(Received for publication, April 28, 1995; and in revised form, July 25, 1995)
From the
The significance of site-specific phosphorylation of cardiac
troponin I (TnI) by protein kinase C and protein kinase A in the
regulation of Ca-stimulated MgATPase of reconstituted
actomyosin S-1 was investigated. The TnI mutants used were T144A,
S43A/S45A, and S43A/S45A/T144A (in which the identified protein kinase
C phosphorylation sites, Thr-144 and Ser-43/Ser-45, were, respectively,
substituted by Ala) and S23A/S24A and N32 (in which the protein kinase
A phosphorylation sites Ser-23/Ser-24 were either substituted by Ala or
deleted). The mutations caused subtle changes in the kinetics of
phosphorylation by protein kinase C, and all mutants were maximally
phosphorylated to various extents (1.3-2.7 mol of phosphate/mol
of protein). Protein kinase C could cross-phosphorylate protein kinase
A sites but the reverse essentially could not occur. Compared to
wild-type TnI and T144A, unphosphorylated S43A/S45A, S43A/S45A/T144,
S23A/S24A, and N32 caused a decreased Ca
sensitivity
of Ca
-stimulated MgATPase of reconstituted actomyosin
S-1. Phosphorylation by protein kinase C of wild-type and all mutants
except S43A/S45A and S43A/S45A/T144A caused marked reductions in both
the maximal activity of Ca
-stimulated MgATPase and
apparent affinity of myosin S-1 for reconstituted (regulated) actin. It
was further noted that protein kinase C acted in an additive manner
with protein kinase A by phosphorylating Ser-23/Ser-24 to bring about a
decreased Ca
sensitivity of the myofilament.
It is
suggested that Ser-43/Ser-45 and Ser-23/Ser-24 in cardiac TnI are
important for normal Ca sensitivity of the
myofilament, and that phosphorylation of Ser-43/Ser-45 and
Ser-23/Ser-24 is primarily involved in the protein kinase C regulation
of the activity and Ca
sensitivity, respectively, of
actomyosin S-1 MgATPase.
In cardiac myocytes, the activation of several types of
receptors, such as
-adrenergic(1, 2, 3, 4, 5) ,
muscarinic(1, 6) , and purinergic (6) dynorphin A(7) ,
endothelin-1(8, 9) , and angiotensin II (10, 11, 12) receptors, stimulates the
hydrolysis of membrane phosphoinositides leading to the generation of
two classes of second messengers, diacylglycerol and inositol
trisphosphate. In many tissues diacylglycerol directly activates both
the conventional Ca
-dependent group of PKC (
)isozymes (
,
,
,
and
) and the novel Ca
-independent group of PKC
isozymes (
,
,
, and
), whereas inositol
trisphosphate, by increasing intracellular Ca
,
indirectly activates Ca
-dependent PKC isozymes (for a
review, see (13) ). It is worth noting that PKC-
and
PKC-
, atypical members of the Ca
-independent
group, are activated by neither diacylglycerol nor Ca
(13) . Several lines of recent evidence indicate involvement of
PKC in cardiac function and development (14, 15) as
well as differential expression of the PKC isozymes in cardiac myocytes
and
tissue(14, 15, 16, 17, 18) .
However, the complex molecular events mediated by PKC (or more
precisely, by the individual isozymes) that are responsible for cardiac
contractility regulation, for example, remain largely unclear. It has
been reported that phenylephrine (
-adrenergic receptor
agonist) elicited transient negative inotropy followed by sustained
positive inotropy(3, 19, 20, 21) ,
endothelin-1 caused monotonic positive inotropy(22) , whereas
dynorphin A (
-opioid receptor agonist) induced negative
inotropy(7) . All three of these distinct receptor agonists are
believed to act, at least in part, through PKC activation. Furthermore,
phorbol esters (such as TPA), potent and long-acting PKC activators,
produced predominantly negative inotropic effects in various cardiac
preparations(23, 24, 25, 26, 27) .
These seemingly paradoxical observations might reflect certain opposing
factors of PKC activation which include the net effects of
intracellular pH change, the size of intracellular Ca
transient, and the states and species of cellular proteins being
phosphorylated.
One target for PKC in the heart is the contractile
apparatus itself. Cardiac TnI and TnT from the thin filament have been
shown to be effective substrates for PKC(28) , and some of the
phosphorylation sites in these proteins have been
determined(29, 30) . Phosphorylation of TnI and/or TnT
by PKC resulted in an inhibition of Ca-stimulated
MgATPase of the reconstituted actomyosin complex (31, 32, 33) or in native myofibril
preparations(32, 34) , an effect associated with
altered interactions among the contractile protein
components(32, 33) . PKC also phosphorylated MLC2 (34, 35, 36) and C-protein (34, 35, 36) in myofibrillar and thick
filament preparations. Phosphorylation of MLC2 by PKC or MLC kinase has
been shown to cause further activation of
Ca
-stimulated MgATPase of thick filament-substituted
myofibrils(36) . Because the overall effect of PKC
phosphorylation of myofibrils (which caused phosphorylation of TnI,
TnT, C-protein, and MLC2) was predominantly an inhibition of
Ca
-stimulated MgATPase, it was suggested that the
inhibitory effect of TnI/TnT phosphorylation could override the
stimulatory effect of MLC2 phosphorylation(35, 36) .
Therefore, it appears that the actual activity (inhibition or
activation) of Ca
-stimulated myofibrillar MgATPase
could be regulated by the relative phosphorylation states of these
proteins (i.e. TnI/TnT versus MLC2). The biological
significance of PKC phosphorylation of contractile proteins has been
substantiated by the findings that their in vitro phosphorylation sites were found to be the same as those in
situ as determined by using living cardiomyocytes incubated with
TPA or
-adrenergic agonist(34, 35) .
Previously, we demonstrated that PKC phosphorylated bovine cardiac
TnI at Ser-43/Ser-45, Ser-78, Thr-144, and other undetermined sites (29) and that phosphorylation of these multiple sites was
associated with an inhibition of Ca-stimulated
actomyosin MgATPase(31, 32) . The effect of
phosphorylation of the specific sites was unknown. We (33) and
others (for reviews, see Refs. 37 and 38) have previously reported that
phosphorylation of Ser-23/Ser-24 by PKA resulted in a decreased
Ca
sensitivity of the myofibrillar MgATPase.
Furthermore, Swiderek et al. (30) found that PKC also
phosphorylated bovine cardiac TnI at Ser-23/ Ser-24.
In the present
study we have systematically investigated the functional consequences
of site-specific phosphorylation with the use of TnI point mutants in
which the phosphorylation sites were substituted by Ala residues and a
truncated mutant in which phosphorylation sites were deleted. The
findings indicated that PKC phosphorylation of Ser-43/Ser-45 was
critical for the inhibition of maximal Ca-stimulated
actomyosin S-1 MgATPase, an effect associated with an apparent
decreased affinity of S-1 for the thin filament. Furthermore, we found
that PKC and/or PKA phosphorylation at Ser-23/Ser-24 also resulted in
decreased Ca
sensitivity of the reconstituted
actomyosin complex. The present study is of interest in view of a
recent report from one of our laboratories on a deletion mutant that
lacks the first 32 amino acids specific to cardiac TnI(39) . It
was suggested that the NH
-terminal extension functions
primarily to provide a means, via phosphorylation at Ser-23/Ser-24, for
regulation of the Ca
sensitivity of the contractile
complex. Similarly, recent studies on deletion mutants of skeletal
muscle TnI (40, 41) suggested that the
NH
-terminal domain surrounding Ser-43/Ser-45 (of the
cardiac sequence) functions to anchor TnI to TnC and other components
of the thin filament and thus represents an important site for possible
regulation by phosphorylation.
The abilities of recombinant mouse cardiac wild-type TnI and
various mutants, in which the identified phosphorylation sites for PKC
and PKA were either substituted or deleted, compared to the native
bovine cardiac TnI, MBP, and histone H1, to serve as substrates for PKC
and PKA were examined, and the kinetic constants are summarized (Table 1). It was found that the mutations caused subtle changes
in the substrate activities (indicated by the V/K
ratios) of the
resulting proteins for PKC, i.e. in a decreasing order,
wild-type > T144A > N32
S43A/S45A > S43A/S45A/T144A >
S23A/S24A. The mutations on PKC phosphorylation sites, in contrast,
caused little or no change in substrate activities for PKA, i.e. wild-type
T144A = S43A/S45A
S43A/S45A/T144A. As
expected, mutants S23A/S24A and N32, in which PKA phosphorylation sites
were, respectively, substituted and deleted, were not significantly
phosphorylated by PKA, indicating that PKA was essentially unable to
phosphorylate the PKC sites. Bovine TnI was an inferior substrate,
compared to mouse wild-type TnI, for both PKC and PKA. Although MBP and
histone H1 were effective substrates for PKC in the presence of 0.3 M KCl (which was required for keeping TnI preparations in the
soluble form), they were not appreciably phosphorylated by PKA under
the same conditions. Because MBP and histone H1 were effectively
phosphorylated by PKA in the absence of 0.3 M KCl (albeit
lower than by PKC), it seemed that PKA was more sensitive to salt
inhibition than PKC.
The time-dependent phosphorylation of TnI preparations and MBP by PKC indicated that the initial reaction rates for these proteins were somewhat different, with S23A/S24A being the least effective substrate (Fig. 1). If the reactions were carried out for an extended time (3 h) with high amounts of PKC and ATP, higher phosphorylation extents of 1.3-2.7 mol of phosphate/mol of protein were obtained for all TnI preparations (Fig. 1). The initial phosphorylation rate by PKA was found to be similar for wild-type, S43A/S45A and T144A, but was slightly higher for S43A/S45A/T144A (Fig. 2). For these TnI preparations, phosphorylation of about 0.8-1.4 mol of phosphate/mol of protein was obtained after prolonged incubation with high amounts of PKA and ATP. As expected, S23A/S24A and N32 were not appreciably phosphorylated by PKA.
Figure 1:
Time-dependent
phosphorylation of various native and recombinant proteins by PKC.
Phosphorylation reactions were carried out for up to 30 min in the
presence of 5 µM [-
P] ATP (3
10
cpm), and for an extended period (180 min) in
the presence of 400 µM [
-
P]ATP
(4-8
10
cpm) and a higher amount of PKC for
the determination of maximal phosphorylation. For all cases, 1.5
µM TnI or its mutants were used for phosphorylation. The
findings were confirmed in another set of experiments. See
``Experimental Procedures'' for further
details.
Figure 2: Time-dependent phosphorylation of various native and recombinant proteins by PKA. The phosphorylation conditions were the same as shown in Fig. 3for PKC except PKA replaced PKC. The findings were confirmed in another set of experiments.
Figure 3: Two-dimensional tryptic peptide maps of various recombinant TnI preparations compared with native bovine TnI. The TnI preparations (6 µM) were exhaustively phosphorylated by PKC for 3 h and the phosphorylation values (moles of phosphate/mol of protein) are shown in parentheses. The findings were confirmed, in entirety or in part, in three to six other experiments. See ``Experimental Procedures'' for further details.
Two-dimensional tryptic peptide maps of various TnI
preparations phosphorylated by PKC (Fig. 3) and PKA (Fig. 4) were examined. A pattern of five P-phosphopeptides was readily detected for bovine TnI
phosphorylated by PKC (Fig. 3). We previously determined, using
bovine cardiac TnI, that the phosphorylated residues were Ser-78 in
peptide 1, Ser-43/Ser-45 in peptide 2, and Thr-144 in peptide 3 (29) . Our previous evidence also suggested that peptide 5
contains phosphorylated Ser-23/Ser-24(34) . The identity of the
phosphorylated residue in peptide 4 is unknown. A similar peptide map
was obtained for mouse wild-type TnI phosphorylated by PKC.
Phosphopeptide 1 was absent from maps of mouse TnI preparations because
Ser-78 in the bovine and rat sequences (50) is replaced by a
nonphosphorylatable Arg residue in the mouse sequence(39) .
Substitutions of Ala for the phosphorylatable residues in the mouse TnI
mutants T144A, S43A/S45A, and S43A/S45A/T144A were confirmed by the
absence of corresponding phosphopeptides in their tryptic peptide maps (Fig. 3). For the mouse TnI mutant S23A/S24A, phosphopeptide 5
was absent while phosphorylation at Ser-43/Ser-45 (peptide 2) was
predominant. Similarly, peptide 5 was absent while phosphopeptides 1,
2, 3, and 4 were present of the rat TnI mutant N32. Rat cardiac TnI (50) has Val at position 76 instead of Ala in the bovine
sequence and thus the N32 mutant would produce a tryptic phosphopeptide
(containing Ser-78) with a different mobility than that from bovine TnI (Fig. 3). In comparison to PKC, PKA almost exclusively and
preferentially phosphorylated Ser-23/Ser-24 in all TnI preparations
tested, as confirmed by the lack of phosphopeptide 5 in maps for N32 (Fig. 4) and S23A/S24A (data not shown). A very minor
phosphorylation at Thr-144 (phosphopeptide 3) by PKA was also noted in
all TnI preparations containing that amino acid residue (Fig. 4).
Figure 4: Two-dimensional tryptic peptide maps of various recombinant TnI preparations. The TnI preparations (6 µM) were exhaustively phosphorylated by PKA for 3 h; the phosphorylation values (moles of phosphate/mol of protein) were 1.3-1.5 for all except <0.2 for N32. The findings were confirmed in another set of experiments. See ``Experimental Procedures'' for further details.
We reported previously that PKC phosphorylation of
bovine cardiac TnI at multiple sites resulted in a reduced maximal
Ca-stimulated MgATPase activity of reconstituted
actomyosin and actomyosin S-1(31) . With the use of the
recombinant TnI mutants, we have now determined which of the
phosphorylation sites were responsible for this effect. Since TnI has
been shown to be an important regulator of the pH-dependent
Ca
sensitivity of cardiac
myofilaments(39, 57, 58) , phosphorylation of
TnI might also affect this pH dependence. We have therefore examined
the MgATPase activity of reconstituted actomyosin S-1 at pH 6.5 in
addition to the standard pH 7.0 (Fig. 5), and the kinetic
constants are summarized (Table 2). Phosphorylation by PKC of
wild-type resulted in 62 and 85% reductions in the maximal
Ca
stimulation when the MgATPase activity was assayed
at pH 7.0 and 6.5, respectively. Similarly, PKC-phosphorylated T144A
caused 78 and 80% reductions of the corresponding values, whereas
phosphorylation of S43A/S45A led to much smaller reductions of 25 and
24%, suggesting that phosphorylation at Ser-43/Ser-45, but not Thr-144,
was primarily responsible for the reduced
Ca
-stimulated activity. Phosphorylation of the
S43A/S45A/T144A produced intermediate reductions, i.e. 34 and
48% at pH 7.0 and 6.5. As with Thr-144, phosphorylation at
Ser-23/Ser-24 (the PKA-preferred sites) could not account for the
reduced maximal Ca
-stimulated activity, because PKC
phosphorylation of S23A/S24A and N32 still resulted in marked
reductions (51-68%) of the enzyme activity. PKC phosphorylation
of bovine TnI reduced the maximal Ca
stimulation to
an extent similar to that reported previously(31) , which was
less than that for phosphorylated mouse wild-type TnI (Table 2).
Figure 5:
Effects of PKC phosphorylation of
wild-type TnI and mutants on the Ca-dependent
stimulation of MgATPase of reconstituted actomyosin S-1. The
unphosphorylated and phosphorylated (1.8-2.3 mol/mol) TnI
preparations were used for reconstitution, and the enzyme activity was
assayed in the presence of varying Ca
concentrations
at pH 6.5 or 7.0. Actomyosin S-1 MgATPase activity in the absence of
Ca
was subtracted from values obtained in the
presence of Ca
and was less than 15% of the total
MgATPase activity. The maximal Ca
stimulated MgATPase
activities for reconstituted actomyosin S-1 containing respective
unphosphorylated TnI preparations assayed at pH 7.0 and 6.5 were taken
as 100%. The findings were confirmed in three or four experiments. See
``Experimental Procedures'' for further details. The curves
drawn are the ``best fits'' of the data to the Hill equation
using nonlinear regression.
The mutations at the phosphorylation sites also affected the
Ca sensitivity of the actomyosin MgATPase ( Fig. 5and Table 2). All of the reconstituted actomyosin
S-1 preparations, containing unphosphorylated TnI mutants as well as
wild-type or bovine TnI, exhibited decreased Ca
sensitivity at pH 6.5 compared to pH 7.0, although the extent of
the decrease varied somewhat among the TnI preparations. However, when
assayed at pH 7.0, higher EC
values for
Ca
(i.e. decreased Ca
sensitivity) of 1.6-2.7 µM were noted for
reconstituted actomyosin S-1 preparations containing unphosphorylated
S43A/S45A, S43A/S45A/T144A, S23A/S24A, and N32, but not T144A (1.1
µM), compared to mouse wild-type (1.2 µM) and
bovine TnI (1.1 µM). Qualitatively similar differences in
Ca
sensitivity were also observed when the
preparations were assayed at pH 6.5. The findings suggested the
importance of Ser-23/Ser-24 and Ser-43/Ser-45 in the regulation of the
thin filament Ca
sensitivity. PKC phosphorylation of
wild-type, T144A, and bovine TnI caused up to 3-fold decreases in
Ca
sensitivity at pH 7.0 and 6.5, but the effect was
less pronounced or even undetectable for other TnI mutants,
particularly N32 and S43A/S45A, further supporting the importance of
Ser-23/Ser-24 and Ser-43/Ser-45 in Ca
sensitivity. No
apparent differences were observed in the basal MgATPase activities (in
the absence of added Ca
) among the actomyosin S-1
preparations containing the recombinant TnI proteins, whether
phosphorylated or unphosphorylated (data not shown).
Because PKC
cross-phosphorylated PKA sites (Ser-23/Ser-24) in TnI (Fig. 3)
and phosphorylation at these sites led to a decreased Ca sensitivity of MgATPase ( Fig. 5and Table 2), we
directly compared the effects of PKC and/or PKA phosphorylation of
wild-type TnI (Fig. 6). The kinetic data are summarized (Table 3). A short (15-min) exposure of TnI to PKC caused the
incorporation of 1.1 mol of phosphate/mol of TnI and phosphorylation of
all PKC sites, but only minor phosphorylation of PKA sites
Ser-23/Ser-24 (phosphopeptide 5) was noted. This short exposure to PKC
produced a 32% decrease in maximal Ca
stimulation and
a 2.2-fold increase in the EC
for Ca
. In
contrast, a brief exposure of TnI to PKA, which allowed exclusive
phosphorylation (1.3 mol of phosphate/mol of protein) at Ser-23/Ser24,
resulted in a 2.9-fold increase in the EC
for
Ca
without appreciably affecting the maximal
Ca
activation. When all sites were phosphorylated
(2.3 mol of phosphate/mol of TnI) due to a short exposure to both PKA
and PKC, there was 55% reduction in this activation, accompanied by a
3.9-fold increase in the EC
for Ca
. The
combined effects of the two enzymes were similar to those produced by a
long (2-h) incubation with PKC, yielding phosphorylation (2.3 mol of
phosphate/mol of TnI) at all sites, a 56% reduction of the maximal
Ca
-stimulated activity, and a 6-fold increase in the
EC
for Ca
. These findings are consistent
with the idea that PKC decreased Ca
sensitivity by
cross-phosphorylating the typical PKA sites (Ser-23/Ser-24) or by
phosphorylating certain PKC sites. Moreover, the actions of the two
enzymes could be additive under certain conditions.
Figure 6:
Effects of phosphorylation by PKC and/or
PKA of wild-type TnI on Ca-stimulated MgATPase of
actomyosin S-1. Wild-type TnI was phosphorylated with or without A, PKC for 15 min; B),PKC for 2 h; C, PKA
for 15 min; and D, PKC + PKA for 15 min. For the
individual incubation conditions, the values of phosphorylation
(mol/mol) are shown in parentheses, and the tryptic phosphopeptide maps
are shown in insets. The findings were confirmed in another
set of experiments. See ``Experimental Procedures'' for
further details. The curves drawn are as in Fig. 5.
The results from
the above experiments ( Fig. 5and Fig. 6) suggested that
PKC phosphorylation of TnI affected its interactions with other
components of the thin filament, preventing full
Ca-dependent activation of the complex. We were also
interested in determining if phosphorylation altered interactions of
the thick and thin filaments. Therefore, we next examined the
concentration-dependent effects of the thin filament (regulated actin),
containing various TnI preparations, on the
Ca
-stimulated MgATPase activity of myosin S-1 (Fig. 7). The kinetic data are summarized (Table 4). PKC
phosphorylation of all of the TnI preparations, except S43A/S45A and
S43A/S45A/T144A, markedly increased (up to 4-fold) the K
(i.e decreased apparent affinity) for
the thin filament. Phosphorylation of all of the TnI preparations
except N32 and S43A/S45A also caused a reduction in the V
, with T144A and S23A/S24A providing the
greater (55 and 43%, respectively) reductions compared to other TnI
preparations (2-33%). This evidence suggested that, while
phosphorylation at Ser-43/Ser-45 was primarily responsible for the
majority of the apparent decreased V
and
affinity of myosin S1 for regulated actin, phosphorylation of TnI at
other sites (excluding Thr-144) may also affect these parameters. We
also noted that the K
of myosin S-1 for thin
filaments containing either unphosphorylated S43A/S45A,
S43A/S45A/T144A, S23A/S24A, or N32 was greater than that containing
wild-type or T144A. These findings suggested that substitution of
Ser-43/Ser-45 or Ser-23/Ser-24 by Ala residues or deletion of the
NH
-terminal sequence containing Ser-23/Ser-24 caused a
decreased affinity of S-1 for the thin filament. In agreement with the
observations of Tobacman and Adelstein(56) , we also observed
that, for all preparations of regulated actin studied, the addition of
Ca
altered primarily the V
of
the MgATPase activity and not the K
of myosin
S-1 for regulated actin (data not shown). In additional experiments, we
found that the binding of 0.3 µM myosin S-1 to 20
µM regulated actin, in the presence of 2.1 mM ATP, was reduced 26-30% in preparations containing
PKC-phosphorylated bovine TnI, wild-type TnI, and T144A, compared to a
10% reduction for the phosphorylated S43A/S45A (data not shown). These
findings underscored the importance of phosphorylation at Ser-43/Ser-45
in regulating the interactions of TnI with other components of the thin
filament and ultimately interactions of the thick and thin filaments.
Figure 7:
Activation of
Ca-stimulated MgATPase of reconstituted actomyosin
S-1 by regulated actin. The unphosphorylated and phosphorylated
(1.8-2.3 mol/mol) TnI preparations were used to reconstitute
regulated actin, and MgATPase of actomyosin S-1 was assayed at pH 7.0
in the presence of varying concentrations of regulated actin. The
enzyme activity in the absence of Ca
was subtracted
from the activity in the presence of Ca
(40
µM) and was less than 10% of the total enzyme activity.
The findings were confirmed in two other experiments. See
``Experimental Procedures'' for further details. The curves
drawn are the ''best fits`` of the data to the Hill equation
using nonlinear regression.
Point mutations of PKC and PKA phosphorylation sites and a
deletion of PKA phosphorylation sites in cardiac TnI did not bring
about gross changes in the general properties of the mutants. The
mutants retained their abilities to reconstitute into functional
actomyosin S-1, which demonstrated typical activation by Ca and pH-dependent Ca
sensitivity of MgATPase.
Certain observations concerning phosphorylation of the mutants by PKC
seemed noteworthy. Although mutations altered phosphorylation kinetics
as expected, the extents of phosphorylation that we observed for the
mutants were quite surprising. If one assumed Ser-23/Ser-24,
Ser-43/Ser-45, Thr-144, and the unidentified site in phosphopeptide 4
in mouse wild-type TnI could be exhaustively phosphorylated, a
stoichiometry of six could be expected. However, we failed to obtain a
maximal value that was higher than three. On the other hand, a
stoichiometry similar to that for wild-type was noted for the triple
mutant S43A/S45A/T144A (e.g.Fig. 1). These findings
seemed to support a hypothesis that phosphorylation of multiple sites
in TnI might be mutually regulating. It is conceivable, therefore, that
phosphorylation of certain sites in wild-type could negatively regulate
or even restrict further phosphorylation of other sites, resulting in
phosphorylation that was much lower than the theoretical value. In
contrast, absence of preferred sites such as in mutant S43A/S45A/T144A
could lead to an extensive and rapid cross-phosphorylation of the PKA
sites (Ser-23/Ser-24) (e.g.Fig. 3), resulting in
phosphorylation that was higher than expected. This
``plasticity'' concerning multisite phosphorylation might be
of special significance, in light of the complex roles played by TnI
(and perhaps TnT as well) in the regulation of contractile activity.
The PKC phosphorylation sites in cardiac TnI are located in specific
functional domains of the protein. Studies on fast skeletal muscle TnI
have indicated that two functional domains exist in the
protein(40, 41, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71) .
The NH-terminal domain of TnI binds strongly to the
COOH-terminal domain of TnC and serves to anchor TnI to the other
components of the Tn complex. The COOH-terminal half of TnI contains a
region, residues 96-116, corresponding to residues 130-150
in the bovine and mouse cardiac sequences(39, 50) ,
that can bind to Tm-actin and inhibit the actomyosin MgATPase activity,
as well as bind to
TnC(40, 41, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71) .
Talbot and Hodges (61) identified the minimal
``inhibitory'' sequence within this region to be comprised of
residues 105-114, corresponding to residues 139-148 in the
bovine cardiac sequence(50) . In the absence of
Ca
, this inhibitory domain of TnI remains bound to
Tm-actin. When Ca
binds to TnC, a conformational
change in the Tn complex occurs causing the inhibitory region, along
with the remainder of the COOH-terminal region of TnI, to bind to
certain sites in the NH
- and COOH-terminal domains of TnC,
leading to removal of inhibition by
TnI(40, 41, 67) . Within this inhibitory
sequence, cardiac TnI has Thr at position 144 in place of Pro in the
skeletal muscle sequences. Using synthetic peptides corresponding to
residues 104-115 of the skeletal muscle sequence and residues
138-149 of the cardiac sequence, Talbot and Hodges (62) showed that Pro or Thr substitutions had no effect on the
inhibitory activity of the peptides. In contrast, Van Eyk and Hodges (64) demonstrated that replacement of these polar residues with
the neutral amino acid Gly caused a 38% loss of inhibitory activity for
the synthetic peptide. We, however, mutated this site in cardiac TnI to
Ala (T144A) and found little or no effects on MgATPase activity.
Because the inhibitory region of TnI contains several basic amino
acids, we suspected that adding a negatively charged phosphate group on
Thr-144 would affect TnI-thin filament interactions sufficiently to
account for the PKC-mediated inhibition of the
Ca
-stimulated MgATPase activity. This, however, was
not the case in that a full inhibitory effect due to phosphorylation by
PKC was retained with the T144A mutant. Because none of our mutants
could be phosphorylated only at Thr-144, we were not able to ascertain
the direct effects of phosphorylation at this site.
Cardiac TnI
differs from fast and slow skeletal muscle TnI primarily in the
NH-terminal region. The presence of a cardiac-specific
extension of 24-36 amino acids at the NH
terminus
suggests that an additional functional domain exists in cardiac
TnI(50, 72) . However, deletion of this region (the
N32 mutant) conferred little or no changes to the inhibition by TnI of
actomyosin MgATPase activity or to overall regulation by Ca
or pH of myofilament function (39) (Table 2).
Reconstituted actomyosin S-1 containing the unphosphorylated S23A/S24A
or N32 mutants did, however, display decreased Ca
sensitivity (Fig. 5, Table 2), suggesting that the
serine residues may be important for normal Ca
sensitivity of the actomyosin complex. A similar effect was
apparent when wild-type TnI was replaced with the N32 mutant in cardiac
myofibril preparations(39) . Furthermore, phosphorylation by
PKA of Ser-23/Ser-24 produced a decreased
Ca
-sensitivity of reconstituted actomyosin and native
myofibrils (34, 37, 38) (Fig. 6).
These results suggest that phosphorylation of Ser-23/Ser-24 alters the
interaction of the NH
-terminal extension with other
functional domains of TnI which regulate the TnI-TnC or TnI-actin
interactions. Although PKA is considered the primary protein kinase for
phosphorylation at these Ser residues, our studies indicated that PKC
can cross-phosphorylate Ser-23/Ser-24 and decrease the Ca
sensitivity of the resulting reconstituted actomyosin complex ( Fig. 5and Fig. 6, Table 2and Table 3). Since
both PKA and PKC can phosphorylate TnI at Ser-23/Ser-24, it was
suggested that PKC, in addition to PKA, could regulate the
Ca
sensitivity of the myofilaments. Simply put, PKA
appeared to regulate primarily the Ca
sensitivity,
whereas PKC modulated both the Ca
sensitivity and
activity of the myofilament MgATPase. It appeared that PKC
phosphorylated more readily Ser-23/Ser-24 (phosphopeptide 5) in mouse
TnI than in bovine TnI (Fig. 3). This difference may account for
the more pronounced decrease in Ca
sensitivity of
MgATPase of actomyosin-containing mouse TnI, compared to bovine TnI,
phosphorylated by PKC when the enzyme was assayed at pH 7.0 (Table 2), and our earlier findings that PKC phosphorylation of
bovine TnI resulted primarily in inhibition of the activity without
consistently affecting the Ca
sensitivity of
actomyosin MgATPase (31, 32, 33) .
As
suggested from studies on skeletal muscle
TnI(40, 41, 59, 65, 66, 67, 68, 69) ,
Ser-43/Ser-45 are located in the ``anchor'' region of cardiac
TnI, residues 33-80(50) , that binds to TnC. Furthermore,
these residues are located in the highly basic segment comprising
residues 37-51 (50) that probably directly interacts with
the acidic protein TnC(59, 68, 69) . The
NH-terminal region of skeletal muscle TnI also interacts
with TnT(70, 71) , implying that this region of TnI
may not be accessible to PKC. However, by using reconstituted Tn, we
demonstrated that these Ser residues were phosphorylated by PKC, even
when TnI was complexed with TnC(29) . Moreover, analysis of
tryptic phosphopeptide maps of TnI from
P-labeled rat
cardiac myocytes treated with phorbol ester or phenylephrine revealed
the presence of a phosphopeptide corresponding to that containing
Ser-43/Ser-45(34) . These results suggested that Ser-43/Ser-45
could be phosphorylated in intact myocytes upon activation of PKC. Due
to the location of these amino acids, it was not surprising to find
that phosphorylation at Ser-43/Ser-45 was responsible for the majority
of the PKC-mediated inhibition of the maximal
Ca
-stimulated MgATPase activity of reconstituted
actomyosin S-1 complex ( Fig. 5and Fig. 6, Table 3and Table 4). Furthermore, simple substitution of
these Ser residues with Ala residues resulted in a significant decrease
in the maximal Ca
-stimulated MgATPase activity (Table 2). We can therefore hypothesize that Ser-43/Ser-45
appeared to be critical for maintaining the maximal
Ca
-stimulated actomyosin MgATPase activity and that
phosphorylation of these residues represents an important mechanism for
regulation (inhibition) of the enzyme activity and, hence, the function
of the contractile apparatus. In addition, the Ala for Ser
substitutions resulted in a decreased Ca
sensitivity
for the reconstituted actomyosin, and phosphorylation of Ser-23/Ser-24
in S43A/S45A could not further decrease the Ca
sensitivity ( Fig. 5and Table 2). This suggested
that Ser-43/Ser-45 may be in the region of TnI which interacts with the
phosphorylated Ser-23/Ser-24 and ultimately, through interactions with
either TnC or TnT, may influence the Ca
sensitivity
of the contractile complex. With the use of additional mutants, we are
currently investigating the more discrete effects of phosphorylation at
Ser-43/Ser-45 on interactions of TnI with other components of the thin
filament, such as binding of TnI to F-actin, TnT and TnC.
Phosphorylation of TnI at Ser-43/Ser-45 also caused a decrease in
the apparent affinity (increased K) of S-1 for
the thin filament, leading to MgATPase inactivation ( Fig. 7and Table 3) and a decrease in the actual binding of S-1
ATP to
the thin filament (data not shown). It seemed that phosphorylation at
Ser-43/Ser-45 would cause a conformational change in TnI such that
TnC
Ca
is prevented from completely
``pulling'' the inhibitory TnI away from certain binding
sites on F-actin, with which myosin interacts. According to the model
of Hill et al.(73) , as modified by
Lehrer(74) , we hypothesize that phosphorylated TnI would then
continue to stabilize a portion of the thin filament in the
``off'' state, preventing the binding of additional
S-1
ATP (i.e. weak binding cross-bridges), thereby
reducing the number of cross-bridges able to isomerize to
``strongly bound'' cross-bridges, and ultimately preventing
the complete myosin head-induced ``off to on'' transition of
Tm. Under the low ionic strength conditions (18 mM KCl) used
in the present study, the binding of S-1
ATP to regulated actin is
only modestly sensitive to Ca
(56, 75) and not inhibited by unphosphorylated
TnI(75) . We would predict, however, that the effects of
phosphorylated TnI would be more pronounced under conditions of
physiological ionic strength (120 mM KCl) where even
unphosphorylated TnI has been shown to affect the binding of
S-1
ATP to the thin filament(76) . We are currently
investigating such possibilities as well as other effects of
phosphorylated TnI on the interactions of the thick and thin filaments.
Adult cardiac tissue is uniquely sensitive to the effects of pH.
Acidotic conditions cause greater reductions in the maximal force,
shortening velocity, and Ca sensitivity of fibers
from adult cardiac muscle, compared to fibers from neonatal cardiac and
slow and fast skeletal
muscles(39, 57, 58, 77, 78, 79) .
These functional effects have been proposed to be the consequence of
pH-sensitive alterations in the binding of Ca
to TnC,
and the differential transmission of the Ca
binding
signal has been suggested to be dependent upon the isoforms of TnI and
TnC(57, 58, 78, 79) . In the present
studies, we found that at pH 6.5, compared to pH 7.0, TnI
phosphorylated by PKC caused greater reductions in the
Ca
-stimulated MgATPase activity of reconstituted
actomyosin S-1 ( Fig. 5and Table 2). This suggested that
effects on myocardial contractility due to phosphorylation of TnI by
PKC may be more pronounced during acidosis.