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Levels of cAMP-dependent protein kinase activity in sea urchin gametes and embryos during the first cell division
Printed in Sweden Copyright 9 1975 by Academic Press, Inc. All rights of reproduction in any form reserved
Experimental Cell Research 96 (1975) 77-80
LEVELS ACTIVITY
OF cAMP-DEPENDENT IN SEA URCHIN
DURING
THE
PROTEIN
GAMETES
FIRST CELL
KINASE
AND EMBRYOS
DIVISION
MARIETTA Y. W. LEE and R. M. IVERSON
Laboratory for Quantitative Biology, University of Miami, Coral Gables, FL, and College of Science, Florida Atlantic University, Boca Raton, FL 33432, USA
SUMMARY The levels of protein kinase activity in the gametes of the sea urchin Lytechinus variegatus were determined on a per sperm and per egg basis, The levels of protein kinase were also determined in sea urchin embryos through the first cell cycle following fertilization. The enzyme activity, which decreased the first 5 min, increased rapidly during the S phase of the cell cycle but decreased during metaphase and cell division. The timing of the fluctuation was constant with different substrates, although the magnitude of the activity varied. The periodic change in protein kinase levels may be correlated with histone phosphorylation or tubulin polymerization.
We have previously reported the presence of a cyclic 3',5t-adenosine monophosphate (cAMP)-dependent protein kinase which phosphorylates histones and protamine in the sperm, oocyte and developing embryo of the sea urchin, Lytechinus variegatus [1]. The level of protein kinase in the sperm (expressed as U/mg soluble protein) were 30-fold higher than those of the oocytes or gastrula embryos [1]. In this work we have determined the levels of protein kinase on a unit cell basis. A number of studies have focused on the phosphorylation of histones during the cell cycle [2-4]. These studies indicate that histone phosphorylation occurs during the S phase, and appears to be directed toward the lysine-rich histones. MATERIALS AND METHODS The gametes from the local sea urchin (Lytechinus variegatus) were obtained by injecting 1 ml of
isotonic KCI (0.56 M) into the perivisceral cavity, The male sea urchin was then placed aboral surface down on a Syracuse watch glass into which the sperm was shed [6]. The eggs from the female sea urchin were collected in a 50 ml plastic beaker filled with Millipore-filtered sea water (MSW). The eggs were washed twice by sedimentation and replaced each time with fresh MSW.
Levels o f protein kinase activity per egg and per sperm One ml of dry packed sperm from one male sea urchin was diluted 1:100 in a volumetric flask with distilled water. An aliquot was counted in an AO Spencer hemacytometer under a phase contrast microscope (10• eye piece and 43x objective). Another 1 ml of sperm cells was homogenized and assayed for protein kinase activity. The eggs from one female sea urchin were also diluted 1:100 in a volumetric flask with two drops of formaldehyde and distilled water. Methylene blue (1%) was added to stain the eggs. One ml of the diluted sample was filtered through a Millipore filter assembly onto a Millipore filter (HA 0.45 /zm pore size). The eggs were then counted under a dissecting microscope [7]. One ml of the undiluted suspension was also taken for protein kinase assay. The 20000 g precipitate of the sperm and egg were redissolved in buffer. To each precipitate enough 3 M TCA was added to give a final TCA concentration of 0,3 M. DNA determinations were done according to the method of Burton [8].
Exptl CellRes 96 (1975)
78
Lee and lverson
Table 1. Levels of protein kinase per gamete and per mg of gamete DNA -cAMP
Sperm egg % of enzyme activity in sperm/egg
+cAMP
U/gamete
U/mg DNA
U/gainete
U/rag DNA
0.00044 0.0113
769 5 650
0.00206 0.036
3 552 18 000
3.8%
13.4%
5.7%
19.7%
Cell cycle studies Eggs from a single female sea urchin were fertilized using the sperm from a single male urchin. Polyspermy was minimized by washing the eggs immediately after fertilization. The zygotes were grown in a 250 ml separatory funnel at room temperature (23~ with constant aeration. At designated time intervals, samples were removed, spun down by a hand centrifuge and immediately frozen in an acetone dry ice solution. Two volumes of cells were suspended per 1 vol of TMD buffer (50 mM TrisHC1, 5 mM magnesium chloride, 1 mM dithiothreitol, pH 7.4). The resultant homogenates were centrifuged at 20000 g for 30 min. The supernatants were assayed for protein kinase activities using salmon protamine sulfate or calf thymus histone f2b as the substrate on the same day as homogenization. Protein kinase assays were done as previously described [1]. Protein concentration was determined by the method of Lowry et al. [9]. One unit of protein kinase activity was defined as that amount of enzyme which transfers 1 pmole of 32p from T-32p-ATP to protein substrate per minute. The developmental stages and degree of synchrony were determined, using a phase contrast microscope on living embryos and on embryos fixed in fresh ethanol : acetic acid (3 : 1) solution,
R E S U L T S AND DISCUSSION
Levels of protein kinase in sea urchin gametes on a unit cell basis The previously [1] reported levels of protein kinase activity in sea urchin gametes were determined on the basis of specific activity (U/mg soluble protein). Preliminary to the cell cycle studies the levels were determined on a unit cell basis. This was done in order to have some idea of Exptl Cell Res 96 (1975)
how much of the activity found in the washed embryos might be due to the sperm, since excess sperm not removed by the washing procedure might contribute significantly to the basal protein kinase levels. As can be seen in table 1, the total enzyme activity of the sperm was 3.8 and 5.7% that of the oocytes (minus and plus cAMP, respectively) on a per gamete basis. Although the specific activity of the enzyme in the sperm is some 30-fold higher than in the oocytes, a single sperm contained only about 1/20th of the amount of activity of an egg. It was concluded that in the experiments on the cell cycle which followed, the sperm not removed by washing should not contribute significantly to the measured enzyme activity.
Protein kinase levels during the first cell cycle The results of the first experiment in which cAMP-dependent protein kinase levels were determined during the first cell cycle are shown in fig. 1. In these two independent experiments the protein kinase activities were determined with protamine sulfate as the substrate in the presence and absence of cAMP. It is seen that the activity increases during the first 5-20 min stage, and then declines between 20 and 40 rain. Metaphase occurred at about 35 rain, and cytokinesis at about 45 min. In order to examine the possibility that a particulate form of the enzyme was present, the cell debris obtained after centrifugation of the crude homogenate was extracted with 0.1% Triton X-100. A small amount of activity was found, with specific activities similar to those obtained in the soluble fraction. However, the total amount of activity never amounted to more than 10-15 % of the soluble activity and did not alter the pattern of the results. It may
Protein kinase activity in sea urchin g a m e t e s
200[
4/'.\
1 ~
oA
9
13
200f
20
,oo / \
e~:
\.
40
60
0
I
20
I
40
I
60
.
I
80
Figs 1, 2. Abscissa: time after fertilization (rain); ordinate: protein kinase (U/rag protein); S.E.M. Fig. 1. Cell cycle s t u d y of protein kinase activity of the s e a urchin after fertilization. 20000 g supern a t a n t in O - - O , absence of c A M P ; Q - - O , 5• 10 6 M c A M P . M e t a p h a s e occurred at about 35 min, cytokinesis at about 45 rain. 100 /zg s a l m o n protamine sulfate was used as the substrate per 100 /zl of incubation mixture. (A) and (B) are results of two separate experiments.
be noted that these results were expressed in terms of specific activities. When the data was examined in terms of total activities per unit volume of packed cells, the same pattern of activities was noted. In order to confirm the fluctuation of protein kinase activity during the cell cycle, the experiment was repeated but this time samples were taken in duplicate at closer time intervals, and histone f2b was used as the substrate. The results are shown in fig. 2. A clearly defined peak of activity is seen between 10 and 30 min. The activity rose sharply at the streak stage and early prophase, and decreased during metaphase and telophase. This fluctuation in protein kinase activity is also seen to be much more p r o n o u n c e d than that observed in earlier experiments (fig. 1). During this peak of increased activity there was a 4-fold response to cAMP. Cycloheximide at 5 • 10-3 M failed to inhibit this increase in e n z y m e activity. It may be noted that cAMP levels have also been shown to fluctuate during the cell cycle. Zeilig et al. [10] observed a fluctua6--751815
9
,5ol-,/\
.50
0
79
sperm unione{ streakstage aster pr162
metephose
telophase
Fig. 2. Protein kinase activity of the sea urchin e m b r y o during the first division cycle. T h e e m b r y o s were frozen and thawed before homogenation, 9 1 6 9 in the absence of c A M P ; O - - O , in the presence of 5• -n M c A M P . 100 ~g of calf t h y m u s histone f2b was used as the substrate per 100 /zl of incubation mixture. A b o u t l0 rain after fertilization, a sperm aster c a n be seen as a spherical region with clear rays extending from a clear-center. T h e n a clear streak appears in the egg slightly above the equator at about 20-30 rain. This is k n o w n as the streak stage. The observations were m a d e at 23~
tion of c A M P levels in synchronized H e L a cells. Recently Yasumasu et al. [11] have observed periodic changes in the content of c A M P with close relation to the cleavage cycle in the sea urchin H e m i c e n t r o t u s pulcherrimus. Since the regulation of protein kinase activity is thought to be primarily the c A M P activation mechanism, our results are somewhat surprising, for they suggest that an additional level of regulation in terms of changes in the total e n z y m e activity may also be important. This change in protein kinase levels may be added to the list of biochemical events which occur during the cell division. But, these studies do not reveal the mechanism of this change; the fact that cycloheximide did not prevent this increase in activity suggests that this was not due to de novo e n z y m e synthesis. A second possibility is the release of a particulate form of the enzyme; this possibility seems unlikely since detergent extraction of the 20000 g Exptl Cell Res 96 (1975)
80
L e e and lverson
precipitate failed to release sufficient additional activity to account for these changes. A third possibility is that changes in the levels may be mediated through changes in the levels of a protein kinase inhibitor, as has been isolated from rabbit skeletal muscle [12]. The relevance of the peak in protein kinase during the S phase obviously indicates the need for further study. This peak may be correlated with histone phosphorylation, but a second possibility which has not been previously considered arises from studies in this laboratory, which showed a similar fluctuation in tubulin synthesis during the cell cycle of the sea urchin embryo [13]. This protein, which is a subunit of the mitotic apparatus and of cilia, has been a mystery in terms of how the self-assembly of the subunits is controlled. Recently, it has been shown that tubulin from rat brain [14] is a phosphoprotein. Eipper suggested that tubulin phosphorylation may be involved in selfassembly [14]. It may be speculated that protein phosphorylation plays a role in triggering the formation of the mitotic apparatus.
Exptl Cell Res 96 (1975)
The authors thank Dr Peter Luykx for his assistance in determining the degree of synchrony of the embryos.
REFERENCES 1. Lee, M Y W & Iverson, R M, Exp cell res 75 (1972) 300. 2. Chalkley, R, Balhorn, R, Granner, D D, Johnson, G & Oliver, D R, Miami winter symp, vol. 5, pp. 251-277. Academic Press, New York (1973). 3. Langan, T A, Miami winter syrup, vol. 5, pp. 287-292. Academic Press, New York (1973). 4. Shephard, G R, Noland, B J & Hardin, J M, Arch biochem biophys 143, 1 (1971). 5. Iverson, R M & Cohen, G H, The cell cycle, pp. 299-313. Academic Press, New York (1969). 6. Costello, D P, Davidson, M E, Egger, A, Fox, M H & Henley, C, Methods for obtaining and handling marine eggs and embryos, p. 184. Marine Biology Lab, Woods Hole, Mass. (1957). 7. Chamberlain, J. Personal communication. 8. Burton, K, Methods in enzymol 12 (1968) 163. 9. Lowry, O H, Rosebrough, N J, Farr, A L & Randall, R J, J biol chem 193 (1951) 265. 10. Zeilig, C E, Johnson, R A, Friedman, D L & Sutherland, E W, J biol chem 55 (1973) 296. 11. Yasumasu, I, Fujiwara, A & Ishida, K, J biochem biophys res comm 54 (1973) 628. 12. Walsh, D A, Ashby, C D, Gonzalez, C, Calkins, D, Fischer, E H & Krebs, E G, J cell biol 246 (1971) 1977. 13. Meeker, G L & lverson, R M, Exp cell res 64 (1971) 129. 14. Eipper, B A, Proc natl acad sci US 69 (1972) 2283. Received March 4, 1975 Revised version received April 30, 1975
Experimental Cell Research 96 (1975) 77-80
LEVELS ACTIVITY
OF cAMP-DEPENDENT IN SEA URCHIN
DURING
THE
PROTEIN
GAMETES
FIRST CELL
KINASE
AND EMBRYOS
DIVISION
MARIETTA Y. W. LEE and R. M. IVERSON
Laboratory for Quantitative Biology, University of Miami, Coral Gables, FL, and College of Science, Florida Atlantic University, Boca Raton, FL 33432, USA
SUMMARY The levels of protein kinase activity in the gametes of the sea urchin Lytechinus variegatus were determined on a per sperm and per egg basis, The levels of protein kinase were also determined in sea urchin embryos through the first cell cycle following fertilization. The enzyme activity, which decreased the first 5 min, increased rapidly during the S phase of the cell cycle but decreased during metaphase and cell division. The timing of the fluctuation was constant with different substrates, although the magnitude of the activity varied. The periodic change in protein kinase levels may be correlated with histone phosphorylation or tubulin polymerization.
We have previously reported the presence of a cyclic 3',5t-adenosine monophosphate (cAMP)-dependent protein kinase which phosphorylates histones and protamine in the sperm, oocyte and developing embryo of the sea urchin, Lytechinus variegatus [1]. The level of protein kinase in the sperm (expressed as U/mg soluble protein) were 30-fold higher than those of the oocytes or gastrula embryos [1]. In this work we have determined the levels of protein kinase on a unit cell basis. A number of studies have focused on the phosphorylation of histones during the cell cycle [2-4]. These studies indicate that histone phosphorylation occurs during the S phase, and appears to be directed toward the lysine-rich histones. MATERIALS AND METHODS The gametes from the local sea urchin (Lytechinus variegatus) were obtained by injecting 1 ml of
isotonic KCI (0.56 M) into the perivisceral cavity, The male sea urchin was then placed aboral surface down on a Syracuse watch glass into which the sperm was shed [6]. The eggs from the female sea urchin were collected in a 50 ml plastic beaker filled with Millipore-filtered sea water (MSW). The eggs were washed twice by sedimentation and replaced each time with fresh MSW.
Levels o f protein kinase activity per egg and per sperm One ml of dry packed sperm from one male sea urchin was diluted 1:100 in a volumetric flask with distilled water. An aliquot was counted in an AO Spencer hemacytometer under a phase contrast microscope (10• eye piece and 43x objective). Another 1 ml of sperm cells was homogenized and assayed for protein kinase activity. The eggs from one female sea urchin were also diluted 1:100 in a volumetric flask with two drops of formaldehyde and distilled water. Methylene blue (1%) was added to stain the eggs. One ml of the diluted sample was filtered through a Millipore filter assembly onto a Millipore filter (HA 0.45 /zm pore size). The eggs were then counted under a dissecting microscope [7]. One ml of the undiluted suspension was also taken for protein kinase assay. The 20000 g precipitate of the sperm and egg were redissolved in buffer. To each precipitate enough 3 M TCA was added to give a final TCA concentration of 0,3 M. DNA determinations were done according to the method of Burton [8].
Exptl CellRes 96 (1975)
78
Lee and lverson
Table 1. Levels of protein kinase per gamete and per mg of gamete DNA -cAMP
Sperm egg % of enzyme activity in sperm/egg
+cAMP
U/gamete
U/mg DNA
U/gainete
U/rag DNA
0.00044 0.0113
769 5 650
0.00206 0.036
3 552 18 000
3.8%
13.4%
5.7%
19.7%
Cell cycle studies Eggs from a single female sea urchin were fertilized using the sperm from a single male urchin. Polyspermy was minimized by washing the eggs immediately after fertilization. The zygotes were grown in a 250 ml separatory funnel at room temperature (23~ with constant aeration. At designated time intervals, samples were removed, spun down by a hand centrifuge and immediately frozen in an acetone dry ice solution. Two volumes of cells were suspended per 1 vol of TMD buffer (50 mM TrisHC1, 5 mM magnesium chloride, 1 mM dithiothreitol, pH 7.4). The resultant homogenates were centrifuged at 20000 g for 30 min. The supernatants were assayed for protein kinase activities using salmon protamine sulfate or calf thymus histone f2b as the substrate on the same day as homogenization. Protein kinase assays were done as previously described [1]. Protein concentration was determined by the method of Lowry et al. [9]. One unit of protein kinase activity was defined as that amount of enzyme which transfers 1 pmole of 32p from T-32p-ATP to protein substrate per minute. The developmental stages and degree of synchrony were determined, using a phase contrast microscope on living embryos and on embryos fixed in fresh ethanol : acetic acid (3 : 1) solution,
R E S U L T S AND DISCUSSION
Levels of protein kinase in sea urchin gametes on a unit cell basis The previously [1] reported levels of protein kinase activity in sea urchin gametes were determined on the basis of specific activity (U/mg soluble protein). Preliminary to the cell cycle studies the levels were determined on a unit cell basis. This was done in order to have some idea of Exptl Cell Res 96 (1975)
how much of the activity found in the washed embryos might be due to the sperm, since excess sperm not removed by the washing procedure might contribute significantly to the basal protein kinase levels. As can be seen in table 1, the total enzyme activity of the sperm was 3.8 and 5.7% that of the oocytes (minus and plus cAMP, respectively) on a per gamete basis. Although the specific activity of the enzyme in the sperm is some 30-fold higher than in the oocytes, a single sperm contained only about 1/20th of the amount of activity of an egg. It was concluded that in the experiments on the cell cycle which followed, the sperm not removed by washing should not contribute significantly to the measured enzyme activity.
Protein kinase levels during the first cell cycle The results of the first experiment in which cAMP-dependent protein kinase levels were determined during the first cell cycle are shown in fig. 1. In these two independent experiments the protein kinase activities were determined with protamine sulfate as the substrate in the presence and absence of cAMP. It is seen that the activity increases during the first 5-20 min stage, and then declines between 20 and 40 rain. Metaphase occurred at about 35 rain, and cytokinesis at about 45 min. In order to examine the possibility that a particulate form of the enzyme was present, the cell debris obtained after centrifugation of the crude homogenate was extracted with 0.1% Triton X-100. A small amount of activity was found, with specific activities similar to those obtained in the soluble fraction. However, the total amount of activity never amounted to more than 10-15 % of the soluble activity and did not alter the pattern of the results. It may
Protein kinase activity in sea urchin g a m e t e s
200[
4/'.\
1 ~
oA
9
13
200f
20
,oo / \
e~:
\.
40
60
0
I
20
I
40
I
60
.
I
80
Figs 1, 2. Abscissa: time after fertilization (rain); ordinate: protein kinase (U/rag protein); S.E.M. Fig. 1. Cell cycle s t u d y of protein kinase activity of the s e a urchin after fertilization. 20000 g supern a t a n t in O - - O , absence of c A M P ; Q - - O , 5• 10 6 M c A M P . M e t a p h a s e occurred at about 35 min, cytokinesis at about 45 rain. 100 /zg s a l m o n protamine sulfate was used as the substrate per 100 /zl of incubation mixture. (A) and (B) are results of two separate experiments.
be noted that these results were expressed in terms of specific activities. When the data was examined in terms of total activities per unit volume of packed cells, the same pattern of activities was noted. In order to confirm the fluctuation of protein kinase activity during the cell cycle, the experiment was repeated but this time samples were taken in duplicate at closer time intervals, and histone f2b was used as the substrate. The results are shown in fig. 2. A clearly defined peak of activity is seen between 10 and 30 min. The activity rose sharply at the streak stage and early prophase, and decreased during metaphase and telophase. This fluctuation in protein kinase activity is also seen to be much more p r o n o u n c e d than that observed in earlier experiments (fig. 1). During this peak of increased activity there was a 4-fold response to cAMP. Cycloheximide at 5 • 10-3 M failed to inhibit this increase in e n z y m e activity. It may be noted that cAMP levels have also been shown to fluctuate during the cell cycle. Zeilig et al. [10] observed a fluctua6--751815
9
,5ol-,/\
.50
0
79
sperm unione{ streakstage aster pr162
metephose
telophase
Fig. 2. Protein kinase activity of the sea urchin e m b r y o during the first division cycle. T h e e m b r y o s were frozen and thawed before homogenation, 9 1 6 9 in the absence of c A M P ; O - - O , in the presence of 5• -n M c A M P . 100 ~g of calf t h y m u s histone f2b was used as the substrate per 100 /zl of incubation mixture. A b o u t l0 rain after fertilization, a sperm aster c a n be seen as a spherical region with clear rays extending from a clear-center. T h e n a clear streak appears in the egg slightly above the equator at about 20-30 rain. This is k n o w n as the streak stage. The observations were m a d e at 23~
tion of c A M P levels in synchronized H e L a cells. Recently Yasumasu et al. [11] have observed periodic changes in the content of c A M P with close relation to the cleavage cycle in the sea urchin H e m i c e n t r o t u s pulcherrimus. Since the regulation of protein kinase activity is thought to be primarily the c A M P activation mechanism, our results are somewhat surprising, for they suggest that an additional level of regulation in terms of changes in the total e n z y m e activity may also be important. This change in protein kinase levels may be added to the list of biochemical events which occur during the cell division. But, these studies do not reveal the mechanism of this change; the fact that cycloheximide did not prevent this increase in activity suggests that this was not due to de novo e n z y m e synthesis. A second possibility is the release of a particulate form of the enzyme; this possibility seems unlikely since detergent extraction of the 20000 g Exptl Cell Res 96 (1975)
80
L e e and lverson
precipitate failed to release sufficient additional activity to account for these changes. A third possibility is that changes in the levels may be mediated through changes in the levels of a protein kinase inhibitor, as has been isolated from rabbit skeletal muscle [12]. The relevance of the peak in protein kinase during the S phase obviously indicates the need for further study. This peak may be correlated with histone phosphorylation, but a second possibility which has not been previously considered arises from studies in this laboratory, which showed a similar fluctuation in tubulin synthesis during the cell cycle of the sea urchin embryo [13]. This protein, which is a subunit of the mitotic apparatus and of cilia, has been a mystery in terms of how the self-assembly of the subunits is controlled. Recently, it has been shown that tubulin from rat brain [14] is a phosphoprotein. Eipper suggested that tubulin phosphorylation may be involved in selfassembly [14]. It may be speculated that protein phosphorylation plays a role in triggering the formation of the mitotic apparatus.
Exptl Cell Res 96 (1975)
The authors thank Dr Peter Luykx for his assistance in determining the degree of synchrony of the embryos.
REFERENCES 1. Lee, M Y W & Iverson, R M, Exp cell res 75 (1972) 300. 2. Chalkley, R, Balhorn, R, Granner, D D, Johnson, G & Oliver, D R, Miami winter symp, vol. 5, pp. 251-277. Academic Press, New York (1973). 3. Langan, T A, Miami winter syrup, vol. 5, pp. 287-292. Academic Press, New York (1973). 4. Shephard, G R, Noland, B J & Hardin, J M, Arch biochem biophys 143, 1 (1971). 5. Iverson, R M & Cohen, G H, The cell cycle, pp. 299-313. Academic Press, New York (1969). 6. Costello, D P, Davidson, M E, Egger, A, Fox, M H & Henley, C, Methods for obtaining and handling marine eggs and embryos, p. 184. Marine Biology Lab, Woods Hole, Mass. (1957). 7. Chamberlain, J. Personal communication. 8. Burton, K, Methods in enzymol 12 (1968) 163. 9. Lowry, O H, Rosebrough, N J, Farr, A L & Randall, R J, J biol chem 193 (1951) 265. 10. Zeilig, C E, Johnson, R A, Friedman, D L & Sutherland, E W, J biol chem 55 (1973) 296. 11. Yasumasu, I, Fujiwara, A & Ishida, K, J biochem biophys res comm 54 (1973) 628. 12. Walsh, D A, Ashby, C D, Gonzalez, C, Calkins, D, Fischer, E H & Krebs, E G, J cell biol 246 (1971) 1977. 13. Meeker, G L & lverson, R M, Exp cell res 64 (1971) 129. 14. Eipper, B A, Proc natl acad sci US 69 (1972) 2283. Received March 4, 1975 Revised version received April 30, 1975