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Substrates for protein kinase C in a cell free preparation of rat aorta smooth muscles
Life Sciences, Printed in the
Vol. 42, U.S.A.
Pergamon
pp. 1315-1321
Press
SUBSTRATES FOR PROTEIN KINASE C IN A CELL FREE PREPARATION OF RAT AORTA SMOOTH MUSCLES
Toshio
Nakaki,
Bradley
C. Wise
and De-Maw
Chuang
Laboratory of Preclinical Pharmacology National Institute of Mental Health St. Elizabeths Hospital, Washington, D. C. 20032 (Received in final form February
1,
1988)
Summary
Protein phosphorylation has been studie$in a cell free system of rat aorta smooth muscles. Addition of Ca + caused phosphorylation of 2+The addition of phosphatidylserine or calmodulin several proteins. together with Ca further increased the phosphorylation of proteins with apparent molecular weights of 20 and 92.5 kilodaltons. The activators of protein kinase C, 12-O-tetradecanoylphorbol-13-acetate and t,2-diolein, increased phosphorylation of the protein bands of ei g ht to those increased by phosphatidylserine in ;~;iI=sm;;c~~a~+ whereas the biologically inactive phorbol ester, 4 f$,-phorbol-12,13 hidecanoate (4 &%PDD) failed to change the pattern of protein phosphorylation. These results show that proteins present in smooth muscle of rat aorta with molecular weights of 20 and 92.5 kilodaltons are substrates for protein kinase C. The phorbol ester 12-0-tetradecanoylphorbol-1Zacetate (TPA) causes smooth muscle contraction (l-3) and is mitogenic for smooth muscle cells (4). A current concept of the molecular mechanism of action of TPA is that the effects of phorbol esters are mediated by an activation of protein kinase C (5). Protein kinase C is present in rat aorta (6) and hence TPA may increase protein phosphorylation in aorta smooth muscles. In vitro some contractile constituents of smooth muscle such as myosin light chain -(7), myosin light chain kinase (8,9), vinculin (lo), filamin (10) and troponin (11) are substrates for purified protein kinase C. However, it is still unknown whether these proteins are phosphorylated by protein kinase C -in vivo and whether protein kinase C phosphorylates additional proteins in smooth muscle. Identification of protein substrates for protein kinase C in rat aorta might increase our understanding of the mechanism whereby phorbol esters cause smooth muscle contraction. The present results show that two proteins of apparent molecular weight of 20 and 92.5 kilodaltons are substrates for protein kinase C in extracts of rat aorta smooth muscle. Methods
Chemicals: Markers for the molecular weight determination of proteins were purchased from Bio-Rad (Richmond, CA) and Pharmacia (Piscataway, NJ). 1,2-Diolein, 4UPDD and TPA were purchased from Sigma Chemical Company (St. Louis, MO). The sources of other chemicals used in these experiments were given previously (12). __________x_‘__‘__________________’--___~__~_~~~~~~~~~~~~~_~~~~~~~~~~~~~~~~~~--The present addresses are: T. Nakaki, Department of Pharmacology, Keio University School of Medicine, Tokyo 160, Japan; B.C. Wise, Fidia Georgetown Institute for Neuroscience, Georgetown University, Washington, D.C. 20007. 0024-3025188
$3.00
+ .OO
1316
Protein
Phosphorylation
in
Rat Aorta
Vol.
42,
No.
13,
1988
Phosphorylation in a cell-free system: Male Sprague-Dawley rats (Zivic-Miller, 8-12 weeks old) were decapitated and a 3 cm-segment of thoracic aorta was excised and placed into a physiolo$cal saline solution. Fat and connective tissue were removed and the aorta segment was incubated for 1 hr at 37’C in a calcium-free Krebs Ringer bicarbonate buffer (118 mM NaCI, 4.7 mM KCI, 1.2 mM NaH PO 1.2 mM M&l , 25 11 mM glucose). A mixture of 95% 02 and 5 3o C ‘b’ was continu 2 usly mM NaHC03, bubbled through the buffer. Then the aorta was cut longitudinally ar?d placed on a petri dish with the tunica intima facing upward. The intima was removed by scraping with a razor blade and the remaining aorta was placed in homogenization buffer (250 mM sucrose, 0.2 mM EGTA, 2.5 mM MgC12, 50 mM 2-mercaptoethanol, 25 mM Tris-HCI, pH 7.5). In a typical experiment, five aortas were placed in 1.5 ml of the above buffer. The aortas were minced and homogenized with a motor-driven glass homogenizer immersed in an ice bath. The homogenate was centrifuged at 100 x g for 10 min at 4’C. The pellet was discarded and the resulting supernatant was centrifuged at 100,000 x g for 60 min at 4’C. The supernatant was used for the phosphorylation study, which was carried out in a reaction mixture of 100~1 containing 25 m&I Tris-HCI, pH 7.5, 10 mM MgC12, 0.2 mM EGTA, 6Oyg of soluble proteins, 5yM’)j/P ATP (10 uCi/assay), and when used various concentrations of CaC12 or 20 pg phosphatidylserine. The reaction mixtures were incubated at 30°C for various time periods and the reactions were terminated by addition of 50~11 of sodium dodecyl sulfate (SDS)-stop solution (9% SDS, 30% glycerol, 0.03% bromophenol blue and 125 mM Tris-HCI, pH 6.8) and placed in a boiling water bath for 2 min. The samples were prepared for SDS-polyacrylamide gel electrophoresis and the gels were stained, destained, dried and subjected to autoradiography as described previously (12). Autoradiograms were scanned with a densitometer using visible light. Results Preincubation of aorta with calcium-free Krebs-Ringer bicarbonate buffer was necessary to reduce the high basal level of protein phosphorylation in order to improve the detection of protein phosphorylation elicited by exogenously added calcium (data not shown). At the concentration used (Fig. 1) the addition of Ca 2+ (lane 2) or phosphatidylserine (lane 4) to the cell-free phosphorylation system had no substantial effect on the protein phosphorylation pattern. However, thF+a.ddition of calmodulin (lane 3) or phosphatidylserine (lane 5) in the presence of Ca increased greatly the phosphorylation of proteins with apparent molecular weights of 20 and 92.5 kilodaltons (Fig. 1). It is unknown whether these 20 or 92.5 kilodalton protein bands consist of a The addition of CAMP increased the homogeneous population of protein molecules. phosphorylation of 22, 45 and 110 kilodalton protein bands and the addition of cCMP increased the phosphate incorporation into a 45 kilodalton protein band (data not shown). These effects of cyclic nucleotides were similar to those reported by Rapoport et al. (13). The addition of 0.2 mM Ca2+ In ’ the presence of 0.2 mM EGTA (Fi% 2, lane 2) had no effect on protein phosphorylation. However, addition of I mM Ca increased the phosphorylation of several protein bands (lane 4). These bands include proteins with apparent molecular weights of 16, 20, 3&45, 48, 60 and 92.5 kilodaltons. The addition of phosphatidylserine together with Ca (line 5) resulted in a further increase in the with molecular weights of 16, 20 and 92.5 kilodaltons. In phosphorylation of2yroteins the absence of Ca , phosphatidylserine did not stimulate protein phosphorylation (data not shown). PDD and 1,2-diolein on the phosphorylation of aortic proteins The effects of TPA, 4 are shown in Fig. 3. TPA and 1,2-diolein activate protein kinase C (5,14), whereas 4w TPA (trace 4) andl,2-diolein (trace 3), PDD, a structural analog of TPA, is inactive. but not C&PDD (trace 2), increased significantly the phosphorylation of proteins with apparent molecular weights of 20 and 92.5 kilodaltons.
vol.
42, No.
13,
Protein
1988
Phosphorylation
in Rat Aorta
1317
Discussion The diversity of cellular response mediated by protein kinase C may be explained Several proteins have been found to be by the variety of substrates in a given cell. substrates for protein kinase C in smooth muscles of rat vas deferens (15). However, none of them has a molecular weight similar to those found to be phosphorylated in the smooth muscles of rat aorta. In a cell free preparation of rat aorta and at low concentrations of Ca*+, proteins ‘t$ apparent molecular weights of 20 and 92.5 kilodaltons were phosphorylated in a c”LI + and phosphatidylserine dependent-manner and therefore, are substrates for protein kinase C. TPA or 1,2-diolein which activated protein kinase C (5) also increas?: At higher Ca the phosphorylation of the 20 and 92.5 kilodalton protein bands. co y+entrations, an additional protein band of 16 kilodaltons was phosphorylated in a Ca Although we have not clarified the and phosphatidylserine-dependent manner. molecular characteristics of the 20 and 92.5 kilodalton proteins, it is tempting to speculate that they are myosin light chain and phosphorylase b which have molecular
20I
1234
5
Fig. 1 Autoradiogram showing protein phosphorylation stimulated by Ca*+, calmodulin and phosphatidylserine in the cell free preparation of rat aorta. The 100,000 x g supernatant of rat aorta homogenate (6Opg protein) was incubated for 20 seconds in the presence of 0.2 mM EGTA plus various agents as described in methods. Proteins were separated on a 10% polyacrylamis& and 0.1% SDSfiel. Each lane shows: 1, control; 2, 0.5 mM Ca ; 3, 0.5 mM Ca plus lSyg/ml calmodulin; 4, 200 )Ig/ml ph2;phatidylserine; 5, 200 yg/ml phosphatidylserine plus 0.5 mM Ca .
Protein Phosphorylation in Rat Aorta
1318
Vol. 42, No. 13, 1988
weights of 20 and 92 kilodaltons respectively. The proteins pith apparent molecular weights of 20 and 92.5 kilodaltons are also substrates for Ca +/calmodulin-dependent kinase (Fig. 1). However, it iq+unknown whether the same site in these proteins are -dep+endent enzymes. Since myosin light chain is well phosphorylated by these two Ca known to be a substrate for the Ca /calmodulin dependent myosin light chain kinase (71, one may further suggest that the 20-kilodalton protein detected in the present study is the myosin light chain. Other proteins Ca2+/camodulin-dependent
may
also kinase
be the substrates in the rat aorta
92.5
for extracts.
protein For
kinase example,
92K
66.2
X
i
21.5
14.4
Fig. 2 (6) showing protein 5+scans (A) and densitometri Autoradiograms by Ca and phosphatidylserine. stimulated phosphorylation conditions and processing were the same as Jhose Phosphorylation described i?+Fig. 1. Each trace shows: 1, control; 3 0.2 mM CaL’;24 plus phosphatidyl serine; 4, I mM Ca +; 5, 1 mM Ca 0.2 mM Ca plus phosphatidylserine. Calculation “f the peak area of the 20 kilo&alton protein revealed: 1 mM Ca + alone, 1,400+290*; 1 mM plus phosphatidylserine, 2,300+380**, when control was Ca normalized to 100 in each e eriment-(n=3). *P
C
and the
Vol.
42,
No.
13,
1988
Protein
Phosphorylation
in
Rat
Aorta
1319
phosphorylation of two other proteins with appare$ molecular weignts of 64 and 39 plus phosphatidylserine or Ca kilodaltons was modestly increased by 0.5 mM Ca plus calmodulin (Fig. 1 lanes 5 and 3). The nature of these two proteins was not further characterized. It should
be stressed that in out study the aorta segment was preincubated for one in a calcium-free Krebs Ringer bicarbonate buffer before being used to prepare the cell free system for phosphorylation study. This preincubation was found to The reason for this improvement markedly reduce the level of basal phosphorylation. may be related to the fact that the concentration of extracellular calcium is about one
hr at
37OC
Fig. 3 Densitometric scan of an autoradiogram showing protein phosphorylation stimulated by addition of 33 nM TPA and lOpg/ml 1,2-diolein but not by the addition of 33 nM 4 o( PDD. Phosphorylation conditions and processing were the same as those described in Fig. 1 except that 0.2 mM CaCl and 200 ug/ml phosphatidylserine were used. Each trace shows: 1, control; 2, 4 PDD; 3, 1,2-diolein; 4, TPA. Control condition included 5% ethanol, which was used as the vehicle for TPA, 1,2-diolein and 40( PDD. The left panel (A) shows overall scan of the gel. The right panel (B) shows a magnification of the scan of high molecular weight proteins. Calculation of the peak area of the 20 kilodalton protein revealed the following percentage of control. 1,2-diolein, 174; TPA, 158; 40(PDD, 100. The experiment was repeated three times with similar results.
1320
Protein Phosphorylation in Rat Aorta
Vol. 42, No. 13, 1988
thousand times higher than that of the cation present intracellularly. Thus, preincubation of the aorta in calcium-free buffer would deplete most, if not all of the calcium trapped in the outer surface of the plasma membrane, thereby reducing the high level of basal protein phosphorylation. It is also conceivable that following removal of aorta from the rat, there may have been substantial cell death or injury resulting from the anoxic conditions. An influx of extracellular or release of intracellular calcium may also contribute to the high basal level of protein phosphorylation. Preincubation is, thus, required to restore normal cellular calcium (and other ionic) levels in surviving smooth muscle cells. We have suggested that 1,2-diacylglycerol, a phosphoinositide metabolite formed by phospholipase C, might play an important role in mediating the tonic component of the rat aorta contraction elicited by serotonin (5HT) (3). In fact 5HT elicits smooth muscle contraction (16) and phosphoinositide hydrolysis (17) through activation of pharmacologically identified 5HT2 receptors in rat aorta (18). Moreover 2-nitro-4carboxyphenyl-N,N-diphenylcarbamate, an inhibitor of polyphosphoinositide-specific phospholipase C, abolished both the SHT-stimulated phospholipse C activity and the Furtherrnore, the protein kinase C 5HT-induced tonic phase of contraction (3). activator, TPA, induces the tonic contraction of rat aorta (2,3). In the present study we show that TPA and I,2 diacylglycerol increase the phosphorylation of protein bands with apparent molecular weights of 20 and 92.5 We suggest that the phosphorylation of these two aortic proteins could be kilodaltons. important in mediating the action of vasoactive substances such as angiotensin II, norepinephrine and 5HT which have been shown to activate phosphoinositide hydrolysis in rat aorta (17,19,20). Furthermore, these proteins may possibly be involved in the mitogenic activity of TPA on smooth muscle cells (4). References
I. 2. 3. 4. 5. 6.
7. 8. 9. IO. 11. 12. 13. 14. 15.
H. RASMUSSEN, J. FORDER, I. KOJIMA, and A. SCRIABINE, Biochem. Biophys. Res. Commun. 122 776-784 (1984). N.R. DANTHULmI, and R.C. DETH, Biochem. Biophys. Res. Commun. -125 1103-1109 (1984). T. NAKAKI, B.L. ROTH, D.M. CHUANC, and E. COSTA, J. Pharmacol. Exp. Ther. 234 442-446 (1985) m. OWEN, J. Cell Biol. 101454-459 (1985). M. CASTAGNA, Y. TAKAI, K. KAIBUCHI, K. SANO, U. KIKKAWA, and Y. NISHIZUKA, J. Biol. Chem. 257 7847-7851 (1982). J.F. KUO, R.G.G. ANDERS0KB.C. WISE L. MACKERLOVA, I. SALOMONSSON, N.L. BRACKETT, N. KATOH, M. SHOJI, and R.W. WRENN, Proc. Natl. Acad. Sci. USA 77 7039-7043 (1980). T. E@O, M. NAKA, and H. HIDAKA, Biochem. Biophys. Res. Commun. -105 942-948 (1982). M. IKEBE, M. INAGAKI, K. KANAMARU, and H. HIDAKA, J. Biol. Chem. -260 4547-4550 (1985). M. NISHIKAWA, S. SHIRAKAWA, and R.S. ADELSTEIN, J. Biol. Chem. -260 8978-8983 (1985). S. KAWAMOTO, and H. HIDAKA,
Biochem. Biophys. Res. Commun. -118 736-742 (1984). N. KATOH, B.C. WISE, and J.F. KUO, Biochem. 3.209 189-195 (1983). B.C. WISE, and E. COSTA, J. Neurochem. g 227-234 (1985). R.M. RAPOPORT, M. DRAZNIN, and F. MURAD, Proc. Natl. Acad. Sci. USA -79 6470-6474 (1982). KAWAHARA, T. MORI, and Y. Y. TAKAI, A. KISHIMOTO, Y. IWASA, Y. NISHIZUKA, J. Biol. Chem. 254 3692-3695 (1979). R.W. WRENN, N. KATOH, and J.F. KUO, Biochem. Biophys. Acta -676 266-269 (1981).
Vol. 42,
No.
13,
1988
Protein Phosphorylation in Rat Aorta
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M.L. COHEN, R.W. FULLER, and K.S. WILEY, J. Pharmacol. Exp. Ther. 218 421-425 (1981). and E. COSTA, Neuropharmacology 17. B.L. ROTH, T. NAKAKI, D.M. CHUANC, -23 1223-1225 (1984). Exp. Ther. 18. B.L. ROTH, T. NAKAKI, D.-M. CHUANG, and E. COSTA, J. Pharmacol. 238 480-485 (1986). P.A. VELLETRI, W. LOVENBERG, and M.A. BEAVEN, 3. Biol. Chem. 19. TNABIKA, 16.
260 4661-4670
20.
(1985).
=LEGAN, B, CHERNOW, 389-390 (1985).
J. PARRILLO,
and B.L. ROTH,
Eur. J. Pharmacol.
-110
Vol. 42, U.S.A.
Pergamon
pp. 1315-1321
Press
SUBSTRATES FOR PROTEIN KINASE C IN A CELL FREE PREPARATION OF RAT AORTA SMOOTH MUSCLES
Toshio
Nakaki,
Bradley
C. Wise
and De-Maw
Chuang
Laboratory of Preclinical Pharmacology National Institute of Mental Health St. Elizabeths Hospital, Washington, D. C. 20032 (Received in final form February
1,
1988)
Summary
Protein phosphorylation has been studie$in a cell free system of rat aorta smooth muscles. Addition of Ca + caused phosphorylation of 2+The addition of phosphatidylserine or calmodulin several proteins. together with Ca further increased the phosphorylation of proteins with apparent molecular weights of 20 and 92.5 kilodaltons. The activators of protein kinase C, 12-O-tetradecanoylphorbol-13-acetate and t,2-diolein, increased phosphorylation of the protein bands of ei g ht to those increased by phosphatidylserine in ;~;iI=sm;;c~~a~+ whereas the biologically inactive phorbol ester, 4 f$,-phorbol-12,13 hidecanoate (4 &%PDD) failed to change the pattern of protein phosphorylation. These results show that proteins present in smooth muscle of rat aorta with molecular weights of 20 and 92.5 kilodaltons are substrates for protein kinase C. The phorbol ester 12-0-tetradecanoylphorbol-1Zacetate (TPA) causes smooth muscle contraction (l-3) and is mitogenic for smooth muscle cells (4). A current concept of the molecular mechanism of action of TPA is that the effects of phorbol esters are mediated by an activation of protein kinase C (5). Protein kinase C is present in rat aorta (6) and hence TPA may increase protein phosphorylation in aorta smooth muscles. In vitro some contractile constituents of smooth muscle such as myosin light chain -(7), myosin light chain kinase (8,9), vinculin (lo), filamin (10) and troponin (11) are substrates for purified protein kinase C. However, it is still unknown whether these proteins are phosphorylated by protein kinase C -in vivo and whether protein kinase C phosphorylates additional proteins in smooth muscle. Identification of protein substrates for protein kinase C in rat aorta might increase our understanding of the mechanism whereby phorbol esters cause smooth muscle contraction. The present results show that two proteins of apparent molecular weight of 20 and 92.5 kilodaltons are substrates for protein kinase C in extracts of rat aorta smooth muscle. Methods
Chemicals: Markers for the molecular weight determination of proteins were purchased from Bio-Rad (Richmond, CA) and Pharmacia (Piscataway, NJ). 1,2-Diolein, 4UPDD and TPA were purchased from Sigma Chemical Company (St. Louis, MO). The sources of other chemicals used in these experiments were given previously (12). __________x_‘__‘__________________’--___~__~_~~~~~~~~~~~~~_~~~~~~~~~~~~~~~~~~--The present addresses are: T. Nakaki, Department of Pharmacology, Keio University School of Medicine, Tokyo 160, Japan; B.C. Wise, Fidia Georgetown Institute for Neuroscience, Georgetown University, Washington, D.C. 20007. 0024-3025188
$3.00
+ .OO
1316
Protein
Phosphorylation
in
Rat Aorta
Vol.
42,
No.
13,
1988
Phosphorylation in a cell-free system: Male Sprague-Dawley rats (Zivic-Miller, 8-12 weeks old) were decapitated and a 3 cm-segment of thoracic aorta was excised and placed into a physiolo$cal saline solution. Fat and connective tissue were removed and the aorta segment was incubated for 1 hr at 37’C in a calcium-free Krebs Ringer bicarbonate buffer (118 mM NaCI, 4.7 mM KCI, 1.2 mM NaH PO 1.2 mM M&l , 25 11 mM glucose). A mixture of 95% 02 and 5 3o C ‘b’ was continu 2 usly mM NaHC03, bubbled through the buffer. Then the aorta was cut longitudinally ar?d placed on a petri dish with the tunica intima facing upward. The intima was removed by scraping with a razor blade and the remaining aorta was placed in homogenization buffer (250 mM sucrose, 0.2 mM EGTA, 2.5 mM MgC12, 50 mM 2-mercaptoethanol, 25 mM Tris-HCI, pH 7.5). In a typical experiment, five aortas were placed in 1.5 ml of the above buffer. The aortas were minced and homogenized with a motor-driven glass homogenizer immersed in an ice bath. The homogenate was centrifuged at 100 x g for 10 min at 4’C. The pellet was discarded and the resulting supernatant was centrifuged at 100,000 x g for 60 min at 4’C. The supernatant was used for the phosphorylation study, which was carried out in a reaction mixture of 100~1 containing 25 m&I Tris-HCI, pH 7.5, 10 mM MgC12, 0.2 mM EGTA, 6Oyg of soluble proteins, 5yM’)j/P ATP (10 uCi/assay), and when used various concentrations of CaC12 or 20 pg phosphatidylserine. The reaction mixtures were incubated at 30°C for various time periods and the reactions were terminated by addition of 50~11 of sodium dodecyl sulfate (SDS)-stop solution (9% SDS, 30% glycerol, 0.03% bromophenol blue and 125 mM Tris-HCI, pH 6.8) and placed in a boiling water bath for 2 min. The samples were prepared for SDS-polyacrylamide gel electrophoresis and the gels were stained, destained, dried and subjected to autoradiography as described previously (12). Autoradiograms were scanned with a densitometer using visible light. Results Preincubation of aorta with calcium-free Krebs-Ringer bicarbonate buffer was necessary to reduce the high basal level of protein phosphorylation in order to improve the detection of protein phosphorylation elicited by exogenously added calcium (data not shown). At the concentration used (Fig. 1) the addition of Ca 2+ (lane 2) or phosphatidylserine (lane 4) to the cell-free phosphorylation system had no substantial effect on the protein phosphorylation pattern. However, thF+a.ddition of calmodulin (lane 3) or phosphatidylserine (lane 5) in the presence of Ca increased greatly the phosphorylation of proteins with apparent molecular weights of 20 and 92.5 kilodaltons (Fig. 1). It is unknown whether these 20 or 92.5 kilodalton protein bands consist of a The addition of CAMP increased the homogeneous population of protein molecules. phosphorylation of 22, 45 and 110 kilodalton protein bands and the addition of cCMP increased the phosphate incorporation into a 45 kilodalton protein band (data not shown). These effects of cyclic nucleotides were similar to those reported by Rapoport et al. (13). The addition of 0.2 mM Ca2+ In ’ the presence of 0.2 mM EGTA (Fi% 2, lane 2) had no effect on protein phosphorylation. However, addition of I mM Ca increased the phosphorylation of several protein bands (lane 4). These bands include proteins with apparent molecular weights of 16, 20, 3&45, 48, 60 and 92.5 kilodaltons. The addition of phosphatidylserine together with Ca (line 5) resulted in a further increase in the with molecular weights of 16, 20 and 92.5 kilodaltons. In phosphorylation of2yroteins the absence of Ca , phosphatidylserine did not stimulate protein phosphorylation (data not shown). PDD and 1,2-diolein on the phosphorylation of aortic proteins The effects of TPA, 4 are shown in Fig. 3. TPA and 1,2-diolein activate protein kinase C (5,14), whereas 4w TPA (trace 4) andl,2-diolein (trace 3), PDD, a structural analog of TPA, is inactive. but not C&PDD (trace 2), increased significantly the phosphorylation of proteins with apparent molecular weights of 20 and 92.5 kilodaltons.
vol.
42, No.
13,
Protein
1988
Phosphorylation
in Rat Aorta
1317
Discussion The diversity of cellular response mediated by protein kinase C may be explained Several proteins have been found to be by the variety of substrates in a given cell. substrates for protein kinase C in smooth muscles of rat vas deferens (15). However, none of them has a molecular weight similar to those found to be phosphorylated in the smooth muscles of rat aorta. In a cell free preparation of rat aorta and at low concentrations of Ca*+, proteins ‘t$ apparent molecular weights of 20 and 92.5 kilodaltons were phosphorylated in a c”LI + and phosphatidylserine dependent-manner and therefore, are substrates for protein kinase C. TPA or 1,2-diolein which activated protein kinase C (5) also increas?: At higher Ca the phosphorylation of the 20 and 92.5 kilodalton protein bands. co y+entrations, an additional protein band of 16 kilodaltons was phosphorylated in a Ca Although we have not clarified the and phosphatidylserine-dependent manner. molecular characteristics of the 20 and 92.5 kilodalton proteins, it is tempting to speculate that they are myosin light chain and phosphorylase b which have molecular
20I
1234
5
Fig. 1 Autoradiogram showing protein phosphorylation stimulated by Ca*+, calmodulin and phosphatidylserine in the cell free preparation of rat aorta. The 100,000 x g supernatant of rat aorta homogenate (6Opg protein) was incubated for 20 seconds in the presence of 0.2 mM EGTA plus various agents as described in methods. Proteins were separated on a 10% polyacrylamis& and 0.1% SDSfiel. Each lane shows: 1, control; 2, 0.5 mM Ca ; 3, 0.5 mM Ca plus lSyg/ml calmodulin; 4, 200 )Ig/ml ph2;phatidylserine; 5, 200 yg/ml phosphatidylserine plus 0.5 mM Ca .
Protein Phosphorylation in Rat Aorta
1318
Vol. 42, No. 13, 1988
weights of 20 and 92 kilodaltons respectively. The proteins pith apparent molecular weights of 20 and 92.5 kilodaltons are also substrates for Ca +/calmodulin-dependent kinase (Fig. 1). However, it iq+unknown whether the same site in these proteins are -dep+endent enzymes. Since myosin light chain is well phosphorylated by these two Ca known to be a substrate for the Ca /calmodulin dependent myosin light chain kinase (71, one may further suggest that the 20-kilodalton protein detected in the present study is the myosin light chain. Other proteins Ca2+/camodulin-dependent
may
also kinase
be the substrates in the rat aorta
92.5
for extracts.
protein For
kinase example,
92K
66.2
X
i
21.5
14.4
Fig. 2 (6) showing protein 5+scans (A) and densitometri Autoradiograms by Ca and phosphatidylserine. stimulated phosphorylation conditions and processing were the same as Jhose Phosphorylation described i?+Fig. 1. Each trace shows: 1, control; 3 0.2 mM CaL’;24 plus phosphatidyl serine; 4, I mM Ca +; 5, 1 mM Ca 0.2 mM Ca plus phosphatidylserine. Calculation “f the peak area of the 20 kilo&alton protein revealed: 1 mM Ca + alone, 1,400+290*; 1 mM plus phosphatidylserine, 2,300+380**, when control was Ca normalized to 100 in each e eriment-(n=3). *P
C
and the
Vol.
42,
No.
13,
1988
Protein
Phosphorylation
in
Rat
Aorta
1319
phosphorylation of two other proteins with appare$ molecular weignts of 64 and 39 plus phosphatidylserine or Ca kilodaltons was modestly increased by 0.5 mM Ca plus calmodulin (Fig. 1 lanes 5 and 3). The nature of these two proteins was not further characterized. It should
be stressed that in out study the aorta segment was preincubated for one in a calcium-free Krebs Ringer bicarbonate buffer before being used to prepare the cell free system for phosphorylation study. This preincubation was found to The reason for this improvement markedly reduce the level of basal phosphorylation. may be related to the fact that the concentration of extracellular calcium is about one
hr at
37OC
Fig. 3 Densitometric scan of an autoradiogram showing protein phosphorylation stimulated by addition of 33 nM TPA and lOpg/ml 1,2-diolein but not by the addition of 33 nM 4 o( PDD. Phosphorylation conditions and processing were the same as those described in Fig. 1 except that 0.2 mM CaCl and 200 ug/ml phosphatidylserine were used. Each trace shows: 1, control; 2, 4 PDD; 3, 1,2-diolein; 4, TPA. Control condition included 5% ethanol, which was used as the vehicle for TPA, 1,2-diolein and 40( PDD. The left panel (A) shows overall scan of the gel. The right panel (B) shows a magnification of the scan of high molecular weight proteins. Calculation of the peak area of the 20 kilodalton protein revealed the following percentage of control. 1,2-diolein, 174; TPA, 158; 40(PDD, 100. The experiment was repeated three times with similar results.
1320
Protein Phosphorylation in Rat Aorta
Vol. 42, No. 13, 1988
thousand times higher than that of the cation present intracellularly. Thus, preincubation of the aorta in calcium-free buffer would deplete most, if not all of the calcium trapped in the outer surface of the plasma membrane, thereby reducing the high level of basal protein phosphorylation. It is also conceivable that following removal of aorta from the rat, there may have been substantial cell death or injury resulting from the anoxic conditions. An influx of extracellular or release of intracellular calcium may also contribute to the high basal level of protein phosphorylation. Preincubation is, thus, required to restore normal cellular calcium (and other ionic) levels in surviving smooth muscle cells. We have suggested that 1,2-diacylglycerol, a phosphoinositide metabolite formed by phospholipase C, might play an important role in mediating the tonic component of the rat aorta contraction elicited by serotonin (5HT) (3). In fact 5HT elicits smooth muscle contraction (16) and phosphoinositide hydrolysis (17) through activation of pharmacologically identified 5HT2 receptors in rat aorta (18). Moreover 2-nitro-4carboxyphenyl-N,N-diphenylcarbamate, an inhibitor of polyphosphoinositide-specific phospholipase C, abolished both the SHT-stimulated phospholipse C activity and the Furtherrnore, the protein kinase C 5HT-induced tonic phase of contraction (3). activator, TPA, induces the tonic contraction of rat aorta (2,3). In the present study we show that TPA and I,2 diacylglycerol increase the phosphorylation of protein bands with apparent molecular weights of 20 and 92.5 We suggest that the phosphorylation of these two aortic proteins could be kilodaltons. important in mediating the action of vasoactive substances such as angiotensin II, norepinephrine and 5HT which have been shown to activate phosphoinositide hydrolysis in rat aorta (17,19,20). Furthermore, these proteins may possibly be involved in the mitogenic activity of TPA on smooth muscle cells (4). References
I. 2. 3. 4. 5. 6.
7. 8. 9. IO. 11. 12. 13. 14. 15.
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