Vol. 169, No. 3, 1990 June 29, 1990
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1185-1190
INTRACELLULAR PROTEIN PHOSPHORYLATION IN OAT ( Avena sativa BYPHY!IWHROMEACTION:INVOLVEMENT OF PROTEIN KINASE C Moon-Hwan ParkandQuae *Department
of Biochemistry,
L. ) PRCYTOPLASTS
ChaGl
Chungbuk National University, Republic of Korea
Cheongju 360-763
Received May 21, 1990 SUMMARY: Phosphorylations of two proteins ( 27 K&,32 KDa) in oat cells were dependent on phytochrome action. To determine which kinase system(s) for the phosphorylation of these two proteins are controlled by the phytochrome, involvement of the Ca2+/DG dependent protein kinase (protein kinase C)wasfirst investigated. When a protein kinase C inhibitor (1-(S-isoguinoline sulfonyl)2-methylpiperazine :H-7 ) or the inositol phospholipid metabolic blocker Li+ was added into the cell suspension , respectively, the phosphorylations of these two proteins were substantially reduced. On the other hand,an addition of l-oleoyl-2-acetyl-sn-glycerol (OAG :activator of protein kinase C )orphorbol 12-myristate 13-acetate (TPA :tumor promoting phorbolester Ienhanced the phosphorylations of these proteins. These results suggest that phytochrome action is certainly connected with the protein phosphorylation via the activation of protein kinase C or a similar molecule with protein kinase C. 0 1990
Academic
Press,
Inc.
It was reported that the physiologically active form of phytochrome (Pfr) might initiate the photomorphogenic cell responses by controlling the cytosolic Ca*+ concentration (1,2,11,14). The regulatory role of protein phosphorylation by the calcium dependent protein kinase was also suggested as an important step in the signal transduction in plant systems (3-7). Amolecule similar to protein kinase C (PKC )has been described in plant cells (8),and phosphorylation of proteins changes the activities of various enzymes in plant cells (9). Although protein phosphorylation in plants by PKC has not been dwnstrated yet, it has been reported that phosphorylation by other kinase systems (e.g.,Ca2+/caln-odulin dependent protein kinase ) affectsnumerous developmental processes (10). Protein phosphorylation appears to be a central regulatory mechanism in the signal transduction in both animals and plants, but it is not yet clear whether it interfaces with PI turnover in 1 To whom correspondence should be addressed. Abbreviations : H-7, 1-(5-isoguinoline sulfonyl)-2-methyl piperazine phorbol 12-myristate 13-acetate ; OAG, l-oleoyl-2-acetyl-sn-glycerol; sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
; TPA, SDS-PAGE,
0006-291X/90 $1.50 1185
Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol.
169, No. 3, 1990
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
plants (10). We had reported that the phosphorylations of two proteins (27 KDa, 32 KDa) in oat cells were dependent on the photoreversible behavior of phytochrome (11). However, it has not been determined which kinase system(s) for the phosphorylation of these proteins is controlled by the phytoChrome action. The data obtained in this study suggested the involvement of PKC or a similar molecule in the phosphorylation of two proteins by the phytochrome signal. MATF,RIALS AND METHODS Material5 Cellulase and pectinas ped for the prepar@on of protoplasts were the products of Yakult Honsa. 7 P3orthophosphate ( P:l mCi/ml) was purchased from Amersham, and the rest of the chemicals used in these experiments were obtained from Sigma Chemical Cwy.Cat seed (Avena sativa L.CV Garry)was purchased from Stanford Seed Co., Buffalo, NY, UCoat seedlings Fifty o-rams of oat seed was soaked at 4°C for 24 hours in darkness. The soaked ~2 was spread on wet vermiculite (100 g/tray) in aluminum trays (35x 45 cm), and the trays wlere putinanoat-growing boxmade of-. The oats were grown at 25°C for 5 days in complete darkness. Preparation of protoplasts The preparation of protoplasts was mainly carried out as previously described (12,13). After 5 days,the tips (2 cm)of the coleoptiles were harvested. Harvesting and further handling were performed under a dim green safe light. The washed oat tissue was cut into 1 mn pieces,which were thenplaced in the enzyme solution ( 0.6 M sorbitol, 1 m CaCl , 8.05 % cellulase, 0.5 % macerozyme, pH 6.5 ). After allawing the mixture tz react at 25°C for 7.5hours with gentle shaking (circular motion) , it was centrifuged at 200xg for 5 minutes and then filtered through a nylon mesh ( 100 urn). The enzyme solution was then removed by washing with the following buffer solution ( 0.6 M sorbitol, 0.05 8 BSA, 1 &I CaC12, pH 6.5). After washing, the protoplasts were centrifuged at 1600 rpm for 10 minutes to remove the washing buffer. Sucrose density gradient centrifugations of the pelleted protoplasts were carried out at 1600 rpin for 8 minutes by using a stepwise gradient consisting of buffer I (0.6 M sucrose, 1 nM CaCl , pH 6.5), buffer II (0.S M sucrose, 0.1 M sorbitol, 5 nM Mes, 1 IIN CaC12, pH 25 ),and buffer IlI (0.6 M sorbitol, 5 n@!Mes, 1 mM CaC12, pH 6.5). The protoplasts banded between buffer It andbuffer III. In vivo protein phosphorylation Purified protoplasts wzre suspended in the 32Pi-loadina buffer ( 0.6 M sorbitol, 253s~Mkes, i0 r&l NaIiC03, 2-m KH PO , 25 nW Hepes, i IIN CaCl‘, pH 5.8 ), and then Pi (0.5 Ki) was added to de .&spension. LiCl (50 IT&J)2 or H-7 ( 50 JIM) was added to the suspension under different experimental conditions as described inthe sumnary. The suspension was incubated at 4°C for 2 hours, gentle shaking was carried out evq 10 minutes during the incubation. At the end o 52 the incubation, the excess Piinthe mediumwas removedby washing with Pi-loading buffer ;and then the protoplasts were irra-vted with red light or stimulated by OAG (100 ug/medium 1 ml) or TPA (10 M) (16,17). Irradiation of light and SDS-PAGE 32 Protoplasts incorporated with Pi were irradiated with red light ( 660 run: the monochromatic light was generated by a 150 watt Xe lamp equipped in the spectrofluorometer, Hitachi MPF 3000), and the effect of the light was observed as a function of irradiation time. Reaction was t erminated by adding SDS-PAGE sample buffer (0.125 M Tris, 4 % SDS, 20 % glycerol, pH 6.5) to the irradiated protoplasts, and the proteins ware reduced by the addition of 10 pl of 2-mercaptoethanol and heating at 100°C for 5 minutes. SDS-PAGE was performed by us1186
Vol.
169, No. 3, 1990
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
ing 2 % stacking gels and 10 % resolving gels. Autoradiography was carried out for 7 days on X-ray film,and phosphorylation densities of the proteins were analyzed by a densitometer (LKB 2202). at -70°C
RESULTSAND DISCUSSION In the previous report
(ll),
we described that the phosphorylations
two proteins ( 27 KDa,32 KDa) were affected by the photoreversible 2+ phytochrome through a change in the cytosolic Ca concentration. investigate
what kinds of calcium dependent protein
of
behavior of In order to
kinase(s) are regulated
phytochrome action, the involvement of protein kinase C or a similar molecule was first checked by adding its inhibitor or activator. If protein kinase C is involved in the phosphorylations of these two proteins by the by the
phytochrome action,
IX generation must be controlled
by phytochrome action.
For the enhancement of CG formation by the red light, mulated by the red light.
PI turnover
According to our unpublished results,
must besti-
breakdown of
PIP2and formation of IP3in oat cell were observed to increase by irradiation of red light.
Figure 1 shows the effect
of the proteins,
where lithium
of lithium
ions inhibit
the activity
1
0
ions on the phosphorylations
Irradiation
2
of inositol-l-rnono-
3
4
Time
Imin
5
1
w Effect of lithium ion on in vivo protein phosphorylation(27 KDa,32KDa) in Pi preloaded oat protoplasts. (A) The oat protoplasts labeled with 32Pi were preinqted with 50 nM LiCl for 2 hours at 4°C. Then the protoplast sample ( 5 x 10 /ml) was irradiated with red light(660 nm) for different Periods of time; lanes A to F are the autoradiogramsof the sampleirradiated with red light for 0,1,2,3,4, and 5 min, respectively. (B) Changesof the phosphorylation densities of the 27-KDaand 32-KCaproteins by lithium ions. l , 27-m protein phosphorylation; 0, 32-m protein phosphorylation. 1187
Vol.
169,
No.
BIOCHEMICAL
3, 1990
.Da
A
B
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
B3w-
A
C
B
C
(H-7) on --in vivo protein Fig.2. Effect of Ca2+/ffi dependent33 K inhibitor 5% preloaded oat protoplasts. phosphorylation (27 KDa,32 K&I) in (A) The oat protoplasts labeled with Pi were preincubatq with 50 pM of H-7 for 2 hours at 4°C. Then the protoplast sample (5 x 10 /ml)was irradiated with red light (660 MI) for 2 min. (lane A, not added H-7, not irradiated; lane B, not added H-7, irradiated; lane C, added H-7, irradiated). (B) Change of the phosphorylation densities of the 27-KLka and 32-KDa proteins by H-7. 0, 27-KDa protein phosphorylation; 19, 32-m protein phosphorylation.
i KDa
A6
C
DE
I=
G
B
H hcubation
[ mln
1
of the membrane permeable OAG on --in vivo protein phosphorypreloaded oat protoplasts. (A) OAG(a stock solution of 0.25 MOAGin ENSO)was addedin the protoplast suspensionto a final concentration of 3 x 10-6. The fi 1 concentration of T /ml) was stimulated CMSOwas fixed at 0.0014 %. !lhe protoplast saqle (5 x 10 by OAGfor different periods of time; lanes A to H are the autoradiogramsof the samplestimulated by OAGfor 0, 20 s, 40 s, 1, 2, 3, 4, and 5 min, respectively. (B) Changeof the phosphorylation densities of the 27-KDaand 32-K&1proteins by GAG.l , 27-KDaprotein phosphorylation; 0, 32-K&1protein phosphorylation.
fg
[email protected]
Time
1188
Vol.
169, No. 3, 1990
BIOCHEMICAL
AND BIOPHYSICAL
1
ABCDEFGH
RESEARCH COMMUNICATIONS
2
lncubatlon
3 Time
4
5
1
[ min
bol ester (TPA) on --in viva protein phosphorylation (27 Pi preloaded oat protoplasts. (A) TPA(a stock sclution of 1 x lO‘*M TPA in acetone) was added in the protoplast sus sion to a final concentration of 1 x 10m7M.The protoplastsample (5 x 10!F/ml) was stimulated by TPA for different periods of time; lanesA to H are the autoradiogramsof the samplestimulated by TPA for 0, 20 s, 40 s, 1, 2, 3, 4, and 5 min, respectively. (B) Changeof the phosphorylation densities of the 27-KDaand 32-K& proteins by TPA. l , 27-KDaprotein phosphorylation; 0, 32-KDaprotein phosphorylation. g&
~f)ey&~qf
phosphatase that is required for the turnover the degrees of phosphorylation
of the PI cycle.
densities were drastically
As we expected,
reduced regardless
of the irradiation time of red light,in comparison with the ones of the control not containing lithium ion (11). The phenomenonof the decreasing phosphorylation degree with respect to the irradiation the dephosphorylation volvement of protein
time of red light
by phosphatase. To obtain direct kinase C in the phosphorylation
might be due to
evidence for the inof the two proteins,
an
inhibitor (H-7) for Ca*+/DG dependent protein kinase was added to the cell suspension. The degrees of phosphorylations for two proteins were substantially reduced by addition
of H-7 (Fig.2.C),as
compared to those in the absenceof
this inhibitor (Fig.2.B).However,by this data alone, it is difficult to definitely conclude that protein kinase C is involved in the protein phosphorylation, because there have been a few reports (15,18)describing that H-7 does not show absolute specificity for protein kinase C and it can also inhibit Ca'+lcalnodulin dependent protein
kinase.
membranepermeable DGderivative phorylation
To overcome this problem, OAG, which is a , was added into
degrees were analyzed as a function
the oat cells of its
and the phos-
incubation
time (Fig.3
As expected, the phosphorylation degrees of the two proteins were drastically enhanced with the incubation time. Figure 4 shows the phorbol ester (TPA) enhancement effect on the phosphorylation of proteins, in which significant obserof phosphorylation was also observed. In the case of TPA addition,the 1189
).
Vol.
169,
vation
No.
that
the that of Even though or a similar was carried formed soon.
3, 1990
BIOCHEMICAL
the phosphorylation
AND
BIOPHYSICAL
RESEARCH
degree of the 32-KDa protein
COMMUNICATIONS
was higher
than
the 27-KDa protein can not be interpreted properly at this time. an --in vivo assay to determine any involvement of protein kinase C molecule for the protein phosphorylation by phytochrome action out, --in vitro studies to obtain direct evidence should be per-
ACKNCWLEDGMENT This work was supported Foundation.
by a grant
from the Korea Science and Engineering
REFERENCES 1.
2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.
Kronenberg, G.H.M., and Kendrick, R.E. (1986) Phytochrome: The physiology of action. In Photomorphogenesis in Plants, (R.E. Kendrik and G.H.M. Kronenberg, Eds ), pp= 99-114. Hepler, P-K., and Wayne, R.O. (1985) Annu.Rev.Plant Physiol.36,397-439. Veluthambi, K., and Foovaiah, B.W. (1984) Plant Physiol.76,359-365. Mcfadden, J.J., and Poovaiah, B.W. (1988) Plant Physiol.86,332-334. Blower, D.P., Boss, W-F., and Trewavas, A.J. (1988) Plant P.hysio1.86,505 -509. Chen, Y.R., and Roux, S.J. (1986) Plant Physio1.81,609-613. Veit Otto, and Eberhard Schzer (1988) Plant Cell Physiol.29,1115-1121. Harmon, A.C., Putnam-Evans, c . , and Cannier, M.J. (1987) Plant Fhysiol. 83,830-837. Ranjeva, R., and Boudet, A.M. (1987) Annu.Rev.Plant Physio1.38,73-93. Morse, M-J., Satter, R-L., Crain,R.C., and Cot&, G.G. (1989) Physiolcqia Plantarum 76,118-121. Park, M.H., and Chae, C!. (1989) Biochem. Biophys. Res. Conunun.l62,9-14. Goldstein, A-H., and Hunziker, A.D. (1985) Plant Physiol.77,1013-1015. Edwards, G-E., Robinson, S.P., Tyler, N.J.C., and Walker, D.A. (1978) Plant Physiol.62,1313-1319. Cl-=, Q., Park, H.J.,and Hong, S.D. (1990) Biochim. Biophys. Acta 1051, 115-122. Nishizuka, Y. (1984) Science 225,1365-1370. owl, z., and Kiss, Z (1986) FEBS Lett.195,33-37. Nishizuka, Y. (1989) Cancer 63,1892-1903. Robert R. Rando (1988) FASEB&8,2348-2355.
1190
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1185-1190
INTRACELLULAR PROTEIN PHOSPHORYLATION IN OAT ( Avena sativa BYPHY!IWHROMEACTION:INVOLVEMENT OF PROTEIN KINASE C Moon-Hwan ParkandQuae *Department
of Biochemistry,
L. ) PRCYTOPLASTS
ChaGl
Chungbuk National University, Republic of Korea
Cheongju 360-763
Received May 21, 1990 SUMMARY: Phosphorylations of two proteins ( 27 K&,32 KDa) in oat cells were dependent on phytochrome action. To determine which kinase system(s) for the phosphorylation of these two proteins are controlled by the phytochrome, involvement of the Ca2+/DG dependent protein kinase (protein kinase C)wasfirst investigated. When a protein kinase C inhibitor (1-(S-isoguinoline sulfonyl)2-methylpiperazine :H-7 ) or the inositol phospholipid metabolic blocker Li+ was added into the cell suspension , respectively, the phosphorylations of these two proteins were substantially reduced. On the other hand,an addition of l-oleoyl-2-acetyl-sn-glycerol (OAG :activator of protein kinase C )orphorbol 12-myristate 13-acetate (TPA :tumor promoting phorbolester Ienhanced the phosphorylations of these proteins. These results suggest that phytochrome action is certainly connected with the protein phosphorylation via the activation of protein kinase C or a similar molecule with protein kinase C. 0 1990
Academic
Press,
Inc.
It was reported that the physiologically active form of phytochrome (Pfr) might initiate the photomorphogenic cell responses by controlling the cytosolic Ca*+ concentration (1,2,11,14). The regulatory role of protein phosphorylation by the calcium dependent protein kinase was also suggested as an important step in the signal transduction in plant systems (3-7). Amolecule similar to protein kinase C (PKC )has been described in plant cells (8),and phosphorylation of proteins changes the activities of various enzymes in plant cells (9). Although protein phosphorylation in plants by PKC has not been dwnstrated yet, it has been reported that phosphorylation by other kinase systems (e.g.,Ca2+/caln-odulin dependent protein kinase ) affectsnumerous developmental processes (10). Protein phosphorylation appears to be a central regulatory mechanism in the signal transduction in both animals and plants, but it is not yet clear whether it interfaces with PI turnover in 1 To whom correspondence should be addressed. Abbreviations : H-7, 1-(5-isoguinoline sulfonyl)-2-methyl piperazine phorbol 12-myristate 13-acetate ; OAG, l-oleoyl-2-acetyl-sn-glycerol; sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
; TPA, SDS-PAGE,
0006-291X/90 $1.50 1185
Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol.
169, No. 3, 1990
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
plants (10). We had reported that the phosphorylations of two proteins (27 KDa, 32 KDa) in oat cells were dependent on the photoreversible behavior of phytochrome (11). However, it has not been determined which kinase system(s) for the phosphorylation of these proteins is controlled by the phytoChrome action. The data obtained in this study suggested the involvement of PKC or a similar molecule in the phosphorylation of two proteins by the phytochrome signal. MATF,RIALS AND METHODS Material5 Cellulase and pectinas ped for the prepar@on of protoplasts were the products of Yakult Honsa. 7 P3orthophosphate ( P:l mCi/ml) was purchased from Amersham, and the rest of the chemicals used in these experiments were obtained from Sigma Chemical Cwy.Cat seed (Avena sativa L.CV Garry)was purchased from Stanford Seed Co., Buffalo, NY, UCoat seedlings Fifty o-rams of oat seed was soaked at 4°C for 24 hours in darkness. The soaked ~2 was spread on wet vermiculite (100 g/tray) in aluminum trays (35x 45 cm), and the trays wlere putinanoat-growing boxmade of-. The oats were grown at 25°C for 5 days in complete darkness. Preparation of protoplasts The preparation of protoplasts was mainly carried out as previously described (12,13). After 5 days,the tips (2 cm)of the coleoptiles were harvested. Harvesting and further handling were performed under a dim green safe light. The washed oat tissue was cut into 1 mn pieces,which were thenplaced in the enzyme solution ( 0.6 M sorbitol, 1 m CaCl , 8.05 % cellulase, 0.5 % macerozyme, pH 6.5 ). After allawing the mixture tz react at 25°C for 7.5hours with gentle shaking (circular motion) , it was centrifuged at 200xg for 5 minutes and then filtered through a nylon mesh ( 100 urn). The enzyme solution was then removed by washing with the following buffer solution ( 0.6 M sorbitol, 0.05 8 BSA, 1 &I CaC12, pH 6.5). After washing, the protoplasts were centrifuged at 1600 rpm for 10 minutes to remove the washing buffer. Sucrose density gradient centrifugations of the pelleted protoplasts were carried out at 1600 rpin for 8 minutes by using a stepwise gradient consisting of buffer I (0.6 M sucrose, 1 nM CaCl , pH 6.5), buffer II (0.S M sucrose, 0.1 M sorbitol, 5 nM Mes, 1 IIN CaC12, pH 25 ),and buffer IlI (0.6 M sorbitol, 5 n@!Mes, 1 mM CaC12, pH 6.5). The protoplasts banded between buffer It andbuffer III. In vivo protein phosphorylation Purified protoplasts wzre suspended in the 32Pi-loadina buffer ( 0.6 M sorbitol, 253s~Mkes, i0 r&l NaIiC03, 2-m KH PO , 25 nW Hepes, i IIN CaCl‘, pH 5.8 ), and then Pi (0.5 Ki) was added to de .&spension. LiCl (50 IT&J)2 or H-7 ( 50 JIM) was added to the suspension under different experimental conditions as described inthe sumnary. The suspension was incubated at 4°C for 2 hours, gentle shaking was carried out evq 10 minutes during the incubation. At the end o 52 the incubation, the excess Piinthe mediumwas removedby washing with Pi-loading buffer ;and then the protoplasts were irra-vted with red light or stimulated by OAG (100 ug/medium 1 ml) or TPA (10 M) (16,17). Irradiation of light and SDS-PAGE 32 Protoplasts incorporated with Pi were irradiated with red light ( 660 run: the monochromatic light was generated by a 150 watt Xe lamp equipped in the spectrofluorometer, Hitachi MPF 3000), and the effect of the light was observed as a function of irradiation time. Reaction was t erminated by adding SDS-PAGE sample buffer (0.125 M Tris, 4 % SDS, 20 % glycerol, pH 6.5) to the irradiated protoplasts, and the proteins ware reduced by the addition of 10 pl of 2-mercaptoethanol and heating at 100°C for 5 minutes. SDS-PAGE was performed by us1186
Vol.
169, No. 3, 1990
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
ing 2 % stacking gels and 10 % resolving gels. Autoradiography was carried out for 7 days on X-ray film,and phosphorylation densities of the proteins were analyzed by a densitometer (LKB 2202). at -70°C
RESULTSAND DISCUSSION In the previous report
(ll),
we described that the phosphorylations
two proteins ( 27 KDa,32 KDa) were affected by the photoreversible 2+ phytochrome through a change in the cytosolic Ca concentration. investigate
what kinds of calcium dependent protein
of
behavior of In order to
kinase(s) are regulated
phytochrome action, the involvement of protein kinase C or a similar molecule was first checked by adding its inhibitor or activator. If protein kinase C is involved in the phosphorylations of these two proteins by the by the
phytochrome action,
IX generation must be controlled
by phytochrome action.
For the enhancement of CG formation by the red light, mulated by the red light.
PI turnover
According to our unpublished results,
must besti-
breakdown of
PIP2and formation of IP3in oat cell were observed to increase by irradiation of red light.
Figure 1 shows the effect
of the proteins,
where lithium
of lithium
ions inhibit
the activity
1
0
ions on the phosphorylations
Irradiation
2
of inositol-l-rnono-
3
4
Time
Imin
5
1
w Effect of lithium ion on in vivo protein phosphorylation(27 KDa,32KDa) in Pi preloaded oat protoplasts. (A) The oat protoplasts labeled with 32Pi were preinqted with 50 nM LiCl for 2 hours at 4°C. Then the protoplast sample ( 5 x 10 /ml) was irradiated with red light(660 nm) for different Periods of time; lanes A to F are the autoradiogramsof the sampleirradiated with red light for 0,1,2,3,4, and 5 min, respectively. (B) Changesof the phosphorylation densities of the 27-KDaand 32-KCaproteins by lithium ions. l , 27-m protein phosphorylation; 0, 32-m protein phosphorylation. 1187
Vol.
169,
No.
BIOCHEMICAL
3, 1990
.Da
A
B
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
B3w-
A
C
B
C
(H-7) on --in vivo protein Fig.2. Effect of Ca2+/ffi dependent33 K inhibitor 5% preloaded oat protoplasts. phosphorylation (27 KDa,32 K&I) in (A) The oat protoplasts labeled with Pi were preincubatq with 50 pM of H-7 for 2 hours at 4°C. Then the protoplast sample (5 x 10 /ml)was irradiated with red light (660 MI) for 2 min. (lane A, not added H-7, not irradiated; lane B, not added H-7, irradiated; lane C, added H-7, irradiated). (B) Change of the phosphorylation densities of the 27-KLka and 32-KDa proteins by H-7. 0, 27-KDa protein phosphorylation; 19, 32-m protein phosphorylation.
i KDa
A6
C
DE
I=
G
B
H hcubation
[ mln
1
of the membrane permeable OAG on --in vivo protein phosphorypreloaded oat protoplasts. (A) OAG(a stock solution of 0.25 MOAGin ENSO)was addedin the protoplast suspensionto a final concentration of 3 x 10-6. The fi 1 concentration of T /ml) was stimulated CMSOwas fixed at 0.0014 %. !lhe protoplast saqle (5 x 10 by OAGfor different periods of time; lanes A to H are the autoradiogramsof the samplestimulated by OAGfor 0, 20 s, 40 s, 1, 2, 3, 4, and 5 min, respectively. (B) Changeof the phosphorylation densities of the 27-KDaand 32-K&1proteins by GAG.l , 27-KDaprotein phosphorylation; 0, 32-K&1protein phosphorylation.
fg
[email protected]
Time
1188
Vol.
169, No. 3, 1990
BIOCHEMICAL
AND BIOPHYSICAL
1
ABCDEFGH
RESEARCH COMMUNICATIONS
2
lncubatlon
3 Time
4
5
1
[ min
bol ester (TPA) on --in viva protein phosphorylation (27 Pi preloaded oat protoplasts. (A) TPA(a stock sclution of 1 x lO‘*M TPA in acetone) was added in the protoplast sus sion to a final concentration of 1 x 10m7M.The protoplastsample (5 x 10!F/ml) was stimulated by TPA for different periods of time; lanesA to H are the autoradiogramsof the samplestimulated by TPA for 0, 20 s, 40 s, 1, 2, 3, 4, and 5 min, respectively. (B) Changeof the phosphorylation densities of the 27-KDaand 32-K& proteins by TPA. l , 27-KDaprotein phosphorylation; 0, 32-KDaprotein phosphorylation. g&
~f)ey&~qf
phosphatase that is required for the turnover the degrees of phosphorylation
of the PI cycle.
densities were drastically
As we expected,
reduced regardless
of the irradiation time of red light,in comparison with the ones of the control not containing lithium ion (11). The phenomenonof the decreasing phosphorylation degree with respect to the irradiation the dephosphorylation volvement of protein
time of red light
by phosphatase. To obtain direct kinase C in the phosphorylation
might be due to
evidence for the inof the two proteins,
an
inhibitor (H-7) for Ca*+/DG dependent protein kinase was added to the cell suspension. The degrees of phosphorylations for two proteins were substantially reduced by addition
of H-7 (Fig.2.C),as
compared to those in the absenceof
this inhibitor (Fig.2.B).However,by this data alone, it is difficult to definitely conclude that protein kinase C is involved in the protein phosphorylation, because there have been a few reports (15,18)describing that H-7 does not show absolute specificity for protein kinase C and it can also inhibit Ca'+lcalnodulin dependent protein
kinase.
membranepermeable DGderivative phorylation
To overcome this problem, OAG, which is a , was added into
degrees were analyzed as a function
the oat cells of its
and the phos-
incubation
time (Fig.3
As expected, the phosphorylation degrees of the two proteins were drastically enhanced with the incubation time. Figure 4 shows the phorbol ester (TPA) enhancement effect on the phosphorylation of proteins, in which significant obserof phosphorylation was also observed. In the case of TPA addition,the 1189
).
Vol.
169,
vation
No.
that
the that of Even though or a similar was carried formed soon.
3, 1990
BIOCHEMICAL
the phosphorylation
AND
BIOPHYSICAL
RESEARCH
degree of the 32-KDa protein
COMMUNICATIONS
was higher
than
the 27-KDa protein can not be interpreted properly at this time. an --in vivo assay to determine any involvement of protein kinase C molecule for the protein phosphorylation by phytochrome action out, --in vitro studies to obtain direct evidence should be per-
ACKNCWLEDGMENT This work was supported Foundation.
by a grant
from the Korea Science and Engineering
REFERENCES 1.
2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.
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