286
Biochimica et Biophysica Acta. 1133(19t121286-202 ~9 1992ElsevierScience PublishersB.V.All rightsreserved0167-4889/92/$115.011
Diphosphorylation of platelet myosin by myosin light chain kinase Kazuyuki Itoh, T a e k o H a r a a n d N o b u h i k o S h i b a t a Dit'ision of Molectdar Cardiology, The Centerfor Adult Diseases. Osaka. Osaka (Japan)
(Received 23 April 19911 (Revised manuscriptreceived22 August19911 Keywords: Diphosphorylation:Myosin;Platclct; (Human) Recently, one of the authors (K.I.) and other investigators reported that myosin light chain (MLC) of smooth muscle (gizzard, arterial and tracheal) was diphosphorylated by myosin light chain kinase (MLCK) and that diphosphorylated myosin showed a marked increase in the actin-activated myosin ATPase activity in vitro and ex vivo. In this study, we prepared myosin, actin, tropomyosin (human platelet), MLCK (chicken gizzard) and calmodulin (bovine brain) and demonstrated diphosphorylation of MLC of platelet by MLCK in vitro. Our results are as follows. (1) Platelet MLC was diphosphorylated by a relatively high concentration ( > 20 p g / m l ) of MLCK in vitro. As a result of diphosphorylation, the actin-activated myosin ATPase activity was increased 3 to 4-fold as compared to the monophosphorylation. (2) Both di- and monophosphorylation reactions showed similar Ca 2+, KCI, MgCIz-dePendence. Maximal reaclion was seen at [Ca 2+ ] > 10 -6 M, 60 mM KCI and 2 mM MgCI 2. This condition was physiological in activated platelets. (3) Di- and monophosphorylated myosin showed similar Ca 2+. KCI-dependence of ATPase activity but distinct MgCI2-dependence. Diphosphorylated myosin showed maximal ATPase activity at 2 mM MgCI 2 and monophosphorylated myosin showed a maximum at 10 mM MgCI z. (4) The addition of tropom)osin stimulated actin-activated ATPase activity in both di- and monophosphorylated myosin to the same degree. (5) ML-9, a relatively specific inhibitor of MLCK, inhibited the aggregation of human pJatelets induced by thrombin ex vivo in a dose-dependent manner. Moreover, this drug also partially inhibited both di- and monophosphorylation reactions and actin-activated ATPase activity. On the other hand, H-7, a synthetic inhibitor of protein kinase C, had little effect on the aggregation of human platelets induced by thrombin ex vivo. From these results, we conclude that diphosphorylation of platelet myosin by MLCK may play an important role in activated platelets in vivo.
Introduction Phosphorylation of the 20 kDa myosin light chain (MLC) of platelet by Ca2+-calmodulin-dependent myosin light chain kinase (MLCK) is associated with activation and aggregation of platelets and subso.quent release of platelet granules [1]. One of the authors (K.I.) and other investigators reported that MLC in smooth muscle (gizzard, arterial and tracheal) myosin was diphosphorylated by MLCK and that diphosphorylated myosin showed a marked increase in the actin-
activated myosin ATPase activity ir, vitro [2-5] and ex vivo [6,7]. Recently, Ikebe showed diphosphorylation of bovine platelet myosin by MLCK in vitro [8]. He demonstrated that the first and second phosphorylation sites were serine-19 and threonine-18, respectively, and that phosphorylation at the threonine residue markedly increased the actin-activated ATPase activity of myosin. In this study, we prepared myosin, actin, tropomyosin (human platelet), MLCK (chicken gizzard) and calmodulin (bovine brain) and demonstrated another interesting finding about platelet diphosphorylated myosin in calcium, potassium and magnesium chloride dependence in vitro.
Abbreviations: MLC. myosinlight chain; MLCK,myosinlight chain kinase; SDS.sodiumd(~decylsulfate.
Materials and Methods
Correspondence: K. ltoh, Division of Molecular Cardiology, The Center for Adult Diseases.Osaka, Nakamichi, Higashinari-ku,Osaka 537, Japan.
A synthetic relatively specific inhibitor of myosin light chain kinase (MLCK), l-(5-chloronaphthalenesulfonyl)-lh-hexahydro-l,4-diazepine tML -9) [9] and a
287 relatively specific inhibitor of protein kinase C, I-(5isoquinolinesulfonyl)-2-metylpiperazinc dihydrochloride (H-7) were purchased from Seikagaku Kogyo, (Tokyo, Japan). [T-32p]ATP (201)0-31)1)(}Ci/mmol) was from Amersham (Arlington Heights, IL). Thrombin (human, topical grade) and ATP were purchased from Sigma (St. Louis, MO). All other chemicals wcrc reagent grade or better. Human platelet concentrates were obtained from the neighboring Red Cross Blood Center. Myosin was prepared from fresh human platelets by slight modifications of the method of Sellers et al. [10]. Myosin purified by this method was essentially homogeneous as judged by the densitometric scanning of sodium dodecyl sulfate (SDS) polyacrylamide gels and was free from light chain kinase and phosphatase. Actin and tropomyosin were prepared from human platelets as previously described [ 1 I]. Chicken gizzard myosin light chain kinase and bovine brain calmodulin were purified as in Refs. 12 and 13, respectively. The aetin activated myosin ATPase activity was measured by a modification of the method of ReL 14. Briefly, the reaction mixture contained 400 p.g/ml platelet myosin, 200 t t g / m l platelet actin, 0-50 p.g/ml trop,,myosin, 0-100 p.g/ml MLCK, 5 0 / z g / m l of calmodulin, 20 mM imidazole-HCI (pH 7.2), 1 mM D I T, 60-200 mM KCk 0-10 mM MgCI z, 1 mM ATP and the desired submicromolar free Ca 2+ concentration, adjusted by the addition of 185 p.M EGTA. The association constants for CaZ+-EGTA and Ca2+-ATP at pH 7.2 were taken as 6.8.107 and 8.5" 103, respectively [15]. The reaction was started by adding ATP and after incubation at 37°C for 5 min, it was stopped by adding ice-cold trichloroacetic acid. The mixture was centrifuged and the phosphate (Pi) content released into the supernatant was determined by the method of Youngberg and Youngberg [16]. The aetin-activated myosin ATPase activity was calculated by subtracting values obtained with incubation for 4 min from those with incubation for 5 min. The extent of phosphorylation was monitored by alkaline urea/glycerol gel electrophoresis (urea-PAGE) [17] and by the incorporation of 32p. Gels were stained by 0.06% Coomassie brilliant blue R-250, and after destaining were scanned using a UltroSean XL laser densitometer (Pharmacia) attached to a PC-286 VE personal computer (Epson). The extent of phosphorylation was calculated by the following equation: where NP, non-phosphorylated MLC; P, mono-phosphorylated MLC; PP, diphosphorylated MLC Extentof phosphorylation = (2 × Area(PP)+ Area(P)) /(Area(PP) + Area(P)+ Area(NP)) x 100(%)
Human platelet rich plasma was obtained from healthy donor by centrifugation. Platelet aggregation stimulated by thrombin was monitored with a dual aggregation meter (Sicnco) by the turbidimetric method of Born [18]. SDS polyacrylamide gel elcctrophoresis was carried out in the buffer system Laemmli [19]. Protein concentrations were determined by the method of Lowry ct al. [20] with bovine serum albumin as a standard. Results
Fig. I shows the time-course of the phosphorylation of platelet myosin by a high concentration (100 p.g/ml = 0.8 p~M) of MLCK. An autoradiograph (inset b) of the gel in Fig. 1 shows that 32Pi was incorporated totally into the MLC-I (20 kDa myosin light chain). Alkaline urea/glycerol gel electrophoresis (ureaPAGE) ~s a suitable and conventional method for identifying the phosphorylation of MLC and for separating nonphosphorylated (NP), mono-phosphorylated (P) and diphosphorylated MLCs (PP) by the difference of their charges. After more than 2 min of incubation, incorporation of phosphate exceeded 2 mol/mol of myosin and the more rapidly moving diphosphorylated MI.C (PP) was seen in th,.~ urea-PAGE (see Fig. 2A and B). The diphosphorylation reaction occurred slowly and finally 3 mol of phosphate/mol of myosin was incorporated. Under suitable conditions, the phospho-
"
8
2.6 ~
"1.4 - -
o
oo~, i
;
~,
'" !
(nan)
Fig. I. Time-courseof phosphorylationof platelet myosin.Platelet myosin (1 ,u,M) was phosphoqdated at 3(FC in 50 ~l o| reaction mediumconsistingof 50 ram Tris-HCI(pH 7.2), 2 mM MgCI:. I p.M free Ca2., 1 ,u,Mcalmodalin,0.8 ~M myosinlightchain kinaseand 0.1 mM [3,-32plATP. Lane a: 10% SDS polyac~lamidegel clectrophoresis:lane b: autoradiographcorrespondingto lane a.
288
O"-(
JO
0
1
4
100
MLCK(pg/~) B
p--
LC-2--
1
C
1 1
TM
MLCK
1ol (-)
(+) 00 t
(-)
(+) .41 2
estimated by u r e a - P A G E and the radioactivity. Therefore, we estimated the extent of phosphorylatiolt by using u r e a - P A G E in the following experiments. Fig. 2A shows the phosphorylation of platelet my,Jsin and actin-activated A T P a s e activity at varying _~,ILCK concentrations. At high MLCK concentrat.io,, diphosphorylated myosin light chain (PP) w~,s shown in the u r e a - P A G E (Fig. 2B) and diphosphorylated myosin showed a markedly increased A T P a s e activity. As shown in Fig. 2B, a diphosphorylation band (PP) was already seen in the u r e a - P A G E when monophosphorylation occurred 5 0 - 7 0 % , and the extent of PP was increasing as the phosphorylation level increased. Therefore, we assumed that two reactions occurred simultaneously and the diphosphorylation reaction took place more slowly. Fig. 2C shows the effect of tropomyosin on the actin-activated A T P a s e activity at varying phosphorylation levels of myosin. The addition of tropomyosin stimulated actin-activated A T P a s e activity in both diand monophosphorylated myosin to the same degree (1.2-fold increase to control). Fig. 3 shows the free Ca2+-dependence of monoand diphosphorylation of myosin and actin-activated A T P a s e activity. Both mono- and diphosphorylation 200 -~ ,
'~
?,,o o 10~
(-)
(+) 100 3.07
Ir~g/ml) (toolPi/m~myo~n)
Fig. 2. (A) Extent of phosphorylation and actin-activated ATPase activity by varying concentrations of myosin light chain kinase (MLCK). Conditions: 400 #g/ml of platelet myosin, 200/~g/ml of platetet actin, :SUp.g/ml of tropomyosinand 50/zg/ml of calmodulin were incubated with a given concentration of MLCK in a solution containing 20 mM imidazole-HCl(pH 7.7.), 1 mM DTT, 60 mM KCI, I mM MgClz, 1 ,u.Mof free Ca2+ and 1 mM ATP for 5 min at 37°C. (B) NP, P and PP band were clarified in urea-PAGE of (C) Effect of tropomyosinon the actin-acti,'atingATPase activityof platelet myosin in several phosphorylation levels. Condition was same as in A, otherwise in the presence and absence of tropomyosin.
rylation level reached about 3.5 mol of p h o s p h a t e / m o l of myosin (see Fig. 3). Furthermore, a direct correlation was four, d between the extent of phosphorylation
i/
//Ill"FREE
Ca 2+ (M)
Fig. 3. Effects of free calcium concentration on the extent of phosphor/lotion and the actin-activatedATPase activityof platelet myosin in the several phosphorylation levels. Conditions: 400 /zg/ml of platelet myosin, 200 p,g/ml of platelet actin, 50 v,g/ml of tropomyosin and 50/.tg/ml of calmodulin were incubated with given concentration of MLCK in a solution containing 20 mM imidazoleHCI (pH 7.2), 1 ,~tM DTI', 60 mM KCI, 1 mM IvlgClz, given concentration of free Caz+ and 1 mM ATP for 5 min at 37~C. MLCK concentration ( i . D: 100 #g/ml; A. 4:10 v,g/ml; o. o: 2 v,g/ml).
289 r e a c t i o n s of p l a t e l e t m y o s i n by M L C K s h o w e d a m a r k e d d e p e n d e n c e on f r e e C a -'÷ c o n c e n t r a t i o n which involved c a l m o d u l i n a n d a c o n c e n t r a t i o n o f f r e e C a 2 * in t h e m i c r o m o l a r r a n g e ( 2 - 3 t z M ) w a s r e q u i r e d for a m a x i m u m r e s p o n s e . T h e a c t i n - a c t i v a t e d A T P a s e activity also s h o w e d a s i m i l a r d e p e n d e n c e on C a 2+ c o n c e n tration, w h i c h involved ( d i ) p h o s p h o r y l a t i o n o f myosin. It w a s r e p o r t e d that t h e intracellular C a -'+ c o n c e n t r a tion o f t h e platelet w a s 100 n M at rest a n d w a s r a i s e d 3 p , M a f t e r s t i m u l a t i o n by t h r o m b i n [21,22]. T h e r e f o r e , t h e C a 2+ c o n c e n t r a t i o n r a n g e w a s c o m p a r a b l e b e t w e e n in vitro ( d i ) p h o s p h o r y l a t i o n a n d in v!vo activation. Fig. 4 s h o w s t h e K C I - d e p e n d e n c e o f m o n o - a n d diphosphorylation r e a c t i o n s o f m y o s i n a n d actinactivated ATPase activity. Both monoand d i p h o s p h o r y l a t i o n re~'ctions w e r e s i m i l a r o v e r a r a n g e o f K C I c o n c e n t r a t i o n s f r o m 70 to 300 m M . But actina c t i v a t e d A T P a s e activity o f p h o s p h o r y l a t e d and d i p h o s p h o r y l a t e d m y o s i n s h o w e d a m a r k e d KCI d e p e n dence. As the KCI concentration was increased, ATP a s e activity m a r k e d l y d e c r e a s e d a n d b o t h m y o s i n s showed a similar tendency. T h e e f f e c t s o f MgCI,_ c o n c e n t r a t i o n o n the m o n o and diphosphorylation reaction and actin-activated A T P a s e activity o f p l a t e l e t m y o s i n a r e s h o w n in Fig. 5. Both mono- and diphosphorylation reactions showed s i m i l a r M g C I z c o n c e n t r a t i o n d e p e n d e n c e a n d b o t h rea c t i o n s w e r e m a x i m u m at 2 m M M g C I 2. O n t h e o t h e r
5o
t
1~
ILl KCI (raM) Fig. 4. Effects of KCI concentration on the extent of phosphorylation and the actin-activated ATPasc activity of platelet myosin in the several phosphorylation levels. Condilions: 400 p.g/ml of platelet r,lyesin. 2(}fl/tg/ml of platelet actin, 50/.tg/ml of tropomyosin and 50/zg/ml of calmodulin were incubated with given concentration of MLCK in a solution containing 20 mM imidazole-HCI (pH 7.2), 1 mM DTT, given concentration of KCL I mM MgCl,. 1 p,M of flee Ca-" " and t mM ATP for 5 ,nin at 37°C. MLCK concentration ( • , : loll/tg/ml. ,I. O: 4 .u.g/ml).
B
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,
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/'
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/
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"tbi"
or2
=
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MgClz (raM)
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~
~o
Extent of phosphorylation(m~ I~/mol . ~ n )
Fig. 5. (A) Magnesium chloride dependence of the extent of phosphorylation and the actin-activated ATPase activity of platelet myosin in the several phosphowlation levels. Conditions: 400 p,g/ml of platelet myosin, 200 p,g/m] of platelet actin, 50/.Lg/ml of tropomyosin and 50 u.g/ml of calmodulin w e f c incubated with given concentration of MLCK in a solution containing 20 mM imidazole-HCI (pH 7.2), I mM DTr. 60 mM KCI. given concentration of MgCI2, I p.M of free Ca z+ and I mM ATP for 5 rain at 37°C. MLCK concentration ( I , ra: 100 p,g/ml" • , A: 5 p,g/ml; e, o: I p,g/ml). (B) Relationship between the extent of phosphorylation and the actin-activated ATPas¢ activity in varying magnesium chloride concentrations. MgCI 2 concentration (o: 0.5 raM, e: I raM, z~: 2 mM, ra: 5 raM, l : l0 raM).
290 hand, mono- a n d diphosphorylatcd myosin showed different MgCI 2 d e p e n d e n c e of actin-activated A T P a s e activity. Monophosphorylated myosin showed maximal A T P a s e activity at 10 mM MgCI 2, but diphosphorylated myosin showed maximal activity at 1 - 2 mM MgCI~. This difference is shown clearly in Fig. 5B. At a higher ( > 5 raM) MgCI 2 concentration, the slopes of [ A T P a s e / d e g r e e of phosphorylation] were similar between mono- and diphosphorylated states. But at 1 - 2 rnM a d d e d MgCI2, which has been suggested as a physiological range in the activated platele'~s, the slope of [ A T P a s e / d e g r e e of phosphorylation] b e c a m e steep from mono- to diphosphorylated states. T h r o m b i n induces several platelet responses including shape changes, aggregation, secretion a n d clot reA
traction, it has been suggested that contractile proteins have a major role in effccting several of these responses. Therefore, we examined the effect of ML-9, which is reported us a relatively specific inhibitor of M L C K [9], on the aggregation of h u m a n platelcts induced by thrombin ex vivo. As shown in Fig. 6A, ML-9 inhibited the aggregation of h u m a n platelets induced by thrombin ex vivo in a d o s e - d e p e n d e n t manner. Moreover, this d r u g also partially inhibited both monoa n d d i p h o s p h o w ! a : i e n reactions a n d actin-activated A T P a s e activity in vitro. O n the o t h e r hand, H-7, a synthetic inhibitor of protein kinase C h a d iittle effect on the aggregation of h u m a n platelets induced by thrombin ex vivo a n d on the (di)phosphorylation of myosin a n d on the A T P a s c activity (data not shown).
tAu,,, ,0
/
----__
-i0 1
vem¢,e (I. ~ J
tm,n~ re~'~,n 0 5U/ml C E ~ I
~"
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___:.
10
Vmh,cle
.......
Thtomb,n 0 3U/ml
~ . . . . . . . . . .
5'0 ML-9(pM)
lC)0 0
Fig. 6. (A) Effect of ML-9 on the aggregation of human platelet by thrombin. Oivcn concentration of ME-9 or vehicle (ethanol) was pretreated with human platelet rich plasma for 5 rain and stimulated by thrombin. (B) Effect of ML-9 on the extent of phosphorylation and the aclin-activated ATPase activity in platelet myosin. Conditions were the same as in Fig. 2A. Given concen!ration of ML-9 was added in the assay mixture 5 rain before starting incubation. MLCK concenlralion (Ill, El: 100 ~g/ml; e, O: 4/.tg/ml).
291 From these results, we conclude that (di}phosphorylation of platclet myosin may play an important role iq activated platelets in vivo.
Discussion T h e r e are a n u m b e r of reports of diphosphor~'latcd MLC in smooth muscle (gizzard, tracheal a n d arterial) myosin in vitro [2-5] a n d ex vivo [6,7]. Ikebe et al. [8,23] d e m o n s t r a t e d that the diphosphorylation of gizzard a n d platelet myosin by M L C K caused a conformational c h a n g e a n d this is associated with its altered biological properties. In this report, we d e m o n s t r a t e d diphosphorylation of platelet M L C in vitro and comp a r e d mono- a n d diphosphorylation reactions. Suitable conditions for the diphosphorylation reaction can be considered to be physiological in the activated platelet. O n the o t h e r hand, several investigators reported phosphorylation of platelet myosin in vivo. Daniel et al. [l] d e m o n s t r a t e d that myosin existed mainly in the d e p h o s p h o r y l a t e d state in resting platelets a n d that stimulation by thrombin could p r o d u c e a shift to the totally phosphorylated state. N a k a et al. [24] r e p o r t e d that t h r o m b i n - i n d n c e d activation of platelets was associated with M L C phosphorylation by M L C K a n d that phorbol ester-induced activation of platelets was associated with M L C phosphorylation by protein kinase C. In these papers, p h o s p h o p e p t i d e was analyzed by twodimensional peptide m a p p i n g a n d diphosphorylated M L C was not detected. Recently, however, several investigators r e p o r t e d multiple phosphorylated myosin in arterial [6] a n d tracheal [7] s m o o t h muscle a n d that s m o o t h muscle myosin was maximally phosphorylated in the initial p h a s e of contraction a n d the extent of phosphorylation was getting smaller in the later p h a s e (the socalled latch p h e n o m e n o n ) [25]. Moreover, the extent of phosphorylation was affected by the kind of stimuli [26,27]. T h e r e f o r e , there was a lot of interest a b o u t diphosphorylation of platelet M L C in vivo. Most recently, we d e m o n s t r a t e d the diphosphorylation of platelet myosin m the initial p h a s e o f activation by thrombin ex vivo (unpublished data). A n o t h e r interesting finding of diphosphorylated myosin was its m a g n e s i u m d e p e n d e n c e . In this report, we d e m o n s t r a t e d that m o n o - a n d diphosph_-,u, lated myosin showed different MgCI 2 d e p e n d e n c e of actinactivated A T P a s e activity. M o n o p h o s p h o r y l a t e d myosin showed maximal A T P a s e activity at 10 m M MgCI 2, the saute as r e p o r t e d for smooth muscle myosin [28], but diphosphorylated myosin showed maximal activity at 1 - 2 m M MgCI z. This result suggested that diphosphorylated myosin possessed the similar properties with skeletal muscle type myosin, which showed markedly high A T P a s e activity at very low m a g n e s i u m concentration.
Recently, Ikebe [8] reported KCI d e p e n d e n c e of A T P a s e activity of platelet myosin at different phosphorylation levels auc suggested that the formation of the 10S (a folded conformation) was inhibited by second-site phosphorylation. In the present results, we d e m o n s t r a t e d a n o t h e r interesting finding about platelet d i p h o s : b o r y l a t e d myosin in calcium, potassium and magnesium chloride dependence. It is highly probable that platclet MLC is diphosphorylated in vivo and the role of diphosphorylation of myosin remains to be clarified. Acknowledgment We thank Dr. T. Onji for reading the manuscript. References I Daniel. J.L., Molish, I.R. and Holmsen, H. (1981)J. Biol. Chem. 256, 75111-7514. 2 Tanaka. T,, Sobue. K., Owada. K. and Haruka, A. (1985) Biochem. Biophys. Res. Commun. 131,987-993. 3 ltoh. K. and Sohu¢, K. (1987) Seikagaku 7. 499. 4 Cole. H.A., Griffiths. H.S., Patchell, V.B. and Perry, S.V. (1985) FEBS Lelt. 181),165-169. 5 Ikebe. M. and Hartshorne, DJ. (1985}J. Biol. Chem. 260, 10027111031. 6 Haeberle, J.R.. Sutton, T.A. and Trockman, B.A. (1988) J. Biol. Chem. 263. 4424-4429. 7 Colbu~n, J.C., Miehnoff, C.H., Hsu, L.. Slaughter, C.A., Kamm, K.E. and Stull. J.T. (1988) J. Biol. Chem. 263. 19166-19173. S Ikebe, M. (1989) Biochemistry 28. 8750-8755. 9 Saitoh, M., lshikawa. T., Matsushima, S.. Naka. M. and H!daka, H. (1987)J. Biol. Chem. 262, 7796-7801. 10 Sellers, J.R., Soboeiro, M.S., Faust, K., Bengur. A.R. and Harvey, E.V. (1988) Biochemistry 27, 6977-6982. II ,Onji, T.. Takagi. M. and Shibata. N. (1987) Biochem, Binphys. Res. C-mmun. 143. 475-481. 12 Yamazaki, K., Itoh, K., Sobue, K., Moil, T. and Shibata, N. (1987) J. Biochem. 101, I-9. 13 Kakiuchi, S., Sobue. K., Yamazaki, R., Kambayashi,J., Sakon, M. and Kosaki, G. (1981) FEBS Len. 126, 203-207. 14 Itoh. K., Morimoto, S., Shiraishi, T., Taniguchi. K., Onishi, T. and Kumahara, Y. (1988) Biochem. Biophys. Res. Commun. 150, 263-27O. 15 Pershadsingh, H.A.. McDaniel, M.L, Landt, M.. Bry, C.G.. Lacy, P.E. and McDonald, J.M. (Iq80} Nature 288, 492-495. 16 Youngberg, G.E. and Youngberg. M.V. (1930) J. Lab. Med. 16, 158-66. 17 Adelsteir, R.S. and Klee, C.B. (1981) J. Biol. Chem. 256, 750~7509. 18 Born, G.V.R. (1962) Nature 194. 927-929. 19 Laemmli. U.K. (1971})Nature 227, 680-685. 20 Lowry. O.H., Rosebraagh. N.J.. Farr. A.L. and Randall, R.J. (1951) J. Biol. Chem. 36, 265-275. 21 Yamaguchi. A., Yamamoto, N., Kitagawa. H., Tanoue, K. and Yamazaki, H. (1987) FEBS Len. 225, 228-Z':t2. 22 Pollock, W.K. and Rink, T.J. (1986) Biochem. Biophys. Res. Commun. 139, 308-314. Ikebe, M., Koretz. J. and Hartshorne. D.L (1988)J. Biol. Chem. 263, 6432-6437.
292 24 Naka, M., Nishikawa. M., Adelstein, R.S. and Hidaka, H. (1983) Nature 306, 490-492. 25 Rembold C. and Murphy, R. (1990) Am. J. Physiol. 259, C251257. 26 Sasaki, Y., Uchida. T.. and Sasaki, Y. (t989) J. Biochem. 106, 1009-1018.
27 Seto, M., Sasaki. Y. and Sasaki, Y. (1990) Am. J. Physiol. 259. C769-774. 28 |kebe, M.. Barsotti. R.J. and Hartshorne, D.J. (1984) Biochemistry 23, 5062-5068.
Biochimica et Biophysica Acta. 1133(19t121286-202 ~9 1992ElsevierScience PublishersB.V.All rightsreserved0167-4889/92/$115.011
Diphosphorylation of platelet myosin by myosin light chain kinase Kazuyuki Itoh, T a e k o H a r a a n d N o b u h i k o S h i b a t a Dit'ision of Molectdar Cardiology, The Centerfor Adult Diseases. Osaka. Osaka (Japan)
(Received 23 April 19911 (Revised manuscriptreceived22 August19911 Keywords: Diphosphorylation:Myosin;Platclct; (Human) Recently, one of the authors (K.I.) and other investigators reported that myosin light chain (MLC) of smooth muscle (gizzard, arterial and tracheal) was diphosphorylated by myosin light chain kinase (MLCK) and that diphosphorylated myosin showed a marked increase in the actin-activated myosin ATPase activity in vitro and ex vivo. In this study, we prepared myosin, actin, tropomyosin (human platelet), MLCK (chicken gizzard) and calmodulin (bovine brain) and demonstrated diphosphorylation of MLC of platelet by MLCK in vitro. Our results are as follows. (1) Platelet MLC was diphosphorylated by a relatively high concentration ( > 20 p g / m l ) of MLCK in vitro. As a result of diphosphorylation, the actin-activated myosin ATPase activity was increased 3 to 4-fold as compared to the monophosphorylation. (2) Both di- and monophosphorylation reactions showed similar Ca 2+, KCI, MgCIz-dePendence. Maximal reaclion was seen at [Ca 2+ ] > 10 -6 M, 60 mM KCI and 2 mM MgCI 2. This condition was physiological in activated platelets. (3) Di- and monophosphorylated myosin showed similar Ca 2+. KCI-dependence of ATPase activity but distinct MgCI2-dependence. Diphosphorylated myosin showed maximal ATPase activity at 2 mM MgCI 2 and monophosphorylated myosin showed a maximum at 10 mM MgCI z. (4) The addition of tropom)osin stimulated actin-activated ATPase activity in both di- and monophosphorylated myosin to the same degree. (5) ML-9, a relatively specific inhibitor of MLCK, inhibited the aggregation of human pJatelets induced by thrombin ex vivo in a dose-dependent manner. Moreover, this drug also partially inhibited both di- and monophosphorylation reactions and actin-activated ATPase activity. On the other hand, H-7, a synthetic inhibitor of protein kinase C, had little effect on the aggregation of human platelets induced by thrombin ex vivo. From these results, we conclude that diphosphorylation of platelet myosin by MLCK may play an important role in activated platelets in vivo.
Introduction Phosphorylation of the 20 kDa myosin light chain (MLC) of platelet by Ca2+-calmodulin-dependent myosin light chain kinase (MLCK) is associated with activation and aggregation of platelets and subso.quent release of platelet granules [1]. One of the authors (K.I.) and other investigators reported that MLC in smooth muscle (gizzard, arterial and tracheal) myosin was diphosphorylated by MLCK and that diphosphorylated myosin showed a marked increase in the actin-
activated myosin ATPase activity ir, vitro [2-5] and ex vivo [6,7]. Recently, Ikebe showed diphosphorylation of bovine platelet myosin by MLCK in vitro [8]. He demonstrated that the first and second phosphorylation sites were serine-19 and threonine-18, respectively, and that phosphorylation at the threonine residue markedly increased the actin-activated ATPase activity of myosin. In this study, we prepared myosin, actin, tropomyosin (human platelet), MLCK (chicken gizzard) and calmodulin (bovine brain) and demonstrated another interesting finding about platelet diphosphorylated myosin in calcium, potassium and magnesium chloride dependence in vitro.
Abbreviations: MLC. myosinlight chain; MLCK,myosinlight chain kinase; SDS.sodiumd(~decylsulfate.
Materials and Methods
Correspondence: K. ltoh, Division of Molecular Cardiology, The Center for Adult Diseases.Osaka, Nakamichi, Higashinari-ku,Osaka 537, Japan.
A synthetic relatively specific inhibitor of myosin light chain kinase (MLCK), l-(5-chloronaphthalenesulfonyl)-lh-hexahydro-l,4-diazepine tML -9) [9] and a
287 relatively specific inhibitor of protein kinase C, I-(5isoquinolinesulfonyl)-2-metylpiperazinc dihydrochloride (H-7) were purchased from Seikagaku Kogyo, (Tokyo, Japan). [T-32p]ATP (201)0-31)1)(}Ci/mmol) was from Amersham (Arlington Heights, IL). Thrombin (human, topical grade) and ATP were purchased from Sigma (St. Louis, MO). All other chemicals wcrc reagent grade or better. Human platelet concentrates were obtained from the neighboring Red Cross Blood Center. Myosin was prepared from fresh human platelets by slight modifications of the method of Sellers et al. [10]. Myosin purified by this method was essentially homogeneous as judged by the densitometric scanning of sodium dodecyl sulfate (SDS) polyacrylamide gels and was free from light chain kinase and phosphatase. Actin and tropomyosin were prepared from human platelets as previously described [ 1 I]. Chicken gizzard myosin light chain kinase and bovine brain calmodulin were purified as in Refs. 12 and 13, respectively. The aetin activated myosin ATPase activity was measured by a modification of the method of ReL 14. Briefly, the reaction mixture contained 400 p.g/ml platelet myosin, 200 t t g / m l platelet actin, 0-50 p.g/ml trop,,myosin, 0-100 p.g/ml MLCK, 5 0 / z g / m l of calmodulin, 20 mM imidazole-HCI (pH 7.2), 1 mM D I T, 60-200 mM KCk 0-10 mM MgCI z, 1 mM ATP and the desired submicromolar free Ca 2+ concentration, adjusted by the addition of 185 p.M EGTA. The association constants for CaZ+-EGTA and Ca2+-ATP at pH 7.2 were taken as 6.8.107 and 8.5" 103, respectively [15]. The reaction was started by adding ATP and after incubation at 37°C for 5 min, it was stopped by adding ice-cold trichloroacetic acid. The mixture was centrifuged and the phosphate (Pi) content released into the supernatant was determined by the method of Youngberg and Youngberg [16]. The aetin-activated myosin ATPase activity was calculated by subtracting values obtained with incubation for 4 min from those with incubation for 5 min. The extent of phosphorylation was monitored by alkaline urea/glycerol gel electrophoresis (urea-PAGE) [17] and by the incorporation of 32p. Gels were stained by 0.06% Coomassie brilliant blue R-250, and after destaining were scanned using a UltroSean XL laser densitometer (Pharmacia) attached to a PC-286 VE personal computer (Epson). The extent of phosphorylation was calculated by the following equation: where NP, non-phosphorylated MLC; P, mono-phosphorylated MLC; PP, diphosphorylated MLC Extentof phosphorylation = (2 × Area(PP)+ Area(P)) /(Area(PP) + Area(P)+ Area(NP)) x 100(%)
Human platelet rich plasma was obtained from healthy donor by centrifugation. Platelet aggregation stimulated by thrombin was monitored with a dual aggregation meter (Sicnco) by the turbidimetric method of Born [18]. SDS polyacrylamide gel elcctrophoresis was carried out in the buffer system Laemmli [19]. Protein concentrations were determined by the method of Lowry ct al. [20] with bovine serum albumin as a standard. Results
Fig. I shows the time-course of the phosphorylation of platelet myosin by a high concentration (100 p.g/ml = 0.8 p~M) of MLCK. An autoradiograph (inset b) of the gel in Fig. 1 shows that 32Pi was incorporated totally into the MLC-I (20 kDa myosin light chain). Alkaline urea/glycerol gel electrophoresis (ureaPAGE) ~s a suitable and conventional method for identifying the phosphorylation of MLC and for separating nonphosphorylated (NP), mono-phosphorylated (P) and diphosphorylated MLCs (PP) by the difference of their charges. After more than 2 min of incubation, incorporation of phosphate exceeded 2 mol/mol of myosin and the more rapidly moving diphosphorylated MI.C (PP) was seen in th,.~ urea-PAGE (see Fig. 2A and B). The diphosphorylation reaction occurred slowly and finally 3 mol of phosphate/mol of myosin was incorporated. Under suitable conditions, the phospho-
"
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~,
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(nan)
Fig. I. Time-courseof phosphorylationof platelet myosin.Platelet myosin (1 ,u,M) was phosphoqdated at 3(FC in 50 ~l o| reaction mediumconsistingof 50 ram Tris-HCI(pH 7.2), 2 mM MgCI:. I p.M free Ca2., 1 ,u,Mcalmodalin,0.8 ~M myosinlightchain kinaseand 0.1 mM [3,-32plATP. Lane a: 10% SDS polyac~lamidegel clectrophoresis:lane b: autoradiographcorrespondingto lane a.
288
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estimated by u r e a - P A G E and the radioactivity. Therefore, we estimated the extent of phosphorylatiolt by using u r e a - P A G E in the following experiments. Fig. 2A shows the phosphorylation of platelet my,Jsin and actin-activated A T P a s e activity at varying _~,ILCK concentrations. At high MLCK concentrat.io,, diphosphorylated myosin light chain (PP) w~,s shown in the u r e a - P A G E (Fig. 2B) and diphosphorylated myosin showed a markedly increased A T P a s e activity. As shown in Fig. 2B, a diphosphorylation band (PP) was already seen in the u r e a - P A G E when monophosphorylation occurred 5 0 - 7 0 % , and the extent of PP was increasing as the phosphorylation level increased. Therefore, we assumed that two reactions occurred simultaneously and the diphosphorylation reaction took place more slowly. Fig. 2C shows the effect of tropomyosin on the actin-activated A T P a s e activity at varying phosphorylation levels of myosin. The addition of tropomyosin stimulated actin-activated A T P a s e activity in both diand monophosphorylated myosin to the same degree (1.2-fold increase to control). Fig. 3 shows the free Ca2+-dependence of monoand diphosphorylation of myosin and actin-activated A T P a s e activity. Both mono- and diphosphorylation 200 -~ ,
'~
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(+) 100 3.07
Ir~g/ml) (toolPi/m~myo~n)
Fig. 2. (A) Extent of phosphorylation and actin-activated ATPase activity by varying concentrations of myosin light chain kinase (MLCK). Conditions: 400 #g/ml of platelet myosin, 200/~g/ml of platetet actin, :SUp.g/ml of tropomyosinand 50/zg/ml of calmodulin were incubated with a given concentration of MLCK in a solution containing 20 mM imidazole-HCl(pH 7.7.), 1 mM DTT, 60 mM KCI, I mM MgClz, 1 ,u.Mof free Ca2+ and 1 mM ATP for 5 min at 37°C. (B) NP, P and PP band were clarified in urea-PAGE of (C) Effect of tropomyosinon the actin-acti,'atingATPase activityof platelet myosin in several phosphorylation levels. Condition was same as in A, otherwise in the presence and absence of tropomyosin.
rylation level reached about 3.5 mol of p h o s p h a t e / m o l of myosin (see Fig. 3). Furthermore, a direct correlation was four, d between the extent of phosphorylation
i/
//Ill"FREE
Ca 2+ (M)
Fig. 3. Effects of free calcium concentration on the extent of phosphor/lotion and the actin-activatedATPase activityof platelet myosin in the several phosphorylation levels. Conditions: 400 /zg/ml of platelet myosin, 200 p,g/ml of platelet actin, 50 v,g/ml of tropomyosin and 50/.tg/ml of calmodulin were incubated with given concentration of MLCK in a solution containing 20 mM imidazoleHCI (pH 7.2), 1 ,~tM DTI', 60 mM KCI, 1 mM IvlgClz, given concentration of free Caz+ and 1 mM ATP for 5 min at 37~C. MLCK concentration ( i . D: 100 #g/ml; A. 4:10 v,g/ml; o. o: 2 v,g/ml).
289 r e a c t i o n s of p l a t e l e t m y o s i n by M L C K s h o w e d a m a r k e d d e p e n d e n c e on f r e e C a -'÷ c o n c e n t r a t i o n which involved c a l m o d u l i n a n d a c o n c e n t r a t i o n o f f r e e C a 2 * in t h e m i c r o m o l a r r a n g e ( 2 - 3 t z M ) w a s r e q u i r e d for a m a x i m u m r e s p o n s e . T h e a c t i n - a c t i v a t e d A T P a s e activity also s h o w e d a s i m i l a r d e p e n d e n c e on C a 2+ c o n c e n tration, w h i c h involved ( d i ) p h o s p h o r y l a t i o n o f myosin. It w a s r e p o r t e d that t h e intracellular C a -'+ c o n c e n t r a tion o f t h e platelet w a s 100 n M at rest a n d w a s r a i s e d 3 p , M a f t e r s t i m u l a t i o n by t h r o m b i n [21,22]. T h e r e f o r e , t h e C a 2+ c o n c e n t r a t i o n r a n g e w a s c o m p a r a b l e b e t w e e n in vitro ( d i ) p h o s p h o r y l a t i o n a n d in v!vo activation. Fig. 4 s h o w s t h e K C I - d e p e n d e n c e o f m o n o - a n d diphosphorylation r e a c t i o n s o f m y o s i n a n d actinactivated ATPase activity. Both monoand d i p h o s p h o r y l a t i o n re~'ctions w e r e s i m i l a r o v e r a r a n g e o f K C I c o n c e n t r a t i o n s f r o m 70 to 300 m M . But actina c t i v a t e d A T P a s e activity o f p h o s p h o r y l a t e d and d i p h o s p h o r y l a t e d m y o s i n s h o w e d a m a r k e d KCI d e p e n dence. As the KCI concentration was increased, ATP a s e activity m a r k e d l y d e c r e a s e d a n d b o t h m y o s i n s showed a similar tendency. T h e e f f e c t s o f MgCI,_ c o n c e n t r a t i o n o n the m o n o and diphosphorylation reaction and actin-activated A T P a s e activity o f p l a t e l e t m y o s i n a r e s h o w n in Fig. 5. Both mono- and diphosphorylation reactions showed s i m i l a r M g C I z c o n c e n t r a t i o n d e p e n d e n c e a n d b o t h rea c t i o n s w e r e m a x i m u m at 2 m M M g C I 2. O n t h e o t h e r
5o
t
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ILl KCI (raM) Fig. 4. Effects of KCI concentration on the extent of phosphorylation and the actin-activated ATPasc activity of platelet myosin in the several phosphorylation levels. Condilions: 400 p.g/ml of platelet r,lyesin. 2(}fl/tg/ml of platelet actin, 50/.tg/ml of tropomyosin and 50/zg/ml of calmodulin were incubated with given concentration of MLCK in a solution containing 20 mM imidazole-HCI (pH 7.2), 1 mM DTT, given concentration of KCL I mM MgCl,. 1 p,M of flee Ca-" " and t mM ATP for 5 ,nin at 37°C. MLCK concentration ( • , : loll/tg/ml. ,I. O: 4 .u.g/ml).
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Fig. 5. (A) Magnesium chloride dependence of the extent of phosphorylation and the actin-activated ATPase activity of platelet myosin in the several phosphowlation levels. Conditions: 400 p,g/ml of platelet myosin, 200 p,g/m] of platelet actin, 50/.Lg/ml of tropomyosin and 50 u.g/ml of calmodulin w e f c incubated with given concentration of MLCK in a solution containing 20 mM imidazole-HCI (pH 7.2), I mM DTr. 60 mM KCI. given concentration of MgCI2, I p.M of free Ca z+ and I mM ATP for 5 rain at 37°C. MLCK concentration ( I , ra: 100 p,g/ml" • , A: 5 p,g/ml; e, o: I p,g/ml). (B) Relationship between the extent of phosphorylation and the actin-activated ATPas¢ activity in varying magnesium chloride concentrations. MgCI 2 concentration (o: 0.5 raM, e: I raM, z~: 2 mM, ra: 5 raM, l : l0 raM).
290 hand, mono- a n d diphosphorylatcd myosin showed different MgCI 2 d e p e n d e n c e of actin-activated A T P a s e activity. Monophosphorylated myosin showed maximal A T P a s e activity at 10 mM MgCI 2, but diphosphorylated myosin showed maximal activity at 1 - 2 mM MgCI~. This difference is shown clearly in Fig. 5B. At a higher ( > 5 raM) MgCI 2 concentration, the slopes of [ A T P a s e / d e g r e e of phosphorylation] were similar between mono- and diphosphorylated states. But at 1 - 2 rnM a d d e d MgCI2, which has been suggested as a physiological range in the activated platele'~s, the slope of [ A T P a s e / d e g r e e of phosphorylation] b e c a m e steep from mono- to diphosphorylated states. T h r o m b i n induces several platelet responses including shape changes, aggregation, secretion a n d clot reA
traction, it has been suggested that contractile proteins have a major role in effccting several of these responses. Therefore, we examined the effect of ML-9, which is reported us a relatively specific inhibitor of M L C K [9], on the aggregation of h u m a n platelcts induced by thrombin ex vivo. As shown in Fig. 6A, ML-9 inhibited the aggregation of h u m a n platelets induced by thrombin ex vivo in a d o s e - d e p e n d e n t manner. Moreover, this d r u g also partially inhibited both monoa n d d i p h o s p h o w ! a : i e n reactions a n d actin-activated A T P a s e activity in vitro. O n the o t h e r hand, H-7, a synthetic inhibitor of protein kinase C h a d iittle effect on the aggregation of h u m a n platelets induced by thrombin ex vivo a n d on the (di)phosphorylation of myosin a n d on the A T P a s c activity (data not shown).
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Fig. 6. (A) Effect of ML-9 on the aggregation of human platelet by thrombin. Oivcn concentration of ME-9 or vehicle (ethanol) was pretreated with human platelet rich plasma for 5 rain and stimulated by thrombin. (B) Effect of ML-9 on the extent of phosphorylation and the aclin-activated ATPase activity in platelet myosin. Conditions were the same as in Fig. 2A. Given concen!ration of ML-9 was added in the assay mixture 5 rain before starting incubation. MLCK concenlralion (Ill, El: 100 ~g/ml; e, O: 4/.tg/ml).
291 From these results, we conclude that (di}phosphorylation of platclet myosin may play an important role iq activated platelets in vivo.
Discussion T h e r e are a n u m b e r of reports of diphosphor~'latcd MLC in smooth muscle (gizzard, tracheal a n d arterial) myosin in vitro [2-5] a n d ex vivo [6,7]. Ikebe et al. [8,23] d e m o n s t r a t e d that the diphosphorylation of gizzard a n d platelet myosin by M L C K caused a conformational c h a n g e a n d this is associated with its altered biological properties. In this report, we d e m o n s t r a t e d diphosphorylation of platelet M L C in vitro and comp a r e d mono- a n d diphosphorylation reactions. Suitable conditions for the diphosphorylation reaction can be considered to be physiological in the activated platelet. O n the o t h e r hand, several investigators reported phosphorylation of platelet myosin in vivo. Daniel et al. [l] d e m o n s t r a t e d that myosin existed mainly in the d e p h o s p h o r y l a t e d state in resting platelets a n d that stimulation by thrombin could p r o d u c e a shift to the totally phosphorylated state. N a k a et al. [24] r e p o r t e d that t h r o m b i n - i n d n c e d activation of platelets was associated with M L C phosphorylation by M L C K a n d that phorbol ester-induced activation of platelets was associated with M L C phosphorylation by protein kinase C. In these papers, p h o s p h o p e p t i d e was analyzed by twodimensional peptide m a p p i n g a n d diphosphorylated M L C was not detected. Recently, however, several investigators r e p o r t e d multiple phosphorylated myosin in arterial [6] a n d tracheal [7] s m o o t h muscle a n d that s m o o t h muscle myosin was maximally phosphorylated in the initial p h a s e of contraction a n d the extent of phosphorylation was getting smaller in the later p h a s e (the socalled latch p h e n o m e n o n ) [25]. Moreover, the extent of phosphorylation was affected by the kind of stimuli [26,27]. T h e r e f o r e , there was a lot of interest a b o u t diphosphorylation of platelet M L C in vivo. Most recently, we d e m o n s t r a t e d the diphosphorylation of platelet myosin m the initial p h a s e o f activation by thrombin ex vivo (unpublished data). A n o t h e r interesting finding of diphosphorylated myosin was its m a g n e s i u m d e p e n d e n c e . In this report, we d e m o n s t r a t e d that m o n o - a n d diphosph_-,u, lated myosin showed different MgCI 2 d e p e n d e n c e of actinactivated A T P a s e activity. M o n o p h o s p h o r y l a t e d myosin showed maximal A T P a s e activity at 10 m M MgCI 2, the saute as r e p o r t e d for smooth muscle myosin [28], but diphosphorylated myosin showed maximal activity at 1 - 2 m M MgCI z. This result suggested that diphosphorylated myosin possessed the similar properties with skeletal muscle type myosin, which showed markedly high A T P a s e activity at very low m a g n e s i u m concentration.
Recently, Ikebe [8] reported KCI d e p e n d e n c e of A T P a s e activity of platelet myosin at different phosphorylation levels auc suggested that the formation of the 10S (a folded conformation) was inhibited by second-site phosphorylation. In the present results, we d e m o n s t r a t e d a n o t h e r interesting finding about platelet d i p h o s : b o r y l a t e d myosin in calcium, potassium and magnesium chloride dependence. It is highly probable that platclet MLC is diphosphorylated in vivo and the role of diphosphorylation of myosin remains to be clarified. Acknowledgment We thank Dr. T. Onji for reading the manuscript. References I Daniel. J.L., Molish, I.R. and Holmsen, H. (1981)J. Biol. Chem. 256, 75111-7514. 2 Tanaka. T,, Sobue. K., Owada. K. and Haruka, A. (1985) Biochem. Biophys. Res. Commun. 131,987-993. 3 ltoh. K. and Sohu¢, K. (1987) Seikagaku 7. 499. 4 Cole. H.A., Griffiths. H.S., Patchell, V.B. and Perry, S.V. (1985) FEBS Lelt. 181),165-169. 5 Ikebe. M. and Hartshorne, DJ. (1985}J. Biol. Chem. 260, 10027111031. 6 Haeberle, J.R.. Sutton, T.A. and Trockman, B.A. (1988) J. Biol. Chem. 263. 4424-4429. 7 Colbu~n, J.C., Miehnoff, C.H., Hsu, L.. Slaughter, C.A., Kamm, K.E. and Stull. J.T. (1988) J. Biol. Chem. 263. 19166-19173. S Ikebe, M. (1989) Biochemistry 28. 8750-8755. 9 Saitoh, M., lshikawa. T., Matsushima, S.. Naka. M. and H!daka, H. (1987)J. Biol. Chem. 262, 7796-7801. 10 Sellers, J.R., Soboeiro, M.S., Faust, K., Bengur. A.R. and Harvey, E.V. (1988) Biochemistry 27, 6977-6982. II ,Onji, T.. Takagi. M. and Shibata. N. (1987) Biochem, Binphys. Res. C-mmun. 143. 475-481. 12 Yamazaki, K., Itoh, K., Sobue, K., Moil, T. and Shibata, N. (1987) J. Biochem. 101, I-9. 13 Kakiuchi, S., Sobue. K., Yamazaki, R., Kambayashi,J., Sakon, M. and Kosaki, G. (1981) FEBS Len. 126, 203-207. 14 Itoh. K., Morimoto, S., Shiraishi, T., Taniguchi. K., Onishi, T. and Kumahara, Y. (1988) Biochem. Biophys. Res. Commun. 150, 263-27O. 15 Pershadsingh, H.A.. McDaniel, M.L, Landt, M.. Bry, C.G.. Lacy, P.E. and McDonald, J.M. (Iq80} Nature 288, 492-495. 16 Youngberg, G.E. and Youngberg. M.V. (1930) J. Lab. Med. 16, 158-66. 17 Adelsteir, R.S. and Klee, C.B. (1981) J. Biol. Chem. 256, 750~7509. 18 Born, G.V.R. (1962) Nature 194. 927-929. 19 Laemmli. U.K. (1971})Nature 227, 680-685. 20 Lowry. O.H., Rosebraagh. N.J.. Farr. A.L. and Randall, R.J. (1951) J. Biol. Chem. 36, 265-275. 21 Yamaguchi. A., Yamamoto, N., Kitagawa. H., Tanoue, K. and Yamazaki, H. (1987) FEBS Len. 225, 228-Z':t2. 22 Pollock, W.K. and Rink, T.J. (1986) Biochem. Biophys. Res. Commun. 139, 308-314. Ikebe, M., Koretz. J. and Hartshorne. D.L (1988)J. Biol. Chem. 263, 6432-6437.
292 24 Naka, M., Nishikawa. M., Adelstein, R.S. and Hidaka, H. (1983) Nature 306, 490-492. 25 Rembold C. and Murphy, R. (1990) Am. J. Physiol. 259, C251257. 26 Sasaki, Y., Uchida. T.. and Sasaki, Y. (t989) J. Biochem. 106, 1009-1018.
27 Seto, M., Sasaki. Y. and Sasaki, Y. (1990) Am. J. Physiol. 259. C769-774. 28 |kebe, M.. Barsotti. R.J. and Hartshorne, D.J. (1984) Biochemistry 23, 5062-5068.
