(Received for publication, October 19, 1995; and in revised form, December 15, 1995)
From the
The partially purified myosin-bound phosphatase had an
associated protein kinase that phosphorylated the holoenzyme, primarily
on the large (130-kDa) subunit. Phosphorylation of the 130-kDa subunit
resulted in inhibition of phosphatase activity. The major site of
phosphorylation was threonine 654 of the 130-kDa subunit or threonine
695 of the 133-kDa isoform. Phosphorylation of the large subunit did
not dissociate the holoenzyme. Dephosphorylation of the large subunit
was achieved by the holoenzyme, and addition of the catalytic subunit
of the type 2A enzyme did not increase the rate of dephosphorylation.
The associated kinase was inhibited by chelerythrine, with half-maximal
inhibition at approximately 5 µM (in 150 µM ATP). The associated kinase phosphorylated two synthetic peptides,
one corresponding to the sequence flanking the phosphorylated
threonine, i.e. 648-661 of the 130-kDa subunit, and the
other to a known protein kinase C substrate, i.e. a modified
sequence from the autoinhibitory region of protein kinase C. The
associated kinase was activated by arachidonic and oleic acid and to a
lesser extent by myristic acid. The protein kinase that phosphorylated
the 130-kDa subunit and resulted in inhibition of myosin phosphatase
activity was not identified.
Contraction of smooth muscle involves the phosphorylation of
myosin by myosin light chain kinase. The Ca dependence of this system derives from the activation of myosin
light chain kinase by the Ca
-calmodulin
complex(1) . Relaxation, following a reduction in the
intracellular Ca
concentration, involves
dephosphorylation of LC20 by the myosin light chain phosphatase.
In
the simplest model a fixed relationship should exist among the
Ca concentration, myosin phosphorylation, and force.
However, such was not observed in many studies carried out with intact
or skinned smooth muscle preparations(2) , and the need for an
additional component was raised. This could involve independent
mechanisms (3) or alteration of the Ca
dependence of phosphorylation(4) . Use of Ca
indicators showed that the Ca
dependence of
force could vary under different conditions. Usually higher force was
achieved following agonist stimulation, compared with K
depolarization(5, 6, 7, 8) .
Force could also decrease at constant Ca
levels(9, 10) . Subsequently it was shown that
the changes in force reflected parallel changes in myosin
phosphorylation, and thus the balance of myosin light chain kinase and
phosphatase was altered(11, 12, 13) .
One
mechanism by which this can occur is by inhibition of phosphatase
activity. This would increase myosin phosphorylation at a given
Ca concentration and lead to an increased
Ca
sensitivity. Several laboratories made the
observation that GTP
S increased the Ca
sensitivity of contraction, thus implicating a G protein-linked
mechanism(8, 14, 15) . Kitazawa et
al. (12) suggested that this mechanism resulted in
inhibition of phosphatase activity, and Kubota et al. (16) showed that in homogenates of tracheal muscle GTP
S
inhibited the dephosphorylation of heavy meromyosin. The pathway that
leads from receptor to G proteins to the ultimate inhibition of
phosphatase is not established. Arachidonic acid has been suggested as
a possible messenger(11) . This compound dissociates the
trimeric structure of the phosphatase, leading to inhibition (11) and also is released in smooth muscle at concentrations
and at time courses consistent with a physiological role(17) .
Masuo et al. (18) have suggested that the inhibition
of phosphatase activity is linked to activation of PKC. (
)
There are many reports of phosphatases that can
dephosphorylate smooth muscle myosin (reviewed in (19) ), and
clearly there is the possibility that more than one phosphatase is
involved physiologically. In order to restrict the number of
possibilities the assumption was made that the pertinent phosphatase
should bind to myosin. Using myosin or actomyosin, as a source of the
phosphatase, three laboratories obtained similar preparations; namely,
the myosin phosphatase was composed of 3 subunits, 130, 38, and 20
kDa(20, 21, 22) . The 38-kDa subunit is the
PP1 isoform (also referred to as PP1
(20) ) of the
catalytic subunit (21, 23) and the two other subunits
are putative regulatory or target molecules. Because the trimeric
holoenzyme is thought to bind to myosin (20, 21, 22) it is referred to as the
myosin-bound phosphatase (MBP). An important objective is to determine
the function of each subunit and its interactions within the
holoenzyme. Amino sequences have been derived from cloned cDNAs of each
subunit: the 38-kDa subunit(24) ; the gizzard 130-kDa subunit (21) , the rat 110-kDa subunit(25) , and the 20-kDa
subunit(25) . In gizzard, two isoforms of the large subunit
exist, i.e. 130 and 133 kDa, differing by an insert in the
central part of the molecule, residues 512-552(21) . The
130-kDa subunit is the major isoform.
Recently it was shown that
treatment of -toxin-permeabilized portal vein with ATP
S
resulted in increased Ca
sensitivity of force output
and reduced phosphatase activity(26) . This was achieved under
conditions where LC20 thiophosphorylation by myosin light chain kinase
was minimum. However, a number of high molecular weight proteins were
thiophosphorylated, and among these was the large phosphatase subunit,
equivalent to the gizzard 130-kDa subunit(26) . Thus, the
possibility was raised that phosphorylation of the 130-kDa subunit may
be a regulatory mechanism for phosphatase activity. To investigate this
further we examined the isolated gizzard phosphatase and found that a
copurifying kinase phosphorylated the 130-kDa subunit and inhibited
phosphatase activity. These results are presented below.
Figure 1:
Phosphorylation of
MBP. Lane 1, SDS-PAGE of MBP after chromatography on Mono S
HR; lanes 2-9, autoradiograms from a time
course of phosphorylation corresponding to 0, 2.5, 5, 10, 20, 30, 45,
and 60 min, respectively; lanes 10-13, autoradiograms showing time course of digestion by
-chymotrypsin. MBP was phosphorylated for 30 min and digested at
25 °C with
-chymotrypsin (1:1000
-chymotrypsin:MBP (w/w))
for 0, 1, 2, and 3 min (lanes 10-13, respectively). The three arrows indicate the positions of the major phosphatase
subunits at 130, 38, and 20 kDa. The major products of proteolysis (i.e. 72 and 60 kDa) are also indicated by the two
arrows.
Brief digestion of MBP with
-chymotrypsin cleaved the 130-kDa subunit into a C-terminal
fragment of 72 kDa and an N-terminal fragment of 58-60
kDa(33) . Both fragments were phosphorylated (Fig. 1).
Further digestion resulted in an accumulation of
P-labeled
lower molecular weight components.
The P-labeled 130-
and 20-kDa subunits were eluted from gel slices, and their phosphoamino
acid content was determined. As shown in Fig. 2the 130-kDa
subunit contained both phosphoserine and phosphothreonine. The 20-kDa
subunit contained only phosphoserine (data not shown). Phosphotyrosine
was not detected. The C-terminal and N-terminal
-chymotryptic
fragments of the 130-kDa subunit also were analyzed. The 58-kDa
N-terminal part of the molecule contained phosphoserine, and the
C-terminal fragment contained phosphothreonine.
Figure 2:
Phosphoamino acids of the 130-kDa subunit.
Positions of the standard phosphoamino acids are shown for comparison
with the autoradiogram indicating the P-labeled
phosphoamino acids of the 130-kDa subunit, namely phosphoserine and
phosphothreonine.
The time course and stoichiometry of phosphorylation for MBP and its subunits is shown in Fig. 3. For the holoenzyme, a relatively rapid phosphorylation to a level of 1 mol of phosphate/mol of MBP was observed, followed by slower phosphorylation at an additional site(s). The rapid phase of phosphorylation was accounted for by phosphorylation of the 130-kDa subunit (Fig. 3), as phosphorylation of the 20-kDa subunit was relatively slow. These results suggest that there is one preferred site for the endogenous kinase on the 130-kDa subunit and additional secondary site(s).
Figure 3:
Stoichiometry of phosphorylation of MBP
and subunits. MBP () was phosphorylated, and
P
incorporation was measured directly. Phosphorylation of 130-kDa subunit
(
) and the 20-kDa subunit (
) was estimated from bands on
SDS-PAGE (see ``Experimental Procedures''). Error bars indicate S.D. (n = 4).
The stoichiometry of phosphorylation was not
affected by inhibitors of the cAMP-dependent protein kinase, i.e. the heat-stable inhibitor (up to 0.1 mg/ml) and H8 (up to 100
µM at 150 µM ATP). In addition, the myosin
light chain kinase inhibitor, ML9 (up to 300 µM at 150
µM ATP) had no effect. Phosphorylation was not
Ca- and/or calmodulin-dependent. Thiophosphorylation,
using
S-labeled ATP
S, showed a similar time course
(to phosphorylation) and similar stoichiometries. MBP isolated from
fresh chicken gizzards was indistinguishable from the MBP isolated from
frozen gizzards with respect to these phosphorylation profiles. The
phosphorylation was Mg
-dependent, with a maximum rate
at approximately 1 mM MgCl
(lower concentrations
were not tested). The ATP-dependence of phosphorylation was measured
for the linear phase of phosphorylation (at 4 min, see Fig. 3).
The maximum phosphorylation rate was at approximately 300 µM ATP, and the half-maximum rate was at approximately 45 µM ATP. The phosphorylation rate was not affected by increasing ionic
strength, to about 0.5 M NaCl, but was inhibited at 1 M NaCl, to about 40%. Myelin basic protein (0.25 mg/ml), histone III
S (0.25 mg/ml), and
-casein (0.5 mg/ml) were not phosphorylated by
the MBP-associated kinase.
Figure 4:
Effect of phosphorylation on activity of
MBP. At each time point the sample was diluted 100 with 50
mM Tris-HCl (pH 7.5), 0.5 mM dithiothreitol, and 0.1
mg/ml bovine serum albumin and used for phosphatase assays with
P-myosin (
) and
P-LC20 (
). Error bars indicate S.D. (n =
4).
The relationship between the stoichiometry of
phosphorylation and the extent of inhibition was determined. MBP was
phosphorylated to varying levels, and the corresponding phosphatase
activities were assayed with P-myosin and
P-LC20. In addition, the degree of phosphorylation for the
130-kDa subunit was estimated and related to phosphatase activity. As
shown in Fig. 5A, the phosphatase activity was progressively
inhibited as the extent of phosphorylation increased. Maximum
inhibition was achieved at about 1 mol of phosphate/mol of 130 kDa
subunit (Fig. 5B). Similar effects were observed with
thiophosphorylation, using [
S] ATP
S.
Figure 5:
Phosphorylation of MBP and the 130-kDa
subunit and its effect on activity of MBP. Two aliquots were withdrawn
at each point; one was used for estimation of P
incorporation into MBP (A) or the 130-kDa subunit (B), and the second was used for phosphatase assays (as in Fig. 7) using
P-myosin (
), or,
P-LC20 as substrate (
).
Figure 7:
Dephosphorylation of MBP. Time courses of
dephosphorylation of MBP (initially 1.3-1.4 mol of phosphate/mol
of MBP) are shown with the following: MBP alone (); MBP plus 2
nM okadaic acid (
); MBP plus 1 µM okadaic
acid (
); MBP plus 100-ng (0.5 µg/ml) catalytic subunit of PP2A
(
).
The endogenous kinase was removed by gel filtration of MBP (see ``Experimental Procedures''), and the 130-kDa subunit was then thiophosphorylated by the catalytic subunit of the cAMP-dependent protein kinase (10 µg/ml). Approximately 2 mol of thiophosphate/mol 130-kDa subunit were incorporated, but this did not affect phosphatase activity. (Note that after gel filtration of MBP, incubation with ATP for 30 min resulted in less than 20% inhibition of phosphatase activity). Subsequent phosphorylation of the thiophosphorylated 130-kDa subunit by a crude kinase fraction (obtained by gel filtration) inhibited phosphatase activity by 70%. These results suggest that the cAMP-dependent protein kinase did not phosphorylate the inhibitory site(s) on the 130-kDa subunit.
To determine if phosphorylation of MBP caused dissociation of the trimeric subunit structure the thiophosphorylated (approximately 1.5 mol of thiophosphate/mol of MBP) and nonphosphorylated MBP were applied to gel filtration. The elution profiles for the two MBP samples were identical, and there was no indication of dissociation of the catalytic subunit (Fig. 6). The elution profile for the isolated catalytic subunit of PP1 also is shown in Fig. 6.
Figure 6:
Gel filtration of MBP and PP1c. Samples
applied to the Hiprep Sephacryl S-300 16/60 column were as follows:
thiophosphorylated MBP, 1.4 mol of thiophosphate/mol of MBP ();
nonphosphorylated MBP (
) and PP1c (
). The elution profiles
were detected by phosphatase assays using
P-LC20 as
substrate. Arrows indicate (from left) void volume
and elution positions of thyroglobulin (670 kDa),
-globulin (158
kDa), ovalbumin (44 kDa), and myoglobin (17 kDa). The estimated masses
from the elution positions were 240 kDa for MBP and 38 kDa for
PP1c.
In addition, the thiophosphorylated and
nonphosphorylated samples were applied to a monoclonal antibody
affinity column (see ``Experimental Procedures''). The bound
proteins were eluted at low pH and applied to SDS-PAGE. Western blots
were carried out using the monoclonal antibody to the 130-kDa subunit
and a polyclonal antibody to PP1. Both the thiophosphorylated and
nonphosphorylated MBP samples showed a similar subunit composition,
again providing evidence that phosphorylation of MBP did not dissociate
the holoenzyme.
Some kinetics of the inhibitory effect were
determined. MBP was phosphorylated to 1.2-1.3 mol of
phosphate/mol of 130-kDa subunit, and its activity was determined as a
function of either P-LC20 or
P-myosin. The
constants derived from double-reciprocal plots are shown in Table 2. Using
P-LC20 as substrate the only effect
of phosphorylation was a decrease in V
. With
P-myosin as substrate the effect of phosphorylation was
more complex and involved a reduction of V
and
an increase in K
.
The phosphorylated MBP was incubated for 60 min
(conditions as in Fig. 7) and assayed for phosphatase activity
with P-LC20 as substrate. Full recovery of light chain
phosphatase activity occurred when MBP was dephosphorylated.
Figure 8:
Inhibition of MBP-associated kinase by
chelerythrine. Phosphorylation of the 130-kDa subunit was estimated
after 30 min at 30 °C in 30 mM Tris-HCl (pH 7.5), 1 mM MgCl, 0.1 mM EGTA, 0.5 mM dithiothreitol, 150 µM [
-
P]ATP, 1 µM microcystin-LR, 2 µg MBP, and varying concentrations of
chelerythrine. Chelerythrine was dissolved in Me
SO (3
mM stock solution), and all assays contained 4%
Me
SO. In the absence of chelerythrine the level of
phosphorylation was 1.3 mol of phosphate/mol of 130-kDa subunit. Other
values were normalized to this (as 100%). Error bars indicate
± S.D. (n = 3).
Two synthetic peptides were tested as substrates
for the MBP-associated kinase. One is a known PKC substrate, based on
the autoinhibitory region of PKC. The second was a peptide
incorporating the sequence around the phosphorylated Thr of the 130-kDa
subunit, Arg
to Asp
of the 130-kDa subunit,
or 689-702 of the 133-kDa subunit(21) . Both peptides
were phosphorylated by the MBP-associated kinase, as shown in Fig. 9. The peptide from the 130-kDa subunit sequence was
slightly more effective as a substrate, and its rate of phosphorylation
was faster than the
PKC peptide. Addition of excess 130-kDa
peptide to MBP blocked phosphorylation of the 130-kDa subunit. At 300
µM peptide the phosphorylation of MBP was reduced by 50%,
and at 600 µM peptide it was reduced by 90%. As
phosphorylation of MBP was reduced (by added peptide) the inhibition of
MBP phosphatase activity also was reduced. At the higher levels of
peptide (600 µM) no inhibition of MBP was observed.
Figure 9:
Phosphorylation of synthetic peptides by
MBP-associated kinase. Assays with 130-kDa/133-kDa peptide () and
with modified
PKC peptide (
) are shown. The amount of MBP
used (0.5 µg) was chosen to give linear kinetics over this time
course. Conditions were as follows: 30 mM Tris-HCl (pH 7.5), 1
mM MgCl
, 0.5 mM dithiothreitol, 150
µM [
-
P]ATP, 0.5 mg/ml bovine
serum albumin, 1 µM microcystin-LR, and 100 µM peptide.
The MBP-associated kinase phosphorylated the PKC sites on LC20. Incubation of MBP with 5 µM LC20 at 30° for 20 min, under the standard conditions of phosphorylation, phosphorylated approximately 0.3 mol of phosphate/mol of LC20 at the various PKC sites (shown by peptide mapping). LC20 that was thiophosphorylated at S19 by myosin light chain kinase also was phosphorylated by MBP, to a similar stoichiometry.
The effect of various fatty acids on the activity of the MBP-associated kinase was tested. The results are shown in Fig. 10. Arachidonic acid and oleic acid activated the kinase, and these were effective over a range of 200-300 µM fatty acid. Activation by myristic acid was less efficient, and maximum activation occurred at about 1 mM. Phorbol 12-myristate 13-acetate (up to 200 nM) and phosphatidyl serine (to 100 µg/ml) had no effect on kinase activity, either alone or in combination.
Figure 10:
Effect of fatty acids on the activity of
the MBP-associated kinase. Phosphorylation of the 130-kDa subunit was
estimated after 4 min with various concentrations of arachidonic acid
(), oleic acid (
), and myristic acid (
). Conditions
were as in Fig. 9. Fatty acids were dissolved in
Me
SO, and all assays contained 2%
Me
SO.
The above results show that a kinase present in the MBP
preparations phosphorylated the 130-kDa subunit and that
phosphorylation inhibited phosphatase activity. The mechanism of
inhibition is not known, and the interactions among the three subunits
need to be documented before a reasonable model can be presented. Some
general comments, however, can be made. With phosphorylated myosin as
substrate, the phosphorylation of MBP caused a decrease in V and a slight increase in K
to about 60 µM. Both factors may be involved, and
the concentration of phosphorylated LC20 in vivo is expected
to be between 10 and 60 µM. It is unlikely, however, that
dissociation of the trimeric structure of the holoenzyme occurred as a
consequence of phosphorylation since similar elution positions for
phosphorylated and dephosphorylated MBP were obtained on gel
filtration.
The putative inhibitory site is Thr (or
Thr
for the 133-kDa subunit) in the C-terminal half of
the molecule. Although this is the major phosphorylation site it cannot
be concluded that it is the only inhibitory site. Other sites in this
region of the molecule may also be inhibitory, and the contribution of
phosphorylation at the minor sites has not been evaluated. Thus the
assignment of Thr
as the inhibitory site is tentative.
The corresponding residue in rat M110 is Thr
, and this is
flanked by sequences identical to the chicken 130/133-kDa subunits (25) . Phosphorylation of the rat MBP has not been
investigated. The effects induced by phosphorylation are not known, and
an obvious deficiency in this area is that the functions of different
regions of the 130-kDa subunit have not been determined. Only a few
properties have been proposed. It is suggested that the ankyrin repeat
sequences in the N-terminal half may be involved in myosin binding, and
this is based on characteristics of other proteins containing the
ankyrin repeat. Certainly this is not conclusive, but it is known that
the 58-kDa N-terminal fragment binds to myosin(21) . This
fragment also binds the 38-kDa catalytic subunit(23) . Based on
a linear model for the 130-kDa subunit it is difficult to imagine how
phosphorylation at Thr
could affect distant interactions.
Obviously, folding of the 130-kDa subunit is possible, and in addition
the interactions of the 20-kDa subunit should be considered. These may
involve C-terminal interactions since both the 20-kDa (rat and gizzard)
and rat M110 subunits contain C-terminal leucine zipper
sequences(25) . The gizzard 130-kDa subunit does not contain
this sequence. The C-terminal part of the molecule, however, appears to
bind the 20-kDa subunit since the latter is not present with the
58-38-kDa complex (23) . Although the 58-kDa subunit was
phosphorylated by the endogenous kinase, albeit slowly, its phosphatase
activity was not affected. Thus, the focus for the
phosphorylation-dependent inhibition of MBP should be on interactions
involving the C-terminal half of the 130-kDa subunit.
The kinase
responsible for phosphorylation of the 130-kDa subunit is not
identified. Based on the sequence flanking Thr, two
kinases are suggested, namely the cAMP-dependent protein kinase and
PKC. It is unlikely that the former kinase is involved since the
endogenous kinase was not affected by cAMP-dependent protein kinase
inhibitors, nor did phosphorylation of the 130-kDa subunit by
cAMP-dependent protein kinase influence phosphatase activity. The case
for PKC also is not compelling. PKC-like characteristics include
inhibition by chelerythrine, a reasonably specific inhibitor of
PKC(39) , and phosphorylation of known PKC sites, i.e. the
PKC peptide and LC20. But the endogenous kinase was not
activated by phorbol ester or phospholipids, and it was not inhibited
by other PKC inhibitors. It is possible that the endogenous kinase was
subject to proteolysis, and although this may explain the spontaneous
activity in the MBP preparations, it would not account for all of the
observed characteristics. Thus the identity of the endogenous kinase
remains to be established. If this kinase is involved in the
contraction cycle in smooth muscle then it is assumed that its activity
would be regulated. One possibility is that the kinase is the last link
of a signal cascade that results in inhibition of MBP and alteration of
the Ca
sensitivity of myosin phosphorylation.
If
phosphorylation of MBP has a regulatory function then an effective
dephosphorylation mechanism should exist, and this should occur within
the time frame observed for physiological effects. From the available
data it is difficult to assign an accurate time period for the recovery
phase. The time required for restoration of full MBP activity, assuming
this is linked to dephosphorylation of MBP, has not been determined,
but intuitively a period of 1 or 2 min might be the upper limit. The in vitro dephosphorylation rate was slow, and 50%
dephosphorylation required over 20 min. Obviously, the in vivo rate might be higher, due to slightly higher temperature, etc.,
but it should also be considered that another phosphatase is required
for rapid dephosphorylation of MBP. If dephosphorylation by the
PP1 isoform is not adequate, then it is important to determine
which of the other phosphatases might be involved.
In summary, these
results have shown that the large subunit of the trimeric MBP can be
phosphorylated and that this results in inhibition of myosin
phosphatase activity. Previously it was found that incubation of portal
vein preparations with ATPS caused inhibition of phosphatase
activity and thiophosphorylation of several proteins, including the
phosphatase subunit(26) . Thus the in vitro data
support the more physiological experiments and suggest that inhibition
of phosphatase via phosphorylation may play an important regulatory
role in smooth muscle. The identity of the kinase involved is not
known, but it has some characteristics of PKC. Priorities for future
research involve characterization of the kinase and identification of
the presumed link(s) between the agonist-induced membrane event and
activation of the kinase at the level of the contractile apparatus.