European Journal of Pharmacolo~,~, - Molecular Pharmacology. Section. 226 ( 992~.225-231
~-3"~-
'.~'.1992 Elsevier Science Puhlishers B.V. All rights reserved t)922-4106/92/$05.(!0
EJPMOL 90319
Protein kinase C phosphorylates a 15 kDa protein but not phospholamban in intact rat cardiac myocytes Matthias H a r t m a n n J and Jiirgen Schrader Department of Physiolo~% Unirersity ~f DiisseldorJ: 4000 DiisseMorf I, Germany
Received 16 October 1991.revised MS received 5 February 1992.accepted 17 Marcia 1992
In the present study the effects of the protein kinase C activator 12-O-tetradecanoylphorbo[ 13-acetate (TPA) as well as the a- and /3-adrenoceptor agonists methoxaminc and isoproterenol on protein phosphorylation of intact rat cardiac myocytes were invcstigatcd. TPA, isoproterenol and methoxamine were shown to stimulate phosphorylation of a 15 kDa protein. ECs~~for TPA and isoprotercnol were 4 x 10 -~ M and 5 x 10 -'~ M respectively. The time course of phesphorylation by TPA and isoproterenol greatly differcd, revealing a maximal phosphorylation (2.9-fold) after 10 min and 1 min respectively. Cell fractionation showed a significant enrichment of the 15 kDa protein in a crude membrane preparation. While the 15 kDa protein was the only phosphoprotcin stimulated by TPA and methoxamine, isoproterenol additionally enhanced the 32p i incorporation into four proteins corresponding to 6 kDa (phospholamban), 28 kDa, 97 kDa and 140 kDa. Furthermore, dephosphorylation of a 21 kDa substrate upon /9-adrenoceptor stimulation was observed. Phospholamban phosphorylation was effectively (max. 9.1-1bid) stimulated by isoproterenol (ECso of 5 × 10- '~ M), reaching a maximal phosphorylation state within l rain. The present study clearly demonstrates: (1) TPA stimulates the phosphorylation of a membrane-localized 15 kDa protein and this effect can be mimicked by both isoproterenol and methoxamine; (2) TPA, in contrast to isoproterenol, does not change the phosphorylation state of phosphoiamban. Whilst phospholamban under in vitro conditions is known to be a substrate for protein kinase C, it does not appear to be accessible for the enzyme in intact cardiac myocytes.
Protein kinase C; Heart; 15 kDa protein; Phospholamban
1. Introduction Protein phosphorylation has been supposed to be involved in both a- and /3-adrenergic regulation of cardiac contractility by modulation of ion transport mechanisms localized in sarcolemma and sarcoplasmic reticulum (Sperelakis and Wahler, 1988; Movsesian et al., 1984). A sarcolemmal 15 kDa protein has been demonstrated to be phosphorylated upon c~- and /3adrenoceptor stimulation in intact myocardium which was accompanied by analogous changes in mechanical performance (Lindemann, 1986; Presti et al., 1985a). Furthermore, the 15 kDa protein is the only sarcolemmal substrate of protein kinase C, as could be demon-
Correspondence to: Prof. J. Schrader, Physiologisches lnstitut I, Medizinische Einrichtungen der Universitfit Diisseldorf, Moorenstrasse 5, D-4000 Diisseldorf 1, Germany. ~ Present address: Department of Anaesthesiology, Universityof Diissetdorf.
strated in in vitro phosphorylation experiments using highly purified sarcolemmal vesicles (Presti et al., 1985b). Sequence determination of the 15 kDa protein demonstrated multiple separate phosphorylation sites for cAMP-dependent protein kinases and kinase C (Palmer et al., 1991). Furthermore, the latter study revealed a discrepancy between electrophoretic mobility (15 kDa) and the calculated molecular weight (M r = 8409). In vascular smooth muscle a 16 kDa protein, possibly identical with the cardiac 15 kDa protein, has been shown to be phosphorylated by protein kinase C and cAMP-dependent protein kinase (BoulangerSaunier and Stoclet, 1987). The regulator3' protein of Ca2+-uptake into the sarcoplasmic reticulum, phospholamban (Movsesian et al., 1984; Simmerman et al., 1986; Kovacs et al., 1988), consists of five identical 5 - 6 kDa subunits and is effectively phosphorytated upon fl-adrenergic stimulation under in vivo conditions (Le Peuch et al., 198(i; Kranias and Solaro, 1982; Lindemann et al., 1983; Iwasa and Hosey, 1983; Lindemann and Watanabe,
226 1985). In vitro studies furthermore demonstrated that phospholamban serves as a substratc for cAMP-, cGMP- and Ca=+/calmodulin-dependent protein kJnases and kinase C (Tada et al., 1975; Le Pcuch et al., 1979; lwasa and Hosey, 1984; Movsesian et al.. 1984; Raeymaekers et al., 19881. Furthermore, in vitro phosphorylation of phospholamban by protein kinase C was shown to be associated with a stimulation of Ca 2+ uptake by cardiac sarcoplasmic reticulum (Movsesian et al., 1984). The aim of the present study was to investigate the role of kinase C in the phosphory!ation of the sarco!emmal 15 kDa protcin and of phospholamban. For this reason, protein kinasc C of intact 3-'P,-labelled cardiac myoeytes was directly stimulated using the wcll-known activator 12-O-tetradccanoylphorbol 13acetate (TPA) (Castagna et al., 1982) and the resulting phosphor3'lation pattern was compared with that of aand /3-adrcncrgic stimulation.
2. Materials and methods
Zl. Isolation ofcerdiuc myo(ytes Hearts from rats (2511-35(I g)wcrc perfused according to the Langendorff technique with a constant flow of 10 ml/min. The perfusion medium was a Ca-'~-free, high potassium buffer (I) consisting of (raM): NaCI 112.6, KCI 15, KH2PO 4 1.2, MgSO 4 1.2, HEPES 111, glucose 5.5 (pH 7.4; gassed with 10{1~;: O,). After 10 rain of non-recirculating pcrfusion, hearts were perfused in a recirculating manner for an additional 50 rain with 2(1 ml of the same buffer containing 0.1% collagenase and 0.5% albumin. Thereafter, tissue was chopped (Mcllwain, Bachofer) and incubated again for 3(1 rain in a shaking water bath (37°C) in 10 ml of the recirculation buffer. Calcium was continuously administered up to a final concentration of 1 mM (15 rain). Disaggregation of tissue was enhanced by gentle pipetting. Non-disaggregated tissue was removed by sieving through a nylon mesh (2t10 t~m). 1~ remove defect cells and endothelium, the suspension was layered on 10 ml of buffer I1 consisting of (raM): NaCI 125, KCI 2.6, MgSO 4 1.2, HEPES 10, glucose 5.5, CaC12 1, supplemerited with 4% albumin and centrifuged for 75 s at 4(10 rpm (Minifuge, Haereus). Pellet was resuspended in buffer I1 and the centrifugation step was repeated twice under identical conditions. For removal of albumin, the myocyte pellet was layered on buffer 11 (without albumin) and the pellet was resuspended in 1 ml buffer II supplemented with adenosine deaminase (1 U/ml). Contamination of the cardiac myocyte preparation with endothelial cells was excluded by phase contrast microscopy; 70-80% of cardiac my-
ocytes were intact as determined by the rod shape criterion. ZZ Protein phosphorylation experiments Cardiac myocytes were incubated with 200/zCi 32p~ for 60 min and then diluted to a final volume of 8 ml with buffer II. Substances to be te~ted were added to 30{1 p.I aliquots for the indicated time intervals. Reaction was terminated by addition of 150 /xl sodium dodecyl sulfate (SDS)/EDTA-containing buffer consisting of (mM); Tris 30, EDTA 15. SDS 9%, glycerol 15%. Then, 50 ~1 mercaptocthanol (25%) and 10 /~1 bromophenolblue (1 mg/m[) were added. Aliquots containing 50 p.g protein as determined by the method of Bradtord were subjected to electrophoresis. In part, samples were boiled fur 5 ~l~i~l as indicated.
2.3. SDS gel electrophoresis Sampic~ were subjected to 5-15% and 10-20% linear gradient Laemmli gels (Laemmli, 1970). After running the gel, staining (Coomassie R 250 0.125%, methanol 50%, acetic acid 10%), destaining (firstly with methanol 5(t%, acetic acid 10% and secondly with methanol 5%, acetic acid 7%) and equilibration to prevent cracking (methanol 40%, glycerol 5%), gels were dried at 40°C under vacuum. Phosphoproteins were detected using Kodak X-Omat AR film, scanned by use of a laser densitometer (Ultrascan I, LKB, Sweden) and statistically evaluated using the t-test for paired samples. Results are expressed in optical density (OD) and changes in optical density (,.1oo) respectively. The threshold concentration was defined as the minimal concentration capable to significantly enhance protein phosphorylation. Linearity between radioactivity and absorbance was shown in control experiments.
Z4. Cellfractionation Membrane preparation was performed as described (Manatan et al., 1986). In brief, 3ZP~-Iabelled myocytes, pelleted by centrifugation to remove the bulk of radioactivity, were homogenized, (11 with a Branson sonitier (30 bursts; 50% power) and (2) with a pestle homogenizer (ten strokes) in buffer III consisting of (raM): sucrose 250, histidine 30, E D T A 10, disodium hydrogen phosphate 5t) and NaF 25 (pH 7.4). The homogenate was centrifuged at 11,60(/x g for 20 min, the supernatant was then centrifuged at 15,000xg for the same time period. The resulting supernatant was centrifuged at 43,600 x g for 30 min, the pelleted membranes were resuspended in buffer IV (200 Izl) consisting of (raM): KCI 600, histidine 30, EDTA 10, disodium hydrogen phosphate 50 and NaF 25 (pH 7.4).
227 I 8" --
1,5 -
o
1&-
28 kDa 14Q kDa k
08- 6koo 2~kDo
p l
~
a
:
a
a
C
,-----0
TPA
M
ISO
GEL LENGTH {cml
Fig. I. Superimposed densilometrie scans of autoradiograms obtained from intact cell phosphoD, lation experiments in the pre~ence and absence of isoprolerenol. Black areas show increases ill optical density caused by isoproterenol ( 1 0 ~' M): decreases are demonstrafed by hatchings. Time of stimulation with isoprolerenol was 2 min. Samples (50/.tg p r o t e i n / l a n e ) , boiled before run, were resoh'ed by use of a 5 - 1 5 ( ; linear g r a d m n l Laemmtl gel. Note timt the protein c o r r e s p o n d i n g to 21 kDa is d e p h o s p h o w l a t e d upon slimulalion.
Fig. 2. A u l o r a d i o g r a m demonstrating ttte influence of TPA ( ll} ~ M. l(} rain), methoxamine (10 4 M, 5 rain) and isoprotereno] (t;) " M. 2 nain~ on prolein phosphor3'talion of intacl cardiac myocyles resolved I~.~,a 10-20r.~ linear gradient gel. The figure shmss lhe rcgi~m of interest corresponding to Ihe molecular weights ol the 15 kl)a p~,}tcin a n d ph{:'.,pholaraban {PL I ). Sample:; (50 A*g proWin "!me~ were hoiled before electrophoresi~,. (7. conirol: M. mcth*}xaminc: ISO. isoprolerenol: PL 1 , tm~ molecular stale ~f phospholmnban.
The final pellet was resuspended in buffer II1 (200/a.I) and 100/zl S D S / E D T A buffer, 50 #I mercaptocthanol (25%) and 10 /zl bromophenolblue (1%) were added,
All steps were performed on ice. Aliquots containing 20 g g protein were subjected to electrophoresis. In part, samples were bolted for 5 rain as indicated.
Q25
*
.
*
.
13_ O O
--
0
w
0
z
~-3"~-
'.~'.1992 Elsevier Science Puhlishers B.V. All rights reserved t)922-4106/92/$05.(!0
EJPMOL 90319
Protein kinase C phosphorylates a 15 kDa protein but not phospholamban in intact rat cardiac myocytes Matthias H a r t m a n n J and Jiirgen Schrader Department of Physiolo~% Unirersity ~f DiisseldorJ: 4000 DiisseMorf I, Germany
Received 16 October 1991.revised MS received 5 February 1992.accepted 17 Marcia 1992
In the present study the effects of the protein kinase C activator 12-O-tetradecanoylphorbo[ 13-acetate (TPA) as well as the a- and /3-adrenoceptor agonists methoxaminc and isoproterenol on protein phosphorylation of intact rat cardiac myocytes were invcstigatcd. TPA, isoproterenol and methoxamine were shown to stimulate phosphorylation of a 15 kDa protein. ECs~~for TPA and isoprotercnol were 4 x 10 -~ M and 5 x 10 -'~ M respectively. The time course of phesphorylation by TPA and isoproterenol greatly differcd, revealing a maximal phosphorylation (2.9-fold) after 10 min and 1 min respectively. Cell fractionation showed a significant enrichment of the 15 kDa protein in a crude membrane preparation. While the 15 kDa protein was the only phosphoprotcin stimulated by TPA and methoxamine, isoproterenol additionally enhanced the 32p i incorporation into four proteins corresponding to 6 kDa (phospholamban), 28 kDa, 97 kDa and 140 kDa. Furthermore, dephosphorylation of a 21 kDa substrate upon /9-adrenoceptor stimulation was observed. Phospholamban phosphorylation was effectively (max. 9.1-1bid) stimulated by isoproterenol (ECso of 5 × 10- '~ M), reaching a maximal phosphorylation state within l rain. The present study clearly demonstrates: (1) TPA stimulates the phosphorylation of a membrane-localized 15 kDa protein and this effect can be mimicked by both isoproterenol and methoxamine; (2) TPA, in contrast to isoproterenol, does not change the phosphorylation state of phosphoiamban. Whilst phospholamban under in vitro conditions is known to be a substrate for protein kinase C, it does not appear to be accessible for the enzyme in intact cardiac myocytes.
Protein kinase C; Heart; 15 kDa protein; Phospholamban
1. Introduction Protein phosphorylation has been supposed to be involved in both a- and /3-adrenergic regulation of cardiac contractility by modulation of ion transport mechanisms localized in sarcolemma and sarcoplasmic reticulum (Sperelakis and Wahler, 1988; Movsesian et al., 1984). A sarcolemmal 15 kDa protein has been demonstrated to be phosphorylated upon c~- and /3adrenoceptor stimulation in intact myocardium which was accompanied by analogous changes in mechanical performance (Lindemann, 1986; Presti et al., 1985a). Furthermore, the 15 kDa protein is the only sarcolemmal substrate of protein kinase C, as could be demon-
Correspondence to: Prof. J. Schrader, Physiologisches lnstitut I, Medizinische Einrichtungen der Universitfit Diisseldorf, Moorenstrasse 5, D-4000 Diisseldorf 1, Germany. ~ Present address: Department of Anaesthesiology, Universityof Diissetdorf.
strated in in vitro phosphorylation experiments using highly purified sarcolemmal vesicles (Presti et al., 1985b). Sequence determination of the 15 kDa protein demonstrated multiple separate phosphorylation sites for cAMP-dependent protein kinases and kinase C (Palmer et al., 1991). Furthermore, the latter study revealed a discrepancy between electrophoretic mobility (15 kDa) and the calculated molecular weight (M r = 8409). In vascular smooth muscle a 16 kDa protein, possibly identical with the cardiac 15 kDa protein, has been shown to be phosphorylated by protein kinase C and cAMP-dependent protein kinase (BoulangerSaunier and Stoclet, 1987). The regulator3' protein of Ca2+-uptake into the sarcoplasmic reticulum, phospholamban (Movsesian et al., 1984; Simmerman et al., 1986; Kovacs et al., 1988), consists of five identical 5 - 6 kDa subunits and is effectively phosphorytated upon fl-adrenergic stimulation under in vivo conditions (Le Peuch et al., 198(i; Kranias and Solaro, 1982; Lindemann et al., 1983; Iwasa and Hosey, 1983; Lindemann and Watanabe,
226 1985). In vitro studies furthermore demonstrated that phospholamban serves as a substratc for cAMP-, cGMP- and Ca=+/calmodulin-dependent protein kJnases and kinase C (Tada et al., 1975; Le Pcuch et al., 1979; lwasa and Hosey, 1984; Movsesian et al.. 1984; Raeymaekers et al., 19881. Furthermore, in vitro phosphorylation of phospholamban by protein kinase C was shown to be associated with a stimulation of Ca 2+ uptake by cardiac sarcoplasmic reticulum (Movsesian et al., 1984). The aim of the present study was to investigate the role of kinase C in the phosphory!ation of the sarco!emmal 15 kDa protcin and of phospholamban. For this reason, protein kinasc C of intact 3-'P,-labelled cardiac myoeytes was directly stimulated using the wcll-known activator 12-O-tetradccanoylphorbol 13acetate (TPA) (Castagna et al., 1982) and the resulting phosphor3'lation pattern was compared with that of aand /3-adrcncrgic stimulation.
2. Materials and methods
Zl. Isolation ofcerdiuc myo(ytes Hearts from rats (2511-35(I g)wcrc perfused according to the Langendorff technique with a constant flow of 10 ml/min. The perfusion medium was a Ca-'~-free, high potassium buffer (I) consisting of (raM): NaCI 112.6, KCI 15, KH2PO 4 1.2, MgSO 4 1.2, HEPES 111, glucose 5.5 (pH 7.4; gassed with 10{1~;: O,). After 10 rain of non-recirculating pcrfusion, hearts were perfused in a recirculating manner for an additional 50 rain with 2(1 ml of the same buffer containing 0.1% collagenase and 0.5% albumin. Thereafter, tissue was chopped (Mcllwain, Bachofer) and incubated again for 3(1 rain in a shaking water bath (37°C) in 10 ml of the recirculation buffer. Calcium was continuously administered up to a final concentration of 1 mM (15 rain). Disaggregation of tissue was enhanced by gentle pipetting. Non-disaggregated tissue was removed by sieving through a nylon mesh (2t10 t~m). 1~ remove defect cells and endothelium, the suspension was layered on 10 ml of buffer I1 consisting of (raM): NaCI 125, KCI 2.6, MgSO 4 1.2, HEPES 10, glucose 5.5, CaC12 1, supplemerited with 4% albumin and centrifuged for 75 s at 4(10 rpm (Minifuge, Haereus). Pellet was resuspended in buffer I1 and the centrifugation step was repeated twice under identical conditions. For removal of albumin, the myocyte pellet was layered on buffer 11 (without albumin) and the pellet was resuspended in 1 ml buffer II supplemented with adenosine deaminase (1 U/ml). Contamination of the cardiac myocyte preparation with endothelial cells was excluded by phase contrast microscopy; 70-80% of cardiac my-
ocytes were intact as determined by the rod shape criterion. ZZ Protein phosphorylation experiments Cardiac myocytes were incubated with 200/zCi 32p~ for 60 min and then diluted to a final volume of 8 ml with buffer II. Substances to be te~ted were added to 30{1 p.I aliquots for the indicated time intervals. Reaction was terminated by addition of 150 /xl sodium dodecyl sulfate (SDS)/EDTA-containing buffer consisting of (mM); Tris 30, EDTA 15. SDS 9%, glycerol 15%. Then, 50 ~1 mercaptocthanol (25%) and 10 /~1 bromophenolblue (1 mg/m[) were added. Aliquots containing 50 p.g protein as determined by the method of Bradtord were subjected to electrophoresis. In part, samples were boiled fur 5 ~l~i~l as indicated.
2.3. SDS gel electrophoresis Sampic~ were subjected to 5-15% and 10-20% linear gradient Laemmli gels (Laemmli, 1970). After running the gel, staining (Coomassie R 250 0.125%, methanol 50%, acetic acid 10%), destaining (firstly with methanol 5(t%, acetic acid 10% and secondly with methanol 5%, acetic acid 7%) and equilibration to prevent cracking (methanol 40%, glycerol 5%), gels were dried at 40°C under vacuum. Phosphoproteins were detected using Kodak X-Omat AR film, scanned by use of a laser densitometer (Ultrascan I, LKB, Sweden) and statistically evaluated using the t-test for paired samples. Results are expressed in optical density (OD) and changes in optical density (,.1oo) respectively. The threshold concentration was defined as the minimal concentration capable to significantly enhance protein phosphorylation. Linearity between radioactivity and absorbance was shown in control experiments.
Z4. Cellfractionation Membrane preparation was performed as described (Manatan et al., 1986). In brief, 3ZP~-Iabelled myocytes, pelleted by centrifugation to remove the bulk of radioactivity, were homogenized, (11 with a Branson sonitier (30 bursts; 50% power) and (2) with a pestle homogenizer (ten strokes) in buffer III consisting of (raM): sucrose 250, histidine 30, E D T A 10, disodium hydrogen phosphate 5t) and NaF 25 (pH 7.4). The homogenate was centrifuged at 11,60(/x g for 20 min, the supernatant was then centrifuged at 15,000xg for the same time period. The resulting supernatant was centrifuged at 43,600 x g for 30 min, the pelleted membranes were resuspended in buffer IV (200 Izl) consisting of (raM): KCI 600, histidine 30, EDTA 10, disodium hydrogen phosphate 50 and NaF 25 (pH 7.4).
227 I 8" --
1,5 -
o
1&-
28 kDa 14Q kDa k
08- 6koo 2~kDo
p l
~
a
:
a
a
C
,-----0
TPA
M
ISO
GEL LENGTH {cml
Fig. I. Superimposed densilometrie scans of autoradiograms obtained from intact cell phosphoD, lation experiments in the pre~ence and absence of isoprolerenol. Black areas show increases ill optical density caused by isoproterenol ( 1 0 ~' M): decreases are demonstrafed by hatchings. Time of stimulation with isoprolerenol was 2 min. Samples (50/.tg p r o t e i n / l a n e ) , boiled before run, were resoh'ed by use of a 5 - 1 5 ( ; linear g r a d m n l Laemmtl gel. Note timt the protein c o r r e s p o n d i n g to 21 kDa is d e p h o s p h o w l a t e d upon slimulalion.
Fig. 2. A u l o r a d i o g r a m demonstrating ttte influence of TPA ( ll} ~ M. l(} rain), methoxamine (10 4 M, 5 rain) and isoprotereno] (t;) " M. 2 nain~ on prolein phosphor3'talion of intacl cardiac myocyles resolved I~.~,a 10-20r.~ linear gradient gel. The figure shmss lhe rcgi~m of interest corresponding to Ihe molecular weights ol the 15 kl)a p~,}tcin a n d ph{:'.,pholaraban {PL I ). Sample:; (50 A*g proWin "!me~ were hoiled before electrophoresi~,. (7. conirol: M. mcth*}xaminc: ISO. isoprolerenol: PL 1 , tm~ molecular stale ~f phospholmnban.
The final pellet was resuspended in buffer II1 (200/a.I) and 100/zl S D S / E D T A buffer, 50 #I mercaptocthanol (25%) and 10 /zl bromophenolblue (1%) were added,
All steps were performed on ice. Aliquots containing 20 g g protein were subjected to electrophoresis. In part, samples were bolted for 5 rain as indicated.
Q25
*
.
*
.
13_ O O
--
0
w
0
z
