(Received for publication, September 7, 1995; and in revised form, February 2, 1996)
From the
Calponin has been implicated in the regulation of smooth muscle
contraction through its interaction with F-actin and inhibition of the
actin-activated MgATPase activity of phosphorylated myosin. Both
properties are lost following phosphorylation (primarily at serine 175)
by protein kinase C or calmodulin-dependent protein kinase II. To
evaluate further the functional importance of serine 175, wild-type
calponin and three site-specific mutants (S175A, S175D, and S175T) were
expressed in Escherichia coli and compared with calponin
purified from chicken gizzard smooth muscle in terms of actin binding,
actomyosin MgATPase inhibition, and phosphorylation by protein kinase C
and calmodulin-dependent protein kinase II. The affinities of skeletal
muscle F-actin for wild-type and S175T calponins were similar to that
for the tissue-purified protein (K = 0.8, 1.3, and 1.0 µM, respectively),
whereas the affinities for S175A and S175D calponins were much lower (K
= 26.8 and 44.2
µM, respectively). Tissue-purified, wild-type, and S175T
calponins displayed comparable inhibition of the smooth muscle
actin-activated myosin MgATPase, whereas S175A and S175D calponins were
much less effective. Phosphorylation confirmed serine 175 as the
principal site of phosphorylation by both kinases. These results
indicate that the hydroxyl side chain at position 175 of calponin plays
a critical role in the binding of calponin to actin and inhibition of
the cross-bridge cycling rate.
Calponin is a 32-kDa, thin filament-associated protein that has
been implicated in the regulation of smooth muscle
contraction(1, 2, 3, 4) . Through
its interaction with actin, calponin inhibits the actin-activated
MgATPase activity of phosphorylated smooth muscle myosin (1) and inhibits the relative movement of actin and myosin in in vitro motility assays(5, 6) . Exogenous
calponin attenuated Ca-induced contractions of
permeabilized rabbit mesenteric arterial smooth muscle strips in a
concentration-dependent manner(7) . Inhibition of the
actomyosin ATPase occurs predominantly by a reduction of V
rather than an effect on the affinity of
phosphorylated myosin heads for
actin(8, 9, 10) . The inhibitory effect of
calponin can be alleviated by phosphorylation catalyzed by protein
kinase C (PKC) (
)or
Ca
/calmodulin-dependent protein kinase II (CaM kinase
II) (1, 11) and restored following dephosphorylation
by type 2A (12) or 2B protein phosphatases(13) .
Phosphorylation-induced loss of inhibition results from a marked
reduction in the affinity of phosphorylated calponin for
actin(1) . Several examples of phosphorylation of calponin in
intact muscle in response to various contractile stimuli have been
reported(11, 14, 15, 16) , and in
response to phenylephrine (an
-adrenergic agonist
known to trigger activation of PKC) calponin translocates from
cytosolic filamentous structures to a region underlying the
sarcolemma(17) . Other investigators, however, have reported
that calponin is not phosphorylated in intact
muscle(18, 19, 20) . The principal site of
phosphorylation by either kinase is serine 175(11) , although
Nakamura et al. (21) suggested that threonine 184 was
the principal residue phosphorylated by PKC. Mezgueldi et al. (22) have defined the actin-binding domain of calponin as
residues 145-182, which includes serine 175.
To investigate further the importance of serine 175 in the interaction of calponin with actin and inhibition of the actin-activated myosin MgATPase, we have expressed in bacteria wild-type calponin and three site-specific mutants in which serine 175 was replaced by alanine, aspartic acid, or threonine. The following properties of the expressed proteins were compared with calponin purified from chicken gizzard smooth muscle: (i) protein conformation, (ii) actin binding, (iii) inhibition of actin-activated myosin MgATPase activity, and (iv) phosphorylation by PKC and CaM kinase II. The results indicate that serine 175 plays a critical role in the interaction of calponin with actin and ATPase inhibition; in particular, a hydroxyl group in this position is essential for these functions. The results also confirm serine 175 as the principal site of phosphorylation by PKC and CaM kinase II.
Two experimental approaches were used to determine whether or not the conformations of the mutant recombinant calponins were similar to that of wild-type calponin or calponin purified from chicken gizzard smooth muscle: (i) limited proteolysis and (ii) binding of a panel of anti-calponin monoclonal antibodies with distinct epitopes under nondenaturing conditions.
Fig. 1compares the time courses of proteolysis of tissue-purified (A), wild-type (B), S175A (C), S175T (D), and S175D (E) calponins by chymotrypsin. No significant differences are seen between the patterns of proteolysis of the five calponin species. Similar results were obtained with protease K, trypsin, and S. aureus V8 protease (data not shown). As shown in Fig. 2, the time courses of digestion of phosphorylated and unphosphorylated calponins by protease K were not significantly different. Similar results were obtained with trypsin, chymotrypsin, and S. aureus V8 protease (data not shown). We conclude, therefore, that the conformations around the protease-sensitive sites in calponin are conserved in the bacterially expressed wild-type protein, the three site-specific mutants, and phosphorylated calponin. Any alterations in functional properties of the mutants can therefore be assigned directly to the mutation rather than to a gross conformational difference in the mutant protein.
Figure 1: Partial proteolysis of tissue-purified, wild-type, and mutant calponins. Calponins were subjected to limited proteolysis by chymotrypsin as described under ``Experimental Procedures.'' The time courses of digestion were analyzed by 0.1% SDS/7.5-20% polyacrylamide gradient slab gel electrophoresis. A, calponin purified from chicken gizzard; B, bacterially expressed wild-type calponin; C, S175A; D, S175T; E, S175D calponins. The numbers indicate times of digestion (min), and the arrowheads denote the four major fragments generated by proteolysis.
Figure 2: Partial proteolysis of phosphorylated and unphosphorylated calponins. Tissue-purified calponin was incubated in the absence and the presence of PKC under phosphorylating conditions as described under ``Experimental Procedures'' prior to the addition of protease K. Samples were withdrawn at the indicated times (min) after the addition of the protease for SDS-PAGE and autoradiography. A, Coomassie Blue-stained gel; B, autoradiogram.
Fig. 3shows competition binding curves illustrating the binding of four monoclonal antibodies to recombinant wild-type calponin and the two mutants, S175A and S175D, which exhibited functional differences compared with wild-type calponin (see below). The titration curves showed similar blocking patterns for all four monoclonal antibodies by the wild-type and S175A calponins, indicating no detectable structural differences. The S175D mutant showed slightly decreased affinity for monoclonal antibodies CP1, CP3, and CP4 as compared with that of the wild-type or S175A calponins, indicating a minor structural change induced by introduction of a negatively charged residue at position 175. In additional experiments, it was observed that these four and an additional three monoclonal antibodies gave similar binding patterns to solid phase-immobilized wild-type, S175A, and S175T calponins. However, the S175D mutant exhibited lower maximal binding to CP1, 3, 4, 5, and 7 (data not shown).
Figure 3: Epitope competition assays between wild-type recombinant, S175A, and S175D calponins and four specific monoclonal antibodies. Wild-type calponin was coated on microtiter plates and analyzed by enzyme-linked immunosorbant assay for its interaction with monoclonal antibodies CP1, 3, 4, and 8 in the presence of 0.001-10 µg/ml serial dilutions of wild-type, S175A, or S175D calponins. The competition curves were plotted as the binding of monoclonal antibody to the immobilized wild-type calponin versus the concentration of competing protein in solution.
The binding of bacterially expressed calponins to smooth muscle actin or actin/tropomyosin was evaluated by a co-sedimentation assay. Each calponin was mixed with chicken gizzard actin with or without tropomyosin as described under ``Experimental Procedures'' and centrifuged at high speed to separate F-actin and bound proteins from G-actin and unbound proteins. Calponin purified from chicken gizzard (4 µM) bound to smooth muscle actin-tropomyosin as expected (Table 1); 74% of the calponin was recovered in the pellet fraction under these conditions. Wild-type calponin and the S175T mutant showed similar binding to actin/tropomyosin; 71 and 69% of the respective calponins was recovered in the pellet. On the other hand, S175A and S175D showed much reduced binding to actin/tropomyosin; 14% of S175A and 30% of S175D was recovered in the pellet. Similar results were obtained in the absence of tropomyosin; the proportions of wild-type calponin, S175A, S175D, and S175T mutant calponins recovered in the pellet associated with smooth muscle F-actin were 86, 5.6, 33, and 69%, respectively (means of two experiments). Similar results were also obtained with skeletal muscle actin (Table 1); the proportions of tissue-purified calponin, wild-type calponin, S175A, S175D, and S175T mutant calponins recovered in the pellet were 71, 72, 28, 20, and 66%, respectively.
We noticed that the proportion of
sedimented smooth muscle actin was reduced in the presence of the three
calponin mutants compared with tissue-purified or wild-type calponin,
whereas the sedimentation of skeletal muscle actin was unaffected by
the different calponins (Table 1), suggesting that the three
mutant calponins may have a minor effect on smooth muscle actin
polymerization or cause a small degree of actin filament severing
producing short filaments that do not sediment upon high speed
centrifugation. Consequently, skeletal muscle actin/smooth muscle
tropomyosin was chosen as the system to quantify the affinities of
actin for the various calponin species to facilitate accurate
quantification of the amount of actin-bound calponin as the calponin
concentration is varied over the range 1.75-4.5 µM as described under ``Experimental Procedures.'' The
following K values for actin/tropomyosin binding
were determined from the Scatchard plots shown in Fig. 4; 1.0
µM for calponin purified from chicken gizzard, 0.8
µM for wild-type calponin, and 1.3 µM for
S175T. These values are not significantly different, indicating that
the bacterially expressed wild-type protein and the S175T mutant bind
to actin with the same affinity as the native protein. The S175A and
S175D mutants, on the other hand, bound to actin with much lower
affinity; K
= 26.8 µM for
S175A and 44.2 µM for S175D.
Figure 4: Scatchard analysis of the binding of tissue-purified, wild-type, and S175T calponins to F-actin. Actin binding of tissue-purified (A), wild-type (B), and S175T calponin (C) was quantified as described under ``Experimental Procedures,'' and the results are presented as Scatchard plots.
The effects of calponin and its mutants on the actin-activated MgATPase activity of smooth muscle myosin are compared in Fig. 5. As expected, calponin purified from chicken gizzard markedly inhibited the ATPase. A similar degree of inhibition was observed with the bacterially expressed wild-type calponin (20.8% of control ATPase activity was retained at 4 µM and 12.4% at 5 µM calponin) and the S175T mutant (Fig. 5). The S175A and S175D mutants, however, were relatively ineffective in inhibiting the actomyosin ATPase, consistent with their much reduced affinities for actin. SDS-PAGE and autoradiography of samples at the end of the ATPase reactions verified that phosphorylation of the 20-kDa light chain of smooth muscle myosin was unaffected by any of the calponin species (data not shown).
Figure 5:
Concentration dependence of the effects of
tissue-purified and mutant calponins on the actin-activated MgATPase
activity of smooth muscle myosin. Actomyosin ATPase activities were
measured as described under ``Experimental Procedures'' at
the indicated concentrations of chicken gizzard calponin (), S175A
(
), S175D (
), and S175T (
)
calponins.
Fig. 6compares the time courses of phosphorylation of the
various calponin species by PKC. Tissue-purified calponin and
bacterially expressed calponin exhibit similar phosphorylation time
courses with maximal stoichiometry of 1.1 mol P
/mol
calponin under these conditions. No phosphorylation was observed in the
absence of Ca
, phospholipid, and diacylglycerol,
which are required by PKC (a mixture of
,
, and
isoenzymes) for activity. Much less phosphate incorporation into S175A
(
0.3 mol P
/mol) or S175D (
0.4 mol
P
/mol) was observed, consistent with our earlier conclusion
that serine 175 represents the principal site of phosphorylation by
PKC. The S175T mutant is clearly a substrate of PKC, although the
kinetics of its phosphorylation are much slower than for the native or
wild-type protein.
Figure 6:
Phosphorylation of tissue-purified,
wild-type, and mutant calponins by PKC. Chicken gizzard (),
wild-type (
), S175A (
), S175D (
), and S175T
(
) calponins were incubated with PKC as described under
``Experimental Procedures,'' and phosphate incorporation was
quantified at the indicated times. Phosphorylation of the
tissue-purified protein was also measured in the absence of
Ca
, phospholipid, and diacylglycerol (
). The
results are representative of eight independent
experiments.
In the case of phosphorylation by CaM kinase II (Fig. 7), again the native and wild-type proteins are very
similar, and phosphorylation required the presence of Ca and calmodulin. The S175A and S175D mutants were very poor
substrates of this enzyme, even poorer than for PKC. The S175T mutant,
however, proved to be as good a substrate for CaM kinase II as the
native or wild-type protein.
Figure 7:
Phosphorylation of tissue-purified,
wild-type, and mutant calponins by CaM kinase II. A, chicken
gizzard (), wild-type (
), S175A (
), S175D (
),
and S175T (
) calponins were incubated with CaM kinase II as
described under ``Experimental Procedures,'' and phosphate
incorporation was quantified at the indicated times. Phosphorylation of
the tissue-purified protein was also measured in the absence of
Ca
and calmodulin (
). B,
autoradiogram of duplicate samples subjected to SDS-PAGE showing the
time courses of phosphorylation of caldesmon (CaD) and the
indicated calponin species (CaP). Caldesmon served as a useful
internal control. The results are representative of six independent
experiments. CG, chicken gizzard.
Serine 175 has previously been implicated in the binding of
calponin to actin and inhibition of the actin-activated myosin MgATPase (11) . Phosphorylation by PKC or CaM kinase II occurs
predominantly at this site with substoichiometric phosphorylation
occurring at other sites: Thr, Thr
,
Ser
, Ser
, and Thr
by PKC and
Ser
, Ser
, and Ser
by CaM
kinase II. Ser
is located within the actin-binding domain
(residues 145-182) defined by Mezgueldi et
al.(22) . It should be noted, however, that Nakamura et al. (21) concluded that Thr
is the
major site of phosphorylation by PKC. To address the functional
importance of Ser
directly, we have expressed and
characterized three site-specific mutants of calponin and compared
their properties with those of bacterially expressed wild-type calponin
and calponin purified from chicken gizzard smooth muscle. Ser
was mutated (i) to alanine to assess the importance of the
hydroxyl side chain and to determine whether or not this residue indeed
represents the principal phosphorylation site, (ii) to aspartic acid to
assess the functional consequences of introduction of a negative charge
at this position (i.e. in an attempt to mimic, at least in
part, the effects of phosphorylation), and (iii) to threonine to assess
the importance of a hydroxylated side chain at this position and to
determine whether or not PKC or CaM kinase II is capable of
phosphorylating threonine as well as serine in this position.
Prior to functional characterization of the mutants, it was necessary to verify that bacterial expression (in particular, lack of post-translational modification) and site-specific mutagenesis did not affect the conformation of the protein. Two experimental approaches that are very sensitive to differences in protein conformation were used to address this issue: (i) analysis of the time courses of proteolytic digestion at 0 °C by four proteases with distinct cleavage site specificities and (ii) binding to calponin under nondenaturing conditions of a panel of monoclonal antibodies with distinct epitopes. Both methods indicated that the conformations of recombinant wild-type, S175A, and S175T calponins are indistinguishable from that of the native tissue-purified protein. The S175D mutant appears to have a slightly different conformation, reflected by decreased affinity for specific monoclonal antibodies, although its overall conformation is indistinguishable from that of native calponin, as revealed by protease sensitivity.
Wild-type calponin expressed in E. coli bound to smooth and skeletal muscle actins similarly
to the protein purified from chicken gizzard. The K of skeletal muscle actin for wild-type calponin (0.8
µM) was found to be similar to that for native calponin
(1.0 µM). Consistent with these observations, wild-type
calponin inhibited the actin-activated MgATPase activity of smooth
muscle myosin to a similar extent as did the native protein.
Furthermore, phosphorylation of wild-type and tissue-purified calponins
by PKC and CaM kinase II occurred with similar time courses and maximal
stoichiometries. These results indicate that bacterially expressed
wild-type calponin is structurally and functionally indistinguishable
from the protein purified from smooth muscle, thus confirming and
extending the work of Gong et al.(32) .
The
properties of the S175A mutant are particularly intriguing. This mutant
calponin binds very weakly to smooth or skeletal muscle actins (the K of skeletal muscle actin for S175A was estimated
to be 26.8 µM) and is correspondingly a very poor
inhibitor of the actomyosin ATPase. These results imply that the
hydroxyl side chain in position 175 plays a crucial role in the
interaction of calponin with actin, this interaction mediating the
inhibition of actomyosin ATPase activity(1) . The C-terminal
region of actin (residues 326-355) has been implicated as the
binding site for calponin(22) , and the three most C-terminal
residues appear to be particularly important for binding to
calponin(45) . Clearer understanding of the involvement of
Ser
in the interaction with actin will come from
determination of the three-dimensional structure of calponin and,
ultimately, of the calponin-actin complex.
Phosphorylation of S175A
calponin by PKC and CaM kinase II revealed incorporation of
substoichiometric amounts of phosphate: 0.3 mol P/mol with
PKC and 0.1 mol P
/mol with CaM kinase II. This compares
with stoichiometries of phosphorylation of native and wild-type
calponins of 1.1 and 1.0 mol P
/mol with PKC and CaM kinase
II, respectively. These results strongly support our earlier conclusion
that Ser
represents the principal site of phosphorylation
by these two kinases. The low stoichiometry of S175A phosphorylation
presumably represents phosphate incorporation into minor, nonfunctional
sites identified earlier (11) .
The properties of the S175D
mutant are very similar to those of S175A and substantiate the
conclusions that Ser plays a crucial role in binding to
actin and that it represents the principal site of phosphorylation. As
shown earlier, phosphorylation of calponin dramatically reduces its
affinity for actin and alleviates inhibition of the actomyosin
MgATPase(1) . The similar properties of the S175D mutant might
suggest that the introduction of negative charge at position 175
disrupts the calponin-actin interaction. However, the fact that the
S175A mutant behaves very similarly to S175D and phosphorylated
calponin indicates that the hydroxyl group itself is required for this
interaction. The slightly decreased affinity of S175D calponin for
monoclonal antibodies CP1, 3, and 4 suggests that introduction of
negative charge at this position affects the conformation that together
with loss of the hydroxyl group contributes to the marked reduction in
affinity for actin. Similar effects probably account for the dramatic
consequences of phosphorylation of calponin at Ser
.
The properties of the S175T mutant support the importance of a
hydroxyl-containing side chain at position 175. This mutant binds to
smooth and skeletal muscle actins similarly to the native and wild-type
proteins and has a comparable K for skeletal
muscle actin of 1.3 µM. Consequently, it inhibits the
actin-activated myosin MgATPase to a similar extent as does the native
or wild-type protein. Finally, the S175T mutant is as good a substrate
for CaM kinase II as is tissue-purified or wild-type calponin but not
as good a substrate for PKC, reflecting a preference of PKC for serine
rather than threonine at the site of phosphorylation.
In conclusion, our results indicate that serine 175 of calponin plays a crucial role in the interaction of calponin with actin and inhibition of the actin-activated MgATPase activity of smooth muscle myosin and that this residue is the principal site of phosphorylation by PKC and CaM kinase II. In particular, the integrity of the hydroxyl group in the side chain at position 175 is critically important for high affinity binding to actin.
ACKNOWLEDGEMENTS
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