(Received for publication, March 23, 1995; and in revised form, November 30, 1995)
From the
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.
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) ()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.
Figure 1:
Concentration
dependence of PP1 activation by combined treatment with Fe and Zn
. The PP1
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
in the presence of 0.1 mM (
) or 1 mM (
) FeCl
, or B, with varying
concentrations of FeCl
in the presence of 0.1 mM (
) or 1 mM (
) ZnCl
at 30 °C
for 15 min. The enzyme activity was assayed as in the legend of Table 1.
Figure 2:
Effect of EDTA and P on
Mn
- or Co
-activated PP1. The
PP1
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
(A) or 1 mM CoCl
(B) at 30
°C for 15 min. Further incubations were carried out in the absence
(control,
) and presence of 5 mM EDTA (
), 100
mM KPO
(
), 5 mM EDTA plus 100
mM KPO
(
). 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.
Figure 3:
Gel filtration chromatography of Co
-treated PP1. The PP1
(10 µg)
was preincubated with 1 mM
CoCl
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
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
(
) and for radioactivity (
).
Figure 4:
Deactivation of the
Co-activated PP1 by Fe
+. The
PP1
(5 µg in 45 µl) was incubated with 1 mM
Co
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
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
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
. The control was prepared as described in the
legend of Fig. 3. Fractions of 0.4 ml were collected, assayed
for phosphatase activity (
,
) and radioactivity (
,
). 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.''
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
inhibition (
)and that Co
-activated
PP1 is relatively resistant to P
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
Mn
in 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 (-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
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.