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Their Use in Localizing Myosin Kinase in Nonmuscle Cells (Antibody/Fibroblasts/Stress Fibers) PRIMAL DE LANEROLLE*, ROBERT S

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Their Use in Localizing Myosin Kinase in Nonmuscle Cells (Antibody/Fibroblasts/Stress Fibers) PRIMAL DE LANEROLLE*, ROBERT S Proc. Natd Acad. Sci. USA Vol. 78, No. 8, pp. 4738-4742, August 1981 Biochemistry Characterization of antibodies to smooth muscle myosin kinase and their use in localizing myosin kinase in nonmuscle cells (antibody/fibroblasts/stress fibers) PRIMAL DE LANEROLLE*, ROBERT S. ADELSTEINt, JAMES R. FERAMISCOt, AND KEITH BURRIDGEt *Laboratory of Molecular Cardiology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20205; and tCold Spring Harbor Laboratory, Cold Spring Harbor, New York, 11724 Communicated by Donald S. Fredrickson, April 13, 1981 ABSTRACT Antibodies to myosin light chain kinase, purified with, the contraction ofsmooth muscle (17-19) and the secretion from turkey gizzard smooth muscle, were developed in rabbits and of granules by platelets (5). purified by affinity chromatography on a myosin light chain ki- Myosin light chain kinase, the enzyme that catalyzes the nase-Sepharose 4B column. The purified antibodies crossreact phosphorylation ofmyosin, plays a central role in this regulatory with purified smooth muscle myosin light chain kinase but not with process. Although there is considerable information about the a variety of contractile or cytoskeletal proteins. The antibodies biochemical properties ofthe enzyme, which has been purified inhibit the catalytic activity of smooth musclet myosin light chain from both smooth muscle (20, 21) and nonmuscle cells (22-25), kinase, and there is an inverse relationship between the kinase there is at present little information about its location in these activity and the amount ofantibody present in an assay. Half-max- cells. We have used an immunological approach to localize imal inhibition of myosin kinase activity occurs at an antibody/ myosin light chain kinase in nonmuscle cells. We purified myosin kinase molar ratio of 10:1. The affinity-purified antibodies myosin light chain kinase from turkey gizzard smooth muscle to smooth muscle myosin kinase were used to study the location and raised antibodies to this protein in rabbits. These antibodies of myosin kinase in a variety of nonmuscle cells. Immunofluores- chain kinase is localized were purified, tested for their ability to inhibit the catalytic cence studies indicate that myosin light activity ofmyosin light chain kinase, and then used to determine on microfilament bundles (stress fibers) in cultured fibroblasts. kinase in a variety The stress fiber staining pattern is abolished when the antibodies the intracellular location ofmyosin light chain are incubated with purified smooth muscle myosin light chain ki- of nonmuscle cells. nase prior to staining cells, while the staining pattern is unaffected when the antibodies are incubated with actin, myosin, a-actinin, METHODS or tropomyosin prior to staining. Moreover, the stress fiber stain- Antibody Production and Purification. Myosin light chain ing pattern is periodic in well-spread gerbil fibroma cells and ex- kinase was purified from turkey gizzard smooth muscle as de- periments have demonstrated that myosin light chain kinase ap- scribed (20). Two rabbits were bled prior to immunization pears to have the same periodic distribution as myosin but an (pre-immune serum), and antibodies to smooth muscle myosin antiperiodic distribution relative to a-actinin. These data indicate light chain kinase were produced by injecting 1 mg of myosin that myosin light chain kinase and its substrate, myosin, are in light chain kinase, emulsified in complete Freund's adjuvant, close proximity and are consistent with the hypothesis that myosin intramuscularly. The rabbits were immunized with 1 mg of light chain kinase regulates actin-myosin interactions in nonmus- myosin kinase at 1-week intervals for 4 weeks. Since both rab- cle cells. bits produced antibodies, they were bled (40 ml per bleed) from an ear vein once a week starting 7 days after the last immuni- The proteins actin and myosin are thought to mediate a variety zation. Pre-immune sera and immune sera (four separate bleeds ofcontractile events in nonmuscle cells (1). These include gen- from each rabbit) were pooled separately, and IgG fractions eral cellular functions such as locomotion (2) and cytokinesis were prepared by adding solid (NH4)2SO4 to the sera. The pro- (3) and specialized cell functions such as phagocytosis in mac- tein precipitating at 0-33% (NH4)2SO4 saturation was collected rophages (4) and secretion in platelets (5). Intrinsic to these by centrifugation, dialyzed exhaustively in 150 mM NaCl/5 mM functions is the ability ofmyosin to hydrolyze MgATP and gen- NaNJ20 mM Tris HCl, pH 7.5, and stored at -700C. erate force when it interacts with actin. Antibodies to myosin light chain kinase were purified by af- One mechanism for regulating the interaction of actin and finity chromatography ofthe immune IgG fraction. Turkey giz- myosin in nonmuscle and smooth muscle cells is through the zard smooth muscle myosin light chain kinase (15 mg) was cou- reversible phosphorylation of myosin (for review, see ref. 6). pled to 10 ml of CNBr-activated Sepharose 4B (Pharmacia) as Phosphorylation has been shown to have two effects: (i) It is recommended by the manufacturer. Immune IgG was applied required for, or markedly enhances, the actin-activated Mg2e- to myosin light chain kinase-Sepharose 4B beads equilibrated ATPase activity ofmyosin (7-14) and (ii) it stabilizes bipolar fil- in 150 mM NaCl/20 mM Tris HCl, pH 7.5 (buffer A). The beads aments of myosin (15, 16), which are required for the devel- were washed with 10 vol of the same buffer, then with 10 vol opment of tension. of 3 M LiCl/20 mM Tris'HCl, pH 7.5, and eluted with 6 M Experiments with intact smooth muscle strips and with blood guanidine HCl/l M NaCl/20 mM Tris HCl, pH 7.5. After di- have confirmed the relationship between myosin alysis in buffer A, the purified antibodies were concentrated by platelets dialysis in 80% sucrose in buffer A, redialyzed in buffer A/5 mM phosphorylation and contractile activity. These studies have the or simultaneously NaN3, and stored at -700C. All steps in the preparation of shown that myosin is phosphorylated before, IgG fraction and the purification of the antibodies were per- formed at 100C. The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertise- ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. Abbreviation: P/NaC1, phosphate-buffered saline. 4738 Biochemistry: de Lanerolle et aL Proc. Nati Acad. Sci. USA 78 (1981) 4739 Phosphorylation Assays. An isolated fraction of myosin light was prepared from turkey gizzard smooth muscle myosin 100 chains s6 as described for skeletal muscle myosin (26). Calmodulin was 80 removed from the light chain fraction by ion exchange chro- matography on DEAE-Sephacel (20). Porcine brain calmodulin .61._co~ 60 was purified as described (27). Kinase assays were performed g at room temperature as described (20). Inhibition of myosin 40 kinase activity was studied by incubating the kinase with the 0 E various antibody fractions for 2 min before initiating the phos- cc 20 phorylation reaction. Cell Culture Methods. Gerbil fibroma cells (CCL 146) and 1.0 10 100 1000 primary chicken embryo fibroblasts were cultured in Dul- IgG, log pmol becco's modified Eagle's medium/9% fetal calf serum. Human fibroblasts (clone GRC 161D7.9) were the generous gift of J. FIG. 2. Inhibition of purified myosin light chain kinase activity R. Smith (Alton Jones Center) and were grown in the same by affinity-purified myosin light chain kinase antibodies. A standard medium supplemented with nonessential amino acids. amount of purified smooth muscle myosin light chain kinase (0.77 Cells grown on glass pmol) was incubated with various amounts of purified antibodies (*) Indirect Immunofluorescence Studies. or pre-immune IgG (v). The kinase was incubated with the antibodies coverslips were washed with phosphate-buffered saline (PJ for 2 min prior to initiating a reaction that gave linear incorporation NaCl) pH 7.3 and fixed in 3.5% formalin in PjNaCl for 10 min of radioactivity into smooth muscle myosin light chains. The reaction at room temperature. The coverslips were rinsed in PJNaCl was terminated by the addition of trichloroacetic acid at 3 min and the and then extracted in acetone at -200C for 5 min. They were extent of 32p incorporation was determined. Kinase activity is ex- rinsed again in PjNaCl and then stained with the relevant an- pressed as percent of maximum, using the extent of myosin light chain tibodies. The affinity-purified myosin light chain kinase anti- phosphorylation in the absence of added immunoglobulin as 100%. body was used at 90 ,.g/ml. Ra~bbit antibodies to myosin were band pass interference filter 546 ± 2 nm) excitation filter and raised by immunization with chicken gizzard myosin and were an LP590 barrier filter, and fluorescein was analyzed by using similar to those described by Gordon (28). The properties of a Zeiss dichroic excitation filter BP485/20 and barrier filter rabbit antibodies to smooth muscle a-actinin and guinea pig LP520. antibodies to smooth muscle tropomyosin have been described Miscellaneous Techniques. Protein concentrations were de- (29). The myosin light chain kinase antibody was adsorbed with termined as described by Bradford (30) or by Lowry et aL (31) purified myosin light chain kinase, actin, tropomyosin, vimen- using suitable proteins as standards. Antibody crossreactivity tin, or myosin prior to staining some cells. The antibody fraction was studied by double-diffusion precipitation reactions on was incubated with an equal volume ofprotein solution (1-2 mg/ 0.75% agar gels. ml) for 1 hr at 40C and then centrifuged at 13,000 X g for 5 min prior to staining. The coverslips were incubated with the an- RESULTS tibodies in a humidified atmosphere at 37TC for 30 min. After Antibodies to myosin light chain kinase were purified by affinity washing in PjNaCl, the coverslips were stained with fluores- chromatography of the immune IgG fraction on a myosin light cein isothiocyanate-labeled goat anti-rabbit IgG diluted 1:25.
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