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Calpain abolishes the effect of filamin on the actomyosin system in platelets
Biochimica et Biophysica Acta
912 (1987) 283-286
283
Elsevier BBA 30202
BBA Report
Calpain abolishes the e f f e c t of filamin on the a c t o m y o s i n s y s t e m in platelets T o y o s h i Onji, M i c h i k o T a k a g i a n d N o b u h i k o S h i b a t a Division of Molecular Cardiology, The Center for Adult Diseases, Osaka (Japan)
(Received 15 September1986) (Revised manuscript received9 January 1987)
Key words: Filamindegradation;Actomyosin;Calpain; Calciumion; Plateletactivation Piatelet filamin was shown to cross-link F-actin and inhibit actomyosin ATPase activity. Filamin was also shown to be degraded by calpain (calcium-activated neutral proteinase; CANP) when the platelet was activated. The consequences of the proteolysis of fUamin on the actomyosin system have been investigated. When degraded by calpain in the presence of Ca2+, filamin loses its ability to cross-link F-aetin. Under the same conditions, its inhibitory effects on the superprecipitation and ATPase activity of actomyosin are abolished. The result suggests that the degradation of filamin is favorable for the contraction of the activated platelets.
Platelets are highly motile cells. The mechanism of their motility has bee the subject of research in this laboratory. We found that the interaction between actin and myosin is enhanced by tropomyosin in platelet [1,2]. Subsequently, we reported that another actin-binding protein, filamin, is capable of inhibiting actomyosin ATPase activity [3]. Filamin is reported to be degraded by calpain when the platelet is activated [4]. The physiological significance of this phenomenon has been uncertain. We have suggested that the effect of filamin on actomyosin may be reduced when filamin is degraded by calpain [3]. We tested this hypothesis using partially purified calpain from platelet. Actin, myosin and filamin were all purified from porcine platelets as previously described [1-3]. Calpain was partially purified from the same source by the method of Tsujinaka et al. [5] with a slight modification (ion-exchange chroCorrespondence: T. Onji, Division of Molecular Cardiology, The Center for Adult Diseases, Osaka 1-3-3, Nakamichi, Higashinariku, Osaka 537, Japan.
matography was carried out using QAE on highperformance liquid chromatography). The partially purified calpain was composed of 80 kDa and 90 kDa polypeptides (Fig. lb-8). Its activity was negligible at [Ca 2+ ] of less than 0.1 mM and was maximal at its concentration of 2 mM (calpain II or milli-CANP). The proteolysis of filamin by calpain is shown in Fig. lb. Partially purified calpain degrades filamin within 5 min. The major product is a 190-200 kDa fragment. The proteolysis is observed only in the presence of Ca 2÷. If Ca 2÷ is absent, no proteolysis is found, even after 60 rain of incubation of filamin with calpain (Fig. lb-8). After various times of preincubation, the effect of calpain-treated filamin on the viscosity of platelet F-actin was examined. Filamin increases the viscosity of F-actin markedly (Fig. la; value at zero time). Calpain itself does not affect the viscosity of F-actin after any length of preincubation either in the presence or absence of Ca 2+ (Fig. la; dotted line). However, if filamin is preincubated with calpain in the presence of Ca 2+, the viscosity of F-actin is reduced from 220 s / c m to
0167-4838/87/$03.50 © 1987 Elsevier Science Publishers B.V. (BiomedicalDivision)
284
60 s / c m (Fig. la; solid line with closed circle). On the other hand, if it is preincubated with calpain in the absence of Ca 2+ the capacity of filamin to gelate F-actin is unchanged (Fig. la; solid line with open circle). The 190 kDa fragment remains bound to F-actin after proteolysis (data not presented). These results indicate that filamin loses its property to cross-link F-actin when it is degraded by calpain in the presence of Ca 2+. We have reported that filamin is capable of inhibiting the Mg2+-ATPase activity of platelet actomyosin [3]. Another activity of actomyosin in vitro is superprecipitation. The turbidity of platelet actomyosin increases after the addition of ATP due to a profound reassembly of actin and myosin filaments (Fig. 2a-l). When filamin is added to this system, the superprecipitation takes places for a short period, but the turbidity declines slowly after this period almost to the initial level (Fig. 2a-4). The effect of proteolysis of filamin by
calpain on this phenomenon was then examined. When 2 /~g/ml of calpain is preincubated with filamin for 20 min, the effect of filamin on the superprecipitation is slightly diminished (Fig. 2b-3). When 10 ~ g / m l of calpain is preincubated with filamin which is a sufficient concentration to degrade filamin, the ability of filamin to reduce the superprecipitation is completely abolished (Fig. 2a-2). These results indicate that filamin loses its capacity to affect actin-myosin interaction if it is degraded by calpain. The fact that the 190 kDa fragment remains bound after the proteolysis suggests that the ability of filamin to inhibit the interaction is related to its ability to cross-link F-actin. It is probable that filamin interferes with the interaction of actin filaments and myosin filaments. Similar results are obtained for the Mg 2+ ATPase activity of actomyosin. As was reported, filamin inhibits the actomyosin ATPase activity
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Fig. 1. (a) Effect of the proteolysis of fila/nin on its influence on the viscosity of F-actin. Filamin was preincubated with calpain for different time intervals as indicated on the abscissa. After the preincubation an aliquot was added to G-actin solution. Polymerization was started bY adding MgC12 and
KCI to 2 mM and 100 mM, respectively. The viscosity of the F-actin solution was measured after 20 min by the method of McLean-Fletcher and Pollard [11]. - - , filamin preincubated with calpain; . . . . . . , calpain alone; O, preincubation in the presence of EGTA; O, preincubation in the presence of Ca2+; O, control (actin alone). (b) Degradation of filamin by partially purified calpain. Filamin and calpain were preincubated in the mixture described below. After the preincubation, 10 mM of EGTA was added to each sample and the samples were boiled and then electrophoresed by the method of Laemmli [12]. 1, filamin alone; 2-7, filamin preincubated with calpain in the presence of Ca 2+ (2,5,10,20,40,60 rain, respectively); 8, filamin preincubated with calpain for 60 rain in the presence of EGTA. The inserted numbers indicate the molecular weight in thousands. Preincubation conditions: The concentrations of proteins (determined by the method of Bradford [13]: filamin, 150 ~ g / m l ; partially purified calpain, I5 ~g/ml. Incubation mixture: 100 mM KC1, 1 mM CaC12 or 2 mM EGTA 10 mM imidazole-HCl (pH 7.0) and 1 mM dithiothreitol. After the preincubation at 37°C, the concentrations of CaC12 and EGTA in all the mixtures were adjusted to 1 m M and 10 raM, respectively, and aliquots were transferred to the incubation mixture. Incubation conditions: The protein concentrations: actin, 600 ~g/ml; filamin, 30 p,g/ml; partially purified calpain, 3 p,g/ml. Incubation mixture: 100 mM KCI, 2 mM MgC12, 0.2 mM CaC12, 10 mM EGTA, 10 mM imidazole-HCl (pH 7.0) and 0.2 mM dithiothreitol. Polymerization was started by adding KCI and MgCI2, polymerization proceeded at 25°C and the viscosity was measured after 20 min.
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Time(rain.) Fig. 2. (a) The effect of filamin on the superprecipitation of actomyosin after the degradation by partially purified calpain. Filamin was preincubated as in Fig. 1 for 20 rain. After the preincubation, the aliquots were added to actomyosin. The superprecipitation was initiated by adding ATP. The changes in the absorbance at 660 nm (,aA660) were monitored [14]. Assay conditions: myosin, 100 /xg/ml; actin, 200 lag/ml; filamin, 50/~g/ml; calpain, 2 /~g/ml (3) or 10/~g/ml (2); 100 mM KC1; 5 mM MgCl2; 10 mM imidazole-HC1 (pH 7.0); 0.3 mM CaC12; 2 mM EGTA; 1 mM ATP. The reaction temperature was 25 o C. 1, actomyosin alone; 2, actomyosin plus filarain that had been preincubated with calpain (10/~g/ml) in the presence of Ca2+; 3, same as 2 except that the concentration of calpain was 2/~g/ml; 4, actomyosin plus filamin that had been preincubated without calpain. (b) The effect of filamin on the Mg2+-ATPase activity of actomyosin after degradation by partially purified calpaln. Filamin was preincubated and was added to actomyosin as in (a). The ATPase activity was measured as previously reported [1-3]. The assay conditions were the same as in (a) except that phosphoenolpyruvate and pyruvate kinase were included [1-3]. 1, actomyosin alone; 2, actomyosin plus filamin; 3, actomyosin plus filamin which had been preincubated with calpain (10 #g/ml); 4, actomyosin plus calpain (10 ~g/rnl).
by about 50% (Fig. 2b-2). The inhibition is abolished if filamin is preincubated with calpain in the presence of Ca 2+ (Fig. 2b-3), while calpain itself has no effect on the actomyosin ATPase activity (Fig. 2b-4). This agrees with the result obtained for the superprecipitation activity.
Taken together, the results indicate that when filamin is degraded by calpain in the presence of Ca 2÷, its influences on the actomyosin system are abolished. Although the effect of filamin to gelate F-actin in vitro is established, the functional role of filamin in the platelet has been uncertain. The main problem lies in the difficulty in reconciling its stabilizing effect on F-actin with dynamic movements of platelets. Conceptually, cross-linkage of F-actin might promote the tension development by connecting adjacent filaments. Alternatively, filamin may restrict the tension development of actomyosin either by competing with the myosin molecule or by interfering with the interaction between actin filaments and myosin filaments. These conflicting views are expressed for filamin and other cross-linker proteins [6,7]. Our results support the hypothesis that filamin restricts the development of tension by interfering with the interaction between actin and myosin filaments. On the other hand, Fox [4] established that filamin is degraded markedly by calpain when the platelet is activated. The same authors subsequently found that destruction of filamin is required for the expression of glycoproteins on the platelet membrane [8]. In a previous report, we suggested that the main role of filamin would be to stabilize, not to mobilize, F-actin. The present report supports this view. In addition, it indicates a possible consequence of the degradation of filamin by calpain, namely the release of actomyosin. The hypothesis that filamin is a stabilizer is supported by other facts. (i) Filamin inhibits the effect of tropomyosin, which is an actomyosin enhancer [1,3]; (ii) the effect of filamin to gelate F-actin is antagonized by gelsolin, which is activated by Ca 2÷ [9]; (iii) in smooth muscle, filamin seems to be localized away from the actomyosin domain [10]. The changes in platelets after activation are drastic and irreversible. The main messenger for this change is probably Ca 2÷. When the concentration of Ca 2+ is increased after the activation of platelets, the effect of filamin is antagonized both by calpain and gelsolin. On the other hand, myosin is phosphorylated by Ca2÷-calmodulin dependent kinase. Our results reported here raise the possibility that phosphorylated myosin
286 r e o r g a n i z e s F - a c t i n f i l a m e n t s w h i l e f i l a m i n is b e i n g degraded by calpain.
References 1 0 n j i , T. and Shibata, N. (1982) Biochern. Biophys. Res. Commun. 109, 697-703 2 Nosaka, S., Onji, T. and Shibata, N. (1984) Biochim. Biophys. Acta 788, 290-297 3 0 n j i , T., Takagi, M. and Shibata, N. (1985) Biochem. Biophys. Res. Commun. 132, 307-312 4 Fox, J.E., Reynolds, C.C. and Phillips, D.R. (1983) J. Biol. Chem. 258, 9973-9981 5 Tsujinaka, T., Shiba, E., Kanbayashi, J. and Kosaki, G. (1983) Biochem. Int., 6, 71-74
6 Davrowska, R., Goch, A., Osinska, H., Szpacenko, A. and Sosinski, J. (1985) J. Mus. Res. Cell Motil. 6, 29-42 7 Wagner, P.D. (1984) J. Biol. Chem. 259, 6306-6310 8 Fox, J.E.B. (1985) J. Biol. Chem. 260, 11970-119774 9 Yin, H.L., Zaner, K.S. and Stossel, T.P. (1980) J. Biol. Chem. 255, 9494-9500 10 Small, J.B., Furst, D.O. and DeMey, J. (1986) J. Cell Biol. 102, 210-220 11 MacLean-Fletcher, S.D. and Pollard, T.D. (1980) J. Cell Biol. 85, 414-428 12 Laemmli, U.K. (1970) Nature 227, 680-685 13 Bradford, M.M. (1976) Anal. Biochem. 72, 248-254 14 Ebashi, S. (1961) J. Biochem. 50, 236-241
912 (1987) 283-286
283
Elsevier BBA 30202
BBA Report
Calpain abolishes the e f f e c t of filamin on the a c t o m y o s i n s y s t e m in platelets T o y o s h i Onji, M i c h i k o T a k a g i a n d N o b u h i k o S h i b a t a Division of Molecular Cardiology, The Center for Adult Diseases, Osaka (Japan)
(Received 15 September1986) (Revised manuscript received9 January 1987)
Key words: Filamindegradation;Actomyosin;Calpain; Calciumion; Plateletactivation Piatelet filamin was shown to cross-link F-actin and inhibit actomyosin ATPase activity. Filamin was also shown to be degraded by calpain (calcium-activated neutral proteinase; CANP) when the platelet was activated. The consequences of the proteolysis of fUamin on the actomyosin system have been investigated. When degraded by calpain in the presence of Ca2+, filamin loses its ability to cross-link F-aetin. Under the same conditions, its inhibitory effects on the superprecipitation and ATPase activity of actomyosin are abolished. The result suggests that the degradation of filamin is favorable for the contraction of the activated platelets.
Platelets are highly motile cells. The mechanism of their motility has bee the subject of research in this laboratory. We found that the interaction between actin and myosin is enhanced by tropomyosin in platelet [1,2]. Subsequently, we reported that another actin-binding protein, filamin, is capable of inhibiting actomyosin ATPase activity [3]. Filamin is reported to be degraded by calpain when the platelet is activated [4]. The physiological significance of this phenomenon has been uncertain. We have suggested that the effect of filamin on actomyosin may be reduced when filamin is degraded by calpain [3]. We tested this hypothesis using partially purified calpain from platelet. Actin, myosin and filamin were all purified from porcine platelets as previously described [1-3]. Calpain was partially purified from the same source by the method of Tsujinaka et al. [5] with a slight modification (ion-exchange chroCorrespondence: T. Onji, Division of Molecular Cardiology, The Center for Adult Diseases, Osaka 1-3-3, Nakamichi, Higashinariku, Osaka 537, Japan.
matography was carried out using QAE on highperformance liquid chromatography). The partially purified calpain was composed of 80 kDa and 90 kDa polypeptides (Fig. lb-8). Its activity was negligible at [Ca 2+ ] of less than 0.1 mM and was maximal at its concentration of 2 mM (calpain II or milli-CANP). The proteolysis of filamin by calpain is shown in Fig. lb. Partially purified calpain degrades filamin within 5 min. The major product is a 190-200 kDa fragment. The proteolysis is observed only in the presence of Ca 2÷. If Ca 2÷ is absent, no proteolysis is found, even after 60 rain of incubation of filamin with calpain (Fig. lb-8). After various times of preincubation, the effect of calpain-treated filamin on the viscosity of platelet F-actin was examined. Filamin increases the viscosity of F-actin markedly (Fig. la; value at zero time). Calpain itself does not affect the viscosity of F-actin after any length of preincubation either in the presence or absence of Ca 2+ (Fig. la; dotted line). However, if filamin is preincubated with calpain in the presence of Ca 2+, the viscosity of F-actin is reduced from 220 s / c m to
0167-4838/87/$03.50 © 1987 Elsevier Science Publishers B.V. (BiomedicalDivision)
284
60 s / c m (Fig. la; solid line with closed circle). On the other hand, if it is preincubated with calpain in the absence of Ca 2+ the capacity of filamin to gelate F-actin is unchanged (Fig. la; solid line with open circle). The 190 kDa fragment remains bound to F-actin after proteolysis (data not presented). These results indicate that filamin loses its property to cross-link F-actin when it is degraded by calpain in the presence of Ca 2+. We have reported that filamin is capable of inhibiting the Mg2+-ATPase activity of platelet actomyosin [3]. Another activity of actomyosin in vitro is superprecipitation. The turbidity of platelet actomyosin increases after the addition of ATP due to a profound reassembly of actin and myosin filaments (Fig. 2a-l). When filamin is added to this system, the superprecipitation takes places for a short period, but the turbidity declines slowly after this period almost to the initial level (Fig. 2a-4). The effect of proteolysis of filamin by
calpain on this phenomenon was then examined. When 2 /~g/ml of calpain is preincubated with filamin for 20 min, the effect of filamin on the superprecipitation is slightly diminished (Fig. 2b-3). When 10 ~ g / m l of calpain is preincubated with filamin which is a sufficient concentration to degrade filamin, the ability of filamin to reduce the superprecipitation is completely abolished (Fig. 2a-2). These results indicate that filamin loses its capacity to affect actin-myosin interaction if it is degraded by calpain. The fact that the 190 kDa fragment remains bound after the proteolysis suggests that the ability of filamin to inhibit the interaction is related to its ability to cross-link F-actin. It is probable that filamin interferes with the interaction of actin filaments and myosin filaments. Similar results are obtained for the Mg 2+ ATPase activity of actomyosin. As was reported, filamin inhibits the actomyosin ATPase activity
(a) (b)
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Fig. 1. (a) Effect of the proteolysis of fila/nin on its influence on the viscosity of F-actin. Filamin was preincubated with calpain for different time intervals as indicated on the abscissa. After the preincubation an aliquot was added to G-actin solution. Polymerization was started bY adding MgC12 and
KCI to 2 mM and 100 mM, respectively. The viscosity of the F-actin solution was measured after 20 min by the method of McLean-Fletcher and Pollard [11]. - - , filamin preincubated with calpain; . . . . . . , calpain alone; O, preincubation in the presence of EGTA; O, preincubation in the presence of Ca2+; O, control (actin alone). (b) Degradation of filamin by partially purified calpain. Filamin and calpain were preincubated in the mixture described below. After the preincubation, 10 mM of EGTA was added to each sample and the samples were boiled and then electrophoresed by the method of Laemmli [12]. 1, filamin alone; 2-7, filamin preincubated with calpain in the presence of Ca 2+ (2,5,10,20,40,60 rain, respectively); 8, filamin preincubated with calpain for 60 rain in the presence of EGTA. The inserted numbers indicate the molecular weight in thousands. Preincubation conditions: The concentrations of proteins (determined by the method of Bradford [13]: filamin, 150 ~ g / m l ; partially purified calpain, I5 ~g/ml. Incubation mixture: 100 mM KC1, 1 mM CaC12 or 2 mM EGTA 10 mM imidazole-HCl (pH 7.0) and 1 mM dithiothreitol. After the preincubation at 37°C, the concentrations of CaC12 and EGTA in all the mixtures were adjusted to 1 m M and 10 raM, respectively, and aliquots were transferred to the incubation mixture. Incubation conditions: The protein concentrations: actin, 600 ~g/ml; filamin, 30 p,g/ml; partially purified calpain, 3 p,g/ml. Incubation mixture: 100 mM KCI, 2 mM MgC12, 0.2 mM CaC12, 10 mM EGTA, 10 mM imidazole-HCl (pH 7.0) and 0.2 mM dithiothreitol. Polymerization was started by adding KCI and MgCI2, polymerization proceeded at 25°C and the viscosity was measured after 20 min.
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Time(rain.) Fig. 2. (a) The effect of filamin on the superprecipitation of actomyosin after the degradation by partially purified calpain. Filamin was preincubated as in Fig. 1 for 20 rain. After the preincubation, the aliquots were added to actomyosin. The superprecipitation was initiated by adding ATP. The changes in the absorbance at 660 nm (,aA660) were monitored [14]. Assay conditions: myosin, 100 /xg/ml; actin, 200 lag/ml; filamin, 50/~g/ml; calpain, 2 /~g/ml (3) or 10/~g/ml (2); 100 mM KC1; 5 mM MgCl2; 10 mM imidazole-HC1 (pH 7.0); 0.3 mM CaC12; 2 mM EGTA; 1 mM ATP. The reaction temperature was 25 o C. 1, actomyosin alone; 2, actomyosin plus filarain that had been preincubated with calpain (10/~g/ml) in the presence of Ca2+; 3, same as 2 except that the concentration of calpain was 2/~g/ml; 4, actomyosin plus filamin that had been preincubated without calpain. (b) The effect of filamin on the Mg2+-ATPase activity of actomyosin after degradation by partially purified calpaln. Filamin was preincubated and was added to actomyosin as in (a). The ATPase activity was measured as previously reported [1-3]. The assay conditions were the same as in (a) except that phosphoenolpyruvate and pyruvate kinase were included [1-3]. 1, actomyosin alone; 2, actomyosin plus filamin; 3, actomyosin plus filamin which had been preincubated with calpain (10 #g/ml); 4, actomyosin plus calpain (10 ~g/rnl).
by about 50% (Fig. 2b-2). The inhibition is abolished if filamin is preincubated with calpain in the presence of Ca 2+ (Fig. 2b-3), while calpain itself has no effect on the actomyosin ATPase activity (Fig. 2b-4). This agrees with the result obtained for the superprecipitation activity.
Taken together, the results indicate that when filamin is degraded by calpain in the presence of Ca 2÷, its influences on the actomyosin system are abolished. Although the effect of filamin to gelate F-actin in vitro is established, the functional role of filamin in the platelet has been uncertain. The main problem lies in the difficulty in reconciling its stabilizing effect on F-actin with dynamic movements of platelets. Conceptually, cross-linkage of F-actin might promote the tension development by connecting adjacent filaments. Alternatively, filamin may restrict the tension development of actomyosin either by competing with the myosin molecule or by interfering with the interaction between actin filaments and myosin filaments. These conflicting views are expressed for filamin and other cross-linker proteins [6,7]. Our results support the hypothesis that filamin restricts the development of tension by interfering with the interaction between actin and myosin filaments. On the other hand, Fox [4] established that filamin is degraded markedly by calpain when the platelet is activated. The same authors subsequently found that destruction of filamin is required for the expression of glycoproteins on the platelet membrane [8]. In a previous report, we suggested that the main role of filamin would be to stabilize, not to mobilize, F-actin. The present report supports this view. In addition, it indicates a possible consequence of the degradation of filamin by calpain, namely the release of actomyosin. The hypothesis that filamin is a stabilizer is supported by other facts. (i) Filamin inhibits the effect of tropomyosin, which is an actomyosin enhancer [1,3]; (ii) the effect of filamin to gelate F-actin is antagonized by gelsolin, which is activated by Ca 2÷ [9]; (iii) in smooth muscle, filamin seems to be localized away from the actomyosin domain [10]. The changes in platelets after activation are drastic and irreversible. The main messenger for this change is probably Ca 2÷. When the concentration of Ca 2+ is increased after the activation of platelets, the effect of filamin is antagonized both by calpain and gelsolin. On the other hand, myosin is phosphorylated by Ca2÷-calmodulin dependent kinase. Our results reported here raise the possibility that phosphorylated myosin
286 r e o r g a n i z e s F - a c t i n f i l a m e n t s w h i l e f i l a m i n is b e i n g degraded by calpain.
References 1 0 n j i , T. and Shibata, N. (1982) Biochern. Biophys. Res. Commun. 109, 697-703 2 Nosaka, S., Onji, T. and Shibata, N. (1984) Biochim. Biophys. Acta 788, 290-297 3 0 n j i , T., Takagi, M. and Shibata, N. (1985) Biochem. Biophys. Res. Commun. 132, 307-312 4 Fox, J.E., Reynolds, C.C. and Phillips, D.R. (1983) J. Biol. Chem. 258, 9973-9981 5 Tsujinaka, T., Shiba, E., Kanbayashi, J. and Kosaki, G. (1983) Biochem. Int., 6, 71-74
6 Davrowska, R., Goch, A., Osinska, H., Szpacenko, A. and Sosinski, J. (1985) J. Mus. Res. Cell Motil. 6, 29-42 7 Wagner, P.D. (1984) J. Biol. Chem. 259, 6306-6310 8 Fox, J.E.B. (1985) J. Biol. Chem. 260, 11970-119774 9 Yin, H.L., Zaner, K.S. and Stossel, T.P. (1980) J. Biol. Chem. 255, 9494-9500 10 Small, J.B., Furst, D.O. and DeMey, J. (1986) J. Cell Biol. 102, 210-220 11 MacLean-Fletcher, S.D. and Pollard, T.D. (1980) J. Cell Biol. 85, 414-428 12 Laemmli, U.K. (1970) Nature 227, 680-685 13 Bradford, M.M. (1976) Anal. Biochem. 72, 248-254 14 Ebashi, S. (1961) J. Biochem. 50, 236-241