ANALYTICALBIOCHEMISTRY
(1990)
l&87,94-97
A Versatile Microtiter Assay for the Universal cdc2 Cell Cycle Regulator Bernard
Ducommun
Cold Spring
Received
Harbor
November
and David
Laboratory,
Beach
PO Box 100, Cold Spring
Harbor,
21,1989
A microassay for ~34”~“’ based on the high affinity association between cdc2 and Schizosaccharomyces pombe ~13~~“’ has been developed. p13 purified from Escherichia coli was immobilized on microtiter plates and cellular lysate was incubated in the wells to allow the binding of cdc2 and its associated proteins. ~34”~“’ was assayed either as a histone kinase or by immunological methods. The method was optimized for S. pombe cell extracts but can also be applied to other organisms such as Xenopus oocytes or HeLa cells. This rapid assay allows the specific determination of ~34”~‘~ histone Hl kinase activity in a very large number of samples. 0 1990 Academic Press, Inc.
A mitotic kinase activity also called MPF (M-phase Promoting Factor) has been found in a wide variety of eukaryotic organisms and is responsible for the initiation of mitosis (reviewed in (1)). In its active mitotic form, this kinase activity consists of a stoichiometric complex between ~34, which in fission yeast is the product of the cdc2+ gene and ~63, a cyclin encoded by the cdcl3+ gene (2). cdc2 may complex with either cyclin A or B type proteins (3), both of which are involved in the initiation of mitosis and may confer slightly different properties on the protein kinase. A third cdc2-associated protein, referred to as ~60, was identified in HeLa cells and may play a role in the initiation of DNA replication rather than mitosis (4). Although a variety of proteins have been shown to be phosphorylated in vitro by purified cdc2/cyclin (5,6), to date histone Hl is the best in vitro substrate available (7). Two methods have been used to assay the mitotic histone Hl kinase activity of cdc2. Several investigators determined the level of histone Hl kinase activity in a crude lysate after inhibition of the other kinases such as CAMP and Ca2+-dependent protein kinases that might also phosphorylate histone (8). Others have specifically determined the kinase activity of cdc2 after selective 94
New York 11724
precipitation of the p34/cyclin complexes with anti-cdc2 or anti-cyclin antibodies (3,4,9), or by using pl3-Sepharose (9-11). p13 is the 13-kDa product (12) of the Schizosaccharomyces pombe sucl’ gene, which was isolated as a plasmid-borne sequence that could rescue some temperature-sensitive alleles of cdc2. p13 has been shown to be a subunit of the cdc2 protein kinase (12). The S. pombe sucl+ gene product, expressed in Escherichia coli and coupled to Sepharose, binds ~34”~“~ from many organisms and is a highly selective reagent for the purification of cdc2 and cdc2-associated proteins (7,lO). In this paper, we describe a method that takes advantage of the high affinity of p13 for cdc2, to develop a simplified and rapid assay for cdc2. MATERlALS
AND
METHODS
Materials Microtitration plates (Immobilon 2) were purchased from Dynatech. Bovine serum albumin (BSA)l was from Sigma. Peroxidase-labeled goat anti-rabbit antibodies and substrate detection kit were from Bio-Rad. Histone Hl was from Boerhinger-Mannheim. [T-~~P]ATP was from Amersham. Other chemicals were from Sigma. Methods
A 1-pg sample of purified p13 (S. pombe sucl’ gene product) from a bacterial expression system described by Brizuela et al. (12) was diluted in 100 ~1 of 0.1 M sodium carbonate buffer, pH 9.6, and incubated in each well of a microplate overnight at 4°C. Three washes were performed with PBS (phosphate saline buffer) containing 0.2% Triton X-100. Unoccupied sites on the wells 1 Abbreviations used: BSA, bovine serum saline buffer; PMSF, phenylmethylsulfonyl threitol; TCA, trichloroacetic acid; ABTS, zothiazoline-6-sulfonic acid.
albumin; PBS, phosphate fluoride; DTT, dithio2,2’-azinobis(3-ethylben0003-2697/90
$3.00
Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
MICROASSAY
FOR
cdc2
were saturated by incubation with 3% BSA in PBS for 2 h at room temperature. After a wash, with the buffer described above, the plates could be stored at -20°C for a few weeks or immediately used after two subsequent washes. S. pombe cells were broken using glassbeads and were prepared exactly as described (14) in buffer I (25 mM Tris-Hcl, pH 8.0,lO mM MgC& ,15 mM EGTA, and 0.1% Triton X-100) containing inhibitors of proteases (0.1 mM PMSF, 1 fig/ml leupeptin, 10 pg/ml soybean trypsin inhibitor, 1 pg/ml aprotinin, and 10 pg/ml tosyl phenylalanine chloromethyl ketone) and inhibitors of phosphatases (0.1 mM sodium fluoride, 60 mM P-glycerophosphate, 15 mM paranitrophenylphosphate and 0.1 mM sodium orthovanadate). Full-grown stage VI Xenopus oocytes (15) were prepared as described (16). Oocytes were induced to mature by addition of 1 PM progesterone to the external medium. Metaphase-arrested oocytes were homogenized at 4°C in 1 vol of extraction buffer (17) in the presence of protease inhibitors and centrifuged at 42,000 rpm for 90 min at 4°C in the SW50.1 rotor (Beckman). The supernatants were stored at -80°C until use and diluted in buffer I for the assay. HeLa cell extracts were prepared exactly as described (18) then were diluted in buffer I. Protein was determined as described (19). Samples (usually 200 pg of proteins) were diluted in buffer I and mixed in a 1:l ratio with 1% BSA in PBS (in order to avoid any nonspecific binding to the wells), then incubated in the microwells for the desired time (the optimal time is shown to be 5 h) at 4°C with slow constant shaking. The plates were then washed three times with PBS-0.2% Triton and once with kinase assay buffer (50 mM Tris-HCl, pH 7.4, 10 mM MgC12, and 1 mM DTT) containing inhibitors of phosphatases and proteases at the concentrations indicated above. Each wash consisted of filling the wells with the buffer and removing it by shaking inversion 2 min later. A loo-cl1 aliquot of a mixture containing 0.083 mg/ ml of histone Hl, 10 PM cold ATP, and 2.5 &i of [r3”P]ATP was added to each well and the reaction was allowed to procede for 30 min at the desired temperature (usually 30” C). The reaction was stopped by addition of 10 11 of Laemmli sample buffer (20). The reaction was quantified by spotting 15 ~1 of the reaction mixture on Whatmann 3MM paper followed by TCA precipitation. The paper was incubated first for 10 min in 10% TCA containing 40 mM sodium pyrophosphate then washed three times for 10 min in 5% TCA and briefly rinsed in cold ethanol. Quantitation was done using the AMBIS beta scanner system but can also be done by liquid scintillation. Alternatively, after incubation of the extracts in the microwells, the plates were washed three times with PBS-0.2% Triton X-100 and antibodies against p34 (GS and G6 kindly provided by Giulio Draetta) or cyclin (cdc13.6, kindly provided by Robert Booher) were used to detect the proteins bound to the well. Primary anti-
CELL
CYCLE
95
REGULATOR Coat well with pl3
immunolog~col
FIG. 1. Principle with the cell extract plexes (cdc2, cyclin) ted circle represents cyclin, respectively.
detection
Bind cdc2/cyclm
Kmase
complexes
osscy
of the microassay (1) ~13 coating (2) incubation containing p34 (3) Quantitation of bound comby kinase assay or immunological detection. Dot~13, open and dashed squares are for cdc2 and
bodies were incubated at the dilution of 1:lOOO in 200 ~1 of 3% BSA in PBS at 4°C for 2 h with slow shaking, and after three washes, secondary peroxydase-labeled antibodies against rabbit immunoglobulins were incubated at the dilution of 1:3000 in 200 ~1 of 3% BSA in PBS for 1 h at 4°C in the same condition. After three washes 200 ~1 of ABTS substrate, prepared according to the manufacturer’s instructions, was added and the calorimetric change quantified by absorbance determination at 415 nm. RESULTS
A standard method used to determine the histone Hl kinase activity of cdc2 involves incubation of a cell lysate in the presence of p13 crosslinked to Sepharose beads for several hours, followed by washing of the beads and determination of kinase activity in the presence of exogenous added histone Hl and [T~~P]ATP (4,10,11,14). The new assay reported here is based on the same ability of p13 to bind cdc2, but p13 was adsorbed to a solid support in the wells of a microtitration plate, rather than being crosslinked to Sepharose beads. This method eliminates the need for centrifugation between washes and allows a much large number of samples to be rapidly assayed. Figure 1 summarizes the three steps of the assay: (1) coating of plate with ~13, (2) p34 binding, and (3) kinase assay or immunological detection. Purified pl3 (12) was used to coat microtitration plates (see Materials and Methods) and after blockage of the unoccupied sites, cell lysates were incubated in the wells. After washing, the kinase activity of the p34/cyclin complexes bound to pl3 was determined by addition of histone Hl, cold ATP,
96
DUCOMMUN
AND
BEACH
A 4
,
I
01
I
0.2 protein
0.4
r
I 04 protem
I 0.6 (mg/welll
3
4 tlme(hours)
5
6
7
8
FIG. 3. Histone Hl kinase activity as a function of the incubation time of cell extract in microwells. S. pombe cell extract (200 pg) was added to each well and incubated for the indicated times. Each value is for three independant determinations. Only Q of the kinase reaction was TCA precipitated.
25
02
2
0.5
B
Oh 0
0
,
0.3 (mg/well)
0.8
I IO
FIG. 2. (A) Histone Hl kinase activity for increasing amount of protein from S. pombe, Xenopus metaphase-arrested oocytes, and HeLa extracts. Each value is the mean of three independents determinations and is obtained by TCA precipitation of g of the kinase reaction volume. (B) Relative level of cdc2 and cyclin bound to the well after incubation with increasing amounts of S. pombe protein extracts, immunologically determined by absorbance reading at 415 nm.
and [r3’P]ATP to the microwells. Since cdc2 is the only kinase that can bind to p13 (7,10), it is unnecessary to add inhibitors of others kinases in the reaction mixture. Alternatively, the amount of cdc2/cyclin complexes bound to p13 was quantified by an immunological technique similar to an “ELISA” (see Materials and Methods) using antibodies against either cyclin or cdc2. As an increasing amount of protein from S. pombe cell lysate was.added to the pl3-coated well a corresponding increase in the measurable histone Hl kinase was observed (Fig. 2A). This increase in kinase activity was a function of the amount of cdc2/cyclin complexes bound to pl3-coated wells, as shown using antibodies against cdc2 and cyclin (Fig. 2B). However, because of its highest sensitivity, the immunological detection was saturated for lowest amount of protein extract. Previous studies have demonstrated the generality of p13 binding properties in different species (7,10,11). In order to validate this assay for other organisms we determined the p34 histone Hl kinase activity associated with ~34”~“~ homologs from metaphase-arrested Xenopus oocytes and Hela cells. As in the case of S. pombe
extracts the kinase activity varies as a function of the amount of protein added in each well (Fig. 2A). In order to determine the optimal time of incubation of cell lysates in the wells, microplates were coated with 0.2 mg of S. pombe cell extract per well for various lengths of time and the kinase activity bound to the well was determined. After 5 h the kinase activity reached a plateau (Fig. 3). The incubation time of 5 h was then retained for all further experiments. The variation of the histone Hl kinase activity during the S. pombe cell cycle was assayed using the microplate assay, under the conditions described above. S. pombe temperature-sensitive mutant strain (c&25-22) was synchronized by transfer to a restrictive temperature (36°C) for 4.25 h, then released by a shift down to 25°C (13). Samples were taken every 15 min and cell extracts were prepared for the ~34”~‘~ kinase assay. The synchrony of the culture was determined by the fission plate index (Fig. 4). The histone Hl kinase activity showed
-^
6
%I -‘?, 4 x E e2
0
0
30
120 time (mini
FIG. 4. A culture of S. pombe (cdc25-22 mutant) was 36°C for 4.25 h and released by shift-down to 25’C. taken every 15 min. The cell plate index (dashed line) synchrony of the culture. cdc2 histone Hl kinase was triplicate for each sample in microwells coated with p13 or not coated (closed circles).
transferred to Samples were indicates the determined in (open circles)
MICROASSAY
FOR
cdc2
CELL
cell cycle oscillation similar to previously published results (2,8), peaking after block-release and decreasing as cells were leaving mitosis. The same experiment performed in wells which were not coated with p13 showed a low background and did not show any variation of histone Hl kinase activity during the cell cycle (Fig. 4), indicating that the kinase activity determined was one which bound to ~13.
CYCLE
97
REGULATOR
The present method does allow easy multiple determinations for each sample and for the handling of very large numbers of samples using very low amounts of cell extract (0.2 mg total protein). The technique might, for example, allow the testing of large numbers of pharmacological agents for their ability to interfer with cdc2 activity. ACKNOWLEDGMENTS
DISCUSSION A new microtiter plate assay for ~34’~“~ histone Hl kinase activity was presented. The parameters of the method were optimized for S. pombe protein extracts, but the assay is shown to be applicable with metaphasearrested Xenopus oocytes or HeLa extracts (Fig. 2A). Using an immunodetection method, we show that the amount of histone Hl kinase activity measured is related to the amount of cdc2/cyclin complexes bound to ~13 (Figs. 2A and 2B). Furthermore we show that the kinase activity bound to the wells is due to cdc2 and is not contaminated by nonspecific binding of an unrelated kinase from the cell extract (Fig. 4). The histone Hl kinase activity reached a plateau after 5 h of incubation of the extract in the microwells, indicating a probable saturation of the p13 molecules coated to the wells, An alternative method to assay cdc2 kinase in microtiter plates would have been to coat the wells with antibodies against one of the two known components of the mitotic kinase. But anti-cyclin antibodies are highly specific for one species and the antibodies against a consensus sequence for cdc2 (“PSTAIR” antibodies) have been shown to be inconsistently efficient in immunoprecipitating histone Hl kinase activity (21,22). The p13 reagent was chosen because of its specific and high binding affinity for cdc2 in every organism in which it has been tested, thus providing a universal reagent for measuring p34”d”2 mitotic kinase activity. After synchronization of a S. pombe exponentially growing culture, we used the new assay to illustrate the variation of the cdc2 kinase activity during the cell cycle (Fig. 4). The results were similar to those reported previously (2,8). These observations demonstrate that this assay has a sensitivity comparable to the other techniques usually used for ~34”~“’ histone Hl kinase determination. One objection to the use of p13 as a reagent to follow the cell cycle variation of ~34”~“~ activity is that the affinity of p13 for cdc2 has been described to fluctuate (2). Thus it is important to assess the binding of cdc2 to the microtiter wells by immunological methods.
We thank Guilio Draetta and Robert Booher for their bodies, Ed Harlow and Laurent Meijer for their suggestions, Jessus, James Bischoff, and Ulrich Deuschle for their ments on the manuscript, and Jim Duffy and Phil Renna aration of the figures. This work was supported by NIH D.B. and a Long Island Biological Association Fellowship
gifts of antiCatherine critical comfor the prepGM34607 to to B.D.
REFERENCES 1. Fantes, P. (1988) Trends Genet. 4,10,275-290. 2. Booher, R., Alpha, C., Hyams, J., and Beach, D. (1989) Cell 58, 485-497. 3. Draetta, G., Luca, F., Westendorf, J., Brizuela, L., Ruderman, J., and Beach, D. (1989) Cell 56,829-838. 4. Giordano, A., Whyte, P., Harlow, E., Franza, B. R., Beach, D., and Draetta, G. (1989) Cell 58,981-990. 5. McVey, D., Brizuela, L., Mohr, I., Marshak, D., Gluzman, Y., and Beach, D. (1989) Nature (London) 34 1,503~507. 6. Cisek, L., and Corden, J. (1989) Nature 339,679-684. 7. Brizuela, L., Draetta, G., and Beach, D. (1989) Proc. Natl. Acad. Sci. USA 86,4362-4366. 8. Moreno, S., Hayles, J., and Nurse, P. (1989) Cell 58,361-372. 9. Meijer, L., Arion, D., Golsteyn, R., Pines, J., Brizuela, L., Hunt, T., and Beach, D. (1989) EMBOJ. 8,2278-2282. 10. Pondaven, P., Meijer, L., and Beach, D. (1990) Genes Deo. 4, 917. 11. Dunphy, W. G., Brizuela, L., Beach, D., and Newport, J. (1988) Cell 54,423-431. 12. Brizuela, L., Draetta, G., and Beach, D. (1987) EMBO J. 6,35073514. 13. King, S. M., and Hyams, J. S. (1982) Canad. J. Microbial. 28,261264. 14. Ducommun, B., Draetta, G., Young, P., and Beach, them. Biophys. Res. Commun., in press. 15. Dumont, J. N. (1972) J. Morphol. 16. Jessus, C., Thibier, C., and Ozon, 712. 17. Wu, M., and Gerhart, J. G. (1980) 18. Draetta, G., Brizuela, 50,319-325. 19. 20. 21. 22.
L., Potashkin,
D. (1990)
Bio-
136, X3-180. R. (1987) J. Cell Sci. 87,
705-
Dev. Biol.
79,465477.
J., and Beach,
D. (1987)
Bradford, M. M. (1976) Anal. Biochem. 72,248-254. Laemmii, U. K. (1970) Nature (London) 277,680-685. Pines, J., and Hunter, T. (1989) Cell 58,833-846. Gautier, J., Norhury, C., Lohka, M., Nurse, P., and (1988) Cell 64,433-439.
Maller,
Cell
J.
(1990)
l&87,94-97
A Versatile Microtiter Assay for the Universal cdc2 Cell Cycle Regulator Bernard
Ducommun
Cold Spring
Received
Harbor
November
and David
Laboratory,
Beach
PO Box 100, Cold Spring
Harbor,
21,1989
A microassay for ~34”~“’ based on the high affinity association between cdc2 and Schizosaccharomyces pombe ~13~~“’ has been developed. p13 purified from Escherichia coli was immobilized on microtiter plates and cellular lysate was incubated in the wells to allow the binding of cdc2 and its associated proteins. ~34”~“’ was assayed either as a histone kinase or by immunological methods. The method was optimized for S. pombe cell extracts but can also be applied to other organisms such as Xenopus oocytes or HeLa cells. This rapid assay allows the specific determination of ~34”~‘~ histone Hl kinase activity in a very large number of samples. 0 1990 Academic Press, Inc.
A mitotic kinase activity also called MPF (M-phase Promoting Factor) has been found in a wide variety of eukaryotic organisms and is responsible for the initiation of mitosis (reviewed in (1)). In its active mitotic form, this kinase activity consists of a stoichiometric complex between ~34, which in fission yeast is the product of the cdc2+ gene and ~63, a cyclin encoded by the cdcl3+ gene (2). cdc2 may complex with either cyclin A or B type proteins (3), both of which are involved in the initiation of mitosis and may confer slightly different properties on the protein kinase. A third cdc2-associated protein, referred to as ~60, was identified in HeLa cells and may play a role in the initiation of DNA replication rather than mitosis (4). Although a variety of proteins have been shown to be phosphorylated in vitro by purified cdc2/cyclin (5,6), to date histone Hl is the best in vitro substrate available (7). Two methods have been used to assay the mitotic histone Hl kinase activity of cdc2. Several investigators determined the level of histone Hl kinase activity in a crude lysate after inhibition of the other kinases such as CAMP and Ca2+-dependent protein kinases that might also phosphorylate histone (8). Others have specifically determined the kinase activity of cdc2 after selective 94
New York 11724
precipitation of the p34/cyclin complexes with anti-cdc2 or anti-cyclin antibodies (3,4,9), or by using pl3-Sepharose (9-11). p13 is the 13-kDa product (12) of the Schizosaccharomyces pombe sucl’ gene, which was isolated as a plasmid-borne sequence that could rescue some temperature-sensitive alleles of cdc2. p13 has been shown to be a subunit of the cdc2 protein kinase (12). The S. pombe sucl+ gene product, expressed in Escherichia coli and coupled to Sepharose, binds ~34”~“~ from many organisms and is a highly selective reagent for the purification of cdc2 and cdc2-associated proteins (7,lO). In this paper, we describe a method that takes advantage of the high affinity of p13 for cdc2, to develop a simplified and rapid assay for cdc2. MATERlALS
AND
METHODS
Materials Microtitration plates (Immobilon 2) were purchased from Dynatech. Bovine serum albumin (BSA)l was from Sigma. Peroxidase-labeled goat anti-rabbit antibodies and substrate detection kit were from Bio-Rad. Histone Hl was from Boerhinger-Mannheim. [T-~~P]ATP was from Amersham. Other chemicals were from Sigma. Methods
A 1-pg sample of purified p13 (S. pombe sucl’ gene product) from a bacterial expression system described by Brizuela et al. (12) was diluted in 100 ~1 of 0.1 M sodium carbonate buffer, pH 9.6, and incubated in each well of a microplate overnight at 4°C. Three washes were performed with PBS (phosphate saline buffer) containing 0.2% Triton X-100. Unoccupied sites on the wells 1 Abbreviations used: BSA, bovine serum saline buffer; PMSF, phenylmethylsulfonyl threitol; TCA, trichloroacetic acid; ABTS, zothiazoline-6-sulfonic acid.
albumin; PBS, phosphate fluoride; DTT, dithio2,2’-azinobis(3-ethylben0003-2697/90
$3.00
Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
MICROASSAY
FOR
cdc2
were saturated by incubation with 3% BSA in PBS for 2 h at room temperature. After a wash, with the buffer described above, the plates could be stored at -20°C for a few weeks or immediately used after two subsequent washes. S. pombe cells were broken using glassbeads and were prepared exactly as described (14) in buffer I (25 mM Tris-Hcl, pH 8.0,lO mM MgC& ,15 mM EGTA, and 0.1% Triton X-100) containing inhibitors of proteases (0.1 mM PMSF, 1 fig/ml leupeptin, 10 pg/ml soybean trypsin inhibitor, 1 pg/ml aprotinin, and 10 pg/ml tosyl phenylalanine chloromethyl ketone) and inhibitors of phosphatases (0.1 mM sodium fluoride, 60 mM P-glycerophosphate, 15 mM paranitrophenylphosphate and 0.1 mM sodium orthovanadate). Full-grown stage VI Xenopus oocytes (15) were prepared as described (16). Oocytes were induced to mature by addition of 1 PM progesterone to the external medium. Metaphase-arrested oocytes were homogenized at 4°C in 1 vol of extraction buffer (17) in the presence of protease inhibitors and centrifuged at 42,000 rpm for 90 min at 4°C in the SW50.1 rotor (Beckman). The supernatants were stored at -80°C until use and diluted in buffer I for the assay. HeLa cell extracts were prepared exactly as described (18) then were diluted in buffer I. Protein was determined as described (19). Samples (usually 200 pg of proteins) were diluted in buffer I and mixed in a 1:l ratio with 1% BSA in PBS (in order to avoid any nonspecific binding to the wells), then incubated in the microwells for the desired time (the optimal time is shown to be 5 h) at 4°C with slow constant shaking. The plates were then washed three times with PBS-0.2% Triton and once with kinase assay buffer (50 mM Tris-HCl, pH 7.4, 10 mM MgC12, and 1 mM DTT) containing inhibitors of phosphatases and proteases at the concentrations indicated above. Each wash consisted of filling the wells with the buffer and removing it by shaking inversion 2 min later. A loo-cl1 aliquot of a mixture containing 0.083 mg/ ml of histone Hl, 10 PM cold ATP, and 2.5 &i of [r3”P]ATP was added to each well and the reaction was allowed to procede for 30 min at the desired temperature (usually 30” C). The reaction was stopped by addition of 10 11 of Laemmli sample buffer (20). The reaction was quantified by spotting 15 ~1 of the reaction mixture on Whatmann 3MM paper followed by TCA precipitation. The paper was incubated first for 10 min in 10% TCA containing 40 mM sodium pyrophosphate then washed three times for 10 min in 5% TCA and briefly rinsed in cold ethanol. Quantitation was done using the AMBIS beta scanner system but can also be done by liquid scintillation. Alternatively, after incubation of the extracts in the microwells, the plates were washed three times with PBS-0.2% Triton X-100 and antibodies against p34 (GS and G6 kindly provided by Giulio Draetta) or cyclin (cdc13.6, kindly provided by Robert Booher) were used to detect the proteins bound to the well. Primary anti-
CELL
CYCLE
95
REGULATOR Coat well with pl3
immunolog~col
FIG. 1. Principle with the cell extract plexes (cdc2, cyclin) ted circle represents cyclin, respectively.
detection
Bind cdc2/cyclm
Kmase
complexes
osscy
of the microassay (1) ~13 coating (2) incubation containing p34 (3) Quantitation of bound comby kinase assay or immunological detection. Dot~13, open and dashed squares are for cdc2 and
bodies were incubated at the dilution of 1:lOOO in 200 ~1 of 3% BSA in PBS at 4°C for 2 h with slow shaking, and after three washes, secondary peroxydase-labeled antibodies against rabbit immunoglobulins were incubated at the dilution of 1:3000 in 200 ~1 of 3% BSA in PBS for 1 h at 4°C in the same condition. After three washes 200 ~1 of ABTS substrate, prepared according to the manufacturer’s instructions, was added and the calorimetric change quantified by absorbance determination at 415 nm. RESULTS
A standard method used to determine the histone Hl kinase activity of cdc2 involves incubation of a cell lysate in the presence of p13 crosslinked to Sepharose beads for several hours, followed by washing of the beads and determination of kinase activity in the presence of exogenous added histone Hl and [T~~P]ATP (4,10,11,14). The new assay reported here is based on the same ability of p13 to bind cdc2, but p13 was adsorbed to a solid support in the wells of a microtitration plate, rather than being crosslinked to Sepharose beads. This method eliminates the need for centrifugation between washes and allows a much large number of samples to be rapidly assayed. Figure 1 summarizes the three steps of the assay: (1) coating of plate with ~13, (2) p34 binding, and (3) kinase assay or immunological detection. Purified pl3 (12) was used to coat microtitration plates (see Materials and Methods) and after blockage of the unoccupied sites, cell lysates were incubated in the wells. After washing, the kinase activity of the p34/cyclin complexes bound to pl3 was determined by addition of histone Hl, cold ATP,
96
DUCOMMUN
AND
BEACH
A 4
,
I
01
I
0.2 protein
0.4
r
I 04 protem
I 0.6 (mg/welll
3
4 tlme(hours)
5
6
7
8
FIG. 3. Histone Hl kinase activity as a function of the incubation time of cell extract in microwells. S. pombe cell extract (200 pg) was added to each well and incubated for the indicated times. Each value is for three independant determinations. Only Q of the kinase reaction was TCA precipitated.
25
02
2
0.5
B
Oh 0
0
,
0.3 (mg/well)
0.8
I IO
FIG. 2. (A) Histone Hl kinase activity for increasing amount of protein from S. pombe, Xenopus metaphase-arrested oocytes, and HeLa extracts. Each value is the mean of three independents determinations and is obtained by TCA precipitation of g of the kinase reaction volume. (B) Relative level of cdc2 and cyclin bound to the well after incubation with increasing amounts of S. pombe protein extracts, immunologically determined by absorbance reading at 415 nm.
and [r3’P]ATP to the microwells. Since cdc2 is the only kinase that can bind to p13 (7,10), it is unnecessary to add inhibitors of others kinases in the reaction mixture. Alternatively, the amount of cdc2/cyclin complexes bound to p13 was quantified by an immunological technique similar to an “ELISA” (see Materials and Methods) using antibodies against either cyclin or cdc2. As an increasing amount of protein from S. pombe cell lysate was.added to the pl3-coated well a corresponding increase in the measurable histone Hl kinase was observed (Fig. 2A). This increase in kinase activity was a function of the amount of cdc2/cyclin complexes bound to pl3-coated wells, as shown using antibodies against cdc2 and cyclin (Fig. 2B). However, because of its highest sensitivity, the immunological detection was saturated for lowest amount of protein extract. Previous studies have demonstrated the generality of p13 binding properties in different species (7,10,11). In order to validate this assay for other organisms we determined the p34 histone Hl kinase activity associated with ~34”~“~ homologs from metaphase-arrested Xenopus oocytes and Hela cells. As in the case of S. pombe
extracts the kinase activity varies as a function of the amount of protein added in each well (Fig. 2A). In order to determine the optimal time of incubation of cell lysates in the wells, microplates were coated with 0.2 mg of S. pombe cell extract per well for various lengths of time and the kinase activity bound to the well was determined. After 5 h the kinase activity reached a plateau (Fig. 3). The incubation time of 5 h was then retained for all further experiments. The variation of the histone Hl kinase activity during the S. pombe cell cycle was assayed using the microplate assay, under the conditions described above. S. pombe temperature-sensitive mutant strain (c&25-22) was synchronized by transfer to a restrictive temperature (36°C) for 4.25 h, then released by a shift down to 25°C (13). Samples were taken every 15 min and cell extracts were prepared for the ~34”~‘~ kinase assay. The synchrony of the culture was determined by the fission plate index (Fig. 4). The histone Hl kinase activity showed
-^
6
%I -‘?, 4 x E e2
0
0
30
120 time (mini
FIG. 4. A culture of S. pombe (cdc25-22 mutant) was 36°C for 4.25 h and released by shift-down to 25’C. taken every 15 min. The cell plate index (dashed line) synchrony of the culture. cdc2 histone Hl kinase was triplicate for each sample in microwells coated with p13 or not coated (closed circles).
transferred to Samples were indicates the determined in (open circles)
MICROASSAY
FOR
cdc2
CELL
cell cycle oscillation similar to previously published results (2,8), peaking after block-release and decreasing as cells were leaving mitosis. The same experiment performed in wells which were not coated with p13 showed a low background and did not show any variation of histone Hl kinase activity during the cell cycle (Fig. 4), indicating that the kinase activity determined was one which bound to ~13.
CYCLE
97
REGULATOR
The present method does allow easy multiple determinations for each sample and for the handling of very large numbers of samples using very low amounts of cell extract (0.2 mg total protein). The technique might, for example, allow the testing of large numbers of pharmacological agents for their ability to interfer with cdc2 activity. ACKNOWLEDGMENTS
DISCUSSION A new microtiter plate assay for ~34’~“~ histone Hl kinase activity was presented. The parameters of the method were optimized for S. pombe protein extracts, but the assay is shown to be applicable with metaphasearrested Xenopus oocytes or HeLa extracts (Fig. 2A). Using an immunodetection method, we show that the amount of histone Hl kinase activity measured is related to the amount of cdc2/cyclin complexes bound to ~13 (Figs. 2A and 2B). Furthermore we show that the kinase activity bound to the wells is due to cdc2 and is not contaminated by nonspecific binding of an unrelated kinase from the cell extract (Fig. 4). The histone Hl kinase activity reached a plateau after 5 h of incubation of the extract in the microwells, indicating a probable saturation of the p13 molecules coated to the wells, An alternative method to assay cdc2 kinase in microtiter plates would have been to coat the wells with antibodies against one of the two known components of the mitotic kinase. But anti-cyclin antibodies are highly specific for one species and the antibodies against a consensus sequence for cdc2 (“PSTAIR” antibodies) have been shown to be inconsistently efficient in immunoprecipitating histone Hl kinase activity (21,22). The p13 reagent was chosen because of its specific and high binding affinity for cdc2 in every organism in which it has been tested, thus providing a universal reagent for measuring p34”d”2 mitotic kinase activity. After synchronization of a S. pombe exponentially growing culture, we used the new assay to illustrate the variation of the cdc2 kinase activity during the cell cycle (Fig. 4). The results were similar to those reported previously (2,8). These observations demonstrate that this assay has a sensitivity comparable to the other techniques usually used for ~34”~“’ histone Hl kinase determination. One objection to the use of p13 as a reagent to follow the cell cycle variation of ~34”~“~ activity is that the affinity of p13 for cdc2 has been described to fluctuate (2). Thus it is important to assess the binding of cdc2 to the microtiter wells by immunological methods.
We thank Guilio Draetta and Robert Booher for their bodies, Ed Harlow and Laurent Meijer for their suggestions, Jessus, James Bischoff, and Ulrich Deuschle for their ments on the manuscript, and Jim Duffy and Phil Renna aration of the figures. This work was supported by NIH D.B. and a Long Island Biological Association Fellowship
gifts of antiCatherine critical comfor the prepGM34607 to to B.D.
REFERENCES 1. Fantes, P. (1988) Trends Genet. 4,10,275-290. 2. Booher, R., Alpha, C., Hyams, J., and Beach, D. (1989) Cell 58, 485-497. 3. Draetta, G., Luca, F., Westendorf, J., Brizuela, L., Ruderman, J., and Beach, D. (1989) Cell 56,829-838. 4. Giordano, A., Whyte, P., Harlow, E., Franza, B. R., Beach, D., and Draetta, G. (1989) Cell 58,981-990. 5. McVey, D., Brizuela, L., Mohr, I., Marshak, D., Gluzman, Y., and Beach, D. (1989) Nature (London) 34 1,503~507. 6. Cisek, L., and Corden, J. (1989) Nature 339,679-684. 7. Brizuela, L., Draetta, G., and Beach, D. (1989) Proc. Natl. Acad. Sci. USA 86,4362-4366. 8. Moreno, S., Hayles, J., and Nurse, P. (1989) Cell 58,361-372. 9. Meijer, L., Arion, D., Golsteyn, R., Pines, J., Brizuela, L., Hunt, T., and Beach, D. (1989) EMBOJ. 8,2278-2282. 10. Pondaven, P., Meijer, L., and Beach, D. (1990) Genes Deo. 4, 917. 11. Dunphy, W. G., Brizuela, L., Beach, D., and Newport, J. (1988) Cell 54,423-431. 12. Brizuela, L., Draetta, G., and Beach, D. (1987) EMBO J. 6,35073514. 13. King, S. M., and Hyams, J. S. (1982) Canad. J. Microbial. 28,261264. 14. Ducommun, B., Draetta, G., Young, P., and Beach, them. Biophys. Res. Commun., in press. 15. Dumont, J. N. (1972) J. Morphol. 16. Jessus, C., Thibier, C., and Ozon, 712. 17. Wu, M., and Gerhart, J. G. (1980) 18. Draetta, G., Brizuela, 50,319-325. 19. 20. 21. 22.
L., Potashkin,
D. (1990)
Bio-
136, X3-180. R. (1987) J. Cell Sci. 87,
705-
Dev. Biol.
79,465477.
J., and Beach,
D. (1987)
Bradford, M. M. (1976) Anal. Biochem. 72,248-254. Laemmii, U. K. (1970) Nature (London) 277,680-685. Pines, J., and Hunter, T. (1989) Cell 58,833-846. Gautier, J., Norhury, C., Lohka, M., Nurse, P., and (1988) Cell 64,433-439.
Maller,
Cell
J.
