©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Activation of Protein Phosphatase 1
FORMATION OF A METALLOENZYME (*)

(Received for publication, March 23, 1995; and in revised form, November 30, 1995)

Yanfang Chu (1) Ernest Y. C. Lee (2) Keith K. Schlender (1)(§)

From the  (1)Department of Pharmacology, Medical College of Ohio, Toledo, Ohio 43699 and (2)Department of Biochemistry and Molecular Biology, University of Miami School of Medicine, Miami, Florida 33101

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The recombinant catalytic subunit of protein phosphatase 1 is produced as an inactive enzyme which can be activated by Mn (Zhang, Z., Bai, G., Deans-Zirattu, S., Browner, M. F., and Lee, E. Y. C.(1992) J. Biol. Chem. 267, 1484-1490). In this report, we have investigated the effects of divalent cations on the activity of recombinant catalytic subunit of protein phosphatase 1. Latent phosphatase 1 can be activated by Co or Mn, whereas other metal ions tested including Fe, Zn, Mg, Ca, Cu, or Ni were not effective or were only weakly effective in activating the enzyme. The Mn-stimulated activity was susceptible to inactivation by EDTA; however, the Co-activated phosphatase was stable after dilution and chelation of the Co with excess EDTA. After stable activation of phosphatase 1 using Co, a stoichiometric amount of Co was shown to be tightly bound to phosphatase 1. These findings demonstrate for the first time the generation of a stable metalloenzyme form of phosphatase 1. Fe reversibly deactivated the Co-stimulated activity, but did not displace the bound Co. Interestingly, treatment of the enzyme with a combination of Fe and Zn (but not the individual metal ions) significantly activated phosphatase 1. These results suggest that at least two metal binding sites exist on the enzyme and that protein phosphatase 1 may be an iron/zinc metalloprotein in vivo.


INTRODUCTION

Protein phosphatase 1 (phosphorylase phosphatase), one of the four major Ser/Thr protein phosphatases, has been studied mainly in relation to its central role in the regulation of glycogen metabolism (for reviews, see Bollen and Stalmans(1992), Shenolikar and Nairn(1991), and Shenolikar(1994)). The enzymology of the enzyme is complex and involves multiple forms of the enzyme generated by combinations of a 37-kDa catalytic subunit (PP1) (^1)with different regulatory proteins that may also provide for molecular targeting of the enzyme. Several regulatory subunits have been well characterized, including inhibitor 2, the glycogen binding subunit, a nuclear inhibitory subunit, and myofibril binding subunits (Bollen and Stalmans, 1992; Shimizu et al., 1994; Chen et al., 1994). Most of the previous studies of the isolated catalytic subunit have been of an active enzyme that is independent of metal ions for its activity. However, it has been clear for a number of years that there exists a metal ion dependent form or forms of PP1. In the ATP/Mg-dependent enzyme, which is a 1:1 complex of PP1 with inhibitor 2, PP1 is present as an inactive or latent enzyme that is reversibly stimulated by Mn (Villa-Moruzzi et al., 1984). All recombinant forms of PP1 expressed in Escherichia coli, including the four known isoforms, are dependent on Mn for activity (Zhang et al., 1992, 1993a; Alessi et al., 1993). Zhang et al. (1993b) have suggested that the recombinant enzyme represents the conformer that is present in the PP1-inhibitor 2 complex. We have recently isolated a form of PP1 catalytic subunit from cardiac muscle which is inactive, but can be converted to a stable active form by exposure to Co (Chu et al., 1994).

Thus, there are complex and not completely understood facets of the nature of the differences between these forms of the PP1 catalytic subunit, which are revealed by the effects of divalent cations on its activity. The question of how metals affect phosphorylase phosphatase is an old one. It has long been known that divalent cations, in particular Mn and Co, can activate certain phosphorylase phosphatase preparations (Merlevede and Riley, 1966; Kato and Bishop, 1972; Kato et al., 1975; Ullman and Perlman, 1975; Khatra and Soderling, 1978; Khandelwal and Kasmani, 1980; Brautigan et al., 1980, 1982). It has been suggested that the phosphatase present in these preparations is a metalloenzyme (Burchell and Cohen, 1978; Hsiao et al., 1978; Khatra and Soderling, 1978; Defreyn et al., 1979; Mackenzie et al., 1980). However, attempts to demonstrate the presence of bound metal in enzyme preparations have been negative. Metal analysis of a preparation of liver PP1 by atomic absorption showed only small substoichiometric amounts of Ca, Cd, Co, Cu, Fe, Mg, Mn, Ni, Sn, or Zn (Yan and Graves, 1982). Moreover, activation by Mn of the catalytic subunit of ATP/Mg-dependent protein phosphatase (Villa-Moruzzi et al., 1984) or a preparation containing a high molecular weight form of PP1 (Brautigan et al., 1980) did not show significant incorporation of Mn into the enzyme.

Using recombinant PP1, we have explored the issue of whether it may exist as a metalloprotein. In this study, we demonstrate that the activation of PP1 by Co is associated with a stoichiometric incorporation of Co into the enzyme. We also report that PP1 is activated by a combination of Fe/Zn and we suggest that PP1 may be an iron/zinc metalloenzyme in vivo.


EXPERIMENTAL PROCEDURES

Materials

CoCl(2) was obtained from ICN. Superose-12 was from Pharmacia Biotech Inc. The recombinant PP1 used in these studies was the PP1alpha isoform expressed in E. coli as described by Zhang et al.(1992). [P]Phosphorylase a was prepared as described previously (Killilea et al., 1978).

Preparation of Metal-depleted Recombinant PP1

Homogeneous recombinant PP1alpha stored in the presence of Mn as described by Zhang et al.(1992) was treated with 5 mM EDTA, 100 mM KPO(4), 1 M KCl, 33% glycerol, pH 7.0, for 2 h at 4 °C, and extensively dialyzed against 50 mM MOPS, pH 7.0, 500 mM KCl, 0.2 mM EDTA, and 50% glycerol, in order to remove Mn and KPO(4).

Assay for Phosphatase Activity

PP1 was preincubated with divalent cations as indicated. The preincubated enzyme was then assayed directly (without chelation of the cation) for phosphatase activity by measuring the release of [P]P(i) from [P]phosphorylase a (Chu et al., 1994). One unit of phosphatase activity was defined as 1 µmol of [P]P(i) released/min.

Determination of Co^2Binding to PP1

Latent PP1 (10 µg) was incubated in 50 µl of 50 mM MOPS, pH 7.0, 500 mM KCl, 0.2 mM EDTA, 50% glycerol, and 1 mMCoCl(2) (specific radioactivity, 600 dpm/pmol) for 15 min at 30 °C. Then the sample was diluted 5-fold with 50 mM MOPS, pH 7.0, 2 mM EDTA, 0.5 mg of BSA/ml. The bound Co was separated from unbound Co on a Superose-12 gel filtration column equilibrated with 50 mM MOPS, pH 7.0, 150 mM KCl, 0.2 mM EDTA, 1 mM DTT, 10% glycerol at 4 °C.

Protein Determinations

Protein concentration was determined by the method of Bradford(1976) using BSA as a standard.


RESULTS

Effects of Divalent Cations on Activation of PP1

After metal depletion with EDTA/P(i) (see ``Experimental Procedures'') recombinant PP1 required divalent metal cations for activity (Table 1). Of the cations examined individually, only Co and Mn were efficient in activating the enzyme. Treatment of PP1 with 1 mM Ca, Cu, Fe, Ni, or 5 mM Mg did not activate the enzyme, while 1 mM Zn had only a slightly stimulating effect (Table 1). Similar results were obtained when metal-dependent PP1 purified from bovine heart myofibrils (Chu et al., 1994) was tested under the same conditions (data not shown). As shown in Table 1, PP1 was activated to a greater extent by 1 mM Mn than 1 mM Co when the preincubation was carried out in the presence of 150 mM KCl. It is worth noting that Co and Mn were equally effective in the activation of PP1 when the preincubation was carried out in the presence of 500 mM KCl, indicating that Co-activation was more dependent on ionic strength than Mn (data not shown). Very surprisingly, combined treatment of PP1 with Zn and Fe significantly activated the enzyme (Table 1). Preincubation with other metal ion combinations had little or no effect on phosphatase activity. As shown in Fig. 1, maximal activation of the enzyme by Zn in the presence of 0.1 or 1 mM Fe, or by Fe in the presence of 0.1 or 1 mM Zn, was achieved at a metal concentration of 1 mM. The A was approximately 0.1 mM for either cation. Activation by the combination of Zn/Fe did not appear to be sensitive to ionic strength (data not shown).




Figure 1: Concentration dependence of PP1 activation by combined treatment with Fe and Zn. The PP1alpha was diluted with 50 mM MOPS, pH 7.0, 150 mM KCl, 0.5 mg BSA/ml, 1 mM DTT, then incubated: A, with varying concentrations of ZnCl(2) in the presence of 0.1 mM (circle) or 1 mM (bullet) FeCl(2), or B, with varying concentrations of FeCl(2) in the presence of 0.1 mM (up triangle) or 1 mM () ZnCl(2) at 30 °C for 15 min. The enzyme activity was assayed as in the legend of Table 1.



Effect of EDTA and P(i) on Co^2- or Mn-activated PP1

The reversibility of the activation of recombinant PP1 was investigated by studying the effects of EDTA and P(i) on Mn- or Co-activated PP1. More than 80% of the Mn-stimulated activity was reversed by chelation of the Mn with 5 mM EDTA (Fig. 2A). It was noted that much of the Mn-stimulated activity was lost simply by dilution of the Mn (data not shown). Mn-activated PP1 was totally inactivated within 5 min by 100 mM P(i). Thus, the effect of Mn on PP1 was properly characterized as a stimulation of the enzyme. Activation by Fe/Zn was also reversed by chelation of the metal ions (data not shown). Co-activated PP1 was resistant to EDTA or P(i) treatment (Fig. 2B). Incubation at 30 °C for up to 2 h with 5 mM EDTA did not reverse Co activation. Inorganic phosphate (100 mM) or a combination of 100 mM P(i) and 5 mM EDTA only partially reversed the Co-activated PP1 even after 2 h incubation. These results indicate the effect of Co on PP1 is an activation, as we previously observed with the latent cardiac PP1 (Chu et al., 1994).


Figure 2: Effect of EDTA and P(i) on Mn- or Co-activated PP1. The PP1alpha was diluted with 50 mM MOPS, pH 7.0, 0.5 M KCl, 0.2 mM EDTA, 1 mM DTT, 0.5 mg BSA/ml. The enzyme was preincubated with 1 mM MnCl(2) (A) or 1 mM CoCl(2) (B) at 30 °C for 15 min. Further incubations were carried out in the absence (control, circle) and presence of 5 mM EDTA (bullet), 100 mM KPO(4) (), 5 mM EDTA plus 100 mM KPO(4) (box). At indicated intervals, an aliquot of the reaction was diluted with 50 mM imidazole-HCl, pH 7.4, 1.2 mg of theophylline/ml, 1 mM DTT, 0.5 mg of BSA/ml, and assayed for phosphatase activity.



Evidence for a Stable Co^2bulletPP1 Complex

Although Co-activated PP1 is stable, it is not clear whether the Co activation involved formation of a stable CobulletPP1 complex or a transient binding of Co which results in the induction of a stable active conformation. Therefore, we examined the Co-binding properties of PP1 by direct binding studies using Co. For analysis of Co binding, the sample of recombinant PP1alpha was depleted of endogenous Mn by EDTA/P(i) treatment (see ``Experimental Procedures''). The enzyme was incubated with CoCl(2) and passed through a Superose-12 gel filtration column to separate free and bound Co. After chromatography, PP1 was fully activated, and Co was associated with the enzyme (Fig. 3). The fact that the bound Co was not removed by chromatography in the presence of EDTA indicates the formation of a stable metalloenzyme complex. This is supported by a calculated stoichiometry of 0.93 mol of Co bound/mol of PP1 for the fractions in which the enzyme activity and radioactivity co-eluted. These results imply that Co is incorporated into PP1 during the enzyme activation and PP1 contains one stable Co-binding site.


Figure 3: Gel filtration chromatography of Co-treated PP1. The PP1alpha (10 µg) was preincubated with 1 mMCoCl(2) in the presence of 0.5 M KCl as described under ``Experimental Procedures.'' The activated enzyme was chromatographed on a Superose-12 column (1.0 times 30 cm) which was equilibrated at 4 °C with 50 mM MOPS, pH 7.0, 150 mM KCl, 0.2 mM EDTA, 1 mM DTT, and 10% glycerol. Fractions of 0.4 ml were collected and assayed for phosphatase activity (circle) and for radioactivity (up triangle).



Effect of Fe^2 on Co^2-activated PP1

Fe itself had very little effect on activation of PP1. However, as shown in the insert of Fig. 4, Fe could inactivate the Co-activated PP1. The possibility that displacement of Co by Fe is responsible for the enzyme inactivation was examined by determining the Co content of the enzyme before and after treatment with Fe. From Fig. 4, it can be seen that Co remained bound, even though phosphatase activity was almost completely lost. The enzyme activity was fully recovered when the Fe was chelated by EDTA indicating that the Fe effect was reversible (data not shown). The fact that Co was not displaced by Fe after the enzyme inactivation suggested that Co and Fe do not share a common binding site.


Figure 4: Deactivation of the Co-activated PP1 by Fe^2+. The PP1alpha (5 µg in 45 µl) was incubated with 1 mMCo in 50 mM MOPS, pH 7.0, 0.5 M KCl, 0.2 mM EDTA, 25% glycerol for 15 min at 30 °C. Co was chelated with 2 mM EDTA and the enzyme was further incubated with 2 mM FeCl(2) for 15 min at 30 °C. The treated enzyme was separated from unbound Co by passing through a Superose-12 gel filtration column (1.0 times 30 cm), which was equilibrated with the buffer containing 50 mM MOPS, pH 7.0, 150 mM KCl, 10% glycerol, and 0.2 mM FeCl(2). The control was prepared as described in the legend of Fig. 3. Fractions of 0.4 ml were collected, assayed for phosphatase activity (circle, bullet) and radioactivity (up triangle, ). The Fe-treated enzyme is plotted with filled symbols, the control with open symbols. Insert, PP1 was incubated with 1 mM Co for 15 min at 30 °C, then was treated with 1 mM Fe for 15 min at 30 °C. The sample was directly assayed as described under ``Experimental Procedures.''




DISCUSSION

As noted in the Introduction, the issue of whether the catalytic subunit of protein phosphatase 1 is a metalloenzyme is an old issue that has not been satisfactorily resolved to date, although the effects of Mn on the enzyme activity have been well documented (see Bollen and Stalmans(1992) for review). Attempts to show the binding of Mn to PP1 have been negative (Brautigan et al., 1980; Villa-Moruzzi et al., 1984). Our data provide an explanation for this in that Mn binding is reversible, whereas Co binding results in the formation of a stable metalloprotein complex. Thus, the use of Co rather than Mn has proven in this study to be more revealing. The data presented here provides the first direct evidence that PP1 is a metalloenzyme by demonstration of a stable 1:1 complex of enzyme and Co. Previously, direct metal analysis of liver PP1 preparations showed the presence of only substoichiometric amounts of metal (Yan and Graves, 1982). While the formation of a cobalt metalloenzyme form is probably not physiological (see below), the results establish that PP1 has the ability to bind a metal ion in a stable and stoichiometric manner. Our studies show that cobalt ion will be a useful tool for the study of the role of metal ions on PP1 activity.

Although Co can bind to and activate PP1 in vitro, it seems unlikely that PP1 is a Co-bound protein in vivo, because the concentration of Co in tissues is less than 1 µM (Iyengar and Woittiez, 1988). The observations that PP1 purified from rabbit skeletal muscle is susceptible to P(i) inhibition (^2)and that Co-activated PP1 is relatively resistant to P(i) inhibition (see ``Results'') also do not support a role for Co. The fact that the amount of Mn in skeletal muscle ranges from 1 to 2 µM (Versieck, 1985) and that Mn did not show significant binding to PP1 catalytic subunit (Brautigan et al., 1980) do not favor the idea that PP1 is activated by Mnin vivo. Our findings that a combination of Fe/Zn, but not the individual metals, can activate PP1 raises the possibility that PP1 is an iron/zinc metalloenzyme. The activation by Fe/Zn was found to have an A of approximately 0.1 mM for both cations. Fe and Zn are present in skeletal muscle in millimolar and near millimolar concentrations respectively (Versieck, 1985; Iyengar and Woittiez, 1988). Interestingly, metal analysis of a purified preparation of the rabbit liver PP1 revealed that although Zn and Fe were present in substoichiometric amounts, there was considerably more Zn and Fe detected than some other metal cations tested (Yan and Graves, 1982). It is likely that the substoichiometric levels of Zn and Fe in the purified enzyme are due to the loss of metal cation during enzyme purification. This speculation is consistent with the fact that the Fe/Zn co-activated PP1 loses phosphatase activity when the cations are removed by chelation.

Fe itself cannot effectively activate PP1 but it can reversibly inactivate the Co-activated enzyme. Even though the Co-activated enzyme was inactivated in the presence of Fe, the bound Co was not removed. These results indicate that the deactivation does not result from the displacement of Co. It may result from Fe binding at another site and/or an Fe-induced conformational change in the enzyme. These results are consistent with two metal binding sites on PP1. Another family of phosphatases, the mammalian purple acid phosphatases, are metalloproteins (Vincent and Averill, 1990a, 1990b). Comparison of the primary structures of purple acid phosphatases and Ser/Thr protein phosphatases have lead Vincent and Averill to speculate that PP1 and phosphatase 2A are iron/zinc metalloenzymes with active sites isostructural with those of the purple acid phosphatase. Our data provide the first experimental evidence to support the postulate that PP1 is an iron/zinc metalloenzyme. It is interesting to note that we recently established that the catalytic subunit of phosphatase 2A can also exist in a divalent cation-dependent form (Cai et al., 1995).

While the suggestion of Vincent and Averill (1990b) that the Ser/Thr protein phosphatase may contain two metal sites was based on weak sequence homologies of the purple acid protein phosphatase with the Ser/Thr protein phosphatases represented by PP1, protein phosphatase 2A, and protein phosphatase 2B (calcineurin), the recent elucidation of the crystal structure of protein phosphatase 2B has now confirmed the existence of iron and zinc in the active site (Griffith et al., 1995). While this manuscript was under review, the crystal structure of recombinant PP1 (alpha-isoform) was reported (Goldberg et al., 1995). This structure shows the presence of two metal ions in the catalytic site. Since the enzyme was prepared in the presence of Mn, the ions were presumed to be Mn. These studies confirm our findings that PP1 is a metalloenzyme and strengthen the view that PP1 may have bound zinc and iron ions at the active site. Given the structural similarities of protein phosphatase 2B with PP1 and protein phosphatase 2A, it seems likely that the latter will possess similar metal ion sites. On the other hand, Zhuo et al.(1993, 1994) reported that Mn or Ni activation of a bacteriophage Ser/Thr protein phosphatase ( PPase) had an apparent single K(m) for each of the divalent metals. The latter results are consistent with one metal ion binding site involved in the activation of PPase. Further studies will be necessary to identify the physiologically important metal ions responsible for PP1 activation.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants HL 36576 (to K. K. S.) and DK 18512 (to E. Y. C. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Pharmacology, Post Office Box 10008, Medical College of Ohio, Toledo, OH 43699-0008. Tel.: 419-381-4184; Fax: 419-381-2871; :Schlender{at}gemini.mco.edu.

(^1)
The abbreviations used are: PP1, catalytic subunit of protein phosphatase 1; DTT, dithiothreitol; MOPS, 3-(N-morpholino)propanesulfonic acid; BSA, bovine serum albumin; A, concentration required for one-half maximal activation.

(^2)
L. Cai and K. K. Schlender, unpublished results.


ACKNOWLEDGEMENTS

We thank Martha Heck for her help in processing the manuscript. We wish to thank Drs. Zhongjian Zhang and Sumin Zhao for preparation of the phosphatase 1alpha and Erwin Reimann for his helpful discussion while this work was in progress.


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©1996 by The American Society for Biochemistry and Molecular Biology, Inc.