EXPERIMENTAL

CELL

RESEARCH

188,122-128

(1%))

Inhibition of Mitosis in Fertilized Sea Urchin Eggs by Inhibition of the Cyclic AMP-Dependent Protein Kinase CAROLE L. BROWNE,**~WILLIAM

A. BOWER,* ROBERT E. PALAZZO,~ AND LIONEL I. REBHUN-~

*Department of Biology, Wake Forest University, Winston-Salem, North Carolina 27109; and TDepartment of Biology, University of Virginia, Charlottesville, Virginia 22901

Inhibition of CAMP-dependent protein kinase activity by microinjection of a specific physiologic protein inhibitor into sea urchin eggs inhibits the first cleavage after fertilization. Inhibition apparently occurs at some time prior to or during formation of the mitotic spindle. Measurement of the total protein kinase activity of sea urchin egg homogenates after fertilization showed that CAMP-dependent phosphorylation increases after fertilization and then declines prior to or at the time of the Arst cleavage. It is concluded that a CAMP-dependent phosphorylation plays a significant role in events leading to regulation of mitotic spindle assembly. o 1990 Academic

Press,

Inc.

INTRODUCTION For a number of years evidence has suggested that cyclic AMP, acting through its specific protein kinases, may play a role in the regulation of mitosis. Cyclic AMP has been shown to affect the growth of many, but not all, cell types (Rebhun, 1976; Dumont et al., 1989). Examination of cyclic AMP levels at various phases of the cell cycle in synchronized cells showed that, with some exceptions, CAMP levels were low during the M phase (Sheppard and Prescott, 1972). Identification of a role for CAMP in the regulation of mitosis has been complicated by the fact that CAMP-deficient and CAMP kinase-deficient cell lines have been isolated and generally all progress through the cell cycle normally (Granner et al., 1970; Daniel et al., 1973; Bourne et al., 1975; Coffin0 et al., 1975; Evain et al., 1979; Singh et al., 1981). Nevertheless, microinjection of highly specific inhibitors of the catalytic activity of CAMP-dependent protein kinase during early mitosis delayed or prevented progression of cells through mitosis, suggesting that the catalytic activity of the kinase plays an important physiological role in mitosis (Browne et al., 1987). The facts that CAMP levels do not always correlate directly with cell growth rates 1To whom correspondence dressed. 0014-4827/90

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122

and that cyclic AMP is not absolutely required for normal mitosis in all cells suggest that it is not a universal regulator of cell division (Bourne et al., 1975; Rebhun, 1976). However, the ability of CAMP to stimulate or inhibit mitosis in some cells in culture (Rebhun, 1976; Dumont et al., 1989) and the inhibition of mitosis in cultured cells by CAMP-dependent protein kinase inhibitors indicate that it still may play an important role in many cell types, perhaps by acting as an additional regulatory mechanism which adjusts or fine-tunes a primary regulatory mechanism. Cyclic AMP may act in a number of ways to regulate cell division, such as signalling the initiation of DNA synthesis or the synthesis of specific proteins required for mitosis. Hormones known to raise the intracellular level of CAMP also stimulate the incorporation of [3H]thymidine into DNA (Whitfield et al., 1973), and recent studies have revealed structural genes in eucaryotes whose transcription is regulated by CAMP (Boney et al., 1983; Nagamine and Riech, 1985; Schlicter et al., 1986). Cyclic AMP could also act through regulation of microtubule assembly/disassembly and function. One of the sites at which the CAMP-dependent protein kinase appears to be localized in cells is on the microtubule network. There are two types of CAMP-dependent protein kinases in cells, type I and type II, which differ in their regulatory subunits but whose catalytic subunits appear to be virtually identical (Corbin et al., 1975; Hofmann et al., 1975). The type I and type II CAMP-kinases are differentially distributed within cells and tissues and are independently regulated (Lohmann and Walter, 1984). Using immunocytochemical methods, the regulatory subunit of the type II CAMP-dependent protein kinase has been localized on the microtubule network of both interphase and mitotic epithelial cells and fibroblasts in culture (Browne et al., 1980; Browne et al., 1982) and has been reported to be associated with the centrosome in cultured epithelial cells (Nigg et al., 1985) and in cultured as well as primary cultures of glial and neuronal cells (De Camilli et al., 1986). Sea urchin embryos have served as a particularly useful system for the study of mitosis because large numbers

INHIBITION

OF

CAMP

KINASE

INHIBITS

of synchronously divided cells can be obtained in the first several divisions after fertilization. Cyclic AMP levels have been measured during the first divisions after fertilization in an attempt to correlate CAMP levels with early cleavages. Yasumasu and co-workers have reported a periodic increase in CAMP content in the eggs of Hemicentrotus pulcherrimus through the first several cleavages after fertilization (Yasumasu et al, 1973; Ishida and Yasumasu, 1982). Nath and Rebhun (1973a) reported a similar rise in CAMP content in the fertilized eggs of Strongylocentrotus purpuratus at the first cleavage, although subsequent work with more sophisticated methods could not confirm these changes in CAMP levels (Rebhun, 1976). Lee and Iverson (1975) have reported that the level of CAMP-dependent protein kinase activity in fertilized Lytechinw variegates eggs also fluctuates throughout the first cleavage. However, incubation of S. purpuratus and Lytechinus pi&us eggs in CAMP or CAMP derivatives had no effect on fertilization or early cleavage rates even when intracellular levels of the nucleotide reached more than 100X their normal concentration (Nath and Rebhun, 1973b). In contrast, Ishida and Yasumasu (1982) have reported that exogenous CAMP or dibutyryl CAMP accelerates the cleavage cycle. In this paper we report that inhibition of CAMP-dependent protein kinase activity in fertilized sea urchin eggs inhibits the first cleavage at some time prior to the formation of the mitotic spindle but subsequent to nuclear envelope breakdown. Measurement of the kinase activity of fertilized eggs shows that CAMP-dependent kinase activity rises at a time corresponding to the time at which the mitotic spindle forms and declines prior to cleavage. MATERIALS

AND

METHODS

Sea urchins. Arbaciu punctulata were collected by the Marine Biological Laboratory (Woods Hole, MA). L. variegatus were obtained off the coast of Hollywood, Florida, by Dr. Susan J. Decker. L. pictus was obtained from Marinus Co. (Westchester, CA). L. variegate were maintained at 25”C, and L. pi&us at 16”C, in Instant Ocean (Aquarium Systems, Inc.) in 30-gal. tanks equipped with undergravel and side filters. A. punctuluta were maintained at 22°C in running seawater. To induce the release of gametes, 0.5 to 1.0 ml of 0.5 M KC1 was injected into the body cavity through the peritoneal membrane. Eggs were fertilized and allowed to develop at room temperature. The time to spindle formation and cleavage was determined by polarization and phase contrast microscopic observation, respectively. Data were analyzed by a paired t test.

Microinjection. The jelly coat was removed from eggs by mixing the egg suspension 1:l with lo-* M sodium metaperiodate in seawater (Salmon and Segal, 1980). Sperm was diluted in 2 mM aminotriazole to enhance fertilization and soften the fertilization envelope (Showman and Foerder, 1979). Fertilization envelopes were removedby vacuum filtration of the eggs through a nylon filter. Fertilized eggs were washed several times in artificial Caz+-free seawater before being loaded into the microinjection chamber. The microinjection chamber and Brinkman micromanipulator were assembled essentially as de-

MITOSIS

IN

SEA

URCHIN

EMBRYOS

123

scribed by Kiehart (1982). The chamber was mounted on a Zeiss standard microscope, illuminated with a mercury vapor lamp, and equippedwith polarization optics. The microinjection medium, modified from the permeabilization medium of Supryn~wkz and Mazia (1985), contained 250 mM gluconate, 250 n&f methyl glucamine, 59 n&f Hepes, 5 mM NaCl, 5 mM oxalic acid, 1 PM CaCl,, and 6 mM MgS04. Eggs were injected with the protein inhibitor of the CAMP-dependent protein kinase in injection buffer, or injection buffer alone, along with a drop of Wesson oil. Wesson oil is nontoxic to living cells. It was loaded into the microinjection pipet in approximately equal volume with the injection solution, for the purpose of calibrating the volume of the injected solution. Injected volumes varied slightly, depending on the diameter of the pipet tip, but were in the range 30-100 pl, a maximum of 0.11% of egg volume. The oil droplets remained visible throughout the course of the experiment. The protein kinase inhibitor (PKI) binds reversibly and with high affinity to the free catalytic subunit of the kinase, but not to the holoenzyme (Ashby and Walsh, 1973; Van Patten et al., 1986). By binding at the active site of the catalytic subunit, it acts as a competitive inhibitor of the protein kinase substrates (Demaille et al., 1977; McPherson et al., 1979; Whitehouse and Walsh, 1983). Grove et al. (1987) have demonstrated the specificity of the PKI against CAMP-dependent protein kinase, protein kinase C, and protein phosphatase I using a synthetic 31-amino acid peptide that contains the active fragment of the 75-amino acid skeletal muscle PKI. At concentrations of 12 NM and below, the inhibitor fragment showed no activity against these enzymes. The concentrations of PKI injected in these cells varied between 0.5 mg/ml and 5 mg/ml, corresponding to intracellular concentrations of approximately 0.04 to 0.44 fl. This falls well within the range of specificity reported by Grove et al. (1987) for the enzyme. The PKI used for microinjection of Arbacia eggs was a gift from Dr. Donal Walsh at the University of California at Davis. The PKI used for microinjection of L. variegates and L. pictus was obtained commercially (Sigma). Both preparations were dialyzed against microinjection buffer before use. The synthetic protein kinase inhibitor sequence (Sigma) was used in protein kinase activity assays. Protein kinase activity assay. Cyclic AMP-dependent protein kinase activity of fertilized and unfertilized sea urchin eggs was measured according to the method of Beale et al. (1977). Eggs were homogenized 1:l in buffer containing 20 mM KzHP04 (pH 6.8), 6 m&f Mg acetate, 0.5 mM EDTA, and 0.7 mM EGTA. The protein kinase assay mixture contained 1.5 PM KzHP04 (pH 6.&J), 6 mh4 Mg acetate, 1.0 FM dithiothreitol, 25 nM of [-32P]ATP, 0.05 mg histone, 5 pM CAMP (when used), and 10 ~1 of egg homogenate in a total volume of 60 ~1. Samples were counted in a Beckman liquid scintillation counter. The data were expressed in picomoles of 3zP incorporated per milligram of protein. Purification of the catalytic subunit ofprotein kinuse A. In order to block the activity of microinjected protein kinase inhibitor, the catalytic subunit of the CAMP-dependent protein kinase (type II) was purified from bovine heart tissue by the method of Bechtel et al. (1977). The final purified product appeared as a single band on a 10% silverstained polyacrylamide gel.

RESULTS

Microinjection. Fertilized eggs of A. punctulata and L. pictus were microinjected with the specific inhibitor of the CAMP-dependent protein kinase (PKI) in microinjection buffer. Each egg was simultaneously injected with a drop of Wesson oil of comparable volume. Matching controls were injected with buffer and oil. The eggswere illuminated only during periodic observations, to prevent unnecessary temperature increases in the mi-

124

BROWNE Arbacia

punctulata

Lytechinus

pictus

80

.a al 5

6

0.5 nlQ/rnl

5.0 ma/m1

601.5 mg/ml

g 40&?I 0 BP zo-

0,

0 mglml m-30 31.45

0 mg/ml

I 7.0-30 314

46-S

time

injected

20.30 31-4s 46-M

after

fertilizetion

20-30 3145

*0.3ll 3145 46.&l

(min)

FIG. 1. The percentage of eggs injected with the specific protein inhibitor of the CAMP-dependent protein kinase that failed to cleave within 100 min after fertilization. Microscopic examination of inhibitor-injected cells revealed that those cells that failed to divide also failed to form a mitotic spindle, although occasionally a birefringent spot or streak was observed in the spindle area.

croinjection chamber. The eggs were observed with polarization optics, and the times elapsed until the formation of the mitotic spindle and until cleavage were compared with those of uninjected eggs in the same chamber. The eggs were injected at various times, beginning 20 min after fertilization, until the time of spindle formation. Eggs were observed until cleavage, or for a maximum of 90-100 min after fertilization, depending on the species. Control eggs in the microinjection chamber cleaved as rapidly and as synchronously as eggs from the same fertilization that were maintained in a beaker of seawater with constant gentle stirring. There was no significant difference in the time elapsed to spindle formation or to cleavage in eggs injected with buffer alone compared to that of uninjected eggs, indicating that the injection process did not injure the eggs over the course of the experiments (Fig. 1). Nuclear envelope breakdown was observed in 50% of the control Arbacia eggs at approximately 37 min after fertilization and metaphase spindles at 45 min, and cleavage occurred at 61.4 + 2.3 min. In the microinjection experiments, nuclear envelope breakdown was observed in 50% of the control L. picks eggs at 44 min after fertilization and metaphase spindle formation by 55 min., and cleavage occurred at 69.6 + 3.4 min. Data is reported only for those experiments in which the eggs were well synchronized, i.e., at least 80% of the eggs cleaved within a 5-min period and 95% within 10 min. Fertilized Arbacia eggs microinjected with PKI and observed for 90 min after fertilization were significantly delayed in the time elapsed until cleavage, compared to buffer-injected and uninjected controls (Table 1). Arb&u eggs were injected with 0.5 or 1.5 mg/ml of PKI. L. pictus eggs injected with 5 mg/ml of PKI were observed for up to 100 min after fertilization because of the longer time until the first cleavage compared to that for Arbuciu. Microinjection of PKI delayed the time to cleavage compared to that for uninjected and buffer-injected controls in this species as well (Table 1).

ET

AL.

While all concentrations of inhibitor tested were effective in inhibiting cleavage, in general the sooner that the inhibitor was injected, the greater the effect. In eggs injected with 1.5 and 5.0 mg/ml PKI, there was an apparent decrease in the extent of inhibition of cleavage when the PKI was injected later than 45 min after cleavage. The eggs were not followed through subsequent divisions because of a gradual loss of synchrony in eggs in the microinjection chamber. Increasing the concentration of PKI injected above 5 mg/ml did not seem to increase the inhibition of either mitotic spindle formation or cleavage (not shown). A portion of the eggs injected with PKI did not cleave at all during the 90- or lOO-min observation period (Fig. 1). Arbacia eggs injected at 20-30 min after fertilization showed the greatest number of uncleaved eggs. While eggs injected later were delayed in division, fewer remained uncleaved. The greatest percentage of L. picks eggs which did not cleave was in the group injected at 31-45 min after fertilization. Since metaphase spindles were observed in Arbuciu at approximately 45 min after fertilization and in L. p&us at approximately 55 min after fertilization, it appears that inhibition was greatest prior to the formation of the mitotic spindle. Because of the variation in the time to spindle formation and cleavage in different populations of eggs, the data do not show exactly when inhibition occurred relative to spindle formation. No nucleus was observed in the cells that did not cleave, indicating that nuclear envelope breakdown had occurred. However, microscopic examination of inhibitor-injected cells revealed that those cells that failed to divide also failed to form a mitotic spindle, although occasionally a birefringent spot or streak was observed in the spindle area. Preincubation of the PKI with the catalytic subunit of protein kinase A eliminated the delay in cleavage exhibited when PKI was injected alone (Table 2). PKI and catalytic subunit were incubated in a ratio of approximately 1:l (75 pg PKI, 70 pg catalytic subunit) in 50 ~1 microinjection buffer for 15 min prior to microinjection, for a final concentration of PKI of 1.5 mg/ml. This ratio provided a significant excess of catalytic subunit based upon the ratio of PKI to catalytic subunit that demonstrated no discernable effect of PKI on catalytic activity in the in vitro protein kinase activity assay. In microinjection experiments, L. variegutus nuclear envelope breakdown was observed at 45-50 min, metaphase spindles at 55 min, and early cleavage at 63.8 +- 5.8 min after fertilization (Table 2). The time to cleavage of microinjetted eggs was compared to that of uninjected control eggs in the same chamber by a paired t test. While PKI microinjected alone between 35 and 45 min after fertilization significantly delayed cleavage, PKI preincubated with an excess of catalytic subunit had no apparent effect on the time to cleavage. This supports the assumption that the observed PKI inhibition of mitosis is work-

INHIBITION

OF

CAMP

KINASE

INHIBITS

TABLE Microinjection

of the Protein

Kinase

Inhibitor

MITOSIS

IN

of CAMP-Dependent

Species

Time injected after fertilization (min)

URCHIN

125

EMBRYOS

1 Protein X time

PKI injected bdml)

SEA

Kinase

into Fertilized

Sea Urchin

Eggs

t.43cleavage (min)

Injected

Uninjected

n

P

A. punctuluta

0

20-30

59.8

59.1

12

ns

L. pictu.s

0 0.5 0.5 0.5 1.5 1.5 1.5 0

31-45 20-30 31-45 46-55 20-30 31-45 46-55 20-30

58.2 82.9 72.8 78.7 80.9 75.4 65.3 83.2

56.3 59.4 61.8 60.8 64.0 62.0 62.7 81.6*

186 18 12 20 17 9 11

0.:5 0.025 0.025 0.005 0.005 ns ns

05.0 5.0 5.0 5.0

31-45 20-30 31-45 46-55 56-66

97.1 71.5 95.8 83.0 76.6

73.8 74.3 75.3 72.3 72.0

138 11 13 7

o.o”d”5 0.0005 0.05 ns

Note. This particular batch of eggs cleaved significantly later than is usual for L. picks. However, as in the other experiments, there was no significant difference in time to cleavage between injected and uninjected controls.

ing through

inhibition

of the catalytic

subunit

of protein

kinase A. Measurement of protein kinase activity. The protein kinase activity of fertilized L. pictus and L. variegates eggs was determined through the first cleavage by the method of Beale et al. (1977). Eggs were homogenized at lo- or 15min intervals after fertilization and the phosphotransferase activity determined in the presence of histone 2b, a substrate for the CAMP-dependent protein kinase. Each sample was assayed in the presence of added CAMP, PKI, or HzO. The data were calculated as picomoles of 32P incorporated per milligram of total protein. Before homogenization of the eggs, a sample was examined for evidence of nuclear envelope breakdown, spindle formation, and cleavage. In L. pictus, the mean

TABLE

2

Microinjection of Protein Kinase Inhibitor Blocked by Preincubation with the Catalytic Subunit of Protein Kinase A into Fertilized L. uariegutw Eggs Time injected after fertilization (min) Uninjected control Blocked PKI Uninjected control Blocked PKI Uninjected control 1.5 mg/ml PKI

X time to cleavage * se

n

P

30-3s

64.69 + 1.27 65.81 f 1.45

16 16

ns

40-45

64.14 + 2.23 63.79 f 0.93

14 14

ns

35-45

62.83 f 1.05 72.22 + 2.27

18 18

Inhibition of mitosis in fertilized sea urchin eggs by inhibition of the cyclic AMP-dependent protein kinase.

Inhibition of cAMP-dependent protein kinase activity by microinjection of a specific physiologic protein inhibitor into sea urchin eggs inhibits the f...
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