Interaction of Calmodulin with Histones

Transcription

1 THE JOURN4L OF BOLOGCAL CHEMSTRY Vol No. 4, ssue of February 25. pp , 1981 Prrnted in U.S.A nteraction of Calmodulin with Histones ALTERATON OF HSTONE DEPHOSPHORYLATON* (Received for publication, September 17, 1980, and in revised form, October 30, 1980) Donald J. Wolff, James M. Ross, Peter N. Thompson, Margaret A. Brostrom, and Charles 0. Brostrom From the Department of Pharmacology, College of Medicine and Dentistry of New Jersey, Rutgers Medical School, Piscataway, New Jersey The Ca +-dependent regulator protein (CDR), also frequently termed calmodulin, was determined to influence the dephosphorylation of mixed calf thymus histones or purified histones 1,2A, or 2B by a partially purified bovine brain phosphoprotein phosphatase. CDR increased the rate of dephosphorylation of mixed histones more than 20-fold. With increasing concentrations of mixed histones as substrate, a proportionate increase of CDR concentration was required to maintain maximal expression of histone phosphatase activity. Mixed histones suppressed the activation by CDR of a bovine brain cyclic nucleotide phosphodiesterase activity, with activation being restored by increased quantities ofcdr. Dephosphorylation of casein and phosphorylase a by the phosphatase preparation was not affected by CDR. These observations support the interpretation that the effects of CDR on histone dephosphorylation are substrate-directed. The rates of dephosphorylation of histones 1,2A, and 2B by the phosphatase were 4- to 12-fold more rapid at low (sub-micromolar) concentrations of free Ca2+ than at high (200 PM) Ca2+ in incubations containing CDR, but they were unaffected by Ca2+ in incubations without CDR. The addition of stoichiometric quantities of calmodulin increased the apparent K,,, of the phosphatase for the various histones 2- to g-fold, while maximal velocities were 4- to d-fold higher at low than at high added Ca2+. The inhibitory effect of Ca2+ on histone dephosphorylation was immediately reversible by che- lation of Ca2+ with EDTA. Ca2+-dependent inhibition of histone 1 or 2B phosphatase activities was also produced by rabbit skeletal muscle troponin C, but not by rabbit skeletal muscle parvalbumin, by poly(l-aspartate) or poly@-glutamate). The phosphorylated fragment from the NH,-terminal region of either H2A (generated by treatment with N-bromosuccinimide) or H2B (generated by treatment with cyanogen bromide) was dephosphorylated by the phosphatase, with the rates of dephosphorylation being reduced 3- to 6-fold by Ca in incubations containing CDR. An extensive body of evidence supports the premise that cyclic AMP and Ca function as intracellular regulators of diverse physiological processes. The regulatory effects of cyclic AMP are mediated largely, if not exclusively, in mammalian systems through the phosphorylation of selected pro- * This work was supported in part by United States Public Health Service Grants NS and NS The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked aduertisenent in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. teins by a cyclic AMP-dependent protein kinase (1). These effects are terminated by the combined actions of cyclic nucleotide phosphodiesterase which hydrolyzes cyclic AMP and by phosphoprotein phosphatases which dephosphorylate the protein substrates of the protein kinase. The intracellular mediator(s) of the effects of Ca2+ are less well understood. A growing body of data supports the conclusion that a Ca - binding protein of unbiquitous distribution in eukaryotes serves as an important intracellular receptor for Ca2+ and as a multifunctional Ca -dependent regulator (CDR). CDR, also frequently termed calmodulin, has been shown to regulate cyclic nucleotide metabolism both at the level of camp synthesis by adenylate cyclase (2) and degradation by an isozymic form of cyclic nucleotide phosphodiesterase (3-5). CDR has been reported to stimulate the active transport system for Ca2+ in the plasmalemma of erythrocytes (6, 7). The stimulatory effect of Ca2+ on myosin light chain kinase, believed to control contraction in smooth muscle (8), has been reported to be mediated by CDR (9,lO). n membrane systems derived from diverse tissues, CDR has been shown to be required for a Ca2+-dependent autophosphorylation of certain proteins which vary from tissue to tissue (11). t is not clear whether these effects of CDR are mediated by stimulation of a protein kinase, inhibition of a protein phosphatase, or by direct interaction with the substrate proteins of the membranes. The promotion of glycogen catabolism by Cap+ may be mediated by CDR by activation of phosphorylase b kinase (12) and glycogen synthase kinase-2 (13, 14). These observations support the contention that the regulatory properties of Ca2+ intracellularly are produced in part by the phosphorylation of selected proteins in a manner mediated by CDR. These selected proteins are frequently different from the proteins phosphorylated by the cyclic AMP-dependent protein kinase. The regulatory effects of cyclic AMP and Ca2+ are related to one another in a complex manner that appears to vary from one system to another (15). n some systems Ca2+ and camp are functionally supportive, in others functionally independent or antagonistic. The possibility exists that not only protein kinase activities, but also protein phosphatase activities are regulated by Caz+ acting in conjunction with CDR. We, therefore, decided to evaluate the possibility that CDR could affect the dephosphorylation of phosphorylase a, casein, or mixed histones, on the premise tha; these substances might substi tute for, or serve as, the natural substrates of endogenous phosphoprotein phosphatase activities subject to regulation by Ca2+ and CDR. t was hoped that such studies might reveal new inter-relationships between protein phosphorylation and The abbreviations used are CDR, calcium-dependent regulator (calmodulin); EGTA, ethylene glycol bis (P-aminoethyl ether)- N,N,N,N -tetraacetic acid; DNP, dinitrophenyl.

2 dephosphorylation in the context of regulation by Ca2+ and CAMP. The observations presented in this report establish that CDR modulates in vitro the dephosphorylation of mixed histones, histone 1, 2A, and 2B by a brain phosphatase activity. The effects are elicited by interaction of CDR with the histone substrates. No effects of CDR on phosphorylase a or casein phosphatase activities were observed. EXPERMENTAL PROCEDURES RESULTS Histone Phosphatase Modulated Activity by CDR 1847 dentification and Partial Purification of a CDR-stimuluted Mixed Histone Phosphatase Actiuity-n order to astase activities as measured immediately after gel filtration sess whether CDR exerted an effect on phosphoprotein phoswere commonly 50 to 60% of applied activity as measured phatase activity in a crude extract of bovine cerebral cortex, with each substrate. After pooling of the fractions and subsethe dephosphorylation of P-labeled casein, mixed histones, quent storage of the preparation for 2 days at 4 C, a selective and phosphorylase a was monitored in incubations conducted loss of CDR-stimulated histone phosphatase activity occurred, with EGTA or with Ca2+ and CDR (Table, miniprint). These indicating that this activity exhibited markedly different staprocedures had been previously found to be effective in idenbility properties from the other phosphoprotein phosphatase tifying ea2+. CDR effects on cyclic nucleotide phosphodiesteractivities present in the sample. This finding was confirmed ase (5) and adenylate cyclase (2) activities from bovine cerein a study of the thermal stability of the DEAE-cellulose bral cortex. While no effect of added CDR and Ca2+ was purified phosphoprotein phosphatase (peak 1) (Fig. 3, miniobserved with either phosphorylated casein or phosphorylase print). The CDR-stimulated histone phosphatase activity was a as substrate, a 3-fold stimulation of activity was noted when progressively inactivated by 5-min exposure to temperatures phosphorylated mixed histones were employed. n incubations between 45 and 70 C prior to assay in contrast to the higher of mixed histones containing Ca (100 p ~ but ) without added stabilities found for the other activities. The histone phospha- CDR, no stimulation of activity was observed. Phosphatase tase activity measured in the absence of added CDR, the activity in incubations containing both EGTA and CDR was casein and phosphorylase a phosphatase activities were each the same as that seen in incubations containing both Ca2+ and activated by pretreament at elevated temperatures for 5 min, CDR (data not shown). Thus, while it was observed that CDR with maximal activation occurring at 60 C. At temperatures stimulated the dephosphorylation of mixed histones, the effect in excess of 60 C inactivation of each of these activities of CDR was independent of Ca2+ concentration under these occurred. conditions of measurement. Tissue extracts from rat brain, n order to explore further the nature of this activation liver, kidney, heart, and lung also possessed mixed histone event a sample of DEAE-cellulose-purified phosphatase (peak phosphatase actvity which was stimulated 4- to 6-fold by CDR 1) was heated to 60 C for 5 min, applied to a column of AC- (data not shown). Only modest differences of enzyme specific 34, and the eluted fractions assayed for casein, phosphorylase activity were found among these phosphatase sources. a, CDR-dependent and CDR-independent histone phospha- The CDR-stimulated histone phosphatase activity of crude tase activities (Fig. 4, miniprint). Two peaks of activity were brain extract has been partially purified as detailed under observed for the heated sample as measured with any of the Experimental Procedures. n brief, the enzyme from a crude three substrates. The first peak eluted at the exclusion volume extract of brain was found to bind to protamine coupled of the column and contained 17, 13, and 11%, respectively, of covalently to Sepharose and to be eluted from the gel by buffers containing a high ionic strength. The eluted enzyme the applied histone, casein, and phosphorylase a phosphatase activities. The second peak of activity eluted at a volume was fractionated by an ammonium sulfate procedure, being characteristic of a protein of approximately 30,000 molecular soluble at 30% of saturation but insoluble at 50% of saturation. weight and possessed 100, 86, and 8176, respectively, of the DEAE-cellulose column chromatography of the ammonium histone, casein, and phosphorylase a phosphatase activities. sulfate-fractionated material resolved the activity into two n order to evaluate whether low the molecular weight form peaks of activity eluting at approximately 0.17 M and 0.27 M NaCl (Fig. 1, miniprint). Peak 1 possessed 25% of the activity of phosphatase was derived from heat-induced dissociation of originally present in the crude extract (Table 11, miniprint) a presumed catalytic subunit from the native 120,000 molecwith a 43-fold increased specific activity. Peak 1 possessed ular weight form of enzyme, or from latent activity not originally expressed in the preparation, a sample of DEAE-cellu- 16% of the activity present in the crude extract with a 56-fold lose-purified phosphatase (peak 1) was subjected to gel fdtraincreased specific activity. n freshly prepared crude extracts tion on AC-34 and samples of the eluted fractions assayed not subjected to protamine-sepharose or ammonium sulfate treatment, two forms of CDR-stimulated histone phosphatase either without pretreatment or following a pretreatment at activity were similarly resolved by DEAE-celulose chroma- 60 C for 5 min (Fig. 5, miniprint). As assayed with casein, tography (data not shown). The partial purification procedure phosphorylase a, or mixed histone with or without CDR a single peak of activity (120,000 failed to resolve the casein and phosphorylase a M,) was discerned regardless phosphatase activities from the histone phosphatase activity. Both forms of whether or not the sample had been heated. Samples of column fractions treated at 60 C exhibited a uniformly in- Portions of this paper (including Experimental Procedures, Tables, 11, and V to V, and Figs. 1 to 5) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from The Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, Md Request Document No. 80M-1986, cite author(s), and include a check or money order for $7.20 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press. and 1 of CDR-stimulated histone phosphatase activity could readily be stored at -20 C in the lyophilized state without detectable loss of activity over a 3-month period. Physical Properties of CDR-dependent Histone Phosphatase Actiuities-When DEAE-cellulose purified histone phosphatase activity (peak 1) was subjected to gel filtration on a column of AC-34, a single peak of phosphatase activity as measured with phosphorylated mixed histones with or without CDR, casein, or phosphorylase a was observed (Fig. 2, miniprint). The elution volume of these activities when compared with those of standard molecular weight markers indicated a molecular weight of 120,000. Recoveries of phospha- creased phosphatase activity relative to samples maintained at 4 C as measured with casein, phosphorylase a, or mixed histones without CDR, and uniformly decreased phosphatase activity when measured with mixed histones containing CDR. A sample of DEAE-celulose-purified phosphatase (peak 1) was treated at 50 C for 5 min. A sample of the treated enzyme examined for phosphatase activity using phosphorylase a, casein, or mixed histones with or without CDR revealed only

3 1848 Histone Phosphatase Modulated Activity by CDR a 20% loss of activity relative to an untreated control at each condition of assay. When the treated phosphatase was subjected to gel fitration on a column of AC-34, a single peak of phosphatase activity as measured at the four substrate conditions was observed eluting at a volume characteristic of a protein of 70,000 M, (data not shown). Recovery of applied activity was approximately 85% regardless of the substrate used for assay. Kinetic Properties of CDR-dependent Mixed Histone Phosphatase Actiuity-The experiments described above revealed that CDR stimulated mixed histone dephosphorylation by an activity present in tissue extracts, without establishing whether CDR interacted with the phosphatase or with the histone substrate. n order to evaluate whether CDR exerted its effects by forming a complex with the histone substrate to allow dephosphorylation to occur more readily, the concentrations of CDR required to produce optimal stimulation of activity were measured at different substrate concentrations. n incubations containing 95,313, and 950 pg/ml of phosphorylated mixed histones, maximal histone phosphatase activities were observed at concentrations of CDR of 100,300, and 1,000 pg/ml as measured with DEAE-cellulose-purified peak 1 phosphatase (Fig. 6). Thus, maximal activity was observed at equivalent weight concentrations of CDR and histone sub- strate; at ratios above and below equivalency, a progressively decreasing degree of stimulation was observed. At concentrations of phosphorylated mixed histones of 95,313, and 950 pg/ ml, the activities of histone phosphatase measured without added CDR were 120, 130, and 141 pmol of Pi released/min/ mg, and were thus largely independent of substrate concentration. n the presence of optimal CDR, these values were elevated 6.5-, 13-, and 22-fold, respectively, indicating that the degree of stimulation by CDR increased with increasing substrate concentration. The substrate concentration dependence of the histone phosphatase activity of the DEAE-cellulose-purified enzyme (peak 1) was determined without added CDR or with added CDR at concentrations equivalent by weight to histone substrate. With variation of the concentrations of a histone-cdr complex in the incubation (a constant ratio of histone to CDR was added), linear double reciprocal plots of activity were obtained, whereas if histone concentration was varied relative to a constant CDR concentration, complex nonlinear plots were obtained that were not readily interpretable (data not shown). n incubations conducted without CDR, an apparent K,,, of 0.06 mg/ml of phosphorylated mixed histones was obtained with a maximal velocity of 140 pmol of Pi released from substrate/min/mg of enzyme protein. n incubations conducted with CDR, an apparent K,,, of 0.4 mg/ml of phosphorylated mixed histones was determined with a maximal velocity of 2,440 pmol of Pi released from substrate/min/mg of enzyme protein. These kinetic constants were somewhat variable when determined with different preparations of mixed histones and likely reflected variability in the ratios of the individual histones comprising the preparation. The observation that the CDR concentration required for optimal stimulation of histone phosphatase activity increased in direct proportion to the concentration of histone substrate in the incubation was in accord with the interpretation that complex formation occurs between CDR and histone in a manner that increases accessibility of the phosphatase to the phosphorylation sites on the histone. f CDR forms complexes with histones, the addition of histone to incubations containing CDR and CDR-dependent cyclic nucleotide phosphodiesterase would be expected to compromise the effectiveness of phosphodiesterase activation by CDR. The effect of a fixed concentration of mixed histones on the CDR concentration dependence of bovine brain cyclic nucleotide phosphodiesterase was determined (Fig. 7, Panel A). The presence of histones (0.083 mg/ml) had no effect on cyclic nucleotide phosphodiesterase activity measured in the absence of added CDR, but suppressed the stimulation by CDR in a manner that could as LOG [CDR], Fg /ml FG. 6. CDR concentration dependence of histone phosphatase activity at various tixed substrate concentrations. Standard incubations were constructed at 95 (.), 313 (A), and 950 (0) pg/ ml of phosphorylated mixed histones (Sigma 11-AS) containing the indicated concentrations of CDR. Reactions were conducted at standard conditions following initiation with DEAE-cellulose peak 1 purified phosphatase (0.75 pg). Values on the ordinate are expressed as the percentage of the maximal increment of activity attributable to CDR at the given suhstrate concentration. Activities for incubations without added CDR were 120, 132, and 141, and in incubations at optimal CDR, 782, 1692, and 3031 pmol of phosphate released/min/ mg of phosphatase as measured at 95, 313, and 950 pg/ml of mixed histone substrate, respectively. = LOG CDR,ng LOG [HSTONE], ng FG. 7. The effect of mixed histones (Sigma 11-AS) on the CDR activation of bovine brain cyclic nucleotide phosphodiesterase. Panel A, the effect of a fixed concentration of histone on the CDR concentration dependence of bovine brain cyclic nucleotide phosphodiesterase. Cyclic nucleotide phosphodiesterase activity was measured at standard conditions as described under Experimental Procedures in incubations conducted without (A) or containing (0) 82.5 pg/ml of mixed histone (Sigma 11-AS) at the indicated amounts of added CDR. ncubations contained 64 microunits of CDR-dependent phosphodiesterase. Panel B, the effect of increasing histone concentration on cyclic nucleotide phosphodiesterase activity measured at a fixed, limiting concentration of CDR. Cyclic nucleotide phosphodiesterase activity was measured at standard conditions either without (A) or with the addition of 15 ng of CDR (8) at the indicated concentrations of histone. Values for cyclic nucleotide phosphodiesterase activity are expressed as the micromoles of cyclic GMP hydrolyzed/min/ml of enzyme in incubations containing 64 microunits of CDR-dependent phosphodiesterase.

4 be overcome by increasing the CDR Concentrations added to the incubations. The effect of increasing histone concentration on cyclic nucleotide phosphodiesterase activity was measured in incubations without added CDR or in incubations contain- ing a fixed limiting concentration of CDR (Fig. 7, Panel B). n incubations without added CDR no effect of histone was observed on basal cyclic nucleotide phosphodiesterase activity at concentrations ranging 0.07 to 20 pg/d of mixed histones, while in incubations containing CDR, a histone concentrationdependent inhibition of CDR-dependent activity was observed. These observations are consistent with the interpretation that histones form complexes with CDR that are ineffective in stimulating cyclic nucleotide phosphodiesterase activity. The kinetic characteristics of the CDR-dependent histone phosphatase activity of DEAE-cellulose (peak 11) enzyme has been studied in less detail than peak. t is clear, however, that the CDR-activation curve of peak 1 histone phosphatase activity with phosphorylated mixed histones as substrate is biphasic as has been observed for peak enzyme (data not shown). The optimal concentration of CDR required for stimulation of the phosphatase also increased with increasing substrate concentration. This effect of CDR on the histone phosphatase activity of peak 1 is presumably also exerted by complex formation between CDR and the histone substrate. All further kinetic studies described in this report were conducted with the DEAE-cellulose-purified (peak 1) enzyme preparation. Kinetic Characteristics of CDR-dependent H, H2A, and H23 Phosphatase Activities-The CDR concentration dependence of H1 phosphatase activity was measured in incubations containing either 2.2 or 11 p~ H1 in the presence of either 1 mm EGTA or 1 mm Ca2+ (Fig. 8, Panel A). n incubations containing EGTA and either 2.2 or 11 p~ H, a 25% increase of H1 phosphatase activity occurred as quantities of CDR were elevated to values below stoichiometric equivalency with H1; as quantities of CDR substantially in molar excess to H1 were added a modest inhibition of phosphatase activity occurred. n incubations containing Ca", a greater than 6-fold inhibition of H1 phosphatase activity occurred as Histone Phosphatase Modulated Activity by CDR 1849 quantities of CDR in molar excess to H1 substrate were attained. Thus in incubations containing excess CDR, histone 1 dephosphorylation proceeded 3.5- to 5-fold more rapidly at submicromolar concentrations of free Ca2' (1 mm EGTA) than in incubations containing 1 mm added Ca2+. n incubations without added CDR, dephosphorylation rates were independent of the free Ca2+ concentration. The CDR concentration dependence of H2A phosphatase activity was measured in incubations containing either 2.6 or 10.4 PM H2A in the presence of either 200 PM EGTA or 200 p~ Ca'+ (Fig. 8, Panel B). As measured in the presence of EGTA and 10.4 p~ H2A, an approximately 3-fold activation of activity occurred as quantities of CDR stoichiometric with H2A were attained, while in incubations containing 2.6 p~ H2A a more modest 1.8-fold activation occurred at much lower concentrations of CDR. As measured in the presence of 200 p~ Ca", a biphasic response to added CDR was observed. n incubations containing 10.4 p~ H2A and 200 p~ Ca2+ an approximately 2-fold activation of activity occurred as CDR concentrations were elevated to approximately 5 p ~ followed, by a rapid decline of activity as stoichiometric equivalency between CDR and H2A was attained. At concentrations of CDR in excess of H2A, phosphatase activities were more than 10-fold higher in incubations containing EGTA than in incu- bations containing Ca2+. n incubations containing 2.6 p~ H2A and 200 p~ Ca2+, a 1.6-fold activation occurred at concentrations of CDR up to 1 p ~ and, a marked inhibition occurred as stoichiometric equivalency was attained. No further changes of H2A phosphatase activity occurred as measured with either EGTA or Ca", as CDR amounts in excess of stoichiometric equivalency were added. At this condition H2A phosphatase activity was approximately 12-fold higher in incubations containing EGTA than in incubations containing Ca". The CDR concentration dependence of H2B phosphatase activity was examined in incubations containing either 3 or 12 p~ H2B at 200 p~ EGTA or 200 p~ Ca2+ (Fig. 8, Panel C). As measured with 12 p~ H2B and EGTA, a %fold activation of phosphatase activity occurred at concentrations of CDR providing stoichiometric equivalency while at 3 p~ H2B and EGTA a more modest, 1.5-fold stimulation occurred. n incu- 30 w 2 25 w z a. ln W 0-0 z 10 8 a z [CDR].pM,pM FG. 8. The CDR concentration dependence of histone phos- thymus histone 2A as substrate. The reactions were initiated by phatase activity. A, H phosphatase activity. H phosphatase ac- addition of 0.15 pg of phosphatase. C, H2B phosphatase activity. H2B tivity was measured at standard conditions in incubations containing phosphatase activity was measured at standard conditions in incu- 1 mm EGTA (A, A) or 1 mm Ca2+ (0, W) at the indicated concentra- bations containing 200 p~ EGTA (0,O) or 200 p~ Ca2+ (A, A) at the tions of CDR employing either 2.2 (A, B) or 11 (A, 0) p~ phosphor- indicated concentration of CDR employing either 3 p~ (0, A) or 12 ylated calf thymus histone 1 fraction as substrate. The reactions were p~ (0, A) phosphorylated calf thymus histone 2B. ncubations coninitiated by addition of 1.5 pg of phosphatase preparation. B, H2A taining 3 ~ L M H2B and 200 p~ EGTA and all incubations containing phosphatase activity. H2A phosphatase activity was measured at 2 p~ H2B were initiated with 0.15 pg of phosphatase, while incubastandard conditions in incubations containing 200 PM EGTA (0, 0) tions containing 3 p~ H2B and 200 p~ Ca" were initiated with 0.75 or 200 PM Ca2+ (A, A) at the indicated concentrations of CDR pg of phosphatase. employing either 2.6 PM (0, A) or 10.4 p~ (0, A) phosphorylated calf

5 1850 Histone Phosphatase Activity Modulated by CDR bations containing 12 p~ H2B and 200 p~ Ca", a biphasic response to increased CDR concentration was observed (Panel C) as had been noted when H2A has been employed as substrate (Panel B). n incubations containing 3 p~ H2B and 200 pm Ca2+, increased concentrations of CDR produced a concentration-dependent inhibition of activity. As measured at 12 p~ H2B with molar equivalents of CDR, phosphatase activities were 7-fold higher in incubations containing EGTA than in incubations containing Ca2+. n incubations containing 3 p~ H2B with molar equivalents of CDR, phosphatase activities were 13-fold higher in incubations containing EGTA than in incubations with Ca2+. As CDR concentrations were elevated in excess of molar equivalency to H2B, no further changes of activity were discerned irrespective of the free Ca2+ concentrations present. The effects of increasing concentrations of NaCl on H2B (Fig. 9, Panel A) and H2A (Fig. 9, Panel B) phosphatase activities were measured in incubations without CDR or with CDR at either 200 p~ EGTA or 200 p~ Ca". CDR modified both H2B and H2A phosphatase activities, regardless of the free Ca'+ concentration, at concentrations of NaCl ranging from 20 to 200 mm. However, the nature of the CDR-induced modification was dependent on free Ca2+ concentrations in that a stimulation of activity occurred at 200 p~ EGTA and an inhibition of activity resulted at 200 p~ Ca2+. These effects of CDR were largely eliminated by NaCl concentrations approaching 400 mm. n the absence of CDR, the H2B and H2A phosphatase activities responded biphasically to increased Concentrations of NaC1, with maximal activity expressed at the physiological range of concentrations (100 to 200mM). The presence of Ca2+ and CDR had little, if any, effect on either H2A or H2B phosphatase activities without added NaC1; as NaCl concentrations were elevated to mm, phosphatase activities with Ca2+ and CDR declined, while activities without CDR were elevated. n incubations containing CDR and EGTA, both H2A and H2B phosphatase activities responded biphasically, with maximal activity expressed at 40 mm NaC1. At the physiological range of ionic strength, both H2A and H2B phosphatase activities were stimulated approximately 2-fold with EGTA and inhibited 4- to 6-fold with Ca2+ in incubations with CDR as compared to incubations without CDR. With H1 as substrate (Fig. 9, Panel C), phosphatase activity was virtually eliminated in a concentra- tion-dependent manner as NaCl concentrations approached 400 mm, whether CDR was present or not. The effects of CDR on the substrate concentration dependence of the phosphatase using histones 1, 2A, or 2B was subsequently examined to detail further the mechanism by which CDR exerts its effects on activity. The histone 1 concentration dependence of phosphatase activity was measured at either 1 mm EGTA or mm Ca2+ in incubations without CDR, or in incubations containing a constant (2-fold) molar excess of CDR relative to the various histone 1 concentrations (Table 111). The presence of CDR in the incubation increased the apparent K,,, for histone 1 from 6 to 14 p~ irrespective of the concentration of free Ca2' present in the incubation. Adjustment of the free Ca2' to submicromolar values with 1 mm EGTA in incubations with CDR elevated the maximal velocity for H1 dephosphorylation more than 3-fold from 1.3 to 4.5 nmol of Pi released/min/mg phosphatase preparation. The histone 2A concentration dependence of phosphatase activity was measured in incubations without CDR, or in incubations maintained at a constant 2.8-fold molar excess of CDR relative to histone 2A with either 200 p~ EGTA or 200 p~ Ca" (Table 111). The addition of saturating concentrations of CDR resulted in a 6-fold elevation of apparent K, for H2A from 8 to 50 p~ regardless of the free Ca2+ concentration of the incubation. As measured in the presence of saturating CDR, the effect of adjustment of Ca2+ concentration from submicromolar values maintained with EGTA to 200 pm was to reduce the maximal velocity of phosphatase activity 15-fold from 133 to 9 nmol of Pi released/min/mg phosphatase preparation. The histone 2B concentration dependence of phosphatase activity has similarly been determined in incubations without CDR or in incubations maintained at a 3.2-fold molar excessof CDR relative to histone 2B, with either 200 pm EGTA or 200 p~ Ca2+ (Table 111). The addition of saturating concentrations of CDR produced a &fold elevation of appar- ent K,,, for H2B from 4 to 25 p ~ As. measured in the presence of saturating CDR, adjustment of free Ca2+ concentrations from the submicromolar values maintained with EGTA to 200 p~ added Ca2+ resulted in the reduction of the maximal velocity from 137 to 13 nmol of Pi released/min/mg phosphatase preparation. The Effects of Diverse Divalent Cations on CDR-moduluted H and H2B Phosphatase Activities-The effects of z z W m -06;.0.4 U w in W [NoC]. M [NoC], M [NoC]. M FG. 9. The effect of NaCl Concentration on the partially ph% CDR (O), or Ca2' and 12 p~ CDR (A) and the indicated concenpurified histone phosphatase of brain. A, H2B phosphatase ac- tration of NaCl. Reactions were initiated with 1.5, 0.4, and 3 pg of tivity. Standard incubations were conducted with phosphorylated phosphatase preparation, respectively. C, H phosphatase activity. H2B (4.8 p ~ as ) substrate. The incubationscontainedeitheregtastandardincubations were conductedwithphosphorylated H1 (7 without added CDR (B), EGTA with 12 PM CDR (O), orca"and 12 p ~ as ) substrate. The incubations contained Ca" either without p~ CDR (A) and the indicatedconcentrations of NaCl.ReactionsaddedCDR (0) or with 30 p~ addedcdr (A) and the indicated were initiated with 1.5, 0.4, and 3 pg of phosphatase preparation, concentration of NaC1. Reactions were initiated with 1.5 and 7.5 pg of respectively. B, H2A phosphatase activity. Standard incubations were phosphatase preparation respectively. EGTA and Ca2+, where pres- conducted with phosphorylated H2A (5.1 pm) as substrate. The ent, were at 100 pm. incubation contained EGTA without added CDR (a), EGTA with 12

6 Histone Phosphatase Activity Modulated by CDR 1851 diverse cations and chelators on H1 and H2B phosphatase activities have been examined in the presence and absence of CDR in order to evaluate whether EGTA may exert effects on activity unrelated to its chelating properties; to evaluate whether a component of phosphatase requires divalent cations for expression of activity; and to evaluate the role of CDR and its divalent cation binding sites in the modulation of histone phosphatase activity. Standard incubations were constructed containing H1 or H2B with and without CDR from reagents freed of divalent cation contamination by either dialysis against Chelex 100 or by passage over columns of Chelex 100. At divalent cation-free conditions, the addition of CDR stimulated H2B phosphatase activity more than 2-fold (Table V). The addition of EGTA or EDTA had no effect on activity. The addition of 250 p~ Ca2+ or 250 p~ Ca" in excess of EGTA had no effect on activity in incubations without CDR but produced a greater than 3-fold inhibition of H1 and H2B phosphatase activities in the presence of CDR relative to incubations containing CDR without added divalent cation. The addition of 250 PM Mn2+ produced an approximate 2-fold elevation of H1 and H2B phosphatase activities in incubations without CDR and largely eliminated changes of phosphatase activity attributable to CDR. The addition of 1 mm Mg" alone had no appreciable effects on H1 or H2B phosphatase activities, regardless of the presence of CDR. The Ca2+ concentration dependence of H1 and H2B phosphatase activities has been measured in the presence and absence of CDR (Fig. 10, PaneZsA and B). At concentrations ranging from 5 to 500 p ~ Ca2', had no effect on either H1 or H2B phosphatase activities as measured without CDR. n incubations containing H1 (14 PM) with 25 pm CDR a greater than %fold inhibition of activity was produced by concentrations of total added Ca2+ ranging from 10 to 100 PM. n incubations containing H2B (3.8 p ~ with ) 5 p CDR a greater than 3-fold inhibition of activity was produced over the rather narrow range of 10 to 40 p~ total added Ca". The ability to reverse the inhibition by Ca2+ of H2B phos- phatase activity in incubations containing CDR has been explored (Fig. 11). Two incubations containing H2B without or with CDR were constructed from reagents freed of divalent cation contamination. Reactions were initiated by addition of TABLE 111 Kinetic constants of H1, H2A, and H2B phosphatase activities The substrate concentration dependence of the phosphatase purified through the DEAE-cellulose chromatography step was examined in incubations utilizing histones H, H2A, or H2B as substrates. CDR, where added, was premixed with the histone such that a constant ratio of CDR to histone was maintained following dilution of the substrate for assay. Ca2+ and EGTA, where indicated, were present at 1.0 mm. ncubations containing H were conducted at histone con- centrations ranging from 0.4 to 56 p ~ and, with CDR (where added) at a 2.0 molar ratio to histone. Phosphatase contents were 1.5 pg. ncubations containing H2A were conducted at histone concentrations ranging from 0.3 to 52 p~ and with CDR (where added) at a 2.8 molar ratio to histone. Phosphatase contents were 1.5 pg for incubations without CDR, 0.38 pg for incubations with CDR and EGTA, and 3 pg for incubations with CDR and Ca". ncubations containing H2B were conducted at histone concentrations ranging from 1.8 to 28 pm and with CDR (where added) at a 3.2 molar ratio to histone. Phosphatase contents were 1.5 pg for incubations without CDR, 0.3 pg for incubations with CDR and EGTA, and 3.0 pg for incubations with CDR and Ca2+. Histone 1 Histone 2A Histone 2B Condition Km vma* K" vmax K, vmax nmol P,/.M nmol PC/ pm nmol P,/ PM min/mg min/mg min/mg EGTA a EGTA + CDR Ca2+ + CDR phosphatase and samples withdrawn at 2-min intervals to assess the cumulative inorganic phosphate release. At divalent cation-free conditions, H2B phosphatase activity was 4-fod higher in incubations containing CDR. At 10 min, both incubations were adjusted to 50 pm Ca"'. NO effect was observed in incubations without CDR, while a 7-fold inhibition of activity was observed in the incubation containing CDR. At 20 min both incubations were adjusted to 2.7 mm EDTA to reduce free concentrations of Ca2+ to submicromolar values. No effect was seen in incubations without CDR, while an approximate 5-fold stimulation of activity was produced in incubations with CDR. The inhibitory effects of CaZ+ on H1 phosphatase activity measured in the presence of CDR were similarly reversible by EDTA (data not shown). The Effect of Diverse Ca2+-binding Proteins and Polyanionic Polypeptides on H1 and H2B"n order to examine whether the effects on histone dephosphorylation were unique to CDR or were shared with other Ca2+-binding proteins and polyanions, the effect of rabbit skeletal muscle troponin C, rabbit skeletal muscle parvalumin, poly(l-aspartate), and poly(l-glutamate) on H1 or H2B phosphatase activity were examined in the presence of EGTA or 200 PM Ca2' (Table V, miniprint). Addition of CDR to the incubations containing EGTA produced an approximate 1.6-fold activation of H2B phosphatase and little, if any, effect on H1 phosphatase activity, while the addition of CDR to incubations containing Ca" resulted in the inhibition of both activities. Rabbit skeletal muscle troponin C, a Ca2'-binding protein conferring Ca" dependence on muscle contraction and known to be homologous to CDR (37), exhibited virtually identical behavior. Parvalbumin from rabbit skeletal muscle, a high affinity Ca2'- binding protein found in sarcoplasm but of unknown function, had no appreciable effect on either activity as measured with EGTA or Ca2+. Poly(L-aspartate) and poly(l-glutamate) each produced a marked inhibition of the histone phosphatase activities regardless of the concentrations of free Ca2+ present. The Effect of CDR on the Dephosphorylation of H2A and H2B Phosphopeptides-ln order to define more precisely the structural features of the histones responsible for interacting TABLE V The effect of divalent cations on the H and H2B phosphatase activities in the absence andpresence of CDR H1 phosphatase activity was measured in incubations containing H1 (14 phi) and, where indicated, CDR (37.5 p ~ freed ) of divalent cation contamination by dialysis against a slurry of Chelex 100. The incubations were initiated with 7.5 pg of phosphatase preparation and were conducted for 10 min. H2B phosphatase activity was measured in incubations containing H2B (3.8 p ~ and, ) where indicated, CDR (4.6 pm) free of divalent cations. The incubations were initiated with 0.75 pg of phosphatase preparation and were conducted for 15 min. All incubations were conducted in new polypropylene tubes rinsed with deionized water. The incubations contained 25 mm Tris-C1, ph 7.4, freed of divalent cations by passage over a column of Chelex 100, 0.1 M KC1 (spectral grade), and the indicated concentrations of spectral grade cations standardized by atomic absorption spectrophotometry. Histone phosphatase activity Additive 111 H2B -CDR +CDR -CDR +CDR nmol PC released/min/mg None EDTA (250 PM) EGTA (250 p ~ ) EGTA + Ca" (250 p ~ (500 ) Ca2+ (250 pm) Mn2' (250 pm) Mg (1 mm)

7 1852 Histone Phosphatase Activity Modulated by CDR ' 1 ' z 5 \.- Q cn w a z W m log [ CO"] p M FG. 10 (left). The ea2+ concentration dependence of H1 and H2B phosphatase activity measured in the presence and absence of CDR. Hl phosphatase activity (Panel A) was measured in incubations containing H (14 FM) with 25 p~ CDR (A) or without CDR (0) at the indicated concentrations of total added Ca2+. Reactions were initiated with 3 pgof phosphatase diluted in divalent cation-free bovine serum albumin (0.2 mg/ml) and were conducted for 30 min. H2B phosphatase activity (Panel B) was measured in incubations containing H2B (3.8 p ~ with ) 4.6 ~ LCDR M (A) or without CDR (0) at the indicated concentrations of total added Ca2+. Reactions were initiated with 0.38 pg of phosphatase preparation and were conducted for 30 min. CDR was freed of divalent cation contamination by adjustment to 10 mm EDTA followed by dialysis against t.wo changes of deionized water (4000 volumes each) containing a 1% (v/ v) of Chelex 100. H and H2B were each dialyzed against Chelex 100 in deionized water. All incubations contained 25 mm Tris-C, ph 7.4, passed over a column of Chelex 100 and 100 mm spectral grade KCl. O MNUTES FG. 11 (right). Reversibility of the inhibition by Ca2+ of H2B phosphatase activity by chelation. Two incubations of 2 ml initial volume in polypropylene plastic tubes were formulated from reagents freed from divalent cation contamination as described in the legend of Fig. 5. The incubations contained H2B (3.8 p ~ with ) 4.6 p~ CDR (0) or without CDR (A) in 25 mm Tris-C1, ph 7.4, and 0.1 M KC. The incubations were initiated with 7.5pg of phosphatase preparation. At 10 min both incubations were adjusted to a concentration of 50 PM Caz+ and at 20 min to a concentration of 2.75 mm EDTA. Samples (100 pl) were withdrawn from each incubation at 2-min intervals and the inorganic phosphate released was measured as the radioactivity not precipitated by 25% trichloroacetic acid in the presence of 30 mg/ ml of bovine serum albumin. Values on the ordinate are expressed as the cumulative nanomoles of Pi released/mg of added phosphatase preparation calculated from the known specific activity of the substrate. with the CDR and the phosphatase, phosphopeptide fragments of histones 2A and 2B were prepared as described under "Experimental Procedures" and were examined as substrates for the phosphatase in the presence and absence of CDR. Histone 2A was treated with N-bromosuccinimide and two peptide fragments were identified and resolved. N-Bromosuccinimide cleaves proteins at tryptophanyl and tyrosyl peptide bonds (38). The amino acid sequence of calf thymus histone 2A (39) indicates the absence of tryptophan and the presence of Tyr-Ala (residues 39 to 40), Tyr-Leu (50 to 51), and Tyr-Leu (57 to 58) sequences in the molecule. H2A is phosphorylated at serine-19 by cyclic AMP-dependent protein kinase (40). Treatment of phosphorylated calf thymus histone 2A with N-bromosuccinimide resulted in the formation of two peptides, resolvable at an analytic level by slab gel electrophoresis in the system of Laemmli (27) and at a preparative level by gel fitration on Bio-Gel P-60. The nonradioactive fragment when treated with fluorodinitrobenzene and subjected to subsequent hydrolysis in 6 N HC1 yielded a single dinitrophenylated amino acid identified subsequently as DNP-alanine. These data support the assertion that treatment of phosphorylated H2A with N-bromosuccinimide cleaves H2A uniquely at the tyrosylalanyl bond connecting residues 39 and 40. The phosphopeptide isolated from this cleavage would appear therefore to represent the fist 39 residues from the NH2 terminus phosphorylated at serine residue 19. The H2A phosphopeptide was examined as a substrate for the bovine brain phosphatase with and without CDR in the presence of 200 p~ EGTA or 200 p~ Ca2' (Table V, miniprint). H2A phos- phopeptide served as a substrate for the phosphatase and was dephosphorylated at a rate independent of Ca2+ in the absence of CDR. The presence of CDR affected the dephosphorylation rate regardless of the free Ca'+ Concentration; however, dephosphorylation occurred %fold faster in incubations containing EGTA than in incubations containing Ca2+. These behaviors are qualitatively identical with those of the phosphorylated H2A from which the phosphopeptide was derived. While the kinetic constants of the H2A phosphopeptide have not been determined, calculation of the rates of dephosphorylation of H2A from its kinetic constants indicate that a molar concentration equivalent to that measured for the peptide, intact HZA is dephosphorylated approximately 4 times more rapidly. H2B was treated with cyanogen bromide and the major phosphopeptide(s) resolved from uncleaved H2B by gel fiitration on Bio-Gel P-60. Cyanogen bromide cleaves proteins at methionyl residues (41). The amino acid sequence ofcalf thymus histone 2B (42) indicates the presence of Met-Gly (59 to 60) and Met-Asn (62 to 63) in the sequence. H2B can be phosphorylated at Ser-14, Ser-32, and Ser-36 at single or multiple sites by cyclic AMP-dependent protein kinase (43, 44). The precise site of cleavage of H2B by cyanogen bromide was not determined with respect to whether cleavage occurred at one or at both methionine residues. Only modest differences of behavior of these peptides would be expected, however, since the methionine residues are separated only by two amino acids. The phosphopeptide(s) generated by cyanogen bromide treatment served as a substrate for the phosphatase (Table V). n incubations without CDR, the H2R phosphopeptide dephosphorylation rate was unaffected by the concentration of free Ca". The addition of CDR affected the dephosphorylation regardless of free Ca'+ concentration; however, in the presence of EGTA dephosphorylation occurred at a rate 6-

8 Histone Phosphatase Modulated Activity by CDR 1853 s 0 5 h FG. 12: Dephosphorylation of H2B following complexing with H4 or H2A in the presence and absence of CDR. Standard incubations were constructed containing phosphorylated H2B (10.4 p~), 0.1 M NaCl, 0.1 mm EGTA, and the indicated concentrations of H4 (Panel A) orh2a (Panel B). H2B phosphatase activity was measured without CDR (m) or with CDR (A) at 12 p ~ The. reactions were initiated by addition of 0.15 p,g of brain phosphatase purified through the DEAE-cellulose chromatographic step. fold faster than in the presence of Ca2+, Calculation of the rate of dephosphorylation of untreated H2B at a comparable molar concentration reveals that cyanogen bromide cleavage has had virtually no effect on its substrate behavior. The Effect of ea2' on the Dephosphorylation of Phosphorylated Troponin Formed in a Complex with Troponin C or CDR-Troponin C and CDR have been shown in this report to interact with diverse phosphorylated histones to form complexes whose susceptibility to dephosphorylation is dependent on Ca'+. Troponin C, a Ca"-binding protein homologous to CDR (37), is normally formed in a complex with troponin, a protein subject to phosphorylation by CAMP-dependent protein kinase (45). Phosphorylated troponin formed in a complex with either troponin C or CDR was prepared as described under "Experimental Procedures" in order to examine whether such complexes served as substrates for the phosphatase and to explore the possibility that their substrate properties with respect to dephosphorylation would resemble those of histone complexes and be linked to the free Ca" concentration. Phosphorylated troponin formed in a complex with either troponin C or CDR served as a substrate for the phosphatase (Table V, miniprint). For each complex the dephosphorylation occurred 6- to 8-fold faster in incubations containing submicromolar free Ca2' concentrations (buffered with EGTA) than in the presence of 200 ~ Ltotal M added Ca'+. However, at equal concentrations troponin formed in a complex with CDR was dephosphorylated more rapidly than when formed in a complex with troponin C, irrespective of the free Ca2+ concentration present. Phosphorylated troponin uncomplexed to either troponin C or CDR is largely insoluble in water and hence was not examined as a substrate. The Effect of CDR on Histone Phosphatase Activity in Histone Mixtures Recombined in Defined Proportions-A discrepancy between the effects of CDR on histone phosphatase activity as measured with mixed populations of histones and with purified histone components was revealed in our studies. As measured with purified components, dephosphorylation occurred much more rapidly than when measw-ed with mixed populations. As measured with mixed histones, CDR and Ca'' stimulated phosphatase activity, whereas with purified H2B, H2A, or H, activity was inhibited. n an attempt to resolve these discrepancies, the effect of CDR on FG. 13. Dephosphorylation of H2A following complexing with H4 or H2B in the presence and absence of CDR. Standard incubations were formulated containing phosphorylated H2A (9.6 p ~ ) 0.1, M NaC1, 0.1 mm EGTA, and the indicated concentrations of H4 (Panel A) or H2B (Panel B). H2A phosphatase activity was measured without CDR (0) or with CDR (A) at 12 PM. The reactions were initiated by addition of 0.36 pg of brain phosphatase purified through the DEAE-cellulose chromatographic step. histone phosphatase activity was measured with purified components recombined at defined ratios. The effect of recombining increased concentrations of either H4 (Fig. 12, Panel A) or H2A (Fig. 12, Panel B) on H2B phosphatase activity was measured in the absence and presence of CDR. n incubations containing phosphorylated H2B without CDR a greater than 70-fold inhibition of phosphatase activity occurred as concentrations of H4 were elevated to values that were stoichiometrically equivalent with H2B. Similarly, when H2A was recombined with phosphorylated H2B in the absence of CDR, a greater than 8-fold inhibition of activity occurred, as stoichiometric equivalency was attained. When either H4 or H2A was recombined with phosphorylated H2B in the presence of CDR, only 35% inhibition occurred at stoichiometric equivalency. The effect of recombining increased concentrations of either H4 (Fig. 13, Panel A) or H2B (Fig. 13, Panel 3) on H2A phosphatase activity was measured in the absence and presence of CDR. n incubations containing phosphorylated H2A without CDR a greater than 70-fold inhibition of activity occurred as either H4 or H2B concentrations were elevated to stoichiometric equivalency with H2A. n incubations containing CDR the addition of H4 produced only a 50% inhibition as stoichiometric equivalency was attained, while the addition of H2B fully inhibited activity. DSCUSSON The results presented in this report provide evidence for the existence of phosphoprotein phosphatase activities of wide tissue distribution which dephosphorylate histones at rates modulated by the presence of stoichiometric amounts of CDR. One form of the CDR-modulated histone phosphatase (peak ) was purified approximately 40-fold from crude extracts of bovine brain and established to have a molecular mass of 120,000 by the technique of gel filtration on columns of Ultrogel AC-34. This preparation also possessed a mixed histone phosphatase activity measurable without added CDR as well as casein and phosphorylase a phosphatase activities, all of

9 1854 Histone Phosphatase Modulated Activity by CDR which co-eluted with the CDR-dependent histone phospha- cyclic nucleotide phosphodiesterase activity in a concentratase on AC-34. Treatment of this phosphatase preparation at tion-dependent manner which was fully reversible by in- 50 C for 5 min did not alter the ratio of phosphatase activities creased concentrations of CDR (Fig. 7, A and B). These as measured with casein, phosphorylase a, or mixed histones observations are explicable by the formation of CDR-mixed with or without CDR but did modify the gel filtration behavior histone complexes which were ineffective in stimulating cyclic of the phosphatase activities, with all activities eluting at a nucleotide phosphodiesterase, but which serve as substrates volume characteristic of a spherical protein of 70,000 Mr. for the histone phosphatase. Treatment of the DEAE-cellulose-purified phosphatase (peak The effects of CDR on the dephosphorylation of purified ) at 60 C for 5 min produced a putative catalytic subunit of histones 1, 2A, or 2B are similar to those on mixed histone approximately M, 30,000 with activity against all four sub- dephosphorylation in the respect that maximal changes are strates. Phosphatase activity measurements of the untreated observed when CDR is present in amounts stoichiometric enzyme conducted over a wide range of mixed histone concen- with substrate (Fig. 8). CDR affects the dephosphorylation of trations provided linear plots for data placed in the double histones regardless of the concentration of free Cat+; however, reciprocal format. Distinctly different K, values were calcu- the nature of the action (stimulation ' uersus inhibition) is lated for incubations containing CDR as compared to incubations conducted without its addition. Similar kinetic behavior was observed when homogeneous histones 1, 2A, or 2B were used as substrate. A reasonable interpretation of these influenced by the concentration of Ca2+. Thus, the effects of CDR are apparently exerted by CDR.histone complexes which serve as substrates for the phosphatase but which form in a manner independent of Ca". data is that the various phosphatase activities originally re- For histones 1, 2A, and 2B, dephosphorylation rates in the sided on a single species of native enzyme (Mr 120,000) which presence of CDR are 4- to 12-fold slower at 200 PM added Ca2+ on thermal treatment dissociated to form sequentially the 70,000 and 30,000 molecular weight secondary activities. An alternate possibility, given that the preparation is purified only 50-fold from crude extract, is that two or more forms of protein phosphatase co-purified through the DEAE-cellulose chromatographic step (peak ) and were selectively active against one or more of the various substrates. f so, these forms must each elute from columns of DEAE-cellulose at identical salt concentration and must each exhibit identical molecular dimensions as revealed by gel filtration behavior in the untreated condition which change in tandem following thermal pretreatment. The observation of linear Michaelis- Menten kinetic behavior of the preparation as measured with mixed histones or histones 1,2A, or 2B, exhibiting unique K,,, values as measured without or with CDR, can be accommodated by more than one form of enzyme only if either all forms of enzyme had identical substrate specificities with or without CDR or each form exhibited absolute specificity, one form dephosphorylating only free histone, the second form only histone. CDR complexes. The original goal which motivated this series of experiments was a desire to evaluate the possibility that Ca2+ and CDR may affect an enzymatic activity responsible for the dephosthan at submicromolar concentrations of free Ca2' maintained in EGTA buffer systems (Fig. 8). At high histone concentrations dephosphorylation of histones 1, 2A, and 2B proceeds more rapidly in the presence of CDR and EGTA than without CDR, at low histone concentrations, however, the rates are higher with EGTA alone. This behavior is readily explicable in terms of the effects of CDR on the kinetic constants for dephosphorylation. CDR raises the apparent K,,, of the phosphatase for H, H2A, or H2B from 2- to 6-fold regardless of the free concentrations of Ca2+ present (Table 111), in accord with the interpretation that CDR. histone complexes form ternary complexes with the phosphatase less readily than do the corresponding free phosphorylated histones. The maximal velocities observed for the dephosphorylation of the various CDR. histone complexes are clearly higher than the maximal velocities for the dephosphorylation of the respective free phosphorylated histones (Table 111). The inhibitory effect of Ca2+ on the dephosphorylation of histones 1, 2A, or 2B occurred regardless of the substrate concentration, was observed only in incubations containing CDR (Tables 111, V, Fig. 8), and proceeded without altering the apparent K, of the enzyme for substrate (Table 111). t appears unlikely, therefore, that Ca2+ influences the structural features of the CDRahistone phorylation of selected proteins. Phosphorylated mixed his- complex which are involved in forming ternary complexes tones, casein, and phosphorylase a were chosen as substrates in the expectation that these substances might either serve as, or substitute for, the natural substrates of diverse phosphoprotein phosphatase activities. To the extent that phosphorylase a, mixed histones, and casein can serve as alternate substrates for diverse phosphoprotein phosphatase activities, the failure of CDR to affect their dephosphorylation in crude extracts of brain, unless present in concentrations stoichiometric with substrate, supports the conclusion that CDR does not affect phosphoprotein phosphatase activities by direct interaction with enzyme. However, CDR may exert effects on phosphatase activities of more restricted substrate specifcity or influence protein dephosphorylation by direct interaction with the phosphorylated protein substrates. The contention that CDR and histones interact to form complexes is supported by two observations. First, the concentration of CDR required for expression of maximal CDRstimulated mixed histone phosphatase activity increased in direct proportion to the substrate concentration at which the measurements were made (Fig. 6). Second, mixed histones were found to suppress the stimulation of CDR-dependent with the phosphatase. Rather, it appears that the binding of Ca2+ to the CDR component of the CDR-histone complex effects a conformational change in the histone component such that the phosphorylated site has reduced susceptibility to dephosphorylation by the phosphatase. The ability of CDR to interact with phosphorylated histones in such a manner that histone susceptibility to dephosphorylation by the phosphatase becomes coupled to the free concentrations of Cat+ is not a unique property of CDR, but neither is it shared by all ea2+-binding proteins nor by all polyanionic polypeptides. Skeletal muscle troponin C, a Ca2+binding protein believed to confer Cat+-dependence on the contraction of skeletal muscle and known to be homologous to CDR exhibits nearly identical behavior (Table V). By contrast parvalbumin, an homologous Ca2+-binding protein of lower molecular weight and unknown function from skeletal muscle, did not affect histone 1 or 2B dephosphorylation regardless of the free Caz+ concentration. The effect of CDR or troponin C on histone dephosphorylation is more than a function of their polyanionic character. At conditions of high substrate concentration and with chelation of divalent cation the effects of these proteins on histone dephosphorylation are

10 Histone Phosphatase Modulated Activity by CDR 1855 stimulatory, while the effects of the polyanionic polypeptides, complexes between CDR and troponin was observed previpoly(l-aspartate) and poly(l-glutamate), are uniformly inhib- ously, and reported to restore Ca2+ dependence actomyosin itory and independent of Ca2' concentration. ATPase (50). Systems in which CDR confers Ca'+ dependence The effects of divalent cations on histone dephosphoryla- in such a manner will likely require purification for the CDR tion have been examined with and without CDR utilizing requirement to be demonstrated. reagents freed of divalent cations by pretreatment with Chelex The sequence of amino acids in histones is asymmetric. n 100. Without added divalent cation, dephosphorylation of histone 2A, the NH2-terminal residues 1 to 32 are basic, with both histones 1 and 2B occured, indicating that the enzyme a net positive charge of +10 and contain 10 of the helixhad no exogenous divalent cation requirement for catalysis of destabilizing residues, glycine, proline, and serine (51). The the dephosphorylation. Addition of either EDTA or EGTA to COOH-terminal portion of the molecule, residues 117 to 129, the incubations had no effect on H1 or H2B dephosphoryla- is also basic. The central region 33 to 116 is largely apolar. tion as measured either with or without CDR; thus, the Histone 2B is similarly asymmetric (chain length 125 resielevated phosphatase activity observed in the presence of dues). Residues 1 to 31 are basic, while residues 31 to 102 CDR and chelators was not due to direct interaction of these constitute the central apolar core. Histone 1 contains 216 anionic chelators with the cationic substrate. The addition of Mg" did not affect histone dephosphorylation irrespective of residues. The first 40 residues contain about 80% lysine, proline, and alanine, while the central segment (41 to 106) conwhether CDR was present. Mn2+ stimulated histone dephos- tains the apolar and 2 aromatic residues. n water, histones phorylation but presumably acted on the catalytic component adopt primarily a random coil conformation, but with increassince CDR was not required for, did nor it modify appreciably, ing ionic strength, helix formation occurs in the apolar core. the effect of Mn". Alternately, the phosphatase preparation The apolar segments form the secondary and tertiary strucmay be contaminated with a second, Mn"-stimulated phos- ture and are the sites of homo-aggregation in pure histones phatase, as has been described by Li et al. (46), and Antoniw et al. (47) which is normally inactive in the absence of added Mn2+. The inhibitory effects of Ca2+ on histone dephosphorylation occurred in the presence of substoichiometric EGTA, but only in incubations containing CDR. The unique inhibitory effects of Ca2+ occurred at micromolar concentrations of total added Ca2+ (Fig. 10). Free Caz+ concentrations in the and are sites of interaction of histones that form the heterooligomeric complexes that compose the core of nucleosomes (52, 53). The basic regions are the sites of DNA interaction. Histone 1 is phosphorylated at serine 38, histone 2A at serine 19, and H2B at serines 14,32, and 36 by CAMP-dependent protein kinase (40). Thus, for all three histones the phosphorylation sites are found in the basic NH2 terminus region. incubations were clearly much lower than total Ca2+ concentrations since binding of Ca'+ by CDR was significant under these conditions. CDR, which was present in the incubations at micromolar concentrations, binds Ca2+ with high affinity at four cation binding sites (48). The effect of Ca2+ on histone dephosphorylation rates was immediately reversible by chelation of Ca" (Fig. 11). On these bases CDR potentially could couple changes of Ca2+ concentration to altered histone dephosphorylation rates. CDR is widely recognized to possess the property of forming complexes with diverse proteins with consequent modification of their function. Such proteins include adenylate cyclase (2), cyclic nucleotide phosphodiesterase (3, 5), myosin light chain kinase (9, lo), glycogen synthase kinase (13, 14), and diverse membrane proteins (49). Normally such complexes are formed in a Ca"-dependent manner and are reversibly dissociable at submicromolar concentrations of free Ca", These systems commonly possess biological activities that are Ca2+-dependent and for which the Ca'+ dependence is conferred by CDR. The criteria for demonstrating CDR dependence for a given system normally involves (a) the deactivation of the system by removal of endogenous CDR (commonly by dissociation tones as contrasted to measurements with homogenous histones. CDR in the presence of Ca2+, stimulated mixed histone dephosphorylation (Figs. 1, 2, 6) but inhibited dephosphorylation of H1 or 2B (Figs. 8 and 9). The ability of CDR to with chelators and chromatographic fractionation) (6) and the stimulate histone dephosphorylation in mixtures would aprestoration of biological activity and Caz+ dependence by the pear to be explained by its ability to reduce the formation of recombination of the deactivated system with reference CDR. hetero-oligomers. When stoichiometric amounts of H4 or H2A This property of reversible dissociability may not extend to were added to phosphorylated H2B, the dephosphorylation of all CDR-modulated systems. n some systems, CDR may form H2B was completely eliminated. H4 and H2A are known to a complex with polycationic subunits in a manner not depend- form heterocomplexes with H2B (28, 31, 53) and such interent on Ca2' to become an integrated subunit of the system as has been noted for phosphorylase kinase (12). The CDR actions are believed to form the structural basis of the oligomeric nucleosome core particles (52). When CDR was present component of such systems could conceivably bind Ca2+ with Ca2+-dependent conformational changes of CDR transduced into the other subunits with alteration of their biological activity. Clearly, such properties were manifested by CDR complexes with histones 1,2A, and 2B, as well as in complexes with phosphorylated troponin. Either troponin C or CDR formed complexes with phosphorylated troponin which were dephosphorylated by the phosphatase preparation at rates markedly reduced by Ca" (Table V). The formation of such t is apparent that the interactions of CDR with these histones that modify their susceptibility to dephosphorylation is based on ionic interactions between the polyanionic CDR and the polycationic NH2-terminal regions of the histones. This con- clusion is supported by several observations. The effect of CDR on H, H2A, and H2B dephosphorylation are largely eliminated at NaCl concentrations in excess of 400 mm (Fig. 9) but are prominent at physiological values of ionic strength. Phosphorylated H2A cleaved by N-bromosuccinimide at tyrosine residue 39 and thus devoid of the central apolar core and basic COOH-terminal region is dephosphorylated by the phosphoprotein phosphatase at a rate reduced more than 3- fold by Ca2+ in incubations containing CDR (Table V). Similarly, phosphorylated H2B cleaved at methionine(s) 59 and/ or 62 was dephosphorylated at an unchanged rate modulated by CDR relative to the untreated H2B (Table V). The effect of CDR on histone dephosphorylation was markedly different when measured with mixed populations of his- in the incubations, recombination of the histones did not eliminate their susceptibility to dephosphorylation. This behavior is likely due to the formation of alternate complexes between histones and CDR with their phosphorylation sites still accessible to the phosphatase. While complexes of Ca2+. CDR with histone are poorer substrates for the phosphatase than homogenous free histones, they are considerably better substrates than histone hetero-oligomers. t is difficult to predict whether the effects ofca'+ and

11 1856 Histone Phosphatase Modulated Activity by CDR CDR on histone dephosphorylation observed in vitro have applicability in vivo for a number of reasons. The effects of Ca" and CDR on histone dephosphorylation have been measured using histones phosphorylated with a single protein kinase, namely CAMP-dependent protein kinase. The effects ofca'+ and CDR on histone dephosphorylation have been measured using a single phosphoprotein phosphatase preparation with broad substrate specificity which also dephosphorylated casein, phosphorylase a, and troponin, although at appreciably lower rates than was observed for the phosphorylated histones. Finally, the dephosphorylation of histones was measured using isolated histones removed from their native environment in chromatin. Chae et al. (54) have shown that histone 2B in the isolated state is an excellent substrate for CAMP-dependent protein kinase, but when integrated into the native structure of chromatin, H2B is not phosphorylated. ndeed, in our experiments phosphorylated H2B when formed in a complex with H2A or H4 (as it is assumed to occur in nucleosomes) was not dephosphorylated by the phosphatase (Fig. 12). Hence, the phosphorylation site may be inaccessible at this condition. H2B has not been found to be phosphorylated in vivo in Chinese hamster ovary cells during any stage of the cell cycle (55). Similar considerations, however, are not equally applicable to histone 1. H1 is known to be phosphorylated in vivo in rat liver treated with glucagon which elevates CAMP. The same site (serine 38) is phosphorylated in vitro by cyclic AMPdependent protein kinase (56). H1 phosphorylated at this site has been shown in this report to be dephosphorylated more slowly in the presence of Ca2' in incubations containing CDR. Further study will be required to ascertain whether such effects of CDR can occur for H1 integrated into the native structure of chromatin. The interaction of CDR with histones may have functional significance unrelated to their dephosphorylation. Clearly the formation of complexes between CDR and isolated histones or histone hetero-oligomers modifies their structure; such structural changes may alter their biological function and incidently change their susceptibility to dephosphorylation or phosphorylation. However, it is unclear at this time whether the interactions of histones from which DNA has been removed reflect accurately the behavior of the nucleoprotein complex that composes chromatin. Acknowledgments-We would like to thank Carolyn Foster and Stephen Bocckino for help in proofreading the manuscript. REFERENCES. Rubin, C. S., and Rosen, 0. M. (1975) Annu. Rev. Biochem. 45, Brostrom, C. O., Huang, Y.-C., Breckenridge, B. McL., and Wolff, D. J. (1975) Proc. Natl. Acad. Sci. U. 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13 1858 Histone Phosphatase Activity Modulated CDR mterp~lloo of Calmodulln with Htstone8: Alteration of Hlstons Dephosphorylation Donald S. WoUf, James M. Ross. Peter N. Thompson, Margaret A. Brostrom. and Charles 0. Brostrom Experimental Procedures M~terlals - Calf Uvmw mtaed hletons (LAS), calf thymvs hlstone 1-enrlchad fractlon (V-S), calf thymus hletone 2A-enriched fraction (V-S), calf thymus hletone 2Benriched fraction ("5). poly-l-aspadste (m-8). poly-l-glutemate (U-B), andsalmon prolamtoe sulfate and dtoltrophenyl amino adds were obtalned from the Slgma Chemlesl Company. St. Louis. Ma. Atomic absorption standard Bolutlons of CaC12,!dgCz. and MnClp and Spectrapor dlalysls tuhlug (m.w. cutoff 6.000) were purchased from Fisher Sclentlffc company. Spctrographlcally stmdardlzed magnesium sulfate, manganese suuate. calclum c8rbo~mte. andpotaasium ohlorlde were obtained lrom Johneon Matthey Chemlcals. Ltd.. London. England. Cheler-00 was purchased from Blo-Rad reboratorlee. Cyanogen hromlde was obtained from Easrman ocganic Chemleels. N-bromasucelnlmlde was ohmned from Aldricb Chemical Company. Mliwwkee. Wl. SP-Sephadex. Sepharose 6H and Sephadex G-lo0 were ohtalned from Pharmacla Fine Chemieels. plseateway, NJ. Ultrogel AC-34 was obtalned from LKB nstruments. DEAE-eelluloee WE-32) was obtamed from whatman Blochemlcals, Ltd. PhOBphorylase 5 was obtained from rabbit akeletal muscle hy the procedure of Fiaher and Kreba (16). Phosphorylase kimise was prepared from Pabhlt ekeletal muscle as described by Bmstrom et. (17). The catalytic sukmlt of cyclle AMPdependent protem klnaee was prepared from bovine akeleid mwek hy the procedure a1 Beam et &. (la). Hamageneous CDR from hovlne brain was prepared by the pracedure of WoUf et c. (19). CDR-dependent cyel~ nuclean& phnaphodlesterase was prepared from bovine brat" hy he procedure of Brostrom and Wolff (20). Rabblt skeletal muscle psrvalbumln wae prepared 89 described by Chllders and siege1 (21) and ts succe~~fu plrlflcptim conflrmed by csz* -hlndlng proprtleii, maleeulsr welght. gel electrophmetie behavior. and its Chareeteristie ultraviolet absorpflon spei~um as described by Lehky et &, (22). Whole troponin YBB prepared from rabbit skeletal mwde as described by Gremer and Gergely (23). Tr-n 1-C camplexes, troponin 1. and Lroponk C we- prepared from rabblt skeletal musole whole troponin as described by Head and Perry (24). Hlstone eontemlng the three myor Subfractions but free from other htatone components was plppared from H enriched fraetmn (S~gma V-S) by gel filtration on a 2.5 x 160 Em colm of Bio Gel P-60 n 0.01 N HC eankwng 0. M NaCl as descllbed by Bohm et. (25). Homogeneous hlstone 2A or 2B we8 pmpared from R2A enrlched fractlon plgma M-S) or H2B enrlched fractlon (Slgma M-S) by elution of unresolved 2A-2B &turea wlth gradlent.? of guanldlne chloride from AmherUte RC-50 BB deserlbed by fambrawh and Bonner (26). The H2A and HZB &tures were resolved nto fhelr lndlvidual components by gel flltraflon on BO Gel P-60 as described for histone. Homogeneous histone 4 is ohtalned from histone 2Aenrlched fraetlon (Sigma V-s) hy gel flltraflon on BO Gel P-60 a8 described for histone 1 (25). molar concentratlana of histone8 2R and 4 were determlned from extinctlon coefflclenta at 276 nm of 6. 7 x 103 and 5.4 x 103 cm"/mol as descrlhed hy D'Anna and lsenberg (28). The molar eoncentratlons of hlstone and 2A were determlned from mlcrokleldahl analye18 of their nitrogen content (29). the lolow" llltragen contents of histones and 2A (30). and their horn molecular wefghte (31). Pre~aratlon Of phosphorylated slbstrafe. - calf thymuil mued histone. hlstones. 2A. or 2B at 4 mg/ml n 80 mm Trll-C ph mm MgCl 20 mm DTT were ncubated with 1 mm r-c?2p)-atp &rier-ladeled) and 10 u$te of the catalytic