Subunit interactions control protein phosphatase 2A. Effects of limited proteolysis, N-ethylmaleimide, and heparin on the interaction of the B subunit.

Protein phosphatase 2A consists of a heterotrimeric complex composed of a catalytic subunit (C) and two associated subunits (A and B). Limited tryptic digestion of the heterotrimeric ABC form resulted in the selective degradation of the Mr = 55,000 B subunit to a 48-kDa polypeptide. The cleavage sites were determined to be within a 3-7-kDa region of the COOH terminus. Proteolysis led to dissociation of the B subunit from the enzyme complex and correlated with an increase in cardiac myosin light chain, smooth muscle myosin light chain peptide, and Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide) phosphatase activity. Purification of the digestion products and native gel electrophoresis indicated that dissociation of the B subunit was responsible for the increase in phosphatase activity. Kinetic analyses with several substrates revealed that dissociation of the B subunit resulted in a 2-7-fold increase in Vmax and a 1.6-5 fold increase in Km. Proteolytic dissociation of the B subunit increased the sensitivity of protein phosphatase 2A to inhibition by okadaic acid. Inhibition of the trypsinized enzyme was very similar to that observed for the purified AC form of protein phosphatase 2A. Incubation of the ABC complex with N-ethylmaleimide resulted in dissociation of the C subunit and generation of an AB complex. Selective release of the C subunit indicated that the B subunit interacts directly with the A subunit and that one or more free sulfhydryls are required to maintain the heterotrimeric structure of protein phosphatase 2A. Treatment of the enzyme with heparin resulted in an increase in specific activity that was due to the release of the B subunit from the complex. These results provide evidence that the B subunit binds directly to the A subunit to modulate enzyme activity and substrate specificity and that the COOH-terminal region of this protein is important for interaction with the AC complex. Dissociation of the B subunit by polyanionic substances related to heparin may represent a mechanism for regulating the activity of this enzyme.     

Control of protein phosphatase 2A by simian virus 40 small-t antigen.

Soluble, monomeric simian virus 40 (SV40) small-t antigen (small-t) was purified from bacteria and assayed for its ability to form complexes with protein phosphatase 2A (PP2A) and to modify its catalytic activity. Different forms of purified PP2A, composed of combinations of regulatory subunits (A and B) with a common catalytic subunit (C), were used. The forms used included free A and C subunits and AC and ABC complexes. Small-t associated with both the free A subunit and the AC form of PP2A, resulting in a shift in mobility during nondenaturing polyacrylamide gel electrophoresis. Small-t did not interact with the free C subunit or the ABC form. These data demonstrate that the primary interaction is between small-t and the A subunit and that the B subunit of PP2A blocks interaction of small-t with the AC form. The effect of small-t on phosphatase activity was determined by using several exogenous substrates, including myosin light chains phosphorylated by myosin light-chain kinase, myelin basic protein phosphorylated by microtubule-associated protein 2 kinase/ERK1, and histone H1 phosphorylated by protein kinase C. With the exception of histone H1, small-t inhibited the dephosphorylation of these substrates by the AC complex. With histone H1, a small stimulation of dephosphorylation by AC was observed. Small-t had no effect on the activities of free C or the ABC complex. A maximum of 50 to 75% inhibition was obtained, with half-maximal inhibition occurring at 10 to 20 nM small-t. The specific activity of the small-t/AC complex was similar to that of the ABC form of PP2A with myosin light chains or histone H1 as the substrate. These results suggested that small-t and the B subunit have similar qualitative and quantitative effects on PP2A enzyme activity. These data show that SV40 small-antigen binds to purified PP2A in vitro, through interaction with the A subunit, and that this interaction inhibits enzyme activity.     

Parallel purification of three catalytic subunits of the protein serine/threonine phosphatase 2A family (PP2A(C), PP4(C), and PP6(C)) and analysis of the interaction of PP2A(C) with alpha4 protein

The protein serine/threonine phosphatase (PP) type 2A family consists of three members: PP2A, PP4, and PP6. Specific rabbit and sheep antibodies corresponding to each catalytic subunit, as well as a rabbit antibody recognizing all three subunits, were utilized to examine the expression of these enzymes in select rat tissue extracts. PP2A, PP4, and PP6 catalytic subunits (PP2A(C), PP4(C), and PP6(C), respectively) were detected in all rat tissue extracts examined and exhibited some differences in their levels of expression. The expression of alpha4, an interacting protein for PP2A family members that may function downstream of the target of rapamycin (Tor), was also examined using specific alpha4 sheep antibodies. Like the phosphatase catalytic subunits, alpha4 was ubiquitously expressed with particularly high levels in the brain and thymus. All three PP2A family members, but not alpha4, bound to the phosphatase affinity resin microcystin-Sepharose. The phosphatase catalytic subunits were purified to apparent homogeneity (PP2A(C) and PP4(C)) or near homogeneity (PP6(C)) from bovine testes soluble extracts following ethanol precipitation and protein extraction. In contrast to PP2A(C), PP4(C) and PP6(C) exhibited relatively low phosphatase activity towards several substrates. Purified PP2A(C) and native PP2A in cellular extracts bound to GST-alpha4, and co-immunoprecipitated with endogenous alpha4 and ectopically expressed myc-tagged alpha4. The interaction of PP2A(C) with alpha4 was unaffected by rapamycin treatment of mammalian cells; however, protein serine/threonine phosphatase inhibitors such as okadaic acid and microcystin-LR disrupted the alpha4/PP2A complex. Together, these findings increase our understanding of the biochemistry of alpha4/phosphatase complexes and suggest that the alpha4 binding site within PP2A may include the phosphatase catalytic domain.     

Overexpression of human cardiac troponin in Escherichia coli: its purification and characterization

All three subunits of the human cardiac troponin complex (cTn), namely the major isoform of the tropomyosin binding subunit (hcTnT3), the inhibitory subunit (cTnI), and the calcium binding subunit (cTnC), have been coexpressed in Escherichia coli. The cDNAs of each subunit have been cloned into the pSBET vector and transformed into E. coli. The coexpressed subunits assembled within the bacterial cells to form the hcTn complex (hcTnT3.hcTnI.hcTnC). The complex was isolated and purified by three chromatographic steps. Per 6-L cell culture about 10 mg of a highly purified troponin complex showing the expected 1:1:1 molar ratio of hcTnT3:cTnI:cTnC was obtained. Upon phosphorylation by protein kinase A at Ser22 and Ser23 in cTnI, this recombinant troponin complex shows a nearly identical (31)P NMR spectrum to the native one isolated from bovine heart. By measuring the rate of myosin S1 binding to reconstituted thin filaments it was shown that the dependence of the regulation of S1 binding upon calcium concentration and bisphosphorylation was comparable to the native complex.     

The myosin-bound form of protein phosphatase 1 (PP-1M) is the enzyme that dephosphorylates native myosin in skeletal and cardiac muscles

The myosin-bound form of protein phosphatase 1 (PP-1M) and the glycogen-bound form (PP-1G) together account for virtually all the phosphatase activity in rabbit skeletal muscle extracts towards native myosin. PP-1M has a 3-fold higher activity towards native myosin than does PP-1G and accounts for at least 60% of the myosin phosphatase activity in rabbit skeletal muscle. PP-1M accounts for 90% of the myosin phosphatase activity in bovine cardiac muscle, where PP-1G is essentially absent. The high activity of PP-1M towards native myosin appears to arise from interaction of the catalytic subunit with the putative myosin-binding subunit, since chymotryptic digestion liberates a catalytic subunit having the same characteristics as that released by limited proteolysis of PP-1G. Protein phosphatase 2A in skeletal and cardiac muscles is very active towards the isolated myosin P-light chain, but ineffective in dephosphorylating native myosin. The results suggest that PP-1M is the enzyme that dephosphorylates myosin in skeletal and cardiac muscle.     

Coexpression of both alpha and beta subunits is required for assembly of regulated casein kinase II

Casein kinase II is an ubiquitous serine-threonine kinase whose functional significance and regulation in the living cell are not clearly understood. The native enzyme has an oligomeric structure made of two different (alpha and beta) subunits with an alpha 2 beta 2 stoichiometry. To facilitate the study of the structure-activity relationship of the kinase, we have expressed its isolated subunits in a baculovirus-directed insect cell expression system. The resulting isolated recombinant alpha subunit exhibited a protein kinase catalytic activity, in agreement with previous observations [Cochet, C., & Chambaz, E. M. (1983) J. Biol. Chem. 258, 1403-1406]. Coinfection of insect cells with recombinant viruses encoding the two kinase subunits resulted in the biosynthesis of a functional enzyme. Active recombinant oligomeric kinase was purified to near homogeneity with a yield of about 5 mg of enzymatic protein per liter, showing that, in coinfected host cells, synthesis was followed, at least in part, by recombination of the two subunits with an alpha 2 beta 2 stoichiometry. The catalytic properties of the recombinant enzyme appeared highly similar to those previously observed for casein kinase II purified from bovine tissue. Access to the isolated subunits and to their alpha 2 beta 2 association disclosed that the beta subunit is required for optimal catalytic activity of the kinase. In addition, the beta subunit is suggested to play an essential role in the regulated activity of the native casein kinase II. This is clearly illustrated by the observation of the effect of spermine which requires the presence of the beta subunit to stimulate the kinase catalytic activity which is borne by the alpha subunit.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mutation of Tyr307 and Leu309 in the protein phosphatase 2A catalytic subunit favors association with the alpha 4 subunit which promotes dephosphorylation of elongation factor-2.

The cellular location and substrate specificity of the catalytic subunit (C) of protein phosphatase 2A (PP2A) depend on its interaction with A and B subunits. The distribution of epitope-tagged wild-type or mutated C subunits was studied by transient expression in COS-7 cells. Wild-type tagged C expressed at low levels formed ABC trimer and AC dimer like the endogenous C. Single mutations of C at the site of phosphorylation (Y307F) or carboxymethylation (L309Q) resulted in recovery of only AC dimer. Double mutation of both residues resulted in association of C with alpha 4 protein (alpha 4), a novel subunit of PP2A, instead of with A and B subunits. Thus, the distribution of C between ABC trimer, AC dimer, and alpha 4C complexes can be affected by modifications of the C-terminal residues. The alpha 4 protein is a homologue of the yeast Tap42 protein that functions downstream of the TOR protein to regulate protein synthesis. Transient overexpression of FLAG-alpha 4 resulted in increased dephosphorylation of elongation factor 2, but had no effect on phosphorylation of either p70S6 kinase or PHAS-I (eIF4E-BP). Signals that affect phosphorylation or methylation of the C subunit of PP2A may promote subunit exchange and direct phosphatase activity to specific intracellular substrates.     

The control of protein phosphatase-1 by targetting subunits. The major myosin phosphatase in avian smooth muscle is a novel form of protein phosphatase-1.

The major protein phosphatase that dephosphorylates smooth-muscle myosin was purified from chicken gizzard myofibrils and shown to be composed of three subunits with apparent molecular masses of 130, 37 and 20 kDa, the most likely structure being a heterotrimer. The 37-kDa component was the catalytic subunit, while the 130-kDa and 20-kDa components formed a regulatory complex that enhanced catalytic subunit activity towards heavy meromyosin or the isolated myosin P light chain from smooth muscle and suppressed its activity towards phosphorylase, phosphorylase kinase and glycogen synthase. The catalytic subunit was identified as the beta isoform of protein phosphatase-1 (PP1) and the 130-kDa subunit as the PP1-binding component. The distinctive properties of smooth and skeletal muscle myosin phosphatases are explained by interaction of PP1 beta with different proteins and (in conjunction with earlier analysis of the glycogen-associated phosphatase) establish that the specificity and subcellular location of PP1 is determined by its interaction with a number of specific targetting subunits.     

Isolation and characterization of recombinant human casein kinase II subunits alpha and beta from bacteria

cDNA encoding the casein kinase II (CKII) subunits alpha and beta of human origin were expressed in Escherichia coli using expression vector pT7-7. Significant expression was obtained with E. coli BL21(DE3). The CKII subunits accounted for approximately 30% of the bacterial protein; however, most of the expressed proteins were produced in an insoluble form. The recombinant CKII alpha subunit was purified by DEAE-cellulose chromatography, followed by phosphocellulose and heparin-agarose chromatography. The recombinant CKII beta subunit was extracted from the insoluble pellet and purified in a single step on phosphocellulose. From 10 g bacterial cells, the yield of soluble protein was 12 mg alpha subunit and 5 mg beta subunit. SDS/PAGE analysis of the purified recombinant proteins indicated molecular masses of 42 kDa and 26 kDa for the alpha and beta subunits, respectively, in agreement with the molecular masses determined for the subunits of the native enzyme. The recombinant alpha subunit exhibited protein kinase activity which was greatest in the absence of monovalent ions. With increasing amounts of salt, alpha subunit kinase activity declined rapidly. Addition of the beta subunit led to maximum stimulation at a 1:1 ratio of both subunits. Using a synthetic peptide (RRRDDDSDDD) as a substrate, the maximum protein kinase stimulation observed was fourfold under the conditions used. The Km of the reconstituted enzyme for the synthetic peptide (80 microM) was comparable to the mammalian enzyme (40-60 microM), whereas the alpha subunit alone had a Km of 240 microM. After sucrose density gradient analysis, the reconstituted holoenzyme sedimented at the same position as the mammalian CKII holoenzyme.     

Purification and identification of a novel subunit of protein serine/threonine phosphatase 4

The catalytic subunit of protein serine/threonine phosphatase 4 (PP4C) has greater than 65% amino acid identity to the catalytic subunit of protein phosphatase 2A (PP2AC). Despite this high homology, PP4 does not appear to associate with known PP2A regulatory subunits. As a first step toward characterization of PP4 holoenzymes and identification of putative PP4 regulatory subunits, PP4 was purified from bovine testis soluble extracts. PP4 existed in two complexes of approximately 270-300 and 400-450 kDa as determined by gel filtration chromatography. The smaller PP4 complex was purified by sequential phenyl-Sepharose, Source 15Q, DEAE2, and Superdex 200 gel filtration chromatographies. The final product contained two major proteins: the PP4 catalytic subunit plus a protein that migrated as a doublet of 120-125 kDa on SDS-polyacrylamide gel electrophoresis. The associated protein, termed PP4R1, and PP4C also bound to microcystin-Sepharose. Mass spectrometry analysis of the purified complex revealed two major peaks, at 35 (PP4C) and 105 kDa (PP4R1). Amino acid sequence information of several peptides derived from the 105 kDa protein was utilized to isolate a human cDNA clone. Analysis of the predicted amino acid sequence revealed 13 nonidentical repeats similar to repeats found in the A subunit of PP2A (PP2AA). The PP4R1 cDNA clone engineered with an N-terminal Myc tag was expressed in COS M6 cells and PP4C co-immunoprecipitated with Myc-tagged PP4R1. These data indicate that one form of PP4 is similar to the core complex of PP2A in that it consists of a catalytic subunit and a "PP2AA-like" structural subunit.     

Cardiac contractile protein phosphatases. Purification of two enzyme forms and their characterization with subunit-specific antibodies

Two forms of protein phosphatase which dephosphorylate cardiac myosin or myosin light chains and the inhibitory subunit of cardiac troponin were purified from bovine cardiac muscle. The enzymes were composed of subunits of Mr = 63,000, 55,000, and 38,000 in a 1:1:1 molar ratio (PT-1) or Mr = 63,000 and 38,000 in a 1:1 molar ratio (PT-2). Native gel electrophoresis and sucrose gradient sedimentation indicated that activity toward all three substrates was due to a single enzyme species. A monoclonal antibody and polyclonal antiserum directed against an Mr = 38,000 protein phosphatase from this tissue specifically reacted with the Mr = 38,000 subunit of PT-1 and PT-2. The specificity of antibodies for the Mr = 38,000 subunit indicated that it was distinct from the other subunits. The Mr = 63,000 subunits of PT-1 and PT-2 were identical based on mobility on sodium dodecyl sulfate gels and one-dimensional peptide maps. Specificity of antiserum against the Mr = 55,000 subunit of PT-1 showed that this subunit was a distinct protein and not derived from the Mr = 63,000 subunit by proteolysis. PT-2 but not PT-1 could interact with antiserum against the Mr = 38,000 catalytic subunit in competitive immunoassays indicating that the presence of the Mr = 55,000 subunit may alter or mask antigenic site(s). Analysis of the enzymatic properties of PT-1 and PT-2 showed that PT-2 had higher activity with myosin, myosin light chains, and phosphorylase while PT-1 had higher activity with troponin. The results indicate that the presence of the Mr = 55,000 subunit may alter the enzymatic properties of the catalytic subunit.     

Expression and purification of the alpha and beta subunits of Drosophila casein kinase II using a baculovirus vector.

Two recombinant baculoviruses that express the alpha and beta subunits of Drosophila melanogaster casein kinase II, respectively, have been constructed. The expressed proteins are similar to the authentic Drosophila subunits in size and are recognized by antisera raised against the Drosophila holoenzyme. Extracts derived from cells infected with the alpha subunit-expressing virus display elevated casein kinase II activity in vitro. This activity is markedly enhanced in extracts of cells infected with both viruses, or when alpha and beta subunit-containing extracts are mixed in vitro following lysis. Recombinant holoenzyme and the alpha subunit were purified to near homogeneity using phosphocellulose column chromatography. The specific activity of the purified recombinant holoenzyme was very similar to that of the native enzyme, and was fivefold higher than that of the purified free alpha subunit. The Stokes radius of the recombinant holoenzyme was estimated to be 50 A, a value similar to that reported for the native enzyme, whereas the alpha subunit demonstrated a Stokes radius of 26.5 A. Studies using sucrose density gradient centrifugation showed that, under conditions of high ionic strength, the quaternary structure of the purified holoenzyme was tetrameric (like the native enzyme), whereas the structure of the alpha subunit was monomeric. At lower ionic strength the recombinant holoenzyme had a significantly higher sedimentation coefficient, characteristic of the formation of filaments found for the native enzyme. Interestingly, the purified catalytic subunit also displayed a higher S value under conditions of low ionic strength, revealing the formation of alpha subunit aggregates.     

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

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

Characterization of the cardiac holotroponin complex reconstituted from native cardiac troponin T and recombinant I and C.

Cardiac troponin I (cTnI), the inhibitory subunit of cardiac troponin (cTn), is phosphorylated by the cAMP-dependent protein kinase A at two adjacently located serine residues within the heart-specific N-terminal elongation. Four different phosphorylation states can be formed. To investigate each monophosphorylated form cTnI mutants, in which each of the two serine residues is replaced by an alanine, were generated. These mutants, as well as the wild-type cardiac troponin I (cTnI-WT) have been expressed in Escherichia coli, purified and characterized by isoelectric focusing, MS and CD-spectroscopy. Monophosphorylation induces conformational changes within cTnI that are different from those induced by bisphosphorylation. Functionality was assessed by measuring the calcium dependence of myosin S1 binding to thin filaments containing reconstituted native, wild-type and mutant cTn complexes. In all cases a functional holotroponin complex was obtained. Upon bisphosphorylation of cTnI-WT the pCa curve was shifted to the right to the same extent as that observed with bisphosphosphorylated native cTnI. However, the absolute values for the midpoints were higher when recombinant cTn subunits were used for reconstitution. Reconstitution itself changed the calcium affinity of cTnC: pCa50-values were higher than those obtained with the native cardiac holotroponin complex. Apparently only bisphosphorylation of cTnI influences the calcium sensitivity of the thin filament, thus monophosphorylation has a function different from that of bisphosphorylation; this function has not yet been identified.     

Gel-based mass spectrometric analysis of recombinant GABA(A) receptor subunits representing strongly hydrophobic transmembrane proteins

GABA(A) receptors are the major inhibitory transmitter receptors in mammalian brain and are composed of several protein subunits that can belong to different subunit classes, leading to enormous heterogeneity. To establish techniques for the analysis of GABA(A) receptors in complex mixtures such as brain tissue, recombinant receptors composed of alpha1 and His-tagged beta3 subunits expressed in insect cells were purified by affinity chromatography and run on blue native gels. After denaturing, receptors were subjected to one- or two-dimensional electrophoresis in SDS-gels. Proteins were cleaved by multienzyme proteolysis and subjected to nano-ESI-LC-MS/MS. Both GABA(A) receptor subunits were well-separated and unambiguously identified by sequence coverage of 99.1% (alpha1) and 92.9% (beta3).     

Light-driven translocation of the protein phosphatase 2A complex regulates light/dark dephosphorylation of phosducin and rhodopsin

In steps of protein purification of bovine retinal protein phosphatase 2A (PP2A), phosducin dephosphorylation activity peaks coelute with a PP2A enzyme complex, shown by peptide sequence analysis to contain a B' subunit, B56 epsilon. Other PP2A complexes with a slightly larger (56.5 kDa) B' subunit (sequenced to be B56 alpha) or with the B alpha regulatory subunit have no phosducin dephosphorylation activity. Upon exposure to light, a significant increase in the immunoreactive protein level of the A, C, and B56 epsilon PP2A subunits is observed in the cytosolic fraction of mouse retina, the phosducin dephosphorylation of which occurs rapidly. During dark exposure, these subunits translocate to the membrane fraction where rhodopsin is slowly dephosphorylated. This PP2A redistribution occurs in less than 1.5 min and is dependent upon light and not upon an intrinsic circadian rhythm. Forty times more of the A subunit (approximately 20 ng/mouse retina) and 9 times more of the C subunit (approximately 4 ng/mouse retina) than of the B56 epsilon subunit (approximately 0.45 ng/mouse retina) redistribute, which suggests that the predominant form of the PP2A enzyme complex on the membrane in the dark is a dimer, consisting of only A and C subunits. We observe that the dimer favors phosphorylated opsin as a substrate, while the trimer, particularly the enzyme complex with the B56 epsilon subunit, greatly prefers phosphorylated phosducin, with an activity several hundred times those of other substrates that were tested. This light-driven PP2A translocation provides a potential mechanism for efficient dephosphorylation of two critical photoreceptor transduction proteins, cytosolic phosducin and membrane-bound rhodopsin, by the same enzyme.     

Structural characterization of cardiac protein phosphatase with a monoclonal antibody. Evidence that the Mr = 38,000 phosphatase is the catalytic subunit of the native enzyme(s)

The native structures of protein phosphatases have not been clearly established. Several tissues contain high molecular weight enzymes which are converted to active species of Mr approximately 35,000 by denaturing treatments or partial proteolysis. We have used a monoclonal antibody directed against purified bovine cardiac Mr = 38,000 protein phosphatase to determine whether this species is the native catalytic subunit or a proteolytic product of a larger polypeptide. Monoclonal antibody was obtained from a cloned hybrid cell line produced by the fusion of Sp2 myeloma cells with spleen cells from a mouse immunized with phosphatase coupled to hemocyanin. This antibody was specific for the Mr = 38,000 phosphatase as determined by immunoblot analysis of purified enzyme or cardiac tissue extracts after native or sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A single immunoreactive protein of Mr = 38,000 was present in cardiac tissue extracts including extracts prepared from freeze-clamped rat heart rapidly denatured in hot sodium dodecyl sulfate buffer. Precipitation of cardiac extract with 80% ethanol did not alter the Mr of the phosphatase nor did it liberate new immunoreactive material not observed in the extract. Ethanol precipitation caused the dissociation of both phosphatase activity and immunoreactivity from a high Mr form to a form of Mr between 30,000 and 40,000. An immunoreactive protein of Mr = 38,000 was identified in several bovine and rat tissues as well as tissues from rabbits, mice and chickens and human HT-29 cells. From these data we conclude that the Mr = 38,000 cardiac phosphatase is a native catalytic subunit of higher molecular complexes which are dissociated by ethanol precipitation. A very similar, or identical, protein is present in several tissues and species suggesting that this catalytic subunit is a ubiquitous enzyme important in many dephosphorylation reactions.     

Lipid modifications of G protein subunits. Myristoylation of Go alpha increases its affinity for beta gamma

Myristoylated recombinant proteins can be synthesized in Escherichia coli by concurrent expression of the enzyme myristoyl-CoA:protein N-myristoyl-transferase with its protein substrates (Duronio, R.J., Jackson-Machelski, E., Heuckeroth, R.O., Olins, P. O., Devine, C.S., Yonemoto, W., Slice, L. W., Taylor, S. S., and Gordon, J. I. (1990) Proc. Natl. Acad. Sci. U. S.A. 87, 1506-1510). Expression of the G protein subunit Go alpha in this system results in the synthesis of two forms of the protein; these were separated on a column of heptylamine-Sepharose. Purification of the more abundant form of Go alpha yielded a product that has a blocked amino terminus. Chemical analysis of the fatty acids released by acid hydrolysis of the protein revealed myristic acid. The second form of the protein was not myristoylated. Myristoylated and nonmyristoylated recombinant Go alpha were compared with brain Go alpha (which is myristoylated) for their ability to interact with G protein beta gamma subunits. The nonmyristoylated recombinant protein clearly had a reduced affinity for beta gamma, while the myristoylated recombinant protein was indistinguishable from native Go alpha in its subunit interactions. Thus, myristoylation increases the affinity of alpha subunits for beta gamma. We propose that the function of myristoylation of G protein alpha subunits is, at least in part, to facilitate formation of the heterotrimer and the localization of alpha to the plasma membrane.     

The formation and activity of PP2A holoenzymes do not depend on the isoform of the catalytic subunit

The protein phosphatase 2A holoenzyme is composed of one catalytic C subunit, one regulatory/scaffolding A subunit, and one regulatory B subunit. The core enzyme consists of A and C subunits only. The A and C subunits both exist as two closely related isoforms, alpha and beta. The B subunits belong to four weakly related or unrelated families, designated B, B', B", and B"', with multiple members in each family. The existence of two A and two C subunit isoforms permits the formation of four core enzymes, AalphaCalpha, AalphaCbeta, AbetaCalpha, and AbetaCbeta, and each core enzyme could in theory give rise to multiple holoenzymes. Differences between Calpha and Cbeta in expression and subcellular localization during early embryonic development have been reported, which imply that Calpha and Cbeta have different functions. To address the question of whether these differences might be caused by enzymatic differences between Calpha and Cbeta, we purified six holoenzymes composed of AalphaCalpha or AalphaCbeta core enzyme and B subunits from the B, B', or B" families. In addition, we purified four holoenzymes composed of AbetaCalpha or AbetaCbeta and B'alpha1 or B"/PR72. The phosphatase activity of each purified form was assayed using myelin basic protein and histone H1 as substrates. We found that Calpha and Cbeta have identical phosphatase activities when associated with the same A and B subunits. Furthermore, no difference was found between Calpha and Cbeta in binding A or B subunits. These data suggest that the distinct functions of Calpha and Cbeta are not based on differences in enzymatic activity or subunit interaction. The implications for the relationship between the structure and function of Calpha and Cbeta are discussed.