From the Departments of Molecular Physiology and
Biological Physics,
Pathology, and ** Medicine, Health System,
Charlottesville, Virginia, 22908-0736 and the ¶ Department of
Pharmacology and Cancer Biology, Duke University Medical Center,
Durham, North Carolina 27710
Received for publication, April 20, 2001, and in revised form, May 7, 2001
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ABSTRACT |
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Forskolin and 8-bromoguanosine 3'-5'-cyclic
monophosphate (8-Br-cGMP) induce phosphorylation of Ser-13 of telokin
and relaxation of smooth muscle at constant calcium. Comparison with
the effect of wild type with aspartate (D; to mimic phosphorylation)
and alanine (A; non-phosphorylatable) mutants of telokin showed that the S13D mutant was more effective than wild type in relaxing smooth
muscle at constant calcium. The efficacy of the Ser-13A, S12A, and S12D
mutants was not significantly different from that of wild-type telokin.
The effect of neither S13D nor Ser-13A was affected by 8-Br-cGMP,
whereas the effect of wild type, S12A, and S12D was enhanced by
8-Br-cGMP, indicating the specificity of Ser-13 charge modification.
Mutation of Ser-19 (a mitogen-activated protein kinase site) showed the
S19A to be more effective than, and S19D to be not different from,
wild-type telokin. The effect of both mutants was slightly enhanced by
8-Br-cGMP. A truncated (residues 1-142) form lacking the acidic C
terminus had the same relaxant effect as wild-type telokin,
whereas the C-terminal peptide (residues 142-155) had no effect. We
conclude that site-specific modification of the N terminus modulates
the Ca2+-desensitizing effect of telokin on force.
Contraction and relaxation of smooth muscle are dependent mainly
upon the intracellular free Ca2+ concentration
([Ca2+]i). The magnitude of contractile force
developed depends on the level of myosin regulatory light chain
(MLC20)1
phosphorylation that, in turn, is determined by the relative activities
of myosin light chain kinase (MLCK) and smooth muscle myosin light
chain phosphatase (SMPP-1M). The Ca2+ sensitivity of
contraction can be affected by any change in kinase and/or phosphatase
activities that alters this ratio. At a fixed [Ca2+]i, both MLCK and SMPP-1M can be modulated
by second messenger-mediated signaling pathways, resulting in a change
in MLC20 phosphorylation and, consequently, in an increase
or decrease in force developed (reviewed in Refs. 1 and 2). The
Ca2+ sensitivity of the contractile apparatus can be
regulated by a number of agents such as RhoA (3-5), cyclic nucleotides
(6, 7), arachidonic acid (8), and diacylglycerol (8). Dephosphorylation of the regulatory myosin light chains by SMPP-1M can be inhibited by a
G-protein-coupled pathway that can increase the level of MLC20 phosphorylation, with no change in cytosolic calcium
(Ca2+ sensitization) (1, 9, 10), and we suggested that the reverse mechanism, activation of SMPP-1M, could lead to
Ca2+ desensitization. Stimulation of permeabilized smooth
muscle with 8-Br-cGMP relaxes submaximal Ca2+-induced
contractions and can also reverse G-protein-coupled inhibition of
SMPP-1M (6), suggestive of a mechanism mediated by activation of
SMPP-1M.
Telokin, also known as kinase-related protein, is a low molecular mass
(17 kDa) protein whose sequence is identical to the C terminus of MLCK
(11) and is independently expressed at high concentrations in some
smooth muscles (12) through a promoter located within an intron of the
MLCK gene (13). Telokin binds to the S1/S2 region of unphosphorylated
smooth muscle myosin (14, 15) and prevents the folding of the 6 S
myosin into 10 S conformation, thus stabilizing filamentous myosin in
solution (14). Stabilization of unphosphorylated myosin filaments has
been suggested to be a physiological function of telokin in smooth
muscle (14), and its acidic C terminus was believed necessary for high
affinity binding to myosin and filament stabilization. Furthermore, it has been shown (16) that there are at least 6 C-terminal variants of
telokin, differing from each other in the number of C-terminal glutamates (from 0-5), and it was suggested that the heterogeneity of
the C terminus may play an important role in the regulation of
macromolecular protein complexes at the structural level (16).
The concentration of endogenous telokin is high (70-80
µM) in some phasic smooth muscles, and its loss is
accompanied by a linearly related loss of 8-Br-cGMP-induced relaxation
and an enhanced responsiveness to exogenously added native and
recombinant telokin that can relax permeabilized smooth muscle and
reduce MLC20 phosphorylation (17). The relaxant effect of
telokin is potentiated by 8-Br-cGMP and cyclic
GMP-dependent kinase (PKG), and we suggested that
phosphorylation of telokin by PKG (or cAMP-dependent
protein kinase) modulates desensitization of smooth muscle to calcium,
as telokin is the major cytosolic protein phosphorylated during
cGMP-induced relaxation and dephosphorylation of MLC20
(17). Most recently, we have mapped the in vivo site of
phosphorylation of mammalian telokin to Ser-13 in intact,
forskolin-stimulated and permeabilized, 8-Br-cGMP-stimulated rabbit
ileum (18). Therefore, to determine whether Ser-13 is also the
functionally relevant site of telokin phosphorylation, we have produced
mutant telokins with replacement of Ser-13, Ser-12, and Ser-19 with
either an aspartate or alanine to determine their respective
contributions to calcium-independent effects on force and
MLC20 phosphorylation. Additionally we have expressed a
telokin mutant lacking the acidic C terminus to examine the other
proposed functions (14, 19, 20) of telokin.
Tissue Preparation, Force Measurement, and Electron
Microscopy--
Longitudinal ileal smooth muscle was removed from
rabbits anesthetized with halothane and killed by exsanguination
according to approved animal protocols. Small strips (250 µm × 2 mm) were dissected and permeabilized with Triton X-100 (0.1%) in a
Ca2+-free solution containing 1 mM EGTA (G1)
for 30 min. For storage, muscle strips were washed with relaxing
solution (containing 10 mM EGTA and 40 mM
reduced glutathione), placed in the same relaxing solution containing
50% glycerol, and stored at
For in situ labeling of exogenously added telokin,
permeabilized, telokin-depleted strips were mounted in a bubble
chamber, contracted with submaximal calcium, washed briefly in
calcium-free solution containing 0.5 mM ATP, and then
loaded for 10 min in the same solution with [
For electron microscopy, strips of ileal smooth muscle were prepared,
prior to fixation, as described above, fixed overnight in 2%
glutaraldehyde plus 0.1% tannic acid followed by osmium, stained with
uranyl acetate en bloc, and dehydrated and embedded in
Spurr's resin. Non-permeabilized strips served as controls.
Construction of Recombinant Telokin Mutants--
Rabbit telokin
cDNA was obtained as a generous gift from Dr. Paul Herring of
Indiana University (12). The open reading frame was amplified by
polymerase chain reaction (22) using the 5' primer,
GACTccatggTCTCAGGGCTCAGCGGCAGGAAA-3', which introduced an
NcoI site (lowercase letters), and a 3' primer that annealed to the vector. The resultant polymerase chain reaction product was
ligated into pGEX Universal (23) from NcoI to
EcoRI (from the original clone). This placed the telokin
sequence in-frame downstream of the glutathione
S-transferase moiety and an rTEV cleavage site. After
cleavage with rTEV protease (Life Technologies Inc.), the N-terminal
sequence became GAMVSGLSG, which differs from
native telokin only by the first and fourth amino acids (in bold). The
final sequence was confirmed by mass spectroscopy, which determined
that the molecular weight of the purified protein was as predicted.
Polymerase chain reaction mutagenesis (22) was used to introduce Ala
and Asp replacements of codons Ser-13, Ser-12, and Ser-19 or a stop
codon in place of Glu-143. In each case, the entire open reading frame
was sequenced to assure that no unwanted base changes were introduced
by the mutagenesis procedures. Standard molecular biology techniques
were performed as previously described (24).
Telokin was expressed by transforming Escherichia coli
strain BL21(DE3) (Novagen, Madison, WI) with the recombinant plasmids. Strains were grown at 37 °C in minimal defined medium supplemented with 1.1% glucose (25) to an optical density at 650 nm of ~0.5. Protein expression was induced for 2 h by the addition of 1 mM isopropyl thiogalactoside. Cells were broken open by
passing twice through a chilled French press at 18,000 p.s.i. into a
buffer containing 50 mM Tris-HCl, 5 mM EDTA,
10% glycerol, and 1 mM Pefabloc (Centerchem, Stamford, CT)
at pH 8.0. Cellular debris and membranes were removed by a 150,000 × g centrifugation for 1 h. The supernatant was passed
over a 16-ml bed volume glutathione-Sepharose 4B column (Amersham
Pharmacia Biotech) equilibrated with the same buffer. After washing,
the glutathione S-transferase-telokin protein was eluted
with 10 mM glutathione in 50 mM Tris-HCl, pH
8.0. To the eluant, 0.5 mM dithiothreitol, 1 mM
EDTA, and 10 units/mg of protein of rTEV protease was added, and the
suspension was incubated for 4-5 h at room temperature, then overnight
at 4 °C. After exchanging the buffer by dialysis against 30 mM Tris-HCl plus 0.05% In Vitro Site Analysis of Telokin Phosphorylation--
As
described previously (18), mutant telokin was phosphorylated for 2 h with 3 µg of PKG in a buffer containing 25 mM Hepes, pH
7.2, 1 mM MgCl2, 0.1 mM
dithiothreitol, and 0.2 mM [ Myosin LC20 Phosphorylation Measurements--
Myosin
light chain phosphorylation was determined as previously described
(26). Briefly, the same smooth muscle strips used for tension
recordings were rapidly frozen in liquid nitrogen-cooled Freon 22 at
predetermined times and carefully removed from the transducers. Strips
were transferred to 10% trichloroacetic acid in acetone ( Statistics--
A multiple analysis of variance test was
performed where indicated under "Results." All values are the
means ± S.E. unless otherwise indicated, and n is the
number of experiments.
Materials--
Monoclonal anti-telokin antibody was generated at
the University of Virginia lymphoma facility to amino acids 1-27 in
rabbit telokin. Protein kinase G (catalytic subunit) and 8-Br-cGMP were from Calbiochem. Unless noted, all other reagents were from Sigma.
Relaxation of Ca2+-induced Tension by Wild-type (WT)
and Ser-13 Mutant Recombinant Telokins--
As previously shown (17),
recombinant WT telokin relaxed telokin-depleted rabbit ileal smooth
muscle in a dose-dependent manner. In the absence of
8-Br-cGMP, 10 µM WT telokin caused 33 ± 5.6%
relaxation of a submaximal calcium (pCa 6.3)-induced contraction (Fig.
1), and 20 µM WT telokin
relaxed by 49 ± 6.9% (not shown). Preincubation with 8-Br-cGMP
for 5 min potentiated the effect of WT telokin and increased the
relaxation evoked by 10 µM WT telokin to 59 ± 5.5%
(Fig. 1) and 20 µM to 80 ± 2.3% that of the contractile response to submaximal calcium. 10 µM S13D
mutant telokin (which mimics the negative charge on phosphorylated
telokin) relaxed telokin-depleted ileum to 55 ± 5.9% (Fig. 1)
and 20 µM S13D telokin by 76 ± 8.7% (not shown);
8-Br-cGMP did not significantly change the relaxation evoked by S13D
telokin at any concentration (Fig. 1). 10 µM Ser-13A
telokin (to block phosphorylation of Ser-13) relaxed telokin-depleted
ileum by 28 ± 2.4% (Fig. 1) and was not affected by
preincubation with 8-Br-cGMP.
Relaxation by Ser-12 Mutants--
To determine whether
phosphorylation of the residue adjacent to Ser-13, Ser-12 (also
revealed by in situ phosphorylation of the S13D/S19A double
mutant, see below) influenced the relaxant activity of telokin,
we constructed both the S12A and S12D mutants. In the absence of
8-Br-cGMP, the S12A mutant relaxed submaximally contracted
permeabilized ileum by 26.3 ± 1.82%, and its effect was
potentiated to 48.7 ± 1.8% by 8-Br-cGMP (Fig.
2). Similarly, the S12D mutant, in the
absence of 8-Br-cGMP, relaxed calcium-stimulated smooth muscle by
28.8 ± 1.2% and was potentiated to 45.2 ± 2.2% (Fig. 2).
These results are not significantly different from those of WT,
suggesting that Ser-12 is not a physiologically relevant phosphorylation site in the absence of other (i.e. Ser-13)
phosphorylation.
The Effect of the Ser-19 Mutation and in Situ Phosphorylation of
S13D/S19A Telokin--
A telokin mutant with a mutation at the
presumptive mitogen-activated protein kinase site, S19A, resulted in a
53 ± 5.6% relaxation of telokin-depleted strips at 10 µM (Fig. 1), and the S19D mutant induced in only
20.9 ± 9.2% relaxation, suggesting that phosphorylation of
Ser-19 may indeed exert a physiological (inhibition of relaxation) effect. Therefore, we constructed a double mutant with an Asp at
position 13 and an Ala at position 19 (S13D/S19A). 10 µM
S13D/S19A relaxed submaximally contracted ileum by 37 ± 5.4%,
not significantly different from WT. Surprisingly, 8-Br-cGMP and PKG
catalytic subunit increased the relaxation evoked by this double mutant
(10 µM) to 71.2 ± 2% (n = 4) (Fig.
1), suggesting either a separate additional site of phosphorylation of
the S13D/S19A telokin or the contribution of another phosphoprotein
that, most likely, can only interact with telokin that is not
phosphorylated at Ser-19.
To determine whether potentiation of the relaxant effect of S13D/S19A
telokin by 8-Br-cGMP was related to phosphorylation of this mutant at a
site other than Ser-13 or 19, we examined the in situ
phosphorylation of the S13D/S19A mutant. In the absence of exogenously
added telokin, phosphorylation of the small amount of endogenous
telokin that remains in these strips even after storage (<10%), was
detectable both in the presence and absence of 8-Br-cGMP. However, the
lack of an increase in phosphate incorporation into endogenous telokin
in the presence of 8-Br-cGMP (Fig. 3) suggests that this remaining telokin is not a good substrate for PKG or
is already fully phosphorylated. Exogenously added WT, S19A, S19D, and
S13D/S19A telokin were further phosphorylated in the presence of
8-Br-cGMP (Fig. 3), suggesting that indeed, the S13D/S19A double mutant
could be phosphorylated at yet another site.
To identify the site of phosphorylation of the S13D/S19A telokin, the
recombinant double mutant was phosphorylated in vitro and
processed as previously described for site analysis (18, 27). After
in vitro phosphorylation, the only site phosphorylated on
the S13D/S19A double mutant was Ser-12. Interestingly, this site is not
phosphorylated in situ in 8-Br-cGMP or forskolin-stimulated tissues (18) or in recombinant rabbit telokin if only the Ser-13 is
mutated to either S13A or S13D (Fig. 3), suggesting that it is not
simply the unavailability of Ser-13 that makes Ser-12 available to a
kinase but, rather, cooperativity between sites 13 and 19.
Since the Ser-12 residue was phosphorylated in the S13D/S19A double
mutant, we constructed two additional mutants, the S12A/S13D/S19A and
S12D/S13D/S19A triple mutants. Surprisingly, both of these mutants were
still potentiated by 8-Br-cGMP (Fig. 2). That is, in the absence of
8-Br-cGMP, S12A/S13D/S19A relaxed a submaximal calcium contraction by
28.2%, and S12D/S13D/S19A relaxed by 21.0%. However, preincubation
with 8-Br-cGMP increased the relaxation induced by these mutants to
60.7 and 47.0%, respectively (Fig. 2). Furthermore, neither of the
triple mutants was further phosphorylated in situ when
telokin-depleted smooth muscle strips were incubated with
[ MLC20 Phosphorylation Decreases with Telokin-induced,
Ca2+-independent Relaxation--
As shown in Fig. 1, WT
and S13D telokin (10 µM) induced significant relaxation
of stored, telokin-depleted ileum smooth muscles. Therefore, we
examined the phosphorylation of the regulatory light chains of myosin
and found telokin-induced relaxation to be associated with a
proportional decrease in MLC20 phosphorylation. WT-telokin induced a decrease in phosphorylation from 60% in control tissues (at
the plateau of submaximal calcium contraction) to 42%, and S13D
telokin induced a decrease in MLC20 phosphorylation to 26% after 10 min. Resting phosphorylation in the skinned, stored ileum muscles in the absence of calcium was ~22%. This high level of phosphorylation in the absence of calcium may be due to a decrease in
endogenous phosphatase activity in Triton-skinned smooth muscles (28)
and/or the presence of a Ca2+-independent kinase activity.
The Acidic Polyglutamate Tail of Telokin Is Neither Sufficient Nor
Necessary for Telokin-induced Relaxation of Smooth
Muscle--
Polyanionic as well as polybasic peptides can modulate
PP-1C activity in vitro on certain substrates (29, 30), and
it has been suggested (20) that the acid-rich C-terminal region, corresponding to residues Gly-138-Glu-150, is the primary site of
interaction between telokin and myosin. To test whether the polyglutamate C-terminal tail of telokin had similar relaxant properties as the protein itself (i.e. through either a
direct effect on the phosphatase or through competitive binding to
myosin), we synthesized a 14-amino acid peptide corresponding to the C terminus (residues 142-155) of rabbit telokin (MEEGEGEGEEEEEE). The
addition of the acidic C-terminal peptide (20 µM) of
telokin did not relax submaximal calcium-induced contractions of rabbit ileum (n = 3, data not shown) nor did it interfere with
subsequent relaxation induced by 10 µM telokin
(n = 3, data not shown).
In contrast, a truncated telokin without the acidic C terminus relaxed
telokin-depleted ileum at constant calcium in a
dose-dependent manner, with no significant difference from
wild type; 10 µM truncated telokin relaxed a submaximal
calcium contraction by 32.0% and was potentiated to 52.1% in the
presence of 50 µM 8-Br-cGMP, and 20 µM
C-terminally truncated-telokin relaxed 48.2 ± 6.2% in the absence of 8-Br-cGMP (Fig. 4) and was
potentiated to 72.0 ± 3.0%.
Microcystin-induced Contraction Is Unchanged by Telokin--
Since
MLC20 phosphorylation is regulated by both SMPP-1 and MLCK,
we tested the remote possibility that telokin was inhibiting MLCK (31),
although it does not inhibit the rate of MLC20
thiophosphorylation (17). Stored telokin-depleted rabbit ileum strips
were incubated in G1 solution ±10 µM WT telokin for 10 min. Microcystin (5 µM) was added to irreversibly inhibit
SMPP-1M. After 5 min, the muscle was stimulated with submaximal
calcium. Preincubation of stored rabbit ileum with 10 µM
telokin did not slow the rate nor change the amplitude of
calcium-induced contraction in the presence of microcystin used to
irreversibly inhibit SMPP-1 (Fig. 5). Had telokin inhibited MLCK, it would be expected to affect the rate and/or
magnitude of calcium-evoked contraction, even in the presence of a
phosphatase inhibitor.
Triton-skinned, Telokin-depleted Ileum Smooth Muscle Retains Normal
Filament Arrays--
It has been suggested that one major role of
telokin is stabilization of myosin filaments in vivo (14);
therefore, we examined the filament arrays in control and
telokin-depleted, Triton-skinned smooth muscles. Rabbit ileum
longitudinal smooth muscle was permeabilized with 0.1% Triton X-100 as
described under "Experimental Procedures." Western blots for
telokin showed that more than 90% of the telokin was removed within 20 min in Triton X-100. Electron microscopy of thin cross-sections of
skinned, telokin-depleted ileum strips shows a normal number and array
of thick myosin filaments surrounded by thin actin filaments and of
dense bodies (Fig. 6) when compared with
paired, nonpermeabilized preparations. Discontinuity in the plasma
membrane and organelle damage were consistent with the permeabilizing
effects of the detergent.
Our major findings show that 1) mutation of the in vivo
cAMP and/or cGMP kinase site of phosphorylation of telokin (Ser-13) to
an Asp enhances its ability to relax smooth muscles, whereas 2)
mutation of Ser-13 to Ala does not affect its relaxant effect; 3) cGMP
does not potentiate the relaxant activity of either (S13D or S13A)
mutant; 4) mutation of the Ser-12 to either an Asp or an Ala does not
change the activity of telokin; and 5) the acidic C-terminal peptide of
telokin alone does not mimic the relaxant effect of telokin, whereas
the C-terminally truncated mutant is as effective as WT telokin in
evoking relaxation of smooth muscle. We also show that depletion of
endogenous telokin does not lead to the loss or disorganization of
myosin filaments. Taken together, these data suggest that Ser-13 is
indeed a relevant in vivo phosphorylation site associated
with cyclic nucleotide-induced, Ca2+-independent relaxation
and that the acidic C terminus of telokin is neither necessary nor
sufficient for its relaxant effect.
A double mutation of Ser-13 to Asp to mimic phosphorylation
and Ser-19, a putative mitogen-activated protein kinase
site, to a (non-phosphorylatable) Ala, revealed an alternative in
situ phosphorylation site, but the physiological role of
phosphorylation of Ser-19, if any, is less clear. The present results
are consistent with our previous suggestion (17) that cyclic
nucleotide-induced relaxation is mediated at least in part by
phosphorylation of telokin and increased activity of myosin light chain
phosphatase (17). The Ser residue at position 19 (Ser-19) that is a
potential in vivo site of phosphorylation by
mitogen-activated protein kinase (17) as expected did not show an
increase in phosphorylation when intact rabbit ileal smooth muscle was
stimulated with either forskolin or when permeabilized ileal muscle was
stimulated with 8-Br-cGMP (18). Nevertheless, mutations of Ser-19 to
either an Asp or an Ala are consistent with phosphorylation of this
site being a potent inhibitory site (even though the S19D mutant
can be substantially phosphorylated (Fig. 3), presumably on Ser-13 or
Ser-12). Furthermore, it has recently been shown (32) that stimulation
of either chicken carotid arterial smooth muscle or chicken gizzard
with phorbol ester results in an increase in phosphorylation of telokin
at an as yet unidentified site but that only carotid arterial smooth
muscle (and not gizzard) contracts when stimulated with phorbol ester.
These data further suggest differences in the regulation of tonic and
phasic smooth muscle. Unlike phosphorylation of Ser-13, it remains to
be shown whether Ser-19 phosphorylation is relevant in the presence of
physiological concentrations of cGMP in intact muscles or if telokin
may be a target for other signaling pathways.
We should consider the functional effects of the various mutations of
telokin within the context of the native telokin structure, a compact
immunoglobulin fold bordered by an extended N-terminal 32 amino acid
and C-terminal 19 amino acid sequence. Neither the N nor the C terminus
is resolved in either the original crystal structure at 2.8 Å (33) or
a more recent refinement at 1.9-Å resolution (Protein Data Bank code
1FHG), presumably as the result of flexibility of these regions.
The fact that the highly acidic C terminus is neither sufficient nor
necessary for the Ca2+-independent relaxant effect of
telokin does not exclude the possible importance of electrostatic
interactions being involved in such activity, because the truncated
structure is also acidic (calculated Pi, 4.37), with a
significant solvent-exposed acidic surface (33). Truncation of the N
terminus that contains the phosphorylation sites does not eliminate the
relaxant effect of telokin (17), although it abolishes its potentiation
by 8-Br-cGMP. Mutations involving charge modification of exposed acidic
residues of the immunoglobulin fold may be useful in elucidating the
main structural requirement for the relaxant activity of telokin.
Furthermore, circular dichroism studies on the various telokin mutants
revealed changes in the secondary structure, as suggested by a slight
increase in the In the presence of a phosphatase inhibitor, preincubation of relaxed,
telokin-depleted ileal strips with 10 µM wild-type
telokin did not change either the rate or amplitude of
calcium-stimulated contraction, supporting the conclusion (17) that
telokin acts most likely through direct or indirect activation of the
phosphatase rather than inhibition of myosin light chain kinase (31).
However, we have thus far been unable to demonstrate in
vitro a direct effect of telokin on phosphatase activity (18).
This may reflect the inability to recapitulate the correct structural
relationships between myosin filaments, phosphatase regulatory, and
catalytic subunits, and telokin. It is also possible that another
protein(s) is involved in the cascade between phosphorylation of
telokin and relaxation (through activation of the phosphatase) of
smooth muscle.
A variety of proteins in addition to telokin are phosphorylated during
cyclic nucleotide-evoked relaxation including but not limited to HSP 20 and HSP 27 (35, 36), SM-22,3 and
calmodulin.3 The possibility
that one or more of these proteins are acting in conjunction with
telokin to activate the myosin phosphatase is currently under
investigation. However, we know of no direct evidence to show that
these proteins, acting either alone or with telokin, can activate
SMPP-1M. Since exogenous telokin is active in Triton-treated
preparations, membrane proteins phosphorylated by cyclic
nucleotide-activated kinases (34, 37, 38) are unlikely to be relevant
to telokin activity.
The hypothesis that the physiological role of telokin is to stabilize
unphosphorylated myosin filaments through binding via its acidic tail
to myosin (14, 20) is not consistent with the normal myosin filament
arrays (Fig. 6) in tissues skinned with Triton X-100 and depleted of
endogenous telokin (17) and with the lack of effect of removal of the
acidic C terminus on the relaxant effect of telokin (this study). We
cannot, however, rule out the possibility that in vivo
telokin contributes to the structural and functional interactions
between MLCK, SMPP-1M, myosin, and actin because in our preparations
there was a small (<10%) amount of telokin that remained after
skinning. It is unlikely, though, in view of the very low stoichiometry
of remaining telokin to myosin (perhaps less than 5 µM
versus ~52 µM), that the small amount of
telokin remaining in permeabilized preparations is required for
filament stability. In conclusion, we present evidence (through point mutations) that demonstrates the importance of phosphorylation of
the Ser-13 residue of telokin in cyclic nucleotide-induced dephosphorylation of MLC20 and relaxation of smooth muscle.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
20 °C. Details of the solutions used
for studies of permeabilized smooth muscle strips, storage of muscles,
and the time course of depletion of endogenous telokin have been
published (17, 21). For force measurement, strips were removed from the
freezer, washed briefly in G1 solution, and tied with silk
monofilaments to the tips of two fine wires. One wire was fixed, and
the other was connected to a force transducer (SensoNor, AE801). The
strip was mounted in a well on a bubble plate to allow rapid solution
exchange and freezing. Strips were stretched to 1.5× resting length.
All experiments were carried out at room temperature. The telokin
content of paired strips was determined by gel electrophoresis and
Western blotting.
-32P]ATP
(5 mCi/ml). After loading, strips were relaxed by the addition of
either wild-type or mutant (10 µM) telokin in the
presence or absence of 8-Br-cGMP and PKG. After 10 min, strips were
rapidly frozen and homogenized in Laemmli sample buffer and separated by 15% SDS-PAGE. Separated proteins were transferred to polyvinylidene fluoride membranes, air-dried, and placed on film for autoradiography. After autoradiography, membranes were re-wetted and immunoblotted for telokin.
-mercaptoethanol, pH 7.4, the
telokin was purified by passage over a 0.46 × 10-cm BioSepra Q
Hyper D anion exchange column (Marlborough, MA) with elution by a NaCl
gradient or passage over the glutathione column. The telokin-containing
fractions were identified by SDS-polyacrylamide gel electrophoresis.
The buffer was exchanged to 30 mM Pipes (pH 7.1), 165 mM potassium methane sulfonate, 5 mM magnesium
methane sulfonate for the addition of telokin to muscle strips. The
protein was concentrated over a Centricon filter (Millipore Corp.,
Burlingtion, MA). Protein concentrations were determined from the
absorbance at 280 nm using the absorption coefficient of 0.78 (14).
-32P]ATP. The
labeled protein was digested with endolysylpeptidase, and the peptides
were separated by reverse phase chromatography. The labeled
phosphopeptide was identified and further purified over reverse phase
capillary high performance liquid chromatography, and its amino acid
sequence was determined by sequential Edman degradation and radioactive
detection after each Edman cycle.
70 °C)
and stored for a minimum of 12 h and then slowly brought to room
temperature, washed extensively with acetone, and dried. Tissues were
homogenized in a buffer containing 1% SDS, 10% glycerol, 20 mM dithiothreitol, and 5 mg/ml bovine serum albumin. Tissue
homogenates were separated by two-dimensional electrophoresis (pH range
4.5-5.4; 15% SDS-polyacrylamide gel electrophoresis) and transferred
to nitrocellulose membranes as described previously (26). The membranes
were stained with AuroDye (Amersham Pharmacia Biotech), and spots were
quantitated by densitometry. The percentage phosphorylation was
expressed as (P1 + P2)/(U + P1 +P2), where U represents the
unphosphorylated light chain spot and P1 & P2 represent singly and
doubly phosphorylated light chain, respectively.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Summary of relaxation induced by wild-type
and Ser-13 and Ser-19 mutant telokins in the absence and presence of
8-Br-cGMP. Stored, telokin-depleted rabbit ileum strips were
contracted with pCa 6.3 and then 10 µM telokin, either WT
or mutant was added, and strips were allowed to relax for 10 min.
Alternatively, calcium-contracted muscle was incubated with 50 µM 8-Br-cGMP plus 2500 units of PKG for 5 min before
relaxation with telokin. Relaxation by 8-Br-cGMP by itself (no added
telokin) was less than 5%. Percent relaxation is calculated from the
plateau of the pCa 6.3 contraction. n = at least four
for each mutant.
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Fig. 2.
Summary of relaxation induced by Ser-12
telokin mutants in the absence and presence of 8-Br-cGMP. Stored,
telokin-depleted rabbit ileum strips were contracted with pCa 6.3, and
then 10 µM S12A, S12D, S12A/S13D/S19A, or S12D/S13D/S19A
was added, and strips were allowed to relax for 10 min. Alternatively,
calcium-contracted muscles were incubated with 50 µM
8-Br-cGMP plus 2500 units of PKG for 5 min before relaxation with
telokin. Relaxation by 8-Br-cGMP by itself (no added telokin) was less
than 5%. Percent relaxation is calculated from the plateau of the pCa
6.3 contraction. n = at least four for each
mutant.
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Fig. 3.
Autoradiograms showing in situ
labeling of exogenously added telokin mutants in the presence and
absence of 8-Br-cGMP. Telokin-depleted strips were contracted with
pCa 6.3 for 10 min, washed briefly in pCa 6.3 containing 0.5 mM ATP, and incubated for 10 min in the same solution
containing [ -32P]ATP (5 mCi/ml). After loading, strips
were relaxed by the addition of either wild-type or mutant (10 µM) telokin in the presence or absence of 8-Br-cGMP and
PKG. After homogenization, proteins were separated by 15%
SDS-polyacrylamide gel electrophoresis and transferred to
polyvinylidene fluoride membranes, air-dried, and placed on film for
autoradiography. Bottom panel, lanes 9 and
10 show phosphorylation of the endogenous telokin (<10%)
that remains after skinning and storage of smooth muscle.
-32P]ATP in the presence and absence of 8-Br-cGMP and
the mutant protein (Fig. 3). However, we were able to phosphorylate,
in vitro, both mutants (on serine 28) with
cAMP-dependent protein kinase or PKG catalytic subunit.
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Fig. 4.
Truncated telokin lacking the acidic C
terminus relaxes Ca2+-activated rabbit smooth muscle.
Stored rabbit ileum was contracted with submaximal calcium (pCa 6.3) in
the presence of 1 µM calmodulin. C-terminally truncated
telokin lacking the acidic tail (see "Experimental Procedures") was
added at the plateau of contraction, and muscles were allowed to relax
for 10 min before the addition of 50 µM 8-Br-cGMP and
2500 units of PKG.
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Fig. 5.
Recombinant telokin does not affect
Ca2+-activated contractions when SMPP-1 is inhibited by
microcystin. Stored rabbit ileum was treated for 5 min with
microcystin in the absence (panel A) or presence
(panel B) of 10 µM WT-telokin
(WT-TLK) in calcium-free solution containing 1 mM EGTA and then stimulated with submaximal calcium. The
slow contraction induced by microcystin in G1 solution (pCa < 8.0) reflects the activity of a Ca2+-independent kinase.
Results shown are representative of five experiments. Note that neither
the magnitude nor rate of force development upon activation of MLCK
with Ca2+ is inhibited in the presence of WT-TLK.
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Fig. 6.
Transverse section of a portion of a
Triton-skinned, telokin-depleted rabbit ileum smooth muscle. A
normal number and distribution of myosin filaments (large
arrows) surrounded by actin filaments is present. Cytoplasmic and
plasma membrane-bound dense bodies (db) appear normal. The
image is representative of the majority of cells in the total
cross-section of longitudinal muscle. Triton treatment has led to
disruption of large regions of the plasma membrane
(arrowheads) and organelles. Inset, higher
magnification illustrates filament arrays. e.c.s,
extracellular space.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helical content. That is, the WT, S13D, S13A, and
S19D telokin mutants contained no
-helix detectable by
circular dichroism,2 but
mutation of Ser-19 to an alanine increased the predicted
-helical
content to ~25%. Although it is unlikely that a single mutation
changes the overall structure so dramatically, it is very likely that
mutation of the Ser-19 and further mutations (the double and
triple mutants) changed the native structure, allowing further
potentiation by 8-Br-cGMP and PKG perhaps via an interaction with the
phosphatase, myosin, or another protein. Therefore, we are reluctant to
assign a physiological function to the effects of multiple mutations.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant HL48807 (to A. P. S.) and a Natural Sciences and Engineering Research Council of Canada post-doctoral fellowship (to J. A. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ These authors contributed equally.
To whom correspondence should be addressed: Dept. of Molecular
Physiology and Biological Physics, P. O. Box 800736, Charlottesville, VA 22908-0736. Tel.: 434-924-5108; Fax: 434-982-1616; E-mail: aps2n@virginia.edu.
Published, JBC Papers in Press, May 9, 2001, DOI 10.1074/jbc.M103560200
2 L. A. Walker, A. V. Somlyo, and A. P. Somlyo, unpublished observations using JFIT and Dichroprot programs.
3 L. A. Walker, J. A. MacDonald, X. Liu, R. K. Nakamoto, T. A. J. Haystead, A. V. Somlyo, and A. P. Somlyo, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: MLCK, smooth muscle myosin light chain kinase; SMPP-1M, smooth muscle myosin light chain phosphatase; MLC20, 20-kDa regulatory myosin light chain; 8-Br-cGMP, 8-bromoguanosine 3'-5'-cyclic monophosphate; PKG, cGMP-dependent protein kinase; [Ca2+]i, intracellular free calcium concentration; Pipes, 1,4-piperazinediethanesulfonic acid; WT, wild type.
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REFERENCES |
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