- Email: [email protected]
[33] Phosphorylation of nonmuscle myosin and stabilization of thick filament structure
364
MOTILITY APPARATUS IN NONMUSCLE CELLS
[33]
[33]
Phosphorylation of Nonmuscle Myosin and Stabilization of Thick Filament Structure
B y JOHN KENDRICK-JONES, K . A . TAYLOR, a n d J. M . SCHOLEY
I n vitro studies indicate that actomyosin-dependent contractility in vertebrate nonmuscle cells 1-a and in vertebrate smooth muscles 4"5 may be regulated by the level of phosphorylation of a myosin "regulatory" light chain. The widely accepted view is that, in the absence of calcium, this 20,000 molecular weight (Mr) light chain is not phosphorylated and the myosin is unable to interact with actin. Calcium activates a specific calmodulin-dependent kinase 6-8 that phosphorylates the light chain and initiates myosin interaction with actin. Most studies have concentrated on the role of phosphorylation in the regulation of actin-myosin interaction as measured by the actin-activated myosin MgATPase activity. However, evidence a-ll suggests that phosphorylation of the 20,000 Mr light chain also seems to control the assembly of vertebrate smooth muscle and nonmuscle myosins into filaments. The effect of myosin phosphorylation on the stability of vertebrate nonmuscle myosin filaments can be monitored by turbidity measurements, electron microscope observation, and a~p-labeled phosphate incorporation. Similar experiments have been carried out on vertebrate smooth muscle myosin, a
Protein Preparation Myosin was prepared from calf thymus tissue by a procedure based on that of Pollard et al. 12 with the modifications suggested by Yerna et al. z i R. S. Adelstein and M. A. Conti, Nature (London) 256, 597 (1975). 2 M.-J. Yerna, M. O. Aksoy, D. J. Hartshome, and R. D. Goldman, J. Cell Sci. 31, 411 (1978). a j. A. Trotter and R. S. Adelstein, J. Biol. Chem. 254, 8781 (1979). 4 j. V. Small and A. Sobieszek, Eur. J. Biochem. 76, 521 (1977). J. M. F. Sherry, A. Gorecka, M. O. Aksoy, R. Dabrowska, and D. J. Hartshorne, Biochemistry 17, 4411 (1978). 6 R. Dabrowska, J. M. F. Sherry, D. Aromatorio, and D. J. Hartshorne, Biochemistry 17, 253 (1978). r M.-J. Yerna, R. Dabrowska, D. J. Hartshorne, and R. D. Goldman, Proc. Natl. Acad. Sci. U.S.A. 76, 184 (1979). 8 D. R. Hathaway and R. S. Adelstein, Proc. Natl. Acad. Sci. U.S.A. 76, 1653 (1979). a H. Suzuki, H. Onishi, K. Takahashi, and S. Watanabe, J. Biochem. (Tokyo) 84, 1529 (1978).
METHODSIN ENZYMOLOGY,VOL. 85
Copyright© 1982by AcademicPress, Inc. All rightsof reproductionin any formreserved. ISBN 0-12-181985-X
[33]
PHOSPHORYLATION OF NONMUSCLE MYOSIN
365
About 500 g of fresh calf thymus tissue (rapidly cooled in ice-water after removal from the animal and processed within 1 hr) were used for each myosin preparation. The myosin was stored in 0.6 M NaC1, 1 mM MgC12, 25 mM Tris-HC1 buffer, pH 7.5, at 5-15 mg m1-1 in ice and used within 3 days. This procedure reproducibly gave a nonphosphorylated myosin (at least 80-90% dephosphorylated as indicated by 10% polyacrylamide gel electrophoresis in 8 M urea13). Myosin was prepared from pig or human platelets using essentially the same procedure, but with the following minor modification. The washed platelets (25-100 g of packed pellet) were lysed by rapid freezing in liquid nitrogen and thawed in three volumes of extraction buffer containing 0.8 M KC1, 10 mM dithiothreitol, 5 mM EDTA, 1 mM azide, 2.5 mM ATP, 0.01 mg m1-1 lima bean trypsin inhibitor, 25 /~g m1-1 L-l-tosylamide2-phenylethylchloromethyl ketone (TPCK), 1/xg m1-1 leupeptin, 0.2 mM phenylmethylsulfonyl fluoride (PMSF), and 25 mM Tris-HC1 buffer, pH 7.5. Vertebrate smooth muscle myosin was prepared from chicken gizzards by a number of different procedures to exclude the possibility that myosin filament stability was dependent on the method of myosin preparation. Myosin was thus prepared by (a) a similar procedure to that used for preparing thymus myosin; (b) by the method described by Hartshorne et al. 14; and (c) by a procedure modified from that used by Focant and Huriaux 15to prepare myosin from carp and pike skeletal muscle involving ammonium sulfate fractionation of the actomyosin (37-60% fraction) in the presence of 0.6 M KC1, 25 mM imidazole (pH 7.0), 20 m~/MgC12, 5 mM ATP, and 1 mM EGTA. All these myosin preparations were further purified by gel filtration chromatography on Sepharose 4B (90 × 5 cm columns) in 0.6 M KCI, 1 mM EDTA, 25 mM Tris-HCl (pH 7.5), 0.1 mM PMSF, 0.2 mM DTT, 1 mM azide. No difference in the stability of the filaments prepared from these different smooth muscle myosin preparations was observed. In all cases, filament stability at physiological ionic strength and MgATP concentrations was dependent on the level of myosin phosphorylation. Vertebrate smooth muscle light-chain kinase was prepared from ~0 C. F. Shoenberg and M. Stewart, J. Muscle Res. Cell Motil. 1, 117 (1980). H j. M. Scholey, K. A. Taylor and J. Kendrick-Jones, Nature (London) 287, 233 (1980). 12 T. D. Pollard, S. M. Thomas, and R. Niederman, Anal. Biochern. 60, 258 (1976). ~z W. T. Perrie and S. V. Perry, Biochem. J. 119, 31 (1970). ~4 D. J. Hartshorne, A. Gorecka, and M. O. Aksoy, In "Excitation Contraction Coupling in Smooth Muscle" (R. Casteels, T. Godfraind, and J. C. RiJegg, eds.), pp. 377-384. Elsevier/North-Holland Publ., Amsterdam, 1977. ~'~B. Focant and F. Huriaux, FEBS Lett. 65, 16 (1976).
366
MOTILITY APPARATUS IN NONMUSCLE CELLS
[33]
chicken gizzards by the procedures outlined by Aksoy e t al. TM and Dabrowska e t a l . 6 with the following minor modification. After the initial extraction and centrifugation step, the supernatant was adjusted to 0.8 M KC1, 1 mM EGTA, 1 mM MgCI~, 2 mM dithiothreitol, 0.1 mM PMSF, 1 mM sodium azide, and 10 mM Tris-HC1 (pH 7.5) and stirred in ice for 30 min. It was then subjected to ammonium sulfate fractionation to yield the 37-55% saturated ammonium sulfate fraction ("crude tropomyosin" fraction). Using this modification, the step involving gel filtration on Sepharose 4B, described in the above procedures, could be avoided. The light-chain kinase was prepared from calf thymus tissue (-300 g of starting material) by the procedure described by Dabrowska and Hartshorne. lz To minimize proteolysis, 0.5 mM sodium azide, 0.2 mM PMSF, 20 /zg of p-Tosyl-L-arginine methyl ester-HCl (TAME-HCI) per milliliter, and 0.1 mg of lima bean trypsin inhibitor per milliliter were included in the initial extraction medium. Despite these precautions, when the 35-60% saturated ammonium sulfate fraction was chromatographed on Sepharose 4B (90 × 5 cm column) in 0.8M KC1, 1 mM EGTA, 0.2 mM dithiothreitol, 0.2 mM PMSF, 1 mM azide, and 25 mM Tris-HCl (pH 7.5), two peaks of light-chain kinase activity were detected. The peak eluted at about 100,000 Mr contained the calcium-sensitive kinase, and the peak eluted at a slightly lower molecular weight contained a "kinase" that was fully active in the absence of calcium. Only the fractions containing the Ca2÷-dependent kinase were pooled for further purification. The smooth muscle and thymus kinase fractions were further purified on Sepharose 4B-brain calmodulin affinity columns using the procedure outlined by Watterson and Vanaman.IS Kinase activity was measured using DEAE-cellulose-purified gizzard 20,000 Mr light chains 19 and the assay conditions described by Frearson e t a l . so Calmodulin was initially prepared from calf thymus by the method 2 based on conventional procedures, but later a rapid procedure involving a trichloracetic acid precipitation step as initially proposed by Yagi 21 was used. Calf thymus tissue (-500 g) was thoroughly homogenized in 3 volumes of 0.34M sucrose, 5 mM EDTA, 2 mM EGTA, 1 mM dithiothreitol, 1 mM azide, 0.1 mM PMSF, 15 mM Tris-HC1 (pH 7.5) and stirred for 15 min. The unextracted material was removed by centrifugation at 20,000g 16 M. O. Aksoy, D. Williams, E. M. Sharkey, and D. J. Hartshorne, Biochem. Biophys. Res. Commun. 69, 35 (1976). 17 R. Dabrowska and D. J. Hartshorne, Biochem. Biophys. Res. Commun. 85, 1352 (1978). 18 D. M. Watterson and T. C. Vanaman, Biochem. Biophys. Res. Commun. 73, 40 (1976). la R. Jakes, F. Northrop, and J. Kendrick-Jones, FEBS Lett. 70, 229 (1976). z0 N. Frearson, B. W. Focant, and S. V. Perry, FEBS Lett. 63, 27 (1976). 21 M. Yazawa, M. Sakuma, and K. Yagi, J. Biochem. (Tokyo) 87, 1313 (1980).
[33]
PHOSPHORYLATION OF NONMUSCLE MYOSIN
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for 1 hr, and to the supernatant cold 60% (w/v) trichloroacetic acid was added slowly with stirring to a final concentration of 3%. After stirring for 15 min the precipitated protein was collected by centrifugation and dispersed in 25 mM sodium acetate (pH 5.2) by gentle homogenisation, and the pH was adjusted to 5.2 by the addition of sodium hydroxide. With the pH maintained at 5.2, the suspension was stirred for 30 min, then thoroughly homogenized; the undissolved material was removed by centrifugation at 30,000 g for 20 rain. To concentrate the supernatant, trichloroacetic acid was added to a final concentration of 3%, the precipitated protein was collected by centrifugation and dispersed in 25 mM Tris-HC1 buffer (pH 7.5); the pH was adjusted to 7.5 (volume - 5 0 ml). After dialysis against 25 mM Tris-HC1 (pH 7.5), 1 mM MgCl2, 0.5 mM EGTA, solid urea was added to 4 M and the solution was clarified by centrifugation at 40,000 g for 30 rain. The supernatant was chromatographed on a DEAE-cellulose column (40 x 2.54 cm) equilibrated in 2 M urea, 25 mM Tris-HC1 buffer (pH 7.5), 1 mM MgC12, 0.5 mM EGTA, 0.2 mM DTT running buffer. The calmodulin was eluted with a linear gradient (total volume 1400 ml) of 100 mM to 400 mM NaCl in the above running buffer. Calmodulin eluted at - 0 . 2 8 M NaC1 and was further purified by gel filtration on a Sephadex G-100 (95 × 1.6 cm) column equilibrated and run in 25 mM Tris-HC1 (pH 7.5), 1 mM MgC12, 0.5 mM EGTA and stored frozen in the presence of a 1 mM azide. Experimental Procedures The stability of vertebrate nonmuscle myosin filaments was initially monitored by turbidity measurements. Nonphosphorylated thymus myosin (-0.5 mg m1-1) was dialyzed into 0.15 M KC1, 10 mM MgC12, 1 mM EGTA, 0.1 mM dithiothreitol, 25 mM imidazole (pH 7.0) to form filaments. This myosin filament "solution" was incubated at 20° with 25 /zg of calmodulin per milliliter and myosin light-chain kinase purified from either chicken gizzard or calf thymus (100/.tg ml-1), and the stability of the filaments was monitored by measuring the turbidity of the solution at k = 340 nm in a Perkin-Elmer Model 551 double-beam spectrophotometer. A 0.5 mg m1-1 solution of thymus myosin filaments gave an absorption at 340 nm of 0.5-0.6. The turbidity remained constant until [y-32P]ATP (5/zCi/ /zmol) was added to a final concentration of 1 mM, whereupon a rapid drop in turbidity was observed (A34o -- 0.2) suggesting that the myosin filaments had disassembled. The turbidity remained low until the addition of Ca ~+ to a concentration of 2 × l0 -4 M free Ca 2+, which led to a steady increase in turbidity to about the initial level, suggesting that myosin filaments were re-forming. Further additions of ATP (1 mM) had no effect
368
MOTILITY APPARATUS IN NONMUSCLE CELLS
[33]
on the measured turbidity, indicating that the myosin filaments were now stable. The same results were observed regardless of the source of kinase used, i.e., vertebrate smooth muscle or thymus. To verify that the turbidity measurements were monitoring the stability of the myosin filaments, aliquots of the myosin solution were taken at precise times for electron microscope observation. It was found to be essential that the turbidity measurements always be used in conjunction with electron microscope observations in order to verify and to "estimate" the degree of myosin filament disassembly and reassembly. The specimens were prepared for the electron microscope by placing 10/.~l of the myosin solution on carbon-coated 400-mesh grids, allowing the drop to stand for exactly 30 sec, then washing with 13 drops of buffer and 10 drops of 1% uranyl acetate. In the electron microscope, the initial myosin solution contained numerous myosin filaments (bipolar, average length 3380 -+ 560 A) (Fig. 1). After addition of ATP, these filaments were missing and only a dense granular background of dissolved protein could be seen. When Ca 2+ was added, the steady increase in the observed turbidity correlates with the appearance of bipolar filaments (average length 5000 640 ,~) in the electron microscope. These observ~itions suggest that nonphosphorylated thymus myosin filaments, in the absence of Ca 2+, are disassembled by MgATP and are induced to reassemble into filaments when the kinase is activated by Ca 2+. Similar results have been obtained with myosins isolated from human platelets and vertebrate smooth muscle (Fig. 1), TM whereas myosin filaments prepared from vertebrate and invertebrate striated myosins are stable under these conditions. Further confirmation of the "stability" of these nonmuscle myosin filaments, in the presence and in the absence of ATP, can be obtained by following their sedimentation profiles in the analytical ultracentrifuge under the same conditions, but with myosin concentrations in the range 1.5-2.5 mg m1-1. To prove that the nonmuscle myosin filaments are induced to assemble when the myosin is phosphorylated by the calcium-activated calmodulindependent kinase, the level and specificity of phosphate incorporation into the thymus myosin 20,000 Mr light chain during the course of the turbidity measurements was determined by the following procedures. Incorporation of 32p was measured by a Millipore filtration procedure similar to that described by Jakes et al. 19 Aliquots (100/~l) of the myosin solution were removed at precise time intervals [two aliquots were also removed for polyacrylamide gel electrophoresis in sodium dodecyl sulfate (SDS) and in 8M urea] and quenched in 2 ml of ice cold 10% trichloroacetic acid, 1 mM ATP, and 50 mM sodium pyrophosphate. Immediately 0.25 ml of bovine serum albumin (2.5 mg m1-1) were added as a carrier, mixed, and heated at 90 ° for 10 min to ensure complete release of noncovalently bound phos-
[33]
P F I O S P H O R Y L A T I OOF N NONMUSCLE MYOSIN
A
13
IP
2
369
f! /
aL. w
.i
3
FIG. i. Electron micrographs illustrating the stability of (A) vertebrate nonmuscle (thymus) and (B) vertebrate smooth muscle (gizzard) myosin filaments. Row 1: Nonphosphorylated myosin filaments prepared by dialysis against 0.15 M KCI, 10 mM MgCl~, 1 mM EGTA, 0.1 mM dithiothreitol, 25 mM imidazole (pH 7.0) and incubated at 20° in the presence of calmodulin and kinase. The thymus myosin filaments are ~0.35/zm long, whereas those of the vertebrate smooth muscle myosin are -0.75/.tm long. Row 2: After the addition of 1 mM ATP, the filaments have completely disappeared and, in their place, a high concentration of dissolved protein can be seen on the grid. Row 3: After addition of 2 × l0 -4 M free Ca 2+, myosin filaments reappear. Bar represents 1 p.m.
370
M O T I L I T Y A P P A R A T U S IN N O N M U S C L E CELLS
[33]
phate. The samples were cooled in ice, and the protein precipitates were transferred to Millipore filters (type HA, 0.45 /xm), the tubes being thoroughly washed to ensure complete transfer of the precipitated protein. The filters were washed under rapid suction with six 4-ml aliquots of the TCA-ATP-pyrophosphate solution and then rapidly with four 5-ml aliquots of cold distilled water. The partially dried filters (the moist filters ensure that the precipitated protein sticks to the filters during transfer to the scintillation vials) were dispersed in scintillation fluid (Aquasol-2, New England Nuclear) and counted in a Beckman (Model LS7000) counter with the appropriate 32p settings (program I0). The 32p incorporation data indicated that, on addition of calcium, reassembly of the myosin filaments was accompanied by a steady increase in a2p incorporation (-0.8 mol of phosphate incorporated per mole of light chain 20,000 Mr). In the absence of kinase or Ca 2+, there was no significant s2p incorporation or filament formation. The specificity of phosphate incorporation into the 20,000 Mr myosin light chain was checked by polyacrylamide gel electrophoresis and subsequent scanning and autoradiography of the dried gels. Aliquots of the myosin solution taken at the same time points as those for the a2p incorporation measurements were quenched in 0.5% SDS sample buffer and in 3% trichloroacetic acid 9 (for 8 M urea PAGE) and analyzed on 10% acrylamide gels using 0.1% SDS-0.1 M Tris-Bicine (pH 8.1) and 8 M urea0.1 M Tris-glycine (pH 8.6) buffer systems. 13 The relative ratio of the nonphosphorylated to phosphorylated 20,000 Mr light chain content of the myosin was determined by scanning the 8 M urea-acrylamide gels after staining with Coomassie Blue. To check the specificity of 32p incorporation, both types of gels were dried and autoradiographed in cassettes fitted with tungstate intensifying screens (Ilford). There was reasonable agreement between the azp incorporation and the gel analysis, indicating that only the 20,000 Mr light chain is specifically phosphorylated. These experiments, carried out under ionic conditions similar to those that may exist in cells, suggest that, in the resting cell, if the myosin is nonphosphorylated it is not assembled into filaments. Stimulation of the cell leads to an increase in the intracellular free calcium, which activates the Ca~+-calmodulin-dependent kinase, resulting in phosphorylation of the light chain leading to myosin filament assembly. However, we will know whether these in vitro studies on the regulation of myosin filament assembly have any relevance to the situation inside the nonmuscle cell only when procedures have been developed for observing within the living cell the state of the myosin present at rest compared to periods of contractile activity.
MOTILITY APPARATUS IN NONMUSCLE CELLS
[33]
[33]
Phosphorylation of Nonmuscle Myosin and Stabilization of Thick Filament Structure
B y JOHN KENDRICK-JONES, K . A . TAYLOR, a n d J. M . SCHOLEY
I n vitro studies indicate that actomyosin-dependent contractility in vertebrate nonmuscle cells 1-a and in vertebrate smooth muscles 4"5 may be regulated by the level of phosphorylation of a myosin "regulatory" light chain. The widely accepted view is that, in the absence of calcium, this 20,000 molecular weight (Mr) light chain is not phosphorylated and the myosin is unable to interact with actin. Calcium activates a specific calmodulin-dependent kinase 6-8 that phosphorylates the light chain and initiates myosin interaction with actin. Most studies have concentrated on the role of phosphorylation in the regulation of actin-myosin interaction as measured by the actin-activated myosin MgATPase activity. However, evidence a-ll suggests that phosphorylation of the 20,000 Mr light chain also seems to control the assembly of vertebrate smooth muscle and nonmuscle myosins into filaments. The effect of myosin phosphorylation on the stability of vertebrate nonmuscle myosin filaments can be monitored by turbidity measurements, electron microscope observation, and a~p-labeled phosphate incorporation. Similar experiments have been carried out on vertebrate smooth muscle myosin, a
Protein Preparation Myosin was prepared from calf thymus tissue by a procedure based on that of Pollard et al. 12 with the modifications suggested by Yerna et al. z i R. S. Adelstein and M. A. Conti, Nature (London) 256, 597 (1975). 2 M.-J. Yerna, M. O. Aksoy, D. J. Hartshome, and R. D. Goldman, J. Cell Sci. 31, 411 (1978). a j. A. Trotter and R. S. Adelstein, J. Biol. Chem. 254, 8781 (1979). 4 j. V. Small and A. Sobieszek, Eur. J. Biochem. 76, 521 (1977). J. M. F. Sherry, A. Gorecka, M. O. Aksoy, R. Dabrowska, and D. J. Hartshorne, Biochemistry 17, 4411 (1978). 6 R. Dabrowska, J. M. F. Sherry, D. Aromatorio, and D. J. Hartshorne, Biochemistry 17, 253 (1978). r M.-J. Yerna, R. Dabrowska, D. J. Hartshorne, and R. D. Goldman, Proc. Natl. Acad. Sci. U.S.A. 76, 184 (1979). 8 D. R. Hathaway and R. S. Adelstein, Proc. Natl. Acad. Sci. U.S.A. 76, 1653 (1979). a H. Suzuki, H. Onishi, K. Takahashi, and S. Watanabe, J. Biochem. (Tokyo) 84, 1529 (1978).
METHODSIN ENZYMOLOGY,VOL. 85
Copyright© 1982by AcademicPress, Inc. All rightsof reproductionin any formreserved. ISBN 0-12-181985-X
[33]
PHOSPHORYLATION OF NONMUSCLE MYOSIN
365
About 500 g of fresh calf thymus tissue (rapidly cooled in ice-water after removal from the animal and processed within 1 hr) were used for each myosin preparation. The myosin was stored in 0.6 M NaC1, 1 mM MgC12, 25 mM Tris-HC1 buffer, pH 7.5, at 5-15 mg m1-1 in ice and used within 3 days. This procedure reproducibly gave a nonphosphorylated myosin (at least 80-90% dephosphorylated as indicated by 10% polyacrylamide gel electrophoresis in 8 M urea13). Myosin was prepared from pig or human platelets using essentially the same procedure, but with the following minor modification. The washed platelets (25-100 g of packed pellet) were lysed by rapid freezing in liquid nitrogen and thawed in three volumes of extraction buffer containing 0.8 M KC1, 10 mM dithiothreitol, 5 mM EDTA, 1 mM azide, 2.5 mM ATP, 0.01 mg m1-1 lima bean trypsin inhibitor, 25 /~g m1-1 L-l-tosylamide2-phenylethylchloromethyl ketone (TPCK), 1/xg m1-1 leupeptin, 0.2 mM phenylmethylsulfonyl fluoride (PMSF), and 25 mM Tris-HC1 buffer, pH 7.5. Vertebrate smooth muscle myosin was prepared from chicken gizzards by a number of different procedures to exclude the possibility that myosin filament stability was dependent on the method of myosin preparation. Myosin was thus prepared by (a) a similar procedure to that used for preparing thymus myosin; (b) by the method described by Hartshorne et al. 14; and (c) by a procedure modified from that used by Focant and Huriaux 15to prepare myosin from carp and pike skeletal muscle involving ammonium sulfate fractionation of the actomyosin (37-60% fraction) in the presence of 0.6 M KC1, 25 mM imidazole (pH 7.0), 20 m~/MgC12, 5 mM ATP, and 1 mM EGTA. All these myosin preparations were further purified by gel filtration chromatography on Sepharose 4B (90 × 5 cm columns) in 0.6 M KCI, 1 mM EDTA, 25 mM Tris-HCl (pH 7.5), 0.1 mM PMSF, 0.2 mM DTT, 1 mM azide. No difference in the stability of the filaments prepared from these different smooth muscle myosin preparations was observed. In all cases, filament stability at physiological ionic strength and MgATP concentrations was dependent on the level of myosin phosphorylation. Vertebrate smooth muscle light-chain kinase was prepared from ~0 C. F. Shoenberg and M. Stewart, J. Muscle Res. Cell Motil. 1, 117 (1980). H j. M. Scholey, K. A. Taylor and J. Kendrick-Jones, Nature (London) 287, 233 (1980). 12 T. D. Pollard, S. M. Thomas, and R. Niederman, Anal. Biochern. 60, 258 (1976). ~z W. T. Perrie and S. V. Perry, Biochem. J. 119, 31 (1970). ~4 D. J. Hartshorne, A. Gorecka, and M. O. Aksoy, In "Excitation Contraction Coupling in Smooth Muscle" (R. Casteels, T. Godfraind, and J. C. RiJegg, eds.), pp. 377-384. Elsevier/North-Holland Publ., Amsterdam, 1977. ~'~B. Focant and F. Huriaux, FEBS Lett. 65, 16 (1976).
366
MOTILITY APPARATUS IN NONMUSCLE CELLS
[33]
chicken gizzards by the procedures outlined by Aksoy e t al. TM and Dabrowska e t a l . 6 with the following minor modification. After the initial extraction and centrifugation step, the supernatant was adjusted to 0.8 M KC1, 1 mM EGTA, 1 mM MgCI~, 2 mM dithiothreitol, 0.1 mM PMSF, 1 mM sodium azide, and 10 mM Tris-HC1 (pH 7.5) and stirred in ice for 30 min. It was then subjected to ammonium sulfate fractionation to yield the 37-55% saturated ammonium sulfate fraction ("crude tropomyosin" fraction). Using this modification, the step involving gel filtration on Sepharose 4B, described in the above procedures, could be avoided. The light-chain kinase was prepared from calf thymus tissue (-300 g of starting material) by the procedure described by Dabrowska and Hartshorne. lz To minimize proteolysis, 0.5 mM sodium azide, 0.2 mM PMSF, 20 /zg of p-Tosyl-L-arginine methyl ester-HCl (TAME-HCI) per milliliter, and 0.1 mg of lima bean trypsin inhibitor per milliliter were included in the initial extraction medium. Despite these precautions, when the 35-60% saturated ammonium sulfate fraction was chromatographed on Sepharose 4B (90 × 5 cm column) in 0.8M KC1, 1 mM EGTA, 0.2 mM dithiothreitol, 0.2 mM PMSF, 1 mM azide, and 25 mM Tris-HCl (pH 7.5), two peaks of light-chain kinase activity were detected. The peak eluted at about 100,000 Mr contained the calcium-sensitive kinase, and the peak eluted at a slightly lower molecular weight contained a "kinase" that was fully active in the absence of calcium. Only the fractions containing the Ca2÷-dependent kinase were pooled for further purification. The smooth muscle and thymus kinase fractions were further purified on Sepharose 4B-brain calmodulin affinity columns using the procedure outlined by Watterson and Vanaman.IS Kinase activity was measured using DEAE-cellulose-purified gizzard 20,000 Mr light chains 19 and the assay conditions described by Frearson e t a l . so Calmodulin was initially prepared from calf thymus by the method 2 based on conventional procedures, but later a rapid procedure involving a trichloracetic acid precipitation step as initially proposed by Yagi 21 was used. Calf thymus tissue (-500 g) was thoroughly homogenized in 3 volumes of 0.34M sucrose, 5 mM EDTA, 2 mM EGTA, 1 mM dithiothreitol, 1 mM azide, 0.1 mM PMSF, 15 mM Tris-HC1 (pH 7.5) and stirred for 15 min. The unextracted material was removed by centrifugation at 20,000g 16 M. O. Aksoy, D. Williams, E. M. Sharkey, and D. J. Hartshorne, Biochem. Biophys. Res. Commun. 69, 35 (1976). 17 R. Dabrowska and D. J. Hartshorne, Biochem. Biophys. Res. Commun. 85, 1352 (1978). 18 D. M. Watterson and T. C. Vanaman, Biochem. Biophys. Res. Commun. 73, 40 (1976). la R. Jakes, F. Northrop, and J. Kendrick-Jones, FEBS Lett. 70, 229 (1976). z0 N. Frearson, B. W. Focant, and S. V. Perry, FEBS Lett. 63, 27 (1976). 21 M. Yazawa, M. Sakuma, and K. Yagi, J. Biochem. (Tokyo) 87, 1313 (1980).
[33]
PHOSPHORYLATION OF NONMUSCLE MYOSIN
367
for 1 hr, and to the supernatant cold 60% (w/v) trichloroacetic acid was added slowly with stirring to a final concentration of 3%. After stirring for 15 min the precipitated protein was collected by centrifugation and dispersed in 25 mM sodium acetate (pH 5.2) by gentle homogenisation, and the pH was adjusted to 5.2 by the addition of sodium hydroxide. With the pH maintained at 5.2, the suspension was stirred for 30 min, then thoroughly homogenized; the undissolved material was removed by centrifugation at 30,000 g for 20 rain. To concentrate the supernatant, trichloroacetic acid was added to a final concentration of 3%, the precipitated protein was collected by centrifugation and dispersed in 25 mM Tris-HC1 buffer (pH 7.5); the pH was adjusted to 7.5 (volume - 5 0 ml). After dialysis against 25 mM Tris-HC1 (pH 7.5), 1 mM MgCl2, 0.5 mM EGTA, solid urea was added to 4 M and the solution was clarified by centrifugation at 40,000 g for 30 rain. The supernatant was chromatographed on a DEAE-cellulose column (40 x 2.54 cm) equilibrated in 2 M urea, 25 mM Tris-HC1 buffer (pH 7.5), 1 mM MgC12, 0.5 mM EGTA, 0.2 mM DTT running buffer. The calmodulin was eluted with a linear gradient (total volume 1400 ml) of 100 mM to 400 mM NaCl in the above running buffer. Calmodulin eluted at - 0 . 2 8 M NaC1 and was further purified by gel filtration on a Sephadex G-100 (95 × 1.6 cm) column equilibrated and run in 25 mM Tris-HC1 (pH 7.5), 1 mM MgC12, 0.5 mM EGTA and stored frozen in the presence of a 1 mM azide. Experimental Procedures The stability of vertebrate nonmuscle myosin filaments was initially monitored by turbidity measurements. Nonphosphorylated thymus myosin (-0.5 mg m1-1) was dialyzed into 0.15 M KC1, 10 mM MgC12, 1 mM EGTA, 0.1 mM dithiothreitol, 25 mM imidazole (pH 7.0) to form filaments. This myosin filament "solution" was incubated at 20° with 25 /zg of calmodulin per milliliter and myosin light-chain kinase purified from either chicken gizzard or calf thymus (100/.tg ml-1), and the stability of the filaments was monitored by measuring the turbidity of the solution at k = 340 nm in a Perkin-Elmer Model 551 double-beam spectrophotometer. A 0.5 mg m1-1 solution of thymus myosin filaments gave an absorption at 340 nm of 0.5-0.6. The turbidity remained constant until [y-32P]ATP (5/zCi/ /zmol) was added to a final concentration of 1 mM, whereupon a rapid drop in turbidity was observed (A34o -- 0.2) suggesting that the myosin filaments had disassembled. The turbidity remained low until the addition of Ca ~+ to a concentration of 2 × l0 -4 M free Ca 2+, which led to a steady increase in turbidity to about the initial level, suggesting that myosin filaments were re-forming. Further additions of ATP (1 mM) had no effect
368
MOTILITY APPARATUS IN NONMUSCLE CELLS
[33]
on the measured turbidity, indicating that the myosin filaments were now stable. The same results were observed regardless of the source of kinase used, i.e., vertebrate smooth muscle or thymus. To verify that the turbidity measurements were monitoring the stability of the myosin filaments, aliquots of the myosin solution were taken at precise times for electron microscope observation. It was found to be essential that the turbidity measurements always be used in conjunction with electron microscope observations in order to verify and to "estimate" the degree of myosin filament disassembly and reassembly. The specimens were prepared for the electron microscope by placing 10/.~l of the myosin solution on carbon-coated 400-mesh grids, allowing the drop to stand for exactly 30 sec, then washing with 13 drops of buffer and 10 drops of 1% uranyl acetate. In the electron microscope, the initial myosin solution contained numerous myosin filaments (bipolar, average length 3380 -+ 560 A) (Fig. 1). After addition of ATP, these filaments were missing and only a dense granular background of dissolved protein could be seen. When Ca 2+ was added, the steady increase in the observed turbidity correlates with the appearance of bipolar filaments (average length 5000 640 ,~) in the electron microscope. These observ~itions suggest that nonphosphorylated thymus myosin filaments, in the absence of Ca 2+, are disassembled by MgATP and are induced to reassemble into filaments when the kinase is activated by Ca 2+. Similar results have been obtained with myosins isolated from human platelets and vertebrate smooth muscle (Fig. 1), TM whereas myosin filaments prepared from vertebrate and invertebrate striated myosins are stable under these conditions. Further confirmation of the "stability" of these nonmuscle myosin filaments, in the presence and in the absence of ATP, can be obtained by following their sedimentation profiles in the analytical ultracentrifuge under the same conditions, but with myosin concentrations in the range 1.5-2.5 mg m1-1. To prove that the nonmuscle myosin filaments are induced to assemble when the myosin is phosphorylated by the calcium-activated calmodulindependent kinase, the level and specificity of phosphate incorporation into the thymus myosin 20,000 Mr light chain during the course of the turbidity measurements was determined by the following procedures. Incorporation of 32p was measured by a Millipore filtration procedure similar to that described by Jakes et al. 19 Aliquots (100/~l) of the myosin solution were removed at precise time intervals [two aliquots were also removed for polyacrylamide gel electrophoresis in sodium dodecyl sulfate (SDS) and in 8M urea] and quenched in 2 ml of ice cold 10% trichloroacetic acid, 1 mM ATP, and 50 mM sodium pyrophosphate. Immediately 0.25 ml of bovine serum albumin (2.5 mg m1-1) were added as a carrier, mixed, and heated at 90 ° for 10 min to ensure complete release of noncovalently bound phos-
[33]
P F I O S P H O R Y L A T I OOF N NONMUSCLE MYOSIN
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FIG. i. Electron micrographs illustrating the stability of (A) vertebrate nonmuscle (thymus) and (B) vertebrate smooth muscle (gizzard) myosin filaments. Row 1: Nonphosphorylated myosin filaments prepared by dialysis against 0.15 M KCI, 10 mM MgCl~, 1 mM EGTA, 0.1 mM dithiothreitol, 25 mM imidazole (pH 7.0) and incubated at 20° in the presence of calmodulin and kinase. The thymus myosin filaments are ~0.35/zm long, whereas those of the vertebrate smooth muscle myosin are -0.75/.tm long. Row 2: After the addition of 1 mM ATP, the filaments have completely disappeared and, in their place, a high concentration of dissolved protein can be seen on the grid. Row 3: After addition of 2 × l0 -4 M free Ca 2+, myosin filaments reappear. Bar represents 1 p.m.
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M O T I L I T Y A P P A R A T U S IN N O N M U S C L E CELLS
[33]
phate. The samples were cooled in ice, and the protein precipitates were transferred to Millipore filters (type HA, 0.45 /xm), the tubes being thoroughly washed to ensure complete transfer of the precipitated protein. The filters were washed under rapid suction with six 4-ml aliquots of the TCA-ATP-pyrophosphate solution and then rapidly with four 5-ml aliquots of cold distilled water. The partially dried filters (the moist filters ensure that the precipitated protein sticks to the filters during transfer to the scintillation vials) were dispersed in scintillation fluid (Aquasol-2, New England Nuclear) and counted in a Beckman (Model LS7000) counter with the appropriate 32p settings (program I0). The 32p incorporation data indicated that, on addition of calcium, reassembly of the myosin filaments was accompanied by a steady increase in a2p incorporation (-0.8 mol of phosphate incorporated per mole of light chain 20,000 Mr). In the absence of kinase or Ca 2+, there was no significant s2p incorporation or filament formation. The specificity of phosphate incorporation into the 20,000 Mr myosin light chain was checked by polyacrylamide gel electrophoresis and subsequent scanning and autoradiography of the dried gels. Aliquots of the myosin solution taken at the same time points as those for the a2p incorporation measurements were quenched in 0.5% SDS sample buffer and in 3% trichloroacetic acid 9 (for 8 M urea PAGE) and analyzed on 10% acrylamide gels using 0.1% SDS-0.1 M Tris-Bicine (pH 8.1) and 8 M urea0.1 M Tris-glycine (pH 8.6) buffer systems. 13 The relative ratio of the nonphosphorylated to phosphorylated 20,000 Mr light chain content of the myosin was determined by scanning the 8 M urea-acrylamide gels after staining with Coomassie Blue. To check the specificity of 32p incorporation, both types of gels were dried and autoradiographed in cassettes fitted with tungstate intensifying screens (Ilford). There was reasonable agreement between the azp incorporation and the gel analysis, indicating that only the 20,000 Mr light chain is specifically phosphorylated. These experiments, carried out under ionic conditions similar to those that may exist in cells, suggest that, in the resting cell, if the myosin is nonphosphorylated it is not assembled into filaments. Stimulation of the cell leads to an increase in the intracellular free calcium, which activates the Ca~+-calmodulin-dependent kinase, resulting in phosphorylation of the light chain leading to myosin filament assembly. However, we will know whether these in vitro studies on the regulation of myosin filament assembly have any relevance to the situation inside the nonmuscle cell only when procedures have been developed for observing within the living cell the state of the myosin present at rest compared to periods of contractile activity.