosphatases 3 Archaea. The domain Bacteria includes essentially all of the prokaryotrc organisms one encounters m a typical mrcrobrology course E. co& Salmo- nella, Pseudomonas, Rhizobium, Clostridia, Staphylococcus, Bacillus, Ana- baena, and so on. The domain Archaea, on the other hand, IS populated largely by extremophrles that occupy habitats whose heat, acidity, salmtty, or oxygen tension render them hostile, rf not deadly to other hvmg orgamsms. However, rt would be wrong to suppose that the Archaea are simply a set of unusual bacte- ria. Examination of the genes encoding their most fundamentally important macromolecules, ranging from DNA polymerase to ribosomal RNAs, make it clear that the Archaea have much more in common with the Eucarya than they do with the superficially-similar Bacteria (6,7). The earliest detectable branch point in the evolutionary time line resulted m the segregation of the Bacterza away from the organism that eventually gave rise to both the Eucarya and the Archaea. The common progemtor of these latter domains then evolved for a con- siderable period followmg this first btfurcation. As a consequence, many mvesti- gators believe that present day archaeons still possess numerous features reflective of ancient proto-eukaryotes (7). This combmatron of prokaryotrc “srmplrcny” with high relatedness to medically relevant eukaryotes render the Archaea a par- ticularly mtngumg target for the study of protein phosphorylatton phenomena. 1. I. 3. Prokaryotic Protein-Serine/Threonine Phosphatases ldentlfied to Date When one considers that the modification of prokaryotrc proteins by phos- phorylation-dephosphorylatron first was reported nearly 20 yr ago (a-lo), surprisingly little is known about the enzymes responsible for the hydrolyses of phosphoserine and phosphothreonme residues m these organisms. The first prokaryotrc protein-serine/threonme phosphatase to be rdentrfied and charac- terized was the product of the aceK gene in E coli (II). This gene encodes a polypeptide that contains both the protem kmase and protein phosphatase activities responsible for the phosphorylation-dephosphorylation of isocitrate dehydrogenase. Today, AceK remains an anachronism by virtue of its hermaph- roditic structure, and because the sequences of its protein kinase and protein phosphatase domains are unique, exhibiting no srgmticant resemblance to other protein kinases or protein phosphatases (12). The next prokaryote-associated protein-serine/threonine phosphatase to be discovered was ORF 221 encoded by bacteriophage h (13,14). This enzyme, and a potential protein encoded by an open reading frame m bacteriophage $80, exhibit significant sequence homology with the members of the PP1/2A/2B superfamily, one of the two major families of eukaryotic protem-serme/threo- nme phosphatases (15). Whereas this represented the first discovery of a eukaryote-like protein phosphorylatron network component having any asso- 4 Kennel/y ciation with a prokaryotic organism, the mobility and malleability of viral vec- tors begged the question of whether the genes for these protein phosphatases were bacterial in origin. Moreover, it remains unclear to what degree a protein phosphatase from a pathogen can shed light on how bacterial proteins are dephosphorylated under normal physiological cncumstances. More recently, two unambiguously bacterial enzymes have been described that possess protein-serine/threonine phosphatase activity. The first, IphP from the cyanobactermm Nostoc commune (16), is a dual-specificity protein phos- phatase that acts on phosphoseryl, phosphothreonyl, and phosphotyrosyl pro- teins in vitro (17). Like other dual-specific protein phosphatases, IphP contams the characteristic HAT (His-Cys-Xaa@g, or His-kg-Thiolate) active site signature motif characteristic of protein phosphatases capable of hydrolyzing phosphotyrosine (18). The second 1s SpoIIE from BaczlZus subtilu, a bacterial homolog of the second major family of “eukaryotic” protein-serme/threomne phosphatases, the PP2C family (19,20). “Eukaryotic” protein-serine/threonine phosphatases have been uncovered m the Archaea as well. In the author’s laboratory a protein-serine/threonine phos- phatase, PPl -arch, has been purified, characterized, cloned, and expressed from the extreme acidothermophilic archaeon Sulfolobus solfataricus (21,22). This protein 1s a member of the PP1/2A/2B superfamily, with whose eukaryotic members it shares nearly 30% sequence identity (22). Surveys of two other archaeons, which are phylogenetically and phenotypically distinct from S. solfutaricus, the halophile Haloferax volcanii and the methanogen Methano- sarcina thermophda TM- 1, indicate that PP 1 -arch from S. solfataricus 1s the first representative of what may prove to be a widely distributed family of archaeal protein-serine/threonine phosphatases (23,24). This recently has been confirmed at the sequence level through the cloning of a second form of PPl- arch from A4. thermophilu via the polymerase cham reaction (PCR). 1.1.4. Limited Applicability of Cohen’s Scheme to the Classification Prokatyotic Protein-Serine/Threonine Phosphatases Recent experience with prokaryotic protein phosphatases has revealed that Cohen’s criteria for classifying the protem-serme/threonine phosphatases can- not be extrapolated with confidence to prokaryotic enzymes. To briefly review, in the early 198Os, Cohen and coworkers compiled a set of functional attributes characteristic of each of the major protein-serme/threonine phosphatases found in eukaryotes (25). These attributes mcluded their preference for dephosphory- lating the a- vs the P-subunit of phosphorylase kinase, their sensitivity to the heat-stable inhibitor proteins I-l and I-2, and the (m)dependence of their cata- lytic activity on the presence of divalent metal ions such as Mg2+, Mn2+, or Ca2+. In later years sensmvity to potent microbial toxins-such as microcystm- Prokaryotic Phosphatases 5 LR, okadaic acid, and tautomycin-that inhibited the activity of PPl and PP2A were added to the list (26). While this scheme soon was adopted as standard for the classification of eukaryotic protein-serine/threomne phosphatases, attempts to apply it to prokaryotic enzymes have met with mixed success. For example, PPl-arch from S. solfataricus is okadaic acid-insensitive and requires exog- enous divalent metal ions for activity (21). Under Cohen’s scheme, this would classify it as a member of the PP2C family. However, the amino acid sequence of PP 1 -arch clearly places it in the PP 1/2A/2B superfamily (22). The same holds true for another divalent metal ion-dependent, okadaic acid-insensitive PP 1/2A homolog, ORF 22 1 from bacteriophage k (14). 7.2. An Overview of Methods for Assaying, Purifying, and Identifying Clones of a Prokaryotic Protein-Serine/Threonine Phosphatase, PPI-Arch We use [32P]phosphocasein that has been phosphorylated using the catalytic subunit of the CAMP-dependent protein kinase (27) as a general-purpose sub- strate for the assay of protein-serine/threonine phosphatase activity in pro- karyotic organisms. Although it is a eukaryotic phosphoprotein, all of the prokaryotic protein-serine/threonine phosphatases that have been studied (16,17,21-24), as well as the ORF 221 protein-serine/threomne phosphatase from bacteriophage h (14), hydrolyze phosphocasem at a usefully high rate in vitro. Its major virtue resides m the fact that it is readily prepared in quantity by procedures that are simple and economrcal with regard to both effort and expense. The major drawback of phosphocasein is the very high quantity of unlabeled phosphate that is already bound to it, which renders it unsuitable for determining kinetic parameters. However, for routine applications-those requiring knowledge of the relative protein phosphatase activity present in a sample such as surveying cell homogenates or column fractions, screening potential activators or inhibitors, and so on-phosphocasem is entirely suitable. For the assay of PP 1 -arch, a sample of protein phosphatase is incubated with [32P]phosphocasein in the presence of a divalent metal ion cofactor and a pro- tein carrier, bovine serum albumin (BSA). Inclusion of the divalent metal ion cofactor is very important. Every PP1/2A homolog characterized to date in both the Archaea (21,23,24) and bacteriophage h (14) requires divalent metal ions for activity, as does the bacterial PP2C homolog SpoIIE (20). (Eukaryotic PPl is a metalloenzyme (28), but it normally binds divalent metal ions in a sufficiently tenacious manner to render the addition of exogenous cofactors unnecessary.) In the author’s experience, Mn2’ has proven the most efficacious and general cofactor. However, activation by Co*+, N?+, or Mg2+ has been observed on occasion (21,23,24). The assay is terminated by adding trichloro- acetic acid (TCA) and centrifuging. With the assistance of the BSA carrier, the 6 Kennel/y TCA quantitatively precipitates the unreacted [32P]phosphocasem whereas the inorganic [32P]phosphate that was released by the action of the protein phos- phatase remains in the supernatant ltquid. An aliquot of the supernatant is then removed and analyzed for 32P content by liquid scintillation counting. (Meth- ods for verifying that the radioactivity detected is derived from morgamc phos- phate and not small, TCA-soluble phosphopeptides produced by the action of proteolytic enzymes can be found in ref. 21) Purification of PP 1 -arch from S solfaturzcus is a relatively straightforward process mvolvmg ion-exchange chromatography, gel filtration chromatography, and absorption onto and elution from hydroxylapatite. As with many prokary- otic organisms, breaking the cells themselves is a much more arduous task than is typical for most animal cells. In the case of S. solfaturicus, repeated sonica- tion is sufficient, but other organisms may require repeated passage through a French Press or similarly severe methods. Advantage is taken of the fact that S. solfataricus releases a soluble, pea-green pigment upon cell rupture By moni- toring the release of pigment at 400 nm after each somcation cycle, the pomt at which the majority of the cells have been broken open can readily be determmed. The PPl-arch obtamed by the procedure described herein 1s x1000-fold purified over the Soluble Extract. Although this preparation falls somewhat short of absolute homogeneity, the major protein species is PPl-arch, which constitutes 40-70% of the total protem present. The unambiguous identifica- tion of the PP 1 -arch polypeptide, and subsequent determination of its relative abundance, was accomplished by assaymg its catalytic activity m gel slices following polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE) (22). The key to recovering at least a portion of the PPl-arch m an active state followmg electrophoresis is the selection of a much lower temperature, 65°C vs the usual lOO”C, for incubatmg of the pro- tein with SDS Sample Buffer. In addition to identifying and characterizing archaeal protein phosphatases by classic purification, sequencing, and cloning techniques (22) the gene encoding a second member of the PPl-arch family from Ad, thermophdu has been identified using PCR. This was accomplished using primers modeled after regions of PPl-arch from S. solfatarzcus, that are highly conserved with homologous eukaryotic protein phosphatases (see Fig. 1). Included m these primers are 5’ extensions containing nucleotide sequences suitable for anneal- ing the ends of the primers to sites cut by the endonucleases EcoRI or BamHI. This permits the direct cloning of the PCR product(s) into a variety of plasmid vectors. The selectivity of PCR amplification is enhanced by usmg the “touch- down” method (29). The touchdown method is essentially a PCR titration m which the annealing temperature is lowered by one degree every few, usually three, cycles. Under these conditions, the region of DNA that most tightly binds Prokaryo t/c Phospha tases 7 PPl s. solf. QDYVDREPQTGVENLSLIL-KKLIESDENKGKTKIVVLRGNRE PPl rabbit GDYVDRGKQS-LETICLLLAYKI-KYPEN FFLLRONRE PPZA yeast GDYVDRGYYS-VETVSYLVAMKV-RYPHR ITILRGNHE PPZB rat GDYVDRGYFS-IECVLYLWALKIL-YPKT LFLLRGNHE Pruner 1 5’$+ZAAlTCCGGNGA(T/C)TA(T/C)GTNGA(T/C)(A/C)G 3’ EcoRI Prmr 2 5’CGGGATCCG(T/C)TC(A/G)TG(A/G)TTNCCNG(T/G)NA 3’ Fig. 1. Design of degenerate oligonucleotide primers cloning of PP 1 -arch homologs by PCR At top 1s shown the sequence of ammo acids 63-l 04 of PP l-arch from S solfatarzcus aligned with the correspondmg regions of a rabbit PPl, a yeast PP2A, and a rat PP2B (Adapted from ref. 22). The areas of lughly conserved ammo acid sequence used to design the primers are designated with bold lettering. The conserved GDYVDR sequence was used to design pruner 1 and the conserved LRGNHE! sequence was used to design primer 2. Below these protein sequences are gtven the nucleotide sequences of each primer. The underlined portions represent the extensrons added to enable primer 1 to anneal to restnc- tion sites for EcoRI and primer 2 to anneal to restriction sites for BamHI Positions where two bases are enclosed in parentheses indicate that both of the indicated nucleotlde bases were incorporated at that posrtion in the oltgonucleotide sequence, whereas N indicates positions where all four possible nucleotide bases were included. the primers 1s amplified first, and, therefore, constitutes the predommant end product because rt is amplified through several-fold more cycles than the next best match. If three cycles are performed at each temperature and two sequences differ by 2OC in annealing temperature, the higher annealing prod- uct will be amplified (2)6-, or 64-, fold more than the lower annealing product. By scanning through a range of temperatures, the experimentalist gains the selectivity of using the hrghest possible annealing temperature without the risk of overshootmg it completely. It should be noted, however, that PCR is not a panacea. Despite biochemical evidence for the existence of a PPl-arch homolog in the archaeon H volcanii (23), PCR reactions have failed to yield an oligonucleotrde product derived from its gene. 2. Materials 2.1. Assay of PPl-Arch 2.1.1. Preparation of f2P]Phosphoseryl Casein 1. Catalytic subunit of CAMP-dependent protein kinase: 1000 U, from Sigma (St. Louis, MO, cat no P 2645). 8 Kennel/y 2 Casein solution. Autoclaved, hydrolyzed, and partially dephosphorylated casem (5% w/v) from bovine milk from Sigma (cat no C 4765) 3. ATP, 10 mM, pH 7 5 4. [y-32P]ATP, 0 8 mC1 (see Note 1). 5 Buffer A: 50 n&f Tris-HCl, pH 7.0, 1 mA4 dithiothreltol (DTT), 0.1 r&Y EGTA (see Note 2). 6. Buffer B. 60 mA4 magnesium acetate in Buffer A 7. Buffer C: 5% (v/v) glycerol m Buffer A. 8 Stop solution: 100 mA4 sodium pyrophosphate, pH 7 0, 100 WEDTA. 9. A 1.0 x 17 cm column of Sephadex G-25 fine (Pharmacla, Uppsala, Sweden) equilibrated in Buffer C (see Note 3) 2.1.2. Assay of Phosphocasein Phosphatase Activity in Soluble Samples of Protein Phosphatase 1. Buffer D: 50 mMMES, pH 6.5 2. Buffer E: 120 mMMnC12 in Buffer D. 3. Buffer F: 2 mg/mL BSA m Buffer D. 4 TCA, 20% (w/v) 2.1.3. Assay of Phosphocasein Phosphatase Activity in Slices from SDS- Polyacrylamide Gels 1 SDS Sample Buffer 5% (w/v) SDS, 40% (v/v) glycerol, 0 1% (w/v) bromo- phenol blue. 2 Buffer D. 50 mA4 MES, pH 6.5. 3 Buffer G* 0.5 mA4 EDTA in Buffer D. 4 Buffer H: 100 mMMES, pH 6.5,0.66 mg/mL BSA, 40 mMMnC1,. 5. Buffer I: 100 mA4 MES, pH 6 5,0.66 mg/mL BSA, 10 mM EDTA. 2.2. Purification of PP7-Arch from Sulfolobus Solfataricus 1. Buffer J. 20 mMMES, pH 6.5,lOO mA4NaC1,l WEDTA, 1 WEGTA, 1 mA4 DTT, 5 pg/rnL leupeptm, 5 pg/mL soybean trypsin inhibitor, 0 5 mM pheny- lmethylsulfonyl fluoride (PMSF), 0.5 mA4 tosyllysyl chloromethylketone (TLCK), 0.5 mM tosylphenylalanyl chloromethylketone (TPCK) (see Note 4) 2. Buffer K: 10 mMMES, pH 6.5,O 5 mMEDTA, 0 5 pg/mL leupeptin, 0 2 mMPMSF 3. 150 mMNaC1 m Buffer K 4. 400 mMNaC1 m Buffer K. 5. Buffer L* 1 Wsodium phosphate, pH 6.5,0.5 mMEDTA, 0.5 pg/mL leupeptin, 0.2 mM PMSF. 6. Buffer M: 400 mM sodium phosphate, pH 6.5, 0.5 mkf EDTA, 0 5 pg/mL leupeptin, 0.2 mA4 PMSF. 7 Buffer N: 20 mM MES, pH 6 5, 10 mM NaCl, 0.5 mA4 EDTA, 0.5 pg/mL leupeptm, 0.2 mM PMSF. 8. A 10 x 4 cm column (see Note 3) of CM-Trisacryl (Sepracor, Marlborogh, MA) equilibrated in Buffer K. Prokaryotic Phosphatases 9 9 A 6 25 x 30 cm column of DE-52 cellulose (Whatman, Clifton, NJ) equrhbrated in Buffer K 10. A 2.5 x 40 cm column of DE-52 cellulose equilibrated in Buffer K. 11. A 2.5 x 12 cm column of Hydroxylapatrte HT (Bio-Rad, Richmond, CA) equili- brated m Buffer L 12. A 5.0 x 100 cm column of Sephadex G-100 fine (Pharmacra) equrhbrated m Buffer M. 13 An FPLC system (Pharmacra) equipped with a 0 5 x 7 cm column of Mono Q that has been equilibrated in Buffer K. 2.3. Cloning of Phosphatase Genes by PCR 1. The enzymes and buffers of the Perkin-Elmer Cetus GenAmpTM PCR system were used, although PCR reagents from other commerctal sources presumably can be substituted without prejudice to the ultimate results. 2 Oligonucleotide primers 1 and 2 as shown m Fig. 1. 3. Methods 3. I. Preparation of [32P]Phosphocasein 1. Combine the following in a 1.5 mL Eppendorff tube: 100 pL of 5% (w/v) casein (see Note 5), 85 & of buffer B, 10 ) tL of 10 mMATP, and z 0.8 mC!r of [y-32P]ATP. The precise volume of [Y-~~P]ATP added will depend on the concentration of the solution as supplied by the manufacturer as well as the age of the preparation, since 32P has a relatively short halfdlife of 13 d (see Note 6). Make up the total volume to 325 pL with distilled water. 2. Take a vial containing 1000 U of lyophilized catalytic subunit of the CAMP-depen- dent protein kinase. Remove the septum cap. Add 87.5 pL of Buffer A. Agitate gently by hand to dissolve the solid. Let stand for a moment to permit the liquid to drain and collect in the bottom Transfer to the Eppendorff tube from step 1. 3. Rinse residual catalytic subunit from its container by addmg another 87.5 pL of Buffer A and repeating step 2 Securely cap the Eppendorff tube and mix briefly on a Vortex mixer. 4. Incubate for 8-12 h in a 30°C water bath. 5. At the conclusion of the incubation penod, add 50 & of Stop Solution Mix briefly on a Vortex mixer You can store at -2O’C or proceed immediately with the remaining steps. 6. Remove 5 & of the incubation mixture and add to 995 & of distilled water m a 1.5 mL Eppendorff tube. Mix vigorously on a Vortex mixer. Remove three 5 pL portions of the 1:200 diluted incubation mixture, place in individual scintillation vials, then add 1 mL of a water-compatible liquid scintillation fluid such as Eco- Lume (WestChem, Irvine, CA) or Econo-Safe @PI, Mount Prospect, IL). Mea- sure the radioactivity present in a liquid scintillation counter (see Note 7) Thts information is then used to calculate the specific radioactivity of the ATP used to phosphorylate the casein (Assume the cold ATP you added completely accounts for the concentration of total ATP.) Typical specific activities range from l-3 x 10 Kennelly 1016 cpmlmole Please note that it not necessary to try and convert cpm to dpm as long as you use the same scintlllatlon counter and sclntlllatlon fluid for all mea- surements of radioactivity. Under these circumstances, efficiency 1s a constant that cancels itself m all subsequent calculations of moles of product produced, percent sub- strate turnover, and so on 7. Apply the mcubatlon mixture to a 1 0 x 17 cm column of Sephadex G-25 fine that has been equilibrated in Buffer C 8. Develop the column with Buffer C Collect 1 .O mL fractions m numbered 1 5 mL Eppendorff tubes 9 Remove 5 pL ahquots from each fraction, place each m a separate, numbered scmtlllatlon vial, add 1 .O mL of scmtlllatton fluid, and count for radloactivlty. 10 Graph the radloactlvlty present in the aliquots as a function of fraction number. Two peaks of [32P]radloactlvlty should be apparent on the chromatogram. The first peak 1s the [32P]phosphocasem (see Note 8) and the second 1s the unreacted [y-32P]ATP. 11 Store the two or three peak fractions of [32P]phosphocasem at -2O’C The con- centration of casem-bound [32P]phosphate in peak fractions generally ranges from 5-25 w Discard the remaining fractions as radioactive waste. Store the column m a shielded location until needed again (see Note 9). 3.2 Assay of PPl-Arch Activity 1. Thaw a tube of [32P]phosphocasein solution. Mix the contents using a Vortex mixer. Spin briefly m a microcentrifuge to centrifuge the contents into the bot- tom of the tube. This represents an important precaution deslgned to mmimlze the chances of inadvertently contactmg radioactive material that might otherwise be clmgmg to the bottomslde of the cap, or scattering it about the lab while open- ing the tube (see Note 10). Remove 10% of the volume of [32P]phosphocasem required to perform the planned number of assays, and place m an Eppendorff tube. Return the rest of the [32P]phosphocasem stock to the freezer 2 For each assay, combme 5 pL of Buffer E and 5 p.L of Buffer F m a 1 5 mL Eppendorff tube. 3 Add the protein phosphatase sample to be assayed, plus any additional compounds (activators, inhibitors, and so on) you might wish to test, to the Eppendorff tube The volume of the sample plus other addltlons should be 5 15 pL. Make up any unutlhzed portion of tis 15 pL volume, if necessary, with Buffer D. Control assays should substl- tute an equal volume of a suitable buffer m place of the protein phosphatase sample 4 Imtlate the assay by adding 5 pL of [32P]phosphocasem solution, mlxmg briefly on a Vortex mixer, then place in a 25’C water bath (see Notes 11 and 12) This quantity of phosphocasem solution generally yields a final concentration of casein-bound [32P]phosphate of l-4 w. 5. Terminate reaction, generally after a period of 10-90 mm, by addmg 100 pL of 20% (w/v) TCA and mlxmg briefly on a Vortex mixer 6 Pellet precipitated protein by centrifuging at 12,000g for 3 mm m a micro- centrifuge (see Note 10). [...]... Conservatton analysts and structure predictton of the protein serme I threonme phosphatases Sequence stmtlartty with dtadenosme tetraphosphatase from Escherzchza colz suggests homology to the protein phosphatases Eur J Blochem 220,225-237 16 Potts, M , Sun, H , Mockattts, K., Kennelly, P J , Reed, D., and Tonks, N K (1993) A protein- sermeftyrosine phosphatase encoded by the genome of the cyanobactermm... IphP, a cyanobactertal dual-spectfictty protem phosphatase with MAP kinase phosphatase activity Biochemzstry 35, 7566-7572 18 Guan, K and Dixon, J E (1991) Evidence for protein- tyrosine -phosphatase catalysis proceeding via a cysteme-phosphate intermediate J Blol Chem 266, 17,02617,030 19 Bork, P., Brown, N P , Hegyt, H , and Schultz, G (1996) The protein phosphatase 2C (PP2C) superfamily detection ofbacterial... Isolation and cloning of a protein- serinelthreonme phosphatase from an archaeon J Bactenol 177,2763-2769 23 Oxenrtder, K A and Kennelly, P J (1993) A protein- serine phosphatase from the halophihc archaeon Haloferax volcanic Blochem Blophys Res Commun 194, 1330-1335 24 Oxenrtder, K A,, Rasche, M E., Thorstemsson, M V., and Kennelly, P J (1993) An okadaic acid-sensrtive protein phosphatase from the archaeonMethanosarczna... 25 Ingebritsen, T S and Cohen, P (1983) The protein phosphatases involved m cellular regulation 1, Classificatton and substrate specificmes Eur J Bzochem 132,255-261 26 Cohen, P (1991) Classification of protein- serme/threomne phosphatases: Identification and quantificatton m cell extracts Meth Enzymol 201,389-398 27 McGowan, C H and Cohen, P (1988) Protein phosphatase- 2C from rabbit skeletal muscle... paper fibers 8 Calculation 1 U of protein phosphatase releases 1 nmole of phosphate from phosphorylase a per mmute at 30°C Unlts,mC _ (Sample wm - Blank cpm) x 1o x ‘, x 5 T (Total cpm - Blank cpm) Where 10 = phosphorylase concentration m mnoles/mL; T = time of the protem phosphatase assay (5 min m the example); 5 = the fold that protein phosphatase was diluted mto the phosphatase assay 3.5 Method E 3.5.1... 301,53-57 7 Schuchard, M D and Kllhlea, S D (1989)Salt stimulation of the activation of latent protein phosphatase, Fc M, by Mn2+ and Mn/trypsm Bzochem Int 18, 845-849 Protein Phosphate Assays 33 8 Cheng, Q , Ertckson, A K , Wang, Z -X , and Ktlhlea, S D (1996) Sttmulatlon of phosphorylase phosphatase activity of protein phosphatase 2A, by protamme IS ronrc strength dependent and mvolves interaction of protamme... of 10 pL of trypsin inhibitor to each tube at 10 s intervals and vortex mix 5 Remove 10 pL samples for protein phosphatase assays 6 Scale up the Mn/trypsm treatment if necessary for the contmuous spectrophotometric assay (Method E) Protein Phosphate Assays 29 3.3 Method C 3.3.1 Assay for Protein Phosphatase by Changes in Phosphorylase a Activity 1 Carry out the assays m duplicate m labeled tubes Two... one unit of protem phosphatase converts 1 nmole of phosphorylase a to b (equivalent to the release of 1 nmole of phosphate from phosphorylase a) u,mL = (Total A600 - Samples -4600) x 1o x ‘, x 5 Total A6oo T Where 10 = phosphotylase concentration in nmoles/mL; T = time of the protem phosphatase assay (5 mm m example), 5 = the fold that the protein phosphatase was diluted into the phosphatase assay 3.4... into the phosphatase assay 3.4 Method D 3.4.1 Assay for Protein Phosphatase Using 32P-Phosphorylase 1 Carry out assays m duplicate m labeled tubes Also include two tubes for total counts per min (cpm) and two tubes for blank cpm 2 Dilute the protein phosphatase into the appropriate assay buffer Killilea, Cheng, and Wang 30 3 Pipet 10 I.~L of phosphatase samples into the sample tubes Pipet 10 $ of assay... linear salt gradient conastmg of 400 mL of 150 MNaCl in Buffer K and 400 mL of 400 rnMNaC1 in Buffer K Collect fractions, 10 mL, and assay for the presence of protein and protein phosphatase actwity (see Subheading 3.2.) A single peak of protein phosphatase activity that elutes near the midpoint of the gradient should be detected Pool the most active tractions as DE-52 Fraction II (see Fig 2) 12 Dialyze . its protein kinase and protein phosphatase domains are unique, exhibiting no srgmticant resemblance to other protein kinases or protein phosphatases (12). The next prokaryote-associated protein- serine/threonine. tion of protein kmases, protein phosphatases, and their target phosphoprotems. In addition, prokaryotes offer significant advantages as venues for the study of protem kmases and protein phosphatases,. Prokaryotic Protein- SerineiThreonine Phosphatases Peter J. Kennelly 1. Introduction 1.1. Prokaryotic Protein- Serine/Threonine Phosphatases: A Brief Review 1.1.1. Why Study Protein Phosphorylation