Human Platelet Myosin Light Chain Kinase Requires the Calcium
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Proc. Nati. Acad. Sci. USA Vol. 76, No. 4, pp. 1653-1657, April 1979 Biochemistry Human platelet myosin light chain kinase requires the calcium- binding protein calmodulin for activity (calcium-dependent regulator/phosphorylation of nonmuscle contractile protein/affinity chromatography) DAVID R. HATHAWAY AND ROBERT S. ADELSTEIN Section on Molecular Cardiology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20014 Communicated by DeWitt Stetten, Jr., January 17, 1979 ABSTRACT In an actomyosin fraction isolated from human Furthermore, this calcium-binding protein has been shown to platelets, phosphorylation of the 20,000-dalton light chain of be identical to the calcium-dependent regulator* of cyclic AMP myosin is stimulated by calcium and the calcium-binding pro- phosphodiesterase (11). tein calmodulin. The enzyme catalyzing this phosphorylation has been isolated by using calmodulin-affinity chromatography. Although a growing body of evidence suggests that non- Platelet myosin light chain kinase activity was monitored muscle myosin, such as that isolated from platelets, is regulated throughout the isolation procedures by using the 20,000-dalton by light chain phosphorylation, the nature or existence of cal- smooth muscle myosin light chain purified from turkey gizzards cium control mechanisms has not been clarified. In an earlier as substrate. The partially purified myosin kinase requires both study, platelet myosin kinase was found to be active in the ab- calcium and calmodulin for activity and has a specific activity sence of calcium (12). Recently, we isolated, from human of 3.1 ,gmol of phosphate transferred to the 20,000-dalton light platelets, an actomyosin in which chain per mg of kinase per min under optimal assay conditions. fraction phosphorylation of Km values determined for ATP and myosin light chains are 121 the 20,000-dalton myosin light chain is dependent upon calci- AM and 18 ;M, respectively. Of several substrates surveyed as um. Furthermore, this phosphorylation requires not only cal- phosphate acceptors (a-casein, histone II-A, phosphorylase b, cium but also the calcium-binding protein calmodulin. In this protamine, histone V-S, and phosvitin), only the 20,000-dalton report we describe a method for the isolation of a calcium- and myosin light chain is phosphorylated at a significant rate. These calmodulin-dependent myosin light chain kinase from human results suggest that platelet myosin light chain kinase is a cal- platelets. cium-dependent enzyme and that the requirement for calcium is mediated by the calcium-binding protein calmodulin. MATERIALS AND METHODS Despite their nonmuscle origin, platelets possess an abundance Preparation of Platelet Actomyosin. All isolation procedures of the contractile proteins actin and myosin; the combined were performed at 40C. Human platelets were collected by content has been estimated as 10-30% of the total cellular sedimentation of fresh (<24 hr old) platelet concentrates (54 protein (1, 2). Although platelet actin may serve a structural units) at 9000 X g for 10 min. The more rapidly sedimenting role, the function of actomyosin is related to its unique capacity erythrocyte pellet was removed from the bottom of the cen- to generate force and thereby to create tension or to shorten. trifuge tube by aspiration. The platelet pellet (25-35 g) was This feature has been implicated as an important property in- resuspended in 2 vol of 2.5 mM dithiothreitol/150 mM NaCl/3 volved in mediation of cell secretion, shape change, and clot mM citrate buffer, pH 6.8, and sedimented again. At the end retraction (1). of each of two such washes, any remaining erythrocytes were The regulation of actin-myosin interaction in platelets was removed by aspiration. The final platelet pellet was suspended found earlier to involve phosphorylation-dephosphorylation in 2-3 vol of 15 mM Tris-HCl, pH 7.5/0.5 M KC1/2.5 mM di- of the 20,000-dalton light cbain of myosin (3). Phosphorylation thiothreitol/5 mM Na2EDTA. With continuous stirring, the of this subunit is necessary for actin-induced activation of platelet suspension was equilibrated with nitrogen [1500 psi myosin ATPase activity. Moreover, the myosins isolated from (10.4 megapascals)] for 20 min in a Parr bomb. Disruption was smooth muscle (4-6), skeletal muscle prefusion myoblasts (7), effected by opening the release valve and permitting platelets and rabbit alveolar macrophages (J. Trotter and R. S. Adelstein, to flow slowly into an erlenmeyer flask at atmosphenc pressure. unpublished data) have been shown to require phosphorylation The extract was then sedimented at 28,000 X g for 30 min. The for actin-induced activation of myosin ATPase activity. supernatant fraction (80-100 ml) was collected and dialyzed Striated and smooth muscle actomyosins are regulated by overnight against 20 vol of 20 mM Tris-HCl, pH 7.5/2.5 mM calcium. In skeletal and cardiac muscles, the proteins troponin dithiothreitol/5 mM EDTA. The pellet obtained by sedimen- and tropomyosin bind stoichiometrically to actin and, in the tation at 10,000 X g for 20 min was the source of crude acto- absence of calcium, inhibit actin-induced activation of myosin myosin. ATPase activity. The binding of calcium to one component of Preparation of Platelet Myosin Light Chain Kinase. The troponin, troponin C, removes this inhibition (8, 9). In contrast, platelet myosin light chain kinase characterized in this report a principal site of calcium control in smooth muscle is the my- was prepared from 25-35 g of platelets that were washed as osin light chain kinase which catalyzes phosphorylation of the described above. The final, washed pellet was resuspended in 20,000-dalton light chain of myosin (4-6). Recently, the calcium 100 ml of 20 mM Tris-HCI, pH 7.5/2.5 mM dithiothreitol/10 dependence of chicken gizzard myosin kinase has been shown mM EDTA/1 mM ethylene glycol ether)- to be mediated by a 16,500-dalton calcium-binding subunit (10, bis(O-aminoethyl 11). In the absence of this component the kinase is inactive. Abbreviations: NaDodSO4, sodium dodecyl sulfate; EGTA, ethylene glycol bis(3-aminoethyl ether)-N,N'-tetraacetic acid. The publication costs of this article were defrayed in part by page * This protein is also known as CDR. Other names appearing in the charge payment. This article must therefore be hereby marked "ad- literature include "modulator protein" and "phosphodiesterase ac- vertisement" in accordance with 18 U. S. C. §1734 solely to indicate tivator." The preferred term "calmodulin" is used throughout the this fact. text of this paper. 1653 Downloaded by guest on October 1, 2021 1654 Biochemistry: Hathaway and Adelstein Proc. Natl. Acad. Sci. USA 76 (1979) N,N'-tetraacetic acid (EGTA)/phenylmethylsulfonyl fluoride at 75 mg/liter (75 mg in 5 ml of dimethyl sulfoxide added to 1 liter of buffer)/soybean trypsin inhibitor at 0.1 mg/ml. After disruption of whole platelets by nitrogen decompression (see above), the extract was sedimented at 28,000 X g for 30 min and the supernatant was collected for subsequent purification, as X 4: described below. E Kinase Assay. Unless specified otherwise, all kinase assays were performed at room temperature (23-25°C) in a final volume of 0.2 ml containing 20 mM Tris-HCI, pH 7.5, 0.2 mM Ca2+ 20,uM smooth muscle 0. [y-32P]ATP (105 dpm/nmol), 20,000- CL dalton myosin light chain, 10 mM MgCl2, and either 0.1 mM 0.~~~~~~~~~~~~~0 CaCl2 or 1 mM EGTA. The final concentration of calmodulin, unless stated otherwise, was 0.1 AtM. Assays were terminated - ~EGTA by the addition of 50 1l of 50% trichloroacetic acid/10% sodium pyrophosphate, and 32p incorporation into substrate myosin light chains was determined by Millipore filtration of samples followed by liquid scintillation counting of the Millipore filters 10 20 30 40 50 60 (4). Time, min Gel Electrophoresis. Specific phosphorylation of 20,000- FIG. 1. Endogenous phosphorylation ofplatelet actomyosin. Each dalton platelet myosin light chains was confirmed by electro- time point represents 200 jig ofcrude platelet actomyosin incubated phoresis of 150 ,g of actomyosin in cylindrical 7.5% polyac- in 20 mM Tris-HCl, pH 7.5/0.1 mM CaCl2/125 mM KCl/1 MM cal- modulin (CDR)/10 mM MgClJ1 mM [,y-32P]ATP (104 dpm/nmol) rylamide/1% sodium dodecyl sulfate (NaDodSO4) gels with in a final volume of 0.2 ml. The incubations were terminated by the the Fairbanks buffer system (13). The 20,000-dalton light chain addition of 1 ml of 6 M guanidine HCL Samples were dialyzed against was identified after staining with Coomassie blue; slices con- water, applied to 1% NaDodSO4/7.5% polyacrylamide gels, and taining light chains were assayed for 32p incorporation. The stained with Coomassie blue as described (4). 32P incorporation into subunit molecular weight of the platelet myosin light chain the 20,000-dalton myosin light chain was determined by liquid scin- kinase was determined by electrophoresis in 1% NaDodSO4 slab tillation counting of gel slices. gels containing a 5-10% gradient of polyacrylamide. For this procedure, the Laemmli buffer system was used (14). The phorylation of the 20,000-dalton platelet myosin light chain. Fairbanks buffer system, excluding NaDodSO4, was used for The time-dependent incorporation of 32p into the platelet electrophoresis under nondenaturing conditions. Samples of myosin light chains was determined as described in Fig. 1. In platelet myosin kinase purified through affinity chromatog- the absence of calcium (1 mM EGTA), a slow rate of phosphate raphy (0.2 mg/ml) were concentrated 2-fold by dialysis against incorporation was observed. Addition of calcium to a final 100 vol of 20 mM Tris-HCI, pH 7.5/100 mM KCI/5 mM concentration of 0.1 mM resulted in a 2-fold increase in the rate EDTA/30% (wt/vol) glycerol.