Involvement of Myosin Light-Chain Kinase in Endothelial Cell Retraction (Actin/Microfilaments/Cytoskeleton/Calmodulin) ROBERT B

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Proc. Nati. Acad. Sci. USA Vol. 87, pp. 16-20, January 1990 Medical Sciences Involvement of myosin light-chain kinase in endothelial cell retraction (actin/microfilaments/cytoskeleton/calmodulin) ROBERT B. WYSOLMERSKI* AND DAVID LAGUNOFF Department of Pathology, Saint Louis University School of Medicine, 1402 South Grand Boulevard, Saint Louis, MO 63104 Communicated by Earl P. Benditt, September 26, 1989

ABSTRACT Permeabilized bovine pulmonary artery en- light-chain phosphorylation permits activation of myosin dothelial cell monolayers were used to investigate the mecha- ATPase by actin and is thereby believed to initiate the nism of endothelial cell retraction. Postconfluent endothelial contractile events resulting in the shortening and tension cells permeabilized with saponin retracted upon exposure to development of smooth muscle cells (6-8). ATP and Ca2+. Retraction was accompanied by thiophospho- Skinned preparations of smooth muscle in which the mem- rylation of 19,000-Da myosin light chains when adenosine brane barrier to influx of large molecules and charged small 5'-[y-["SSthioltrlphosphate was included in the dium. Both molecules has been destroyed have been important in ana- retraction and thiophosphorylation of myosin liht chains lyzing the biochemical characteristics of the contractile pro- exhibited a graded quantitative dependence on Ca2+. When cess in these cells (6, 7, 9). Similar preparations offibroblasts permeabilized monolayers were extracted in buffer D contain- (10, 11) and epithelial cells (12, 13) have also been studied. ing 100 mM KCI and 30 mM MgCI2 for 30 min, the cells failed We have developed a preparation of permeabilized ECs that to retract upon exposure to ATP and Ca2+, and no thiophos- has provided us with the opportunity to investigate the role phorylation of myosin light chains occurred. The ability both ofATP, Ca2+, calmodulin, and MLCK in the initiation of EC to retract and to thiophosphorylate myosin light chains was retraction. restored by the addition to the permeabilized, extracted cells of myosin light-chain kinase and calmodulin together but not by EXPERIMENTAL PROCEDURES either alone. These studies indicate that endothelial cell retraction, as does smooth muscle contraction, depends on myosin Cell Culture. The bovine pulmonary artery cell linedeveloped light-chain kinase phosphorylation of myosin light chains. by Del Vecchio and Smith (14) was obtained from American Type Culture Collection (CLL-209). Cells were grown in Ea- gle's minimal essential medium supplemented with 2 mM glu- A major function of the endothelial cell (EC) is to serve as a tamine, 10o or 20%o fetal calf serum, penicillin at 50 units/ml, barrier to fluid and solute flux across the blood vessel wall. and streptomycin at 50 gg/ml. Cells were maintained at 370C in Breakdown ofthis barrier leads to increased permeability and a humidified 5% C02/95% air atmosphere. Cells used in these the development of edema. Since the classic report by Majno studies were 7 days postconfluent (2). and Palade (1) describing opening ofintracellularjunctions on Cell Permeabilization. EC cultures were washed with Dul- exposure to histamine, there has been considerable interest becco's phosphate-buffered saline (DPBS) (pH 7.2) and per- in the mechanism underlying this effect. Two broad possi- meabilized with 2 ml of buffer A (20 mM Pipes/10 mM bilities have been entertained, change in the junctions them- imidazole/50 mM KCI/1 mM EGTA/1 mM MgSO4/0.2 mM selves and intrinsic contractile activity of the ECs. dithiothreitol/5 ,g of aprotonin per ml/5 ug of leupeptin per In situ, lining blood vessels and in postconfluent cultures, ml/10 ,ug of soybean trypsin inhibitor per ml/0.1 mM phe- ECs are flattened polygonal cells without obvious polarity, nylmethylsulfonyl fluoride/0.5 mM benzamidine, pH 6.5) possessing a complex actin distribution with a dense periph- containing 25 ,ug of saponin per ml, incubated at 37°C for 10 eral band, stress fibers, and paranuclear filamentous array min, and washed with 2 ml ofbuffer A without saponin. Care (2). Exposure of ECs in culture to histamine (3, 4), ethchlor- was taken not to allow the permeabilized monolayers to dry. vynol (2, 3), or cytochalasin B (3, 5) induces a reversible Permeabilized monolayers were stimulated to retract on retraction of confluent ECs, leaving gaps between the cells, the addition of 100 ,uM free Ca2' and 100 ,uM exogenous ATP which remain attached to one another by thin processes. in buffer B (50 mM KCI/25 mM Pipes/2 mM MgSO4/1 mM Intracellular ATP and extracellular Ca2' have been shown to EGTA/0.2 mM dithiothreitol/5 ,g of aprotonin per ml/5 ,ug be essential for the retraction induced by these agents, and of leupeptin per mj/10 ,ug of soybean trypsin inhibitor per the retraction is accompanied by extensive changes in actin ml/0.1 mM phenylmethylsulfonyl fluoride/0.5 mM benzami- filament distribution (3). These several observations favor dine, pH 7.0) for 10 min at 370C. Retraction was terminated the contraction hypothesis for intercellular gap formation. by removing the buffer containing ATP and adding 3% ECs, like other eukaryotic nonmuscle cells, contain myo- formaldehyde in buffer A. The free Ca2' concentration of sin, actin, and associated proteins involved in cellular motile buffer B was calculated by the method of Bers (15). activities. For our studies, we have used the control of the Rhodamine Phalloidin Staining of Permeabilized Monolay- contractile apparatus in smooth muscle as a model for EC ers. For actin cytochemistry, cells were grown in Coming contraction. Phosphorylation of the 20-kDa light chains of dishes (35 x 10 mm). Monolayers were permeabilized and the myosin is essential for smooth muscle contraction (6, 7). The cells were stimulated to retract as described above. The phosphorylation is catalyzed by the Ca2", calmodulin- monolayers were fixed in freshly prepared 3% formaldehyde dependent enzyme myosin light-chain kinase (MLCK), in buffer A for 30 min at 220C. Fixed monolayers were washed which transfers the y-phosphate from ATP to myosin. The with buffer A and stained with rhodamine phalloidin (Mo-

The publication costs ofthis article were defrayed in part by page charge Abbreviations: EC, endothelial cell; MLCK, myosin light-chain payment. This article must therefore be hereby marked "advertisement" kinase; ATP[y-35S], adenosine-5'-[y-[35S]thio]triphosphate. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 16 Downloaded by guest on September 28, 2021 Medical Sciences: Wysolmerski and Lagunoff Proc. Nati. Acad. Sci. USA 87 (1990) 17 lecular Probes) for visualization of F actin as outlined by CaCI2 was added to give a final free concentration of 0.5 mM Barak et al. (16). CaC12. The kinase was then mixed with phenyl-Sepharose Thiophosphorylation. Permeabilized cultures were incu- preequilibrated with 40 mM Tris HCI, pH 7.4/50 mM NaCI/ bated with 100 ACi of adenosine 5'-[y-[35S]thio]triphosphate 0.5 mM CaCl2/1 mM dithiothreitol for 1 hr at 40C. The (ATP[y-35S]) (Amersham) (1 Ci = 37 GBq) in buffer A under phenyl-Sepharose (Sigma) suspension was centrifuged at varying conditions for 10 min at 370C, washed twice with 1000 x g for 10 min, and the soluble MLCK was removed and buffer A and scraped up in 50 pl of SDS sample buffer (17), dialyzed overnight against three 6-liter changes of 20 mM heated for 3 min at 100'C, and electrophoresed as described Tris HCI, pH 7.4/50 mM NaCI/1 mM EGTA/1 mM dithio- below. threitol. The purified kinase was stored at -70'C in 0.1-ml In some instances, myosin was immunoprecipitated fol- aliquots. lowing the method described by Kawamato and Adelstein Assay for MLCK Activity. MLCK activity was determined (18). The washed cultures were flooded with 600 Al of buffer by incubation of 0.1 ml of column fractions with 0.1 ml of 25 C (25 mM Tris'HCl/100 mM sodium pyrophosphate/100 mM mM Tris-HCI, pH 7.3/5 mM MgC12/50 mM KCI/0.2 mM NaF/250 mM NaCI/10 mM EGTA/5 mM EDTA/1% Noni- det CaC12 excess over the EGTA and EDTA present/5 ,g of P-40/0.2 mM phenylmethylsulfonyl fluoride/0.5 mM calmodulin (Biomedical Technologies, Stoughton, MA) per benzamidine/10 ,g of aprotonin per ml/10 ,ug of leupeptin ml/0.5 mg of isolated myosin light chains per ml/Ly-32PJATP per ml/10 ,g of soybean trypsin inhibitor per ml, pH 8.8) sufficient to tube. with a rubber 10 give 300,000 cpm per Column fractions scraped up policeman, made mM with were incubated at 25°C for 10 min and the was respect to disodium sonicated in a bath and assay ATP, sonicator, terminated by addition of 50%o trichloroacetic acid/109o so- incubated on ice for 20 min. The insoluble particulate mate- dium rial was sedimented at 80,000 x g for 10 min in a Beckman pyrophosphate to give a final concentration of 10% TL-100 ultracentrifuge. The soluble EC extracts were incu- trichloroacetic acid and 2% sodium pyrophosphate. Samples bated with an IgG fraction of rabbit antiserum raised against were heated to 90°C for 20 min, cooled on ice and filtered human platelet myosin and generously provided by Robert through Schliecher and Schuell 0.45-,m filters, washed with Adelstein (National Institutes ofHealth), for 2 hr at 4°C prior 5% trichloroacetic acid/1% sodium pyrophosphate, dried, to the addition of prewashed protein A-Sepharose 4B. After and counted in a Beckman LS1801 scintillation counter to an additional 2-hr incubation, the immune complexes bound assess phosphate incorporation into myosin light chains. The to protein A were collected by centrifugation for 5 min at activity measured was demonstrated to be Ca2+ dependent. 12,800 x g. The pellets were washed twice with buffer C, Cell Extraction and Reconstitution With Purified MLCK. centrifuged through a 10% sucrose cushion, and washed once Permeabilized EC monolayers were extracted in buffer D (40 in DPBS. The pellets were boiled in SDS sample buffer (17) mM Tris-HCI/100 mM KCI/30 mM MgCI2/1 mM dithiothrei- and subjected to electrophoresis on a 5-15% polyacrylamide tol/5 mM EGTA/1 mM EDTA/5 ,ug ofaprotonin per ml/5 ,ug gradient. of leupeptin per ml/10 ,g of soybean trypsin inhibitor per To establish that the 200-kDa protein identified by ml/0.2 mM phenylmethylsulfonyl fluoride/0.5 mM benzami- Coomassie blue staining was the myosin heavy chain, im- dine, pH 7.5) for 30 min to remove MLCK. Extracted munoprecipitated proteins were transferred onto nitrocellu- monolayers were reconstituted with purified MLCK and lose paper from 7% polyacrylamide gel, and the identity of calmodulin in buffer B as described below for 10 min prior to the heavy chains was confirmed by using rabbit antibodies the addition of ATP or ATP[y-35S]. raised against human platelet myosin. SDS/Polyacrylamide Gel Electrophoresis. Gel electrophoresis was carried out in either 5-15% or 7.5-12% gradient RESULTS vertical slab gels by the buffer system of Laemmli (17). Gels Cultures permeabilized with 25 ,ug of saponin per ml exhib- were stained with Coomassie blue, destained, and then ited a complex pattern of actin filaments identifiable by incubated in EN3HANCE (DuPont) for 60 min. Gels were rhodamine phalloidin staining and closely resembling that in exposed to Kodak X-OMAT x-ray film and developed with a intact cells. The dense peripheral band of actin was present Kodak automatic processor. For estimation of molecular but had lost some of its definition. Stress fibers of the cells, mass, either Bio-Rad high molecular mass protein standards like the complex arrangement of paranuclear filamentous or Pharmacia low molecular weight calibration standards actin, were little affected by permeabilization. Exposure of were used. monolayers to either Ca2" or ATP alone (Fig. 1) resulted in Purification ofProteins. Smooth muscle myosin light chains neither cell retraction nor rearrangement of F actin. Perme- and MLCK were prepared from bovine uterus according to abilized EC monolayers exposed to both 100 AM Na-ATP the procedures of Adelstein and Klee (19). For isolation of and 100 ,uM free Ca2+ for 10 min in buffer B containing 2 mM MLCK, we modified the isolation protocol by eliminating the Mg2+ and then fixed and stained with rhodamine phalloidin gel filtration column and applying the dialyzed 40-60%o exhibited extensive cell retraction. The actin stress fibers and ammonium sulfate fraction to a column (2.5 x 30 cm) of the paranuclear array were seen to have contracted centrally, DEAE-Sephacryl equilibrated with 20 mM Tris-HCl, pH leaving only a few strands radiating outward (Fig. 2). Parallel 7.8/1 mM EGTA/1 mM EDTA/1 mM dithiothreitol/0.1 mM changes occur in intact monolayer exposed to platelet acti- phenylmethylsulfonyl fluoride. The column was eluted at a vating factor (Fig. 3). flow rate of 50 ml/hr with a 1200-ml linear NaCl gradient Having demonstrated that permeabilized EC monolayers (30-600 mM) made up in equilibration buffer. Fractions that were responsive to ATP and Ca2 , we sought to determine contained myosin kinase activity were pooled and enough whether stimulation of EC retraction was accompanied by CaC12 and MgSO4 were added to give a final free concentra- phosphorylation of myosin light chains. Fig. 4 shows the tion of 0.5 mM Ca2' and 2 mM Mg2+. The pooled fractions thiophosphorylation pattern from a SDS/polyacrylamide gel were applied to a 12-ml column of calmodulin-Sepharose analysis of immunoprecipitated EC myosin. EC cultures (Sigma) equilibrated with 40 mM Tris HCl, pH 7.4/50 mM incubated with ATP[y-35S] resulted in thiophosphorylation of NaCI/0.5 mM CaC12/2 mM MgCI2/1 mM dithiothreitol. The myosin light chains in a Ca2"-dependent manner. No thio- column was washed with equilibration buffer containing 0.2 phosphorylation of the 19-kDa myosin light chains occurred M NaCl. The kinase was then eluted with equilibration buffer in the presence of EGTA (lane 1), and the extent of thio- containing 2 mM EGTA and 0.2 M NaCl. The peak ofMLCK phosphorylation decreased with decreasing Ca2" concentra- activity from the calmodulin affinity column was pooled, and tion (lanes 2-4). Downloaded by guest on September 28, 2021 18 Medical Sciences: Wysolmerski and Lagunoff Proc. Natl. Acad. Sci. USA 87 (1990)

FIG. 1. Fluorescence micrograph of F-actin filament distribution FIG. 3. Fluorescence micrograph of F-actin distribution in intact in control permeabilized EC monolayer stained with rhodamine EC monolayers incubated with 20 ,M platelet activating factor. phalloidin. Cells were incubated in buffer B containing 100 AM Severe EC retraction comparable to that seen in permeabilized EC Na-ATP for 10 min. No EC retraction results in the absence ofCa2". monolayers treated with ATP and Ca2+ occurs within 15 min with An accumulation of F actin delineates the cell margins; only an loss of the prominent dense peripheral band of actin filament. The occasional gap between adjacent cells is present within the mono- F-actin cytoskeleton retracts centrally, leaving cells attached by layer. The stress fibers and paranuclear array remain intact. (x380.) numerous slender filamentous processes. Neither cell rounding nor detachment from the culture dish was noted. (x380.) To define the mechanism involved in EC retraction, we further investigated the role of MLCK. Cultures were ex- In parallel with the studies of reconstitution of retraction in tracted in buffer D for 30 min at 370C to remove MLCK extracted monolayers, we investigated thiophosphorylation activity. The extraction medium was assayed and found to of myosin light chains. Fig. 8A is a representative autorad- contain MLCK activity (data not shown). Extraction of iogram of the thiophosphorylation pattern of extracted and cultures resulted in only minor changes in the cytoskeleton. reconstituted monolayers. Cultures incubated with ATP[y- Neither cell rounding nor detachment from the culture dish 35S] for 10 min showed no thiophosphorylation of myosin was observed; however, occasional small gaps were evident light chains after a 30-min extraction with buffer D (lane 4). between adjacent cells. Extracted monolayers showed some Reconstitution of extracted monolayers with MLCK and modest loss of F actin staining from the cell periphery, whereas the central microfilament bundles were unaltered. In addition, extraction with buffer D resulted in minimal loss of EC myosin (Fig. 5). When extracted monolayers were incubated with ATP, Ca2", and calmodulin for as long as 20 min, no retraction was evident (Fig. 6). Extracted EC reconstituted with purified MLCK and calmodulin for 10 min at 370C retracted when exposed to ATP and Ca2+ in buffer C (Fig. 7). The F-actin 497 network assumed a collapsed pattern around the nucleus. Permeabilized, extracted ECs reconstituted with the follow- 467 ing combinations-MLCK and Ca2 , MLCK and calmodulin, and calmodulin and Ca2+-and then incubated with ATP failed to retract. 443

430

420.1

1 2 3 4 FIG. 4. Autoradiogram of immunoprecipitated thiophosphorylated myosin light chains of permeabilized ECs. Lanes 2-4, thiophosphorylation of the 19-kDa myosin light chains in the presence of FIG. 2. BC monolayer exposed to 100 ,uM Na-ATP, 100,uM free decreasing free Ca2+ concentration is shown. The apparent increase Ca2+ in buffer B for 10 min. Severe EC retraction of the stained in thiophosphorylation of myosin light chain was confirmed by F-actin cytoskeleton occurs within 10 min without cells detaching comparing the ratio of Coomassie blue-stained myosin heavy chains from the substratum. The actin filaments retract toward the nucleus. to thiophosphorylated light chains. Lanes: 1, 1 mM EGTA; 2, 100,M (x380.) Ca2+; 3, 50 AM Ca2+; 4, 1 ,M free Ca2+. Numbers on right are kDa. Downloaded by guest on September 28, 2021 Medical Sciences: Wysolmerski and Lagunoff Proc. Natl. Acad. Sci. USA 87 (1990) 19

1 2 3 FIG. 5. Radioimmunoblot of immunoprecipitated EC myosin. Immunoprecipitated myosin from 1.5 x 106 ECs was electrophoresed on 7% polyacrylamide gels electrophoretically transferred to nitrocellulose paper and incubated with rabbit anti-human platelet myosin antibodies. Nitrocellulose blots were then incubated with goat anti-rabbit 1251-labeled IgG to visualize myosin heavy chains. Lanes: 1, immunoprecipitated myosin from intact EC monolayers; 2, myosin immunoprecipitated from saponin permeabilized monolayers; 3, myosin immunoprecipitated from EC monolayer extracted FIG. 7. Fluorescence micrograph of MLCK reconstituted mono- with buffer D for 30 min. Extraction procedures used to extract layer. Retraction is reestablished when extracted monolayers are MLCK did not result in significant loss of endogenous EC myosin. incubated with 1 ,uM MLCK/100 ,M free Ca2+/5 ,ug of calmodulin per ml and are then exposed to 100 ,uM ATP in buffer B. There is calmodulin reestablished the thiophosphorylation of the 19- severe EC retraction and F-actin filaments have contracted down kDa myosin light chains in the presence of calcium (lane 5). around the nucleus. No cells have detached from the substratum Extracted monolayers incubated with either MLCK and Ca2+ during this process. (x380.) or MLCK and calmodulin did not thiophosphorylate myosin light chains (lanes 6 and 7). work has accumulated that suggests that in nonmuscle cells To establish that the 19-kDa protein thiophosphorylated as in smooth muscle, myosin light-chain phosphorylation was indeed myosin light chains, we analyzed immunoprecipitated myosin from both extracted and reconstituted monolayers. Figure 8B shows the thiophosphorylation pattern of immunoprecipitated myosin from extracted (lane 1) and reconstituted (lane 2) monolayers. Cultures extracted with buffer D exhibited minimal thiophosphorylation of myosin light chains, while reconstitution with MLCK, Ca2+, and calmodulin reestablished the thiophosphorylation of the 19- kDa myosin light chains (lane 2).

DISCUSSION 67 *...... In 1969 Majno et al. (20), proposed that active contraction of endothelium involving cytoskeletal elements was responsible 6-- 4 43 for the permeability edema induced by histamine. Our own previous studies have shown that agents that cause increased vascular permeability in vivo induce a rearrangement of the actin filaments ofthe EC cytoskeleton in vitro with retraction ..O.30 of the cells (2). We have extended these studies to show that _w-~~~~97 EC retraction and accompanying cytoskeletal changes re- . _ 4~~~~- 20.1 _ quire permissive levels of ATP (3). A considerable body of A 1 2 3 .~~~~.44 5 6 7 B 1 2 FIG. 8. (A) Autoradiogram of thiophosphorylated EC proteins. Lane 1, permeabilized monolayer incubated in the presence of 1 mM EGTA/100 ,uCi of ATP[y-35S]. No thiophosphorylation of myosin light chains occurs in the absence of Ca2+. Lane 2, ECs incubated in the presence of 100 ,uM free Ca2+/100 ,uCi of ATP[y-35S] result in thiophosphorylation of the 19-kDa myosin light chains (arrowhead). Monolayers extracted with buffer D for 15 min (lane 3) or for 30 min (lane 4) lose the ability to thiophosphorylate myosin light chains upon exposure to ATP[y-35S], Ca2+, and calmodulin. Extracted ECs incubated with 1 AM MLCK/100 ,M free Ca2/5A,ug of calmodulin per ml (lane 5) result in thiophosphorylation of endogenous myosin light chains upon exposure to ATP[y-35S]. Extracted monolayers reconstituted with MLCK/calmodulin (lane 6) or MLCK/Ca2+ but no calmodulin (lane 7) alone and then exposed to ATP[y-35S] fail to thiophosphorylate myosin light chains. Numbers on right are kDa. (B) The protein thiophosphorylated by the addition of exogenous MLCK to permeabilized MLCK extracted monolayers was identi- FIG. 6. F-actin distribution in MLCK extracted monolayer. Cells fied as myosin light chains by immunoprecipitation. In the absence were extracted for 30 min in buffer D and then exposed to 100 ,uM of endogenous MLCK, there is only weak thiophosphorylation of ATP/100 ,uM Ca2/5 ,ug of calmodulin per ml/for 10 min. There is myosin light chains (lane 1). Monolayers were reconstituted with 1 some loss of peripheral F actin compared to that in unextracted ,uM MLCK/ATPEy-35S]/100 A4M free Ca2+/5 ,ug ofcalmodulin per ml permeabilized ECs, but there is no evidence of retraction or sub- exhibited strong thiophosphorylation of the identified myosin light stantial loss of F actin. (x380.) chains (lane 2). Downloaded by guest on September 28, 2021 20 Medical Sciences: Wysolmerski and Lagunoff Proc. Natl. Acad. Sci. USA 87 (1990) plays a major role in regulation of actin-myosin-bAsed con- this mechanism is the one invoked by histamine and similarly traction. This regulation of myosin function is mediated by acting agents in intact cells. the Ca2l-activated, calmodulin-dependent enzyme MLCK (6, 7, 9). This is an obvious model for EC retraction; This work was supported by National Institutes of Health Grant calcium-dependent phosphorylation of myosin could initiate Specialized Center of Research Adult Respiratory Failure HL30572. the sequence of events modifying the actin network, which in 1. Majno, G. & Palade, G. E. (1%1) J. Biophys. Biochem. Cytol. turn could account for the formation of retractive gaps 11, 571-606. between cells and increased vascular permeability. In the 2. Wysolmerski, R. B. & Lagunoff, D. (1985) Am. J. Pathol. 119, present study, we performed a series of experiments to 505-512. evaluate the role ofmyosin phosphorylation in EC retraction. 3. Wysolmerski, R. B. & Lagunoff, D. (1988) Am. J. Pathol. 132, To carry out these studies, we developed a permeabilized cell 28-37. 4. Laposata, M., Dovnarsky, D. K. & Shen, H. S. (1982) Blood preparation using saponin. Since it was difficult to delineate 62, 549-556. cell margins in the permeabilized cells by phase-contrast 5. Shasby, M. D., Shasby, S. S., Sullivan, J. M. & Peach, M. J. microscopy, we assessed retraction in terms ofthe change in (1982) Circ. Res. 51, 657-661. cytoskeletal F actin. Addition of ATP and Ca2+ in the 6. Sellers, J. R. & Adelstein, R. S. (1987) in The Enzymes, eds. presence of Mg2+ induced retraction of permeabilized EC Boyer, P. D. & Krebs, E. G. (Academic, Orlando, FL), Vol. cytoskeleton; the extent ofretraction was dependent on both 18, pp. 382-413. the Ca2+ and ATP 7. Stull, J. T., Nunnally, M. H., Mecknoff, C. H. (1986) in The concentrations. Labeling studies with Enzymes, eds. Boyer, P. D. & Krebs, E. G. (Academic, Or- ATP[y-35S] showed that numerous EC proteins were thio- lando, FL), Vol. 17, pp. 114-159. phosphorylated. A prominent band of 19-kDa was thiophos- 8. Kargacin, G. J. & Fay, F. S. (1987) J. Gen. Physiol. 90, 49-73. phorylated in a Ca2+-dependent manner. This band was 9. Kamm, K. E. & Stull, J. T. (1985) Annu. Rev. Pharmacol. positively identified as the myosin light chain by electropho- Toxicol. 25, 593-620. resis of immunoprecipitated myosin (Fig. 4). 10. Holzapfel, G., Wehland, J. & Weber, K. (1983) Exp. Cell Res. Cells in 148, 117-126. monolayers extracted with high salt concentration 11. Masuda, H., Owaribe, K., Hayashi, H. & Hatano, S. (1984) did not retract upon exposure to ATP and Ca2W, and thio- Cell Motil. 4, 315-331. phosphorylation of myosin light chains was correspondingly 12. Broschat, K. O., Stidwill, R. P. & Burgess, D. R. (1983) Cell absent in these preparations. Addition of calmodulin did not 35, 561-571. restore either retraction or thiophosphorylation in the pres- 13. Porrello, K. & Burnside, B. (1984) J. Cell Biol. 98, 2230-2238. ence of ATP and Ca2+, nor did 14. Del Vecchio, P. J. & Smith, J. R. (1981) J. Cell. Physiol. 108, addition .of purified MLCK 337-345. without calmodulin restore retraction or thiophosphoryla- 15. Bers, D. M. (1982) Am. J. Physiol. 242, C404-C408. tion. Addition of MLCK and calmodulin together restored 16. Barak, L. S., Yocum, R. R., Nothnagel, E. A. & Webb, W. W. both retraction (Fig. 7) and thiophosphorylation (Fig. 8). This (1980) Proc. Natl. Acad. Sci. USA 77, 980-984. is the behavior predicted based on the events in smooth 17. Laemmli, U. K. (1970) Nature (London) 227, 680-685. muscle contraction. Several nonmuscle cells exhibit a similar 18. Kawamoto, S. & Adelstein, R. S. (1988) J. Biol. Chem. 263, set ofcontrol mechanisms in which MLCK is believed to play 1099-1102. 19. Adelstein, R. S. & Klee, C. B. (1981) J. Biol. Chem. 256, a pivotal role in the initiation of contractile activity (6, 7, 9). 7501-7509. We have demonstrated the presence of a MLCK controlled 20. Majno, G., Shea, S. M. & Leventhal, H. (1969) J. Cell Biol. 42, contractile mechanism in ECs; the next step is to prove that 647-672. Downloaded by guest on September 28, 2021