©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Thiophosphorylation of the 130-kDa Subunit Is Associated with a Decreased Activity of Myosin Light Chain Phosphatase in -Toxin-permeabilized Smooth Muscle (*)

(Received for publication, June 1, 1995)

Laura Trinkle-Mulcahy (1)(§) Kazuhito Ichikawa (2) David J. Hartshorne (2) Marion J. Siegman (1) Thomas M. Butler (1)(¶)

From the  (1)Department of Physiology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania 19107 and the (2)Muscle Biology Group, University of Arizona, Tucson, Arizona 85721

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Pretreatment of alpha-toxin-permeabilized smooth muscle with ATPS (adenosine 5`-O-(thiotriphosphate)) under conditions resulting in minimal (<1%) thiophosphorylation of the myosin light chain increases the subsequent calcium sensitivity of force output and myosin light chain phosphorylation. The change in calcium sensitivity results at least in part from a 5-fold decrease in myosin light chain phosphatase activity. One of the few proteins thiophosphorylated under these conditions is the 130-kDa subunit of myosin light chain phosphatase. These results suggest that thiophosphorylation of this subunit leads to a decrease in the activity of the phosphatase, and that phosphorylation and dephosphorylation of the subunit may play a role in regulating myosin light chain phosphatase activity.


INTRODUCTION

The calcium sensitivity of force output from smooth muscle is variable(1) , and the mechanism responsible for this has been studied in permeabilized smooth muscles that retain receptor-coupled signal transduction mechanisms(2, 3, 4, 5, 6) . In these preparations, either agonist activation in the presence of GTP or direct activation of G proteins with GTPS (^1)increases myosin light chain phosphorylation at submaximal calcium concentrations(7, 8, 9) . At a constant free calcium concentration, the increase in phosphorylation is correlated with the increase in force, and appears to result from an inhibition of myosin light chain phosphatase activity ( (9) and (10) ; see (11) for review). The mechanism linking receptor activation and inhibition of the myosin light chain phosphatase is not known. Gong et al.(12) have suggested that arachidonic acid may play a role, while Masuo et al.(13) have shown that activation of protein kinase C decreases phosphatase activity.

Several groups (14, 15, 16) have shown recently that the myosin-bound phosphatase of smooth muscle is composed of three subunits of 130, 37-38, and 20 kDa. The 37-38-kDa component is the -isoform of the protein phosphatase type 1 (PP1) catalytic subunit(15) , also referred to as the beta-isoform(14) . The other two subunits are thought to be involved in targeting the phosphatase to its substrate, myosin, and thus are putative regulatory subunits. Binding of the trimeric complex to myosin has been demonstrated(15) , and it has also been shown that the holoenzyme has enhanced activity to its substrate myosin (or HMM) compared to the isolated catalytic subunit(14, 16) . Derived sequences from the cDNAs of the large subunit from gizzard (15) and rat (17) indicate the presence of several potential phosphorylation sites. We report here experiments that suggest phosphorylation of the 130-kDa subunit of myosin light chain phosphatase plays a role in the regulation of its enzymic activity.


EXPERIMENTAL PROCEDURES

Muscle Treatment

Longitudinal strips of rabbit portal vein were dissected and permeabilized as described previously(18) . Briefly, muscles were pretreated with 5 mM DIDS to reduce ecto-ATPase activity and then permeabilized with 4200 units/ml Staphylococcus aureus alpha-toxin (Life Technologies, Inc.) in a relaxing solution containing 10 mM sodium azide as an ecto-ATPase inhibitor. All subsequent solutions contained sodium azide, and the compositions were described previously(18) . Experiments were performed at 20 °C.

Mechanical Measurements

Muscle strips were mounted under isometric conditions at their in vivo length and bathed in stirred solutions (600-800 µl). Active force output was compared to that during an initial activation in pCa 4.5. For inhibition of myosin light chain kinase activity, muscles were bathed in a relaxing solution containing 300 µM ML-9 (19) for at least 5 min before exposure to other ML-9-containing solutions.

Measurement of Phosphatase Activity

The rate constant for myosin light chain phosphatase activity was determined from the time course of decrease in myosin light chain phosphorylation when the muscle was placed in a rigor solution(10) . Muscles were activated for 5 min by transfer to an activating solution (pCa 4.5), by exposure to pCa 6 in the presence of GTPS, or by transfer to a pCa 6 solution containing ML-9 after treatment with ATPS. They were then transferred to a series of rigor solutions and frozen at various times. Myosin light chain phosphorylation was determined by two-dimensional gel electrophoresis(20) , and the time course of dephosphorylation of the light chain was fitted to a single exponential (Sigmaplot, Jandel Scientific).

Identification of Thiophosphorylated Proteins

Muscle strips were incubated in a rigor solution with ML-9 and 100 µM ATPS containing 0.5-10 mCi/ml [S]ATPS for 10 min. Muscles were frozen following several rigor washes to remove the ATPS, and extracted in either 0.5 N HClO(4) or 10% trichloroacetic acid, containing unlabeled ATPS. Proteins were subjected to the following procedures: SDS-PAGE on 7.5-20% gradient gels(21) , SDS-PAGE on low porosity gels for higher molecular weight proteins(22) , and two-dimensional electrophoresis over the pH range 5-7 (2% Pharmalyte (Sigma) 5-7 and 1% Pharmalyte 3-10). Proteins of known pI (Bio-Rad two-dimensional SDS-PAGE standards) were used to calibrate the two-dimensional gels. Autoradiography was performed either with x-ray film on gels using a liquid scintillant enhancer (Autofluor, National Diagnostics Corp.) or with a storage phosphor screen (PhosphorImager, Molecular Dynamics). Monoclonal antibodies (mAb) to the 130-kDa subunit of myosin light chain phosphatase were prepared by conventional methods (23) using the gizzard holoenzyme as antigen. For Western blots, the antimouse immunoglobulin-peroxidase conjugate was recognized either by enhanced chemiluminescence (ECL, Amersham) or colorimetrically with 4-chloro-1-napthol.

The quantity of thiophosphate incorporated into muscle protein was determined from the S present in the acid-insoluble precipitate after pretreatment with 100 µM ATPS (S, 0.5 mCi/ml,). The precipitate was solubilized (Solvable, DuPont NEN), total S dpm measured, and thiophosphate content calculated from the specific activity of [S]ATPS in the labeling solution. Muscle volume was measured as described previously (18) by including 1 mM mannitol (^14C, 0.01 mCi/ml) in the solution prior to freezing the muscle.


RESULTS AND DISCUSSION

Many protein kinases use ATPS to thiophosphorylate a protein substrate, which is then resistant to phosphatase activity(24) . In permeabilized smooth muscle, ATPS treatment results in a calcium-dependent thiophosphorylation of the myosin light chain, which is then a very poor substrate for phosphatase activity (25) . When the light chain is thiophosphorylated, force output is independent of calcium concentration(25) , and the actin-activated myosin ATPase is irreversibly activated(26) . These observations were important in determining the central role that myosin light chain phosphorylation plays in the regulation of smooth muscle contraction. While performing experiments to investigate the factors that control calcium sensitivity of force output, we found that under certain conditions, ATPS pretreatment can increase the calcium sensitivity of contraction in alpha-toxin-permeabilized smooth muscle when there is minimal thiophosphorylation of the myosin light chain.

A typical force trace for an alpha-toxin-permeabilized muscle prior to and following treatment with ATPS is shown in Fig. 1A. Pretreatment with 1 mM ATPS at pCa > 8 in the presence of the myosin light chain kinase inhibitor ML-9 resulted in no increase in force when the muscles were transferred to an ATP-containing relaxing solution. Upon transfer to a pCa 6 solution, however, there was a rapid rise in force to its maximum (106 ± 5% of initial pCa 4.5 contraction) in contrast to a much lower force output observed at the same calcium concentration prior to ATPS treatment (n = 6). If the muscle was placed in pCa 7 after ATPS pretreatment, there was a slow (t 13 min) increase in force to 71 ± 7% of maximum, although there was no force output in this calcium concentration before treatment (n = 4). Fig. 1B shows a similar protocol except that ML-9 was included in the solutions after ATPS treatment. Upon transfer to a pCa 6 solution with ML-9, there was a rapid rise in force which was much greater than that observed at the same calcium concentration in the presence of ML-9 prior to ATPS treatment (n = 19). In this case the enhanced contractile response was usually phasic.


Figure 1: Contractile response to various calcium concentrations prior to and following treatment with ATPS. Typical isometric force traces for muscles activated under various conditions prior to and following treatment with 1 mM ATPS in the presence of 300 µM ML-9 for 10 min at low calcium (pCa > 8). Muscles were initially contracted in pCa 4.5. Openbars indicate exposure to relaxing solution (pCa > 8). PanelA, response to pCa 6 before and after ATPS treatment. PanelB, response to pCa 6 in the presence of ML-9 before and after ATPS treatment. PanelC, response to pCa 7 following ATPS treatment, with subsequent addition of GTPS (100 µM).



Fig. 2shows a summary of the force responses and corresponding myosin light chain phosphorylation measurements in protocols similar to that shown in Fig. 1B. As expected, maximal activation with pCa 4.5 was associated with a high level of light chain phosphorylation, whereas pCa 6 resulted in submaximal force and light chain phosphorylation. Treatment of the muscle with ML-9 reduced the levels of phosphorylation and force elicited by pCa 4.5 and 6. Muscles that were pretreated with 1 mM ATPS for 10 min under low calcium conditions (pCa > 8) in the presence of ML-9 produced no force when transferred to a relaxing solution containing 1 mM ATP. There was no detectable thiophosphorylation of the myosin light chain in two-dimensional gels, and phosphorylation was low (12 ± 4%, n = 5). Subsequent incubation in pCa 6 in the presence of ML-9 resulted in 70% maximum force and myosin light chain phosphorylation. Note the high force and phosphorylation under these conditions compared to pCa 6 with or without ML-9 and pCa 4.5 with ML-9 prior to treatment with ATPS. It is obvious that ATPS pretreatment dramatically increases the calcium sensitivity of myosin light chain phosphorylation.


Figure 2: Force and myosin light chain phosphorylation under various conditions. Force (openbars) and myosin light chain phosphorylation (solidbars) at 5 min for muscles activated under the following conditions: treatment with pCa 4.5 and pCa 6 in both the presence and absence of the myosin light chain kinase inhibitor ML-9 (300 µM), pCa 6 in the presence of 10 µM GTPS, and both low calcium (pCa > 8) and pCa 6 in the presence of ML-9 following treatment with 1 mM ATPS. Data are shown as means ± S.E. for n = 4-7.



For comparison, we also show the calcium-sensitizing effect of GTPS (10 µM) at pCa 6. As expected, both force and myosin light chain phosphorylation are increased compared to pCa 6 alone. This G protein-mediated increase in calcium sensitivity is thought to result primarily from inhibition of myosin light chain phosphatase activity, and we considered the possibility that a similar inhibition of the phosphatase might be the basis for the increase in calcium sensitivity observed with ATPS pretreatment. Therefore, we estimated the rate of myosin light chain phosphatase activity under different conditions by measuring the time course of dephosphorylation of the myosin light chain following removal of ATP (10) . Under these conditions, kinase activity ceases and the subsequent decrease in phosphorylation over time reflects the activity of the phosphatase. The data are shown in Fig. 3.


Figure 3: Time course of myosin light chain dephosphorylation following removal of ATP for muscles activated under various conditions. Fractional light chain phosphorylation for muscles that were transferred to rigor solution (pCa > 8) and frozen at various times after having been activated for 5 min under the following conditions: pCa 4.5 (filledcircles), pCa 6 in the presence of 10 µM GTPS (opencircles), and pCa 6 in the presence of 300 µM ML-9 following pretreatment with 1 mM ATPS (filledtriangles). Data are shown as means ± S.E. for n = 4-7. The curves are single exponential fits to the data (see text).



There is a much slower decrease in myosin light chain phosphorylation in the muscles pretreated with ATPS compared to those activated in pCa 4.5 or in pCa 6 in the presence of 10 µM GTPS. In muscles activated in pCa 6 with ML-9 following the ATPS pretreatment, the phosphatase rate constant (0.0028 ± 0.0003 s) was much lower than those activated in pCa 4.5 (0.016 ± 0.002 s), and even lower than those activated in pCa 6 in the presence of 10 µM GTPS (0.011 ± 0.002 s). The increased calcium sensitivity of force and myosin light chain phosphorylation seen with ATPS pretreatment seems, at least in part, to be due to a more than 5-fold decrease in myosin light chain phosphatase activity.

The decrease in myosin light chain phosphatase activity in the presence of GTPS observed in this study is similar to the 50% decrease reported by others for both GTPS (10, 13) and protein kinase C activators(13) , but there is a much larger decrease in phosphatase rate caused by ATPS pretreatment. Fig. 1C shows an experimental protocol designed to test whether GTPS-mediated calcium sensitization can occur after the phosphatase activity has been decreased by ATPS pretreatment. When the muscle is in pCa 7 after ATPS pretreatment, the force output is less than maximum, and there is little, if any, increase when 100 µM GTPS is added (n = 6). Apparently, the mechanism responsible for GTPS-mediated calcium sensitization cannot decrease phosphatase activity when it is already at a very low rate as a result of ATPS pretreatment.

Control experiments confirmed that the increase in calcium sensitivity was in fact due to ATPS rather than other aspects of the treatment protocol. In the absence of ATPS, there was no change in the calcium sensitivity (n = 4), while treatment with 0.1 and 0.01 mM ATPS gave 0.99 ± 0.04 (n = 3) and 0.55 ± 0.12 (n = 4), respectively, of the response with 1 mM ATPS pretreatment. When 0.1 mM GTPS pretreatment was substituted for ATPS, no enhanced contractile response was observed (n = 4), while subsequent treatment with ATPS elicited the calcium-sensitizing effect.

It is possible that the decrease in myosin light chain phosphatase activity results from thiophosphorylation of a protein by a kinase involved in regulation of the phosphatase. [S]ATPS was used to radiolabel the proteins that are thiophosphorylated under conditions when the phosphatase activity is decreased. The total incorporation of thiophosphate into muscle protein was equivalent to 3.0 ± 0.5 µmol/liter of muscle volume (n = 6). Fig. 4A shows a Coomassie Blue-stained gel of the total homogenate separated by SDS-PAGE and its PhosphorImager autoradiogram; also shown is the scan of the autoradiogram. The bands containing most of the radioactivity were of relatively high molecular mass (>80 kDa), and in this range five or six major components were seen. There was very little radioactivity associated with low molecular weight proteins. Based on the complexity of the total homogenate, it is surprising that a more extensive pattern of thiophosphorylation was not found. In a few cases, labeled proteins of about 28 and 20 kDa were detected. The amount of radioactivity in the 20-kDa region, which would include the myosin light chain, ranged from undetectable to a maximum of 15% of the total counts. This would indicate an upper limit of about 0.5 µM for the amount of thiophosphorylated myosin light chain resulting from ATPS pretreatment. Since the myosin light chain concentration is 50-100 µM(18) , the fraction of myosin light chains thiophosphorylated is very small (<1%).


Figure 4: Separation and identification of thiophosphorylated proteins following treatment with [S]ATPS. PanelA, total muscle homogenate on SDS gel (7.5-20% acrylamide); from left, Coomassie Blue-stained gel, PhosphorImager autoradiogram of the same gel, and a scan of total S counts. PanelB, total muscle homogenate on low porosity SDS gel (same order as in panelA). PanelC, Western blot using mAb to the 130-kDa subunit of myosin light chain phosphatase on low porosity SDS gel; from left, isolated chicken gizzard 130-kDa subunit, total muscle homogenate, and PhosphorImager autoradiogram of the total homogenate lane. Panels D-F, Western blots of IEF-SDS gels using the mAb to the 130-kDa phosphatase subunit. PanelD, total muscle homogenate of a control sample (not treated with ATPS). PanelE, total muscle homogenate of an ATPS-treated sample. PanelF, PhosphorImager autoradiogram of whole muscle homogenate. The inset shows the stained portion of the Western blot, along with a scan of the radioactivity in the same portion of the autoradiogram.



Further analysis of the high molecular weight components was carried out by SDS-PAGE on low porosity gels (Fig. 4B). The Western blot using the mAb to the gizzard 130-kDa subunit of myosin light chain phosphatase and its associated autoradiogram show that one of the thiophosphorylated components (i.e.S-labeled) cross-reacts with the mAb (Fig. 4C). Its mass is slightly higher than the gizzard M130, which is also shown. The Western blots of a control muscle and an ATPS-treated muscle run on two-dimensional gels are shown in Fig. 4(D and E). The control sample, which was frozen in relaxing solution with no exposure to ATPS, showed a simple staining pattern with a major component at pI 5.9-6.0. Following ATPS treatment, several components were seen. They extended toward more acidic values over a pH range of 5.5-6.0, and at least four isoforms were apparent. An autoradiogram of a Western blot of a two-dimensional gel is shown in Fig. 4F. The inset shows the stained portion of the blot along with the autoradiographic scan of the same area. There are several S-labeled components that are superimposable on the staining pattern of the Western blot.

The fact that the 130-kDa subunit of the myosin light chain phosphatase is one of the relatively few proteins that are thiophosphorylated under conditions that result in a large decrease in activity of the enzyme in the permeabilized muscle strongly suggests that the thiophosphorylation of the subunit caused the decrease in activity. If this is the case, then it is likely that phosphorylation and dephosphorylation of the 130-kDa subunit represents an in vivo mechanism for regulation of phosphatase activity that is potentially important for the regulation of smooth muscle contraction. Studies on other cellular phosphatases have demonstrated that phosphorylation of subunits or binding proteins play a role in regulation of activity. Phosphorylation of a site on the targeting subunit of glycogen-associated protein phosphatase 1 causes a decrease in activity by decreasing the affinity of the catalytic subunit for the targeting subunit while phosphorylation of another site increases activity(27) . In addition, phosphorylation of inhibitor 1 and DARPP-32 by the cAMP-dependent protein kinase markedly increases their inhibition of PP1(28) .

In summary, thiophosphorylation of the 130-kDa regulatory subunit of myosin light chain phosphatase is associated with a 5-fold decrease in the activity of the enzyme and a large increase in the calcium sensitivity of force and myosin light chain phosphorylation in alpha-toxin-permeabilized smooth muscle. These results suggest that phosphorylation and dephosphorylation of this subunit may play a role in the regulation of smooth muscle contraction.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grants HL-50586 (to T. M. B.), HL-20984 (to D. J. H.), and HL-23615 (to D. J. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by a Foerderer fellowship and National Institutes of Health Training Grant T35-HL-07599.

To whom correspondence should be addressed: Dept. of Physiology, Jefferson Medical College, 1020 Locust St., Philadelphia, PA 19107. Tel.: 215-955-6583; Fax: 215-955-2073.

^1
The abbreviations used are: GTPS, guanosine 5`-3-O-(thio)triphosphate; ATPS, adenosine 5`-O-(thiotriphosphate); DIDS, 4,4`-diisothiocyanatostilbene-2,2`-disulfonic acid; mAb, monoclonal antibody.


ACKNOWLEDGEMENTS

We thank S. U. Mooers and S. R. Narayan for expert technical assistance.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.