of cyclin A (Murray and Kirschner, 1989). Yet these .... as a model B-type cyclin (Murray et al., 1989; Belmont ...... MacNeill,S. and Philippe,M. (1991) Proc. Natl.
The EMBO Journal vol. 1 1 no. 5 pp. 1 751 - 1761, 1992
Cyclin A- and cyclin B-dependent protein kinases are regulated by different mechanisms in Xenopus egg extracts
Paul R.Clarke', Dagmar Leiss, Michele Pagano2 and Eric Karsentil Cell Biology and 2Differentiation Programmes, European Molecular Biology Laboratory, Postfach 102209, 6900 Heidelberg, FRG 'Corresponding authors Communicated by T.Hunt
Cyclins are proteins which are synthesized and degraded in a cell cycle-dependent fashion and form integral regulatory subunits of protein kinase complexes involved in the regulation of the cell cycle. The best known catalytic subunit of a cyclin-dependent protein kinase complex is p34Cdc2. In the cell, cyclins A and B are synthesized at different stages of the cell cycle and induce protein kinase activation with different kinetics. The kinetics of activation can be reproduced and studied in extracts of Xenopus eggs to which bacterially produced cyclins are added. In this paper we report that in egg extracts, both cyclin A and cyclin B associate with and activate the same catalytic subunit, p34"c"2. In addition, cyclin A binds a less abundant p33 protein kinase related to p34cd"2, the product of the cdk2/Egl gene. When complexed to cyclin B, p34cdc2 is subject to transient inhibition by tyrosine phosphorylation, producing a lag between the addition of cyclin and kinase activation. In contrast, p34cdc2 is only weakly tyrosine phosphorylated when bound to cyclin A and activates rapidly. This finding shows that a given kinase catalytic subunit can be regulated in a different manner depending on the nature of the regulatory subunit to which it binds. Tyrosine phosphorylation of p34Cdc2 when complexed to cyclin B provides an inhibitory check on the activation of the M phase inducing protein kinase, allowing the coupling of processes such as DNA replication to the onset of metaphase. Our results suggest that, at least in the early Xenopus embryo, cyclin A-dependent protein kinases may not be subject to this checkpoint and are regulated primarily at the level of cyclin translation. Key words: cdc2 protein kinase/cyclin/tyrosine phosphorylationlXenopus egg extracts
Introduction During the eukaryotic cell cycle, microtubule dynamics, membrane organization and DNA synthesis are tightly controlled and specific changes are induced at particular points in the cycle. Coordination of these processes is achieved by a regulatory system which responds to checkpoints at major transitions in the cycle (Hartwell and Weinert, 1989). Two key checkpoints are at the GI -S and G2-M phase transitions. In the fission yeast Schizosaccharomyces pombe, both transitions are dependent on the activity of a 34 kDa protein kinase catalytic subunit (D Oxford University Press
encoded by the cdc2 gene, denoted p34cdc2 (Nurse, 1990). The homologue of this gene in the budding yeast Saccharomyces cerevisiae, CDC28, is also essential at the GI-S and G2-M transitions (Reed et al., 1985; Reed and Wittenberg, 1990; Surana et al., 1991). Similarly, in higher eukaryotes, p34cdc2 homologues are required at these phase transitions (Nurse, 1990; Fang and Newport, 1991). The protein kinases of the cdc2 family induce phase transitions by phosphorylating key proteins, which are poorly defined as yet (reviewed in Moreno and Nurse, 1990; Pines and Hunter, 1990b). The temporal regulation of these kinases involves their periodic association with regulatory subunits, the cyclins. Cyclins were initially identified in invertebrate eggs as a class of proteins whose abundance oscillates during the cell cycle, gradually accumulating during interphase and disappearing abruptly at the end of mitosis (Evans et al., 1983). More recently, it has become apparent that different cyclin-like proteins accumulate during the GI and G2 phases (Hunter and Pines, 1991). During G2, two classes of cyclins (A and B types, also called mitotic cyclins) have been recognized, initially on the basis of sequence conservation (Draetta, 1990; Hunt, 1989) and more recently by function (Buendia et al., 1991). B-type cyclins have been found in all eukaryotes examined so far, from yeast to human, and associate tightly with p34cdc2 to form a protein kinase complex that seems to induce metaphase proper (reviewed in Nurse, 1990). The role of cyclin A is less clear. Genetic analysis in yeast has not been possible because, perhaps significantly, cyclin A has not been identified in these organisms. In vertebrates, A- and B-type cyclins have appeared to be functionally similar in some experiments. For example, the injection of mRNA encoding either cyclin A or cyclin B into Xenopus oocytes induces meiotic maturation, although a combination of the two is more effective (Pines and Hunt, 1987; Swenson et al., 1986; Westendorf et al., 1989). However, cyclin A does not appear to be necessary for meiotic maturation of Xenopus oocytes (Minshull et al., 1991). In egg extracts, translation of cyclin B mRNA seems to be sufficient to power several mitotic cycles in the absence of cyclin A (Murray and Kirschner, 1989). Yet these are rather special conditions and the independent evolutionary conservation of A and B type cyclins strongly suggests that they do have distinct functions in the cell cycle. In Drosophila, cyclin A mRNA is abundant in embryos and mutations in the cyclin A gene abolish division in certain cells after exhaustion of the maternal stocks of cyclin A. The cells arrest in G2, even though cyclin B is present, clearly indicating a role for cyclin A in the entry of cells into mitosis, at least at this stage of embryonic development (Lehner and O'Farrell, 1989, 1990b). Recently, cyclin A has been found to produce quite different effects from cyclin B on cell cycle controlled processes such as microtubule nucleation and dynamics (Buendia et al., 1991) and endosome fusion (Thomas et al., 1992), consistent with a role for cyclin A during prophase. Perhaps the most intriguing difference between cyclin A and 1 751
P.R.Clarke et al.
cyclin B is the timing of activation of their associated protein kinases, usually assayed using histone HI as an exogenous substrate. In Xenopus eggs, the kinase activity associated with cyclin A reaches a maximum when the cyclin B-associated kinase activity is just beginning to rise. Cyclin A is degraded before the B-type cyclins and just prior to the breakdown of the nuclear envelope (Minshull et al., 1990). Similarly, in mammalian and insect cells, cyclin A appears earlier in the cell cycle than cyclin B (Lehner and O'Farrell, 1989; Pines and Hunter, 1990a; Whitfield et al., 1990). Cyclin A does not activate the cyclin destruction mechanism like cyclin B, which would otherwise prevent the accumulation of cyclin B after cyclin A (Luca et al., 1991). It is noteworthy that, at least in Xenopus eggs and human cells, the cyclin Aassociated kinase activity increases roughly in parallel with the accumulation of cyclin A, whereas the cyclin B-associated protein kinase activity remains low during G2 even though high levels of cyclin B have already been synthesized (Minshull et al., 1990; Pines and Hunter, 1990a). It has recently become apparent that in addition to its 'mitotic' function, cyclin A also acts during S phase and is present in the nucleus at this stage (Pines and Hunter, 1991; Pagano et al., 1992). In somatic cells, cyclin A associates with p34cdc2 and another closely related protein kinase catalytic subunit, the product of the cdk2 gene (Pines and Hunter, 1990a; Tsai et al., 1991). Cyclin A also associates with the adenovirus transforming protein ElA (Giordano et al., 1989; Pines and Hunter, 1990a) and has been implicated in carcinogenesis (Wang et al., 1990; Hunter and Pines, 1991; reviewed in Moran, 1991). The timing and kinetics of cyclin B -p34cdc2 activation at the entry into mitosis suggests that simple association with cyclin B is not sufficient to activate p34cdc2 and indeed posttranslational modifications are also involved (reviewed in Clarke and Karsenti, 1991). When cyclin B binds to p34cdc2, the kinase subunit becomes tyrosine phosphorylated immediately at an inhibitory site (Tyrl5) (Gould and Nurse, 1989; Solomon et al., 1990; Meijer et al., 1991; Parker et al., 1991), probably catalysed by homologues of the S.pombe weel and/or miki genes (Featherstone and Russell, 1991; Lundgren et al., 1991). An inhibitory threonine phosphorylation site (Thr14) is also present in vertebrate p34cdc2 (Krek and Nigg, 1991a, b; Norbury et al., 1991), although the timing of phosphorylation of this site is less certain. In addition, tight association of cyclin B with p34cdc2 requires phosphorylation at another threonine site (Thrl67 in S.Pombe, Thrl61 in vertebrates) (Ducommun et al., 1991; Gould et al., 1991). Activation at the G2-M transition requires Tyrl5 and presumably Thr14 dephosphorylation. As cyclin B is synthesized during interphase, the inactive and tyrosine phosphorylated form of the cyclin B-p34cdc2 complex accumulates. Tyrosine dephosphorylation and activation of cyclin B-p34cdc2 is under the control of cdc25, which encodes an unusual protein phosphatase (Russell and Nurse, 1986; Dunphy and Kumagai, 1991; Gautier et al., 1991; Kumagai and Dunphy, 1991; Strausfeld et al., 1991; Millar et al., 1991). Tyrosine dephosphorylation can be blocked by unreplicated DNA, suggesting that a checkpoint exists to prevent activation of cyclin B -p34cdc2 and entry into mitosis if DNA replication is not complete (Dasso and Newport, 1990; Enoch and Nurse, 1990; Meijer et al., 1991; Walker and Maller, 1991). The gradual rise in the kinase activity associated with cyclin 1752
A suggests that the regulatory mechanism that keeps the cyclin B-dependent kinase activity suppressed during G2, may not exist or function differently in the cyclin Adependent kinase complex. In this report, we examine this point. We find that in Xenopus egg extracts cyclin A associates predominantly with the same 34 kDa catalytic subunit as cyclin B, namely p34cdc2. Cyclin A also binds specifically to a distinct 33 kDa catalytic subunit, the product of the cdk2 gene, although this is much less abundant, and the majority of the kinase activity associated with cyclin A is due to p34cdc2. However, the regulation of p34cdc2 is different when it is associated with cyclin B and cyclin A; when complexed to cyclin B, p34cdc2 is transiently inhibited by tyrosine phosphorylation, producing a lag before kinase activation, whereas p34cdc2 associated with cyclin A is not inhibited by tyrosine phosphorylation and activates rapidly.
Results In Xenopus egg extracts, cyclin A and cyclin B activate histone Hl kinase with different kinetics In order to dissect the mechanism of protein kinase activation by A- and B-type cyclins, we used extracts of Xenopus eggs blocked in interphase by cycloheximide treatment. This inhibits protein synthesis and prevents the reaccumulation of cyclins after their degradation following egg activation. No cyclin A, Bi or B2 could be detected by specific antibodies in these interphase extracts (not shown) and no
change in protein kinase activity towards histone HI occured when the interphase extracts were incubated at room temperature without exogenous cyclins (Figure 1). Bacterially expressed exogenous cyclins were added to these extracts and their ability to activate p34cdc2-like catalytic subunits was assessed by assaying the level of histone H1 kinase activity produced. We used the truncated sea urchin cyclin BA90, which has been used in several other studies as a model B-type cyclin (Murray et al., 1989; Belmont et al., 1990; Vale, 1991; Glotzer et al., 1991). As found previously by Solomon et al. (1990), high levels of cyclin BA90 activated an histone HI kinase in these extracts to a high level but only after a lag period (Figure 1). This lag was independent of the amount of cyclin added, but was lengthened by reducing the temperature of the incubation or by dilution of the extract (not shown). Activation occurred only above a threshold concentration of cyclin BA90 (50 nM). Above the cyclin threshold, and after the lag period, the rate of activation was similar over at least a 4-fold range of cyclin concentrations (Figure 1). This behaviour reproduces the cyclin threshold and lag period observed for activation of the mitotic protein kinase in vivo (Solomon et al. 1990). In Xenopus, two B-type cyclins have been found (Minshull et al., 1990), although it is not known whether they have different functions and we are currently investigating this. In this study, we have focused on the mechanism of protein kinase activation by A-type cyclins. Human cyclin A (200 nM) also induced an activation of histone H1 kinase in interphase extracts, but there was no lag period (Figure 1). Addition of lower amounts of cyclin A produced lower levels of kinase activity, but there was no absolute threshold in the amount of cyclin A required to activate the histone H 1 kinase (Figure 1). The rate of activation, however, was reduced at low cyclin A concentrations. The kinase activity induced
Regulation of cyclin-dependent protein kinases
A 0
-
.2 *
I
6.
/
2
Sc
:015E E. -E 20
Human
cyclin A
200
100
C
300
[cyclin Al (nM)
B 0.4-
10
0c'a
0.3
.-
2-0.2Sc
20
40
60
80
100
[cyclin AJ (nM) Fig. 2. Initial rates of kinase activation by cyclin A in the concentration range 0-300 nM (A) or 0-100 nM (B) cyclin A. In (B) the mean and Incubation time (mmn) Fig. 1. Activation of protein kinase activity in interphase extracts by bacterially expressed sea urchin cyclin BA9O, human cyclin A or Xenopus cyclin A. Cyclins were diluted in XB and added to an interphase extract in 1/10 of the final volume (10 pi). The final concentrations of cyclins were 20 nM (closed triangles), 50 nM (closed squares), 100 nM (closed circles), 200 nM (open squares). Control incubations were performned without added cyclins (Open circles). At the indicated times, samples (1 pl) were removed into extraction buffer (EB) and assayed for histone H 1 kcinase activity.
by cyclin A was stable upon prolonged incubation in these extracts (not shown). Xenopus cyclin A activates histone Hi kinase with very similar characteristics to human cyclin A (Figure 1), although the level of kinase activity induced was lower. Similarly, an N-terminal truncated form of bovine cyclin A also activates histone Hi kinase rapidly without any lag period (Thomas et al., 1992; T.Hunt, personal communication). These characteristics of kinase activation, ie. no lag period and no cyclin threshold, therefore seem to be common to A-type cyclins and we used human cyclin A in all of the other experiments described in this report. We measured the initial rate of activation of histone Hi kinase at different cyclin A concentrations more precisely by taking samples at shorter intervals. Above 100 nM cyclin A, the initial rate of kinase activation was proportional to concentration of cyclin A (Figure 2A). At lower levels of cyclin A, a concave plot was obtained (Figure 2B). There was no 'all or nothing' response like that observed with cyclin BA9s (Figure 1), although the rate of activation at 25 nM cyclin A was very low.
range of two duplicate experiments are shown.
cyc A+13
.t3} 10
_
K
l
. .c
20
40
60
80
Incubation time (min) Fig. 3. The kinase activity induced by cyclin BAv9O and cyclin A is not additive. 200 nM cyclin BA9O alone (open triangles). 200 nM cyclin A alone (open squares) or cyclin BA9O and cyclin A together (closed squares) were added to interphase extracts and samples removed for the assay of histone Hi kinase activity at the indicated times.
In Xenopus egg extracts, the major kinase catalytic subunit activated by cyclin A and cyclin B is p34cdc2 It has been reported previously that in Xenopus egg extracts, endogenous cyclin A and cyclin B associate with p34cdc2 (Minshull et al., 1990). The identification of the kinase subunit was based on detection with an antibody directed against the PSTAIR sequence conserved in p34cdc2 homologues from other species and ability to bind the
1753
P.R.Clarke et al.
p13sucl protein. However, as Minshull et al. (1990) noted this did not settle the question of whether the same kinase subunit(s) can bind to either cyclin A or cyclin B. Several NotrImrnt 0
-0 -*.
no cyclin + cyclln A ccyclin BISO
Bmrstd - -4- - no cyclin - - a-- * cycln A - ---- cyclin B,9O C
E E 0
%_.I
0
s~
Q0 s
p34cdc2 related proteins have recently been identified in Xenopus that would also fit these criteria (Paris et. al., 1991; T.Hunt, personal communication). If the two cyclins interacted with different catalytic subunits in egg extracts, the activation of histone HI kinase by exogenous A and Btype cyclins should be additive. As shown in Figure 3 this is clearly not the case, suggesting that the two cyclins activate the same protein kinase catalytic subunit. To examine further the relationship between the kinase activities induced by the two cyclins, we used cyclin A or cyclin BA90 coupled to Sepharose beads. These reagents induced histone HI kinase activity efficiently when added to interphase extracts and could be reisolated by low speed centrifugation. We used these beads to deplete specifically cyclin A- or cyclin B-associated kinase catalytic subunits from the extracts. Soluble cyclins were then added to the depleted extracts and the effect on histone HI kinase activity upon subsequent incubation examined (Figure 4). In a control experiment, BSA beads were used which did not significantly alter kinase activation by either cyclin A or cyclin BA90 (Figure 4, upper panel). The activation of histone HI kinase upon addition of soluble cyclin A or cyclin BA90 to extracts was greatly reduced following depletion using cyclin BA90 beads (Figure 4, middle panel). A similar result was obtained with extracts depleted using cyclin A beads (Figure 4, lower panel). In both cases, a small increase in kinase activity was observed when cyclin A was added. This was probably due to the incomplete elimination of p34Cdc2 from the extracts. By immunoblotting depleted extracts with an antibody specific for p34cdc2, we estimated that 10% of the initial amount of p34cdc2 was left in the extract (not shown). Cyclin BA90 did not activate these residual p34cdc2 molecules, probably because of the threshold phenomenon. These results further support the view that, in egg extracts, the major kinase catalytic subunit activated by cyclin A is the same as that activated by cyclin B, that is p34cdc2, but they do not eliminate the possibility that an additional catalytic subunit is activated by cyclin A alone. The nature of the cdc2-like molecules bound to the cyclin A- and cyclin BA90 beads was examined direcfly by Western blotting using antibodies directed against the N-terminal 12 amino acids of starfish p34cdc2 ('anti-N-terminal'), which cross-reacts specifically with Xenopus p34cdc2, or against the PSTAIR region of p34cdc2-like kinases ('anti-PSTAIR'). As shown in Figure 5A, the anti-N-terminal antibody detected a single polypeptide of 34 kDa that bound to the -
10 4 20 30 Incubation time (min) Fig. 4. Effect of the depletion of cyclin BA90- or cyclin A-dependent protein kinases on the subsequent activation of H I kinase activity by cyclin BA90 or cyclin A. Interphase extracts were incubated with cyclin A beads, cyclin BA90 beads or BSA beads for 30 min. Then, the beads were removed by centrifugation. Soluble cyclin A (squares) or cyclin BA90 (triangles) were added to the depleted extracts (broken lines) or untreated control extracts (continuous lines) to a final concentration of 200 nM and aliquots removed for assay of H I kinase activity at the indicated times. BSA- and cyclin BA90 beads depletions were performed in the same experiment and the same control incubations using untreated extracts are shown. Incubations without added cyclins, shown in the upper panel, are omitted in the middle and lower panels for clarity; no activation occured in the absence of exogenous cyclins in any incubation.
A
cycA-beads
cycB\90-beads
PSTAiR' _______-
N-term PSTAI R
N-term 49 5
--*I-
1'
N-term PSTAIR nrti-cdk2 1[cycA- cAlcycA- k 2 |WA Peas cd b6a c s cd k2 i(i s cd k 92
32.5_ 27.5 -_
275_5 *
B 49.5
32 5 ---
185
-
_.
18.5 --*_
Fig. 5. The nature of cdc2-like molecules associated with cyclin A and cyclin BA90. (A) Cyclin A-Sepharose or cyclin BA90-Sepharose beads were incubated with interphase extracts, producing histone HI kinase activity of 14. 1 and 13.2 pmol/min/yl respectively. The cdc2-like molecules bound to each were analysed by Western blotting using anti-N-terminal cdc2 antibody (N-term) or rabbit anti-PSTAIR sequence antibody (PSTAIR). (B) Detection of purified Xenopus cdk2/Egl protein (20 ng) or the cdc2-like molecules bound to cyclin A beads using anti-N-terminal cdc2 antibody (N-term), mouse monoclonal anti-PSTAIR sequence antibody (PSTAIR) or an antibody raised against the Xenopus cdk2/Eg I protein (anti-cdk2). In this case, electrophoresis was performed at low current which increased the resolution of the two major bands containing the PSTAIR sequence. The positions of molecular mass marker proteins (Biorad, prestained low range) are indicated on the left with molecular mass in kDa.
1754
Regulation of cyclin-dependent protein kinases
homologue of cdk2 (also known as EgI, Paris et al., 1991) produced in Escherichia coli and was detected specifically by an antibody raised against Xenopus p33Cdk2 (Figure SB). Bacterially produced p33Cdk2 was not detected by the antiN-terminal antibody (Figure SB). It is very likely therefore that the 33 kDa polypeptide bound to cyclin A is indeed
cyclin A beads. The anti-PSTAIR antibody recognized the same 34 kDa polypeptide as well as an additional and much less abundant polypeptide migrating at 33 kDa. In several experiments, the relative abundance of the 33 kDa polypeptide was estimated to be between 1/5 and 1/20th of the amount of the 34 kDa polypeptide, assuming that these polypeptides are recognized equally well by the anti-PSTAIR antibody (Figures 5 and 7A, and data not shown). In some experiments an additional very faint band was detected at 32 kDa (Figure 5A). Cyclin BA90 beads precipitated the 34 kDa polypeptide but not the other polypeptides that bound to cyclin A and were only recognized by the anti-PSTAIR antibody (Figure 5). In control experiments, none of these polypeptides were detected after blocking the antibodies with the corresponding peptides, nor were they precipitated by BSA beads (not shown). We investigated further the nature of the 33 kDa protein bound by cyclin A beads and detected by the anti-PSTAIR antibody but not the anti-N-terminal antibody. This polypeptide comigrated with the Xenopus -
p33cdk2.
These results demonstrate that cyclin A associates with the same catalytic subunit as cyclin BA90, p34cdc2, but also show that it associates with an additional p34Cdc2-like protein, p33CYa2. The latter binds to cyclin A beads at much lower levels than p34cdc2. Because the depletion of p34cdc2 by cyclin BA90 beads is not complete, we cannot evaluate precisely the possible contribution of p33Cdk2 catalytic subunit to the kinase activity associated with cyclin A. However, it is clear that kinase catalytic subunits other than p34cdc2 contribute only a small proportion of the total histone HI kinase activated by cyclin A in Xenopus egg extracts.
20
Cyclin A does not induce the efficient phosphorylation of p34cdc2 on tyrosine residues During the lag phase and before activation, p34cdc2 bound to cyclin B is maintained in an inactive form by an inhibitory phosphorlation on tyrosine (Solomon et al., 1990). Since no lag phase was apparent during the activation of histone HI kinase by cyclin A, we suspected that there was a difference in the tyrosine phosphorylation of p34cdc2 following its association with cyclin A. Several investigators have shown that dephosphorylation of p34cdc2 on tyrosine when complexed to cyclin B and its concomitant activation, can be blocked by addition to the extract of the tyrosine phosphatase inhibitor, vanadate (Solomon et al., 1990; Dunphy and Kumagai, 1991; Gautier et al., 1991; Strausfeld et al., 1991). As shown in Figure 6 (upper panel) we reproduced this effect in our extracts: in the presence of 2 mM vanadate even high levels of cyclin B could not activate the histone HI kinase at all. At lower concentrations, vanadate partially blocked the activation induced by cyclin B and increased the length of the lag period (not shown; see also Solomon et al., 1990). In contrast, activation of the kinase by addition of 200 nM cyclin A to the extract was only partially suppressed by 2 mM vanadate (29% inhibition at 30 min) and no lag period was induced (Figure 6, lower panel; see also Figures 8C and 9C). At lower concentrations of cyclin A (50 nM), 2 mM vanadate suppressed kinase activation to a greater extent (50% inhibition at 30 min),
15
X 10 e E
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Is 15
c z
I 10 0
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5 20 40 Incubation time (min)
60
Fig. 6. Effect of vanadate ions on kinase activation by cyclin BA90 (upper panel) and cyclin A (lower panel). Extracts were pre-incubated for 10 min with (closed symbols, broken lines) or without (open symbols, continuous lines) 2 mM Na vanadate prior to the addition of cyclins to final concentrations of 200 nM (squares) or 50 nM (triangles) in the case of cyclin A. Control incubations without addition of cyclins were also performed (open circles). Vanadate ions had no effect on histone HI kinase activity in the absence of added cyclins (not shown).
N-term wonjursn t.
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-
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.
-
Fig. 7. Effect of vanadate ions on the modification of cdc2-like molecules bound to cyclin BA90 beads (A) or cyclin A beads (B) and their associated histone HI kinase activities (C). Cyclin beads were incubated for 30 min with interphase extracts in the presence or absence of 2 mM Na vanadate. The electrophoretic migration of the kinase molecules bound to the beads was examined by Western blotting using antibodies directed against the N-terminal of p34cdc2 (N-term) and, in the case of cyclin A beads, the PSTAIR-containing sequence (PSTAIR). Phosphotyrosine was detected using a specific antibody (PhosphoTyr). Histone H 1 kinase activity was assayed after resuspension of a proportion of the beads in extraction buffer (EB).
1755
P.R.Clarke et al.
but still not completely. Kinase activity was stable to prolonged incubation in the absence of vanadate. However, in the presence of vanadate, kinase activity declined slowly after 30 min with 50 nM cyclin A and 50 min with 200 nM cyclin A. The migration of p34cdc2 on SDS-PAGE has been reported by several investigators to be altered by phosphorylation, with tyrosine phosphorylation reducing the mobility (eg. Solomon et al., 1990; Strausfeld et al., 1991). The presence of phosphotyrosine in p34cdc2 can also be assessed using anti-phosphotyrosine antibodies. We examined the kinase molecules bound to cyclin A or B beads in extracts in the presence or absence of vanadate (Figure 7). No phosphotyrosine was detected in the 34 kDa band bound to cyclin BA90 beads in extracts without vanadate, as expected for the active form of the kinase (Figure 7A). When vanadate was added to the extract, the activation of the p34cdc2 upon addition of cyclin BA90 beads was blocked (Figure 7C). This coincided with an upshift in the p34cdc2 band and the appearance of phosphotyrosine (Figure 7A). In contrast, vanadate had little or no effect on the activation of histone HI kinase by cyclin A beads (Figure 7C). No change in the mobility of either p34cdc2 or p33C(k2 associated with cyclin A beads was seen with the addition of vanadate to the extract, nor was any phosphotyrosine detectable (Figure 7B). Although p34cdc2 bound to cyclin BA90 beads could be tyrosine phosphorylated, it remained possible that coupling of the cyclin A to beads had altered its regulatory properties. We therefore examined the migration and tyrosine phosphorylation state of kinase polypeptides in extracts to which soluble cyclins were added, the p34cdc2-like kinases being subsequently retrieved using p13sucl beads (Brizuela et al., 1987; Hindley et al., 1987; Labbe et al., 1989). More than 80 % of the total p34cdc2 was retrieved from the extracts by the pI3sucl beads (not shown). In these experiments, soluble cyclins (A or B) were added to egg extracts in sufficient amounts (200 nM) to maximally activate the histone HI kinase. The phosphorylation state of p34cdc2 molecules bound to these cyclins and retrieved on p1 3sucl beads was then examined by Western blotting using the antiN-terminal antibody (to follow their changes in migration properties, Figure 8A) or the anti-phosphotyrosine antibody (Figure 8B). The kinase activity associated with the pI3sucl beads was also assessed (Figure 8C). In the absence of cyclins, a single band detected by the anti-N-terminal antibody was recovered from interphase extracts. No phosphotyrosine could be detected. Addition of vanadate produced neither a band shift nor tyrosine phosphorylation, confirming previous reports that the association of a B-type cyclin is required for tyrosine phosphorylation of p34cdc2 (Solomon et al., 1990; Meijer et al., 1991). The addition of cyclin BA90 or cyclin A in the absence of vanadate resulted in a strong activation of the histone HI kinase (Figure 8C) and a distinct increase in mobility of the 34 kDa band compared to interphase (Figure 8A). This observation has not been reported before and we do not yet know its exact meaning. The increased mobility suggests that a change in the phosphorylation state of residues other than tyrosine is associated with the activation of p34cdc2 (see Discussion). When vanadate was added to the extract prior to cyclin BA90, activation of the kinase was blocked (Figure 8C) and all of the p34cdc2 migrated more slowly, at a position above 1756
cyclin BA90 +
vanadate
A
49.5-
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IF
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Fig. 8. Effect of cyclins and vanadate ions on the modification of p34cd'2. Interphase extracts were incubated for 30 min alone, or with the addition of 200 nM cyclin A or 200 nM cyclin BA90. Each was done in the presence or absence of 2 mM Na vanadate. The electrophoretic migration of p34cdc2 isolated from the extracts using p 13s"c - Sepharose beads was examined by Western blotting using antibodies directed against the N-terminal of p34cdc2 (A). Phosphotyrosine was detected using a specific antibody (B). Histone HI kinase activity was also assayed on a proportion of the pl3UcI beads (C).
p34cdc2 isolated from interphase extracts. This band was detected strongly by the anti-phosphotyrosine antibody. In complete contrast, vanadate did not block the downshift of p34cdc2 (Figure 8A) and only slightly inhibited the protein kinase activation (Figure 8C) induced by cyclin A addition. Trace amounts of phosphotyrosine were detected in a polypeptide retrieved from the extracts that comigrated exactly with that strongly labelled after cyclin BA90 addition. However, no p34cdc2 was detected by the anti-N-terminal antibody in the corresponding position (Figure 8B), showing that if any p34cdc2 was tyrosine phosphorylated under these conditions, it was a very small proportion of the total amount. Cyclin B usually induces a transient tyrosine phosphorylation of p34cdc2. When tyrosine dephosphorylation was blocked by vanadate, all of the p34cdc2 associated with cyclin B was trapped in the tyrosine phosphorylated form. In the absence of vanadate, tyrosine dephosphorylation occurred and the active form of p34cdc2 without tyrosine phosphate was recovered. The almost complete absence of tyrosine phosphorylation on p34cdc2 associated with cyclin
Regulation of cyclin-dependent protein kinases
p1 3-beads none
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'V................. IL-
1
7--
Ir m
Fig. 10. Activation of histone HI kinase in egg extracts by cyclin BA90, but not by cyclin A, requires a particulate fraction of the extract. Extracts were incubated with the addition of 200 nM cyclin A (open squares, continuous lines) or 200 nM cyclin BA90 (open triangles, continuous lines). Alternatively, aliquots of the same extract were centrifuged at 200 000 g to remove particulate material (Leiss et al., 1992) before addition of the cyclins to the resulting supernatant (filled symbols, broken lines). At the times indicated, aliquots were removed for assay of histone HI kinase activity.
r-ii I I
Fig. 9. Effect of cyclins and vanadate ions on the modification of p34a1c2 during activation of p34c'dC2 by cyclin A. Interphase extracts were incubated in the presence or absence of 2 mM Na vanadate for the indicated times following the addition of 200 nM cyclin A. Control incubations were performed for 30 min without added cyclin or with 200 nM cyclin A. The electrorhoretic migration of p34cd( 2 isolated from the extracts using p13s"u -Sepharose beads was examined by Western blottin using antibodies directed against the N-terminal domain of p34' 2 (A). Phosphotyrosine was detected using a specific antibody (B). Histone HI kinase activity associated with the beads was also assayed (C).
A, even in the presence of vanadate, indicated that cyclin A did not induce tyrosine phosphorylation of p34cdc2. In order to examine further this possibility, we monitored the migration properties and the level of tyrosine phosphorylation of p34cdc2 during its activation following cyclin A addition to an interphase extract (Figure 9). Activation of the kinase correlated with a downshift of the p34cdc2 band. This downshift appeared to be slightly delayed by the presence of vanadate. However, the slowest migrating form of p34cdc2, corresponding to the tyrosine phosphorylated form, was not observed during the time course of activation by cyclin A whether vanadate was present or not (Figure 9A). No tyrosine phosphate was detected on cyclin A-bound p34cdc2 under any condition (Figure 9B). Cyclin B- but not cyclin A-dependent activation of p34cdc2 requires a particulate fraction The finding that cyclin A did not induce tyrosine phosphorylation of p34cdc2 suggested that the regulatory pathway involved in cyclin A-dependent activation of p34cdc2 was different to the cyclin B-dependent pathway. We have previously reported that histone HI kinase activation in egg extracts requires a particulate fraction (Felix et al., 1990). Moreover, the activation of p34cdc2 by exogenous cyclin
BA90 also requires a particulate fraction (Leiss et al., 1992). Apparently, the particulate fraction is required for the tyrosine phosphorylation-dephosphorylation pathway regulating p34cdc2 activation (P.R.Clarke, unpublished results). Therefore we wondered whether the same fraction was essential for the activation of p34cdc2 by cyclin A. As shown in Figure 10, the ability of cyclin A to activate p34cdc2 in the extract was not altered greatly by removal of the particulate fraction by centrifugation. In contrast, the cyclin BA90-induced activation of p34cdc2 was almost completely abolished in the same supernatant.
Discussion In this paper we show that the regulation of the activity of
p34cdc2-like protein kinases by phosphorylation can be modulated by the nature of the cyclin subunit with which they interact. In Xenopus egg extracts, the major kinase catalytic subunit that associates with cyclin A is the same as that which associates with cyclin B, that is p34cdc2. During the activation process, p34cdc2 is apparently stoichiometrically phosphorylated on a tyrosine residue following its interaction with cyclin B but not following its interaction with cyclin A. The very rapid activation of p34cdc2 by exogenous cyclin A in Xenopus egg extracts can be explained by this lack of inhibitory phosphorylation on tyrosine and provides one potential explanation for the different kinetics of activation of the cyclin A- and cyclin B-dependent kinases observed in Xenopus eggs and in human cells. The difference in the kinetics of activation of p34cdc2 by the A- and B-type cyclins and the apparent lack of transient tyrosine phosphorylation of p34cdc2 associated with cyclin A might have been explained if p34cdc2 was stoichiometrically phosphorylated on tyrosine when bound to cyclin A, as when bound to cyclin B, but dephosphorylated very rapidly, such that it was not rate limiting to the activation of the kinase. Our finding that there is little tyrosine 1757
P.R.Clarke et al.
phosphorylation on p34cdc2, even in the presence of high levels of the tyrosine phosphatase inhibitor sodium vanadate, strongly suggests that this is not the case. We have not observed a transient tyrosine phosphorylation of p34cdc2 when it is complexed to cyclin A and even in the presence of vanadate only a very low level of the tyrosine phosphorylated form of p34cdc2 is trapped. These findings indicate that there is a difference in the susceptibility of p34cdc2 to tyrosine phosphorylation when complexed to the two types of cyclin. We suggest that p34cdc2 complexed to cyclin in egg extracts can be viewed to exist in equilibrium between its tyrosine phosphorylated and dephosphorylated forms. When complexed to cyclin B, it is initally essentially all in the tyrosine phosphorylated form and inactive. After a certain lag period, some process, which is as yet unclear but probably involves modulation of the relative activities of the tyrosine kinase and phosphatase, causes a rapid switch in the equilibrium to the dephosphorylated and active form of p34cdc2 (Goldbeter, 1991). However, when it complexes to cyclin A, p34cdc2 is immediately almost entirely in its dephosphorylated and active form. Even when vanadate is added to inhibit the phosphatase and an attempt is made to push the equilibrium to the phosphorylated form, we observe only a partial inhibition of the formation of active p34cdc2 protein kinase. In other words, whereas cyclin B greatly enhances the tyrosine phosphorylation of p34cdc2 when it binds, cyclin A does not. In two other recent reports, some tyrosine phosphorylation of p34cdc2 was detected when it was complexed to exogenous cyclin A (Ducommun et al., 1991; Solomon et al., 1992). However, in these studies the degree of phosphorylation was not apparent. In our experiments to examine tyrosine phosphorylation of p34cdc2 we have used sufficient amounts of cyclin, either soluble or bound to beads, to complex all of the p34cdc2 in the extract in order to examine clearly the stoichiometry of modification. It is possible that when using lower levels of cyclin, a greater proportion of the bound p34cdc2 may be converted into the tyrosine phosphorylated form. Indeed, vanadate appears to have a greater inhibitory effect at a low cyclin A concentration. However, it is technically more difficult to assess precisely the degree of tyrosine phosphorylation at such low cyclin levels. Even at low cyclin A levels, vanadate does not appear to induce a lag period as in the presence of cyclin B, but rather decreases activity throughout the timecourse of activation by cyclin A. It remains a possibility that vanadate has some effect on p34cdc2 activation other than by inhibiting tyrosine dephosphorylation. However, with prolonged incubation in the presence of vanadate, the kinase activity induced by cyclin A reaches a peak and then slowly declines, suggesting that the tyrosine kinase may phosphorylate p34cdc2-cyclin A at a slow rate in the absence of any competing phosphatase activity. The lack or low level of tyrosine phosphorylation of p34cdc2 complexed to exogenously produced cyclin A is supported by other experiments using extracts containing endogenous cyclins. Minshull et al. (1990) found that several modified forms of p34cdc2 and possibly other related kinase subunits (identified by detection with anti-PSTAIR sequence antibody) coimmunoprecipitated with cyclin A and cyclin B. However, the most slowly migrating form found associated with cyclin B (probably the tyrosine phosphorylated form of p34cdc2) was not found in cyclin A
1758
immunoprecipitates (Minshull et al., 1990; T.Hunt, personal communication). Very recently, Galaktionov and Beach (1991) have reported that the activity of two human homologues of the cdc25 tyrosine phosphatase, the enzyme very probably responsible for dephosphorylating p34cdc2, are stimulated in vitro by cyclin B, but not cyclin A (or D1). This result provoked the question of whether a different cdc25 homologue might be involved in the dephosphorylation of kinase catalytic subunits bound to cyclin A. Our results suggest that this is not necessary, at least for p34cdc2-cyclin A. Since cyclin A does not stimulate tyrosine phosphorylation either, cdc25 may not be required to activate p34cdc2 protein kinase in this case. Although tyrosine phosphorylation-dephosphorylation of p34cdc2 is not required for the activation of cyclin A-p34cdc2, other phosphorylation events do appear to be involved. We consistently observe an alteration in the electrophoretic migration of p34cdc2 upon activation by either cyclin A or B. A similar observation has been reported recently by Luca et al. (1991). The increased mobility might suggest a dephosphorylation event. It is possible that p34cdc2 is phosphorylated at a serine/threonine site in interphase extracts, and dephosphorylated at that site when the kinase is activated. One such possible site is Thrl4. However, we have so far been unable to reproduce the downshift of p34cdc2 by treatment with a variety of phosphatases (potato acid phosphatase, alkaline phosphatase, protein phosphatase 2A or cdc25 phosphatase). Conversely, phosphorylation of another threonine residue on p34cdc2 (Thrl6l/167) is required for the tight binding of cyclin A or cyclin B, and activation of either complex. However, it is not yet clear whether phosphorylation at this site can affect the mobility of p34cdc2 on the gel system used in the present study. Our results concerning the proportionality between the rate of activation of cyclin A -p34cdc2 protein kinase and the level of cyclin A (above 100 nM) suggests that whatever the modification is, it is probably not rate limiting to kinase activation under these conditions. The non-linear relationship between low levels of cyclin A and the rate of kinase activation suggests that activation may be more complex than simple association between cyclin A and p34cdc2. The fact that p34cdc2 can be activated by cyclin A without a tyrosine phosphorylated intermediate demonstrates that this is not an obligatory step in the pathway of p34cdc2 activation. This conclusion is supported by other results indicating that tyrosine phosphorylation may not always occur during the activation of p34cdc2 complexed to cyclin B either. Indeed, Ferrell et al. (1991) did not detect tyrosine phosphorylated p34cdc2 during the second to twelfth cell cycles in early Xenopus embryos. However, it was not clear from this study whether the apparent lack of phosphorylation was due to very rapid dephosphorylation or incapacity of the tyrosine kinase to phosphorylate p34cdc2. The ability of p34cdc2 associated with cyclin A in Xenopus egg extracts to activate even when tyrosine dephosphorylation is inhibited suggests that the activity of this kinase may not be subject to the same checkpoint signals as the cyclin B - p34cdc2 complex. In extracts that undergo multiple mitotic divisions powered by endogenously synthesized cyclins, Dasso and Newport (1990) found that activation of the histone HI kinase occured in two phases. There was an initial 'ramp' of activity that increased
Regulation of cyclin-dependent protein kinases
relatively slowly, followed by a sudden increase in activity. The peak of activity could be inhibited by unreplicated DNA (added nuclei in the presence of aphidicolin to block DNA replication) but not the initial ramp of activity. This suggests that there may be two kinase activities differing in their sensitivity to unreplicated DNA. Very recently, Walker and Maller (1991) have provided evidence that, in similar experiments, cyclin A-dependent kinase activity is not suppressed by the presence of unreplicated DNA under conditions where cyclin B-dependent kinase activity is. In Xenopus egg extracts, we have shown that the great majority of the kinase activity that associates with cyclin A is due to p34cdc2. This appears to be somewhat different from the situation in cultured mammalian cell lines. In these cells, the major kinase subunit associated with cyclin A appears to be the product of the cdk2 gene, although a fraction of cyclin A interacts with p34cdc2 (Pines and Hunter, 1990; Tsai et al., 1991; Pagano et al., 1992). Indeed, it may be of interest to study systematically the nature of the catalytic subunits associated with cyclin A during embryonic development, cell differentiation and transformation. It is not yet clear whether p33cdk2 is regulated by tyrosine phosphorylation, although we did not observe any tyrosine phosphorylated form bound to cyclin A beads in the presence of vanadate. In cultured cells, p33cdk2 does not appear to be highly tyrosine phosphorylated and p34cdc2 associated with cyclin A does not appear to contain tyrosine phosphate during G2, although it does during early S phase (Pagano et al., 1992). Such somatic cells differ from early embryos in having distinct S and M phases separated by GI and G2, and this may alter the regulation of cyclin A-dependent protein kinases. For instance, cyclin A is nuclear during S phase in somatic cells (Pines and Hunter, 1991; Pagano et al., 1992), whereas in egg extracts there are no nuclei. The precise role of tyrosine phosphorylation in regulating the kinase subunits associated with cyclin A in somatic cells will require further investigation. It is possible that tyrosine phosphorylation of p34cdc2 and other cyclin-dependent protein kinases may be involved in checkpoints other than at the G2-M transition. Cyclin A- and cyclin B-associated protein kinases show similar substrate specificities in vitro. For instance, they both phosphorylate well histone H1, used to assay their activities in this study. However, some minor differences are found (Minshull et al., 1990). Recently, the role of cyclindependent protein kinases in the control of specific cellular processes has been examined in this laboratory. Using functional assays, cyclin A and cyclin B have been found to have quite different effects on microtubule nucleation by centrosomes (Buendia et al., 1992), microtubule dynamics (Verde,F., Dogterom,M., Stelzer,E., Karsenti,E. and Leibler,S., in preparation) and vesicle fusion (Thomas et al., 1992). Differences between the substrates of cyclin A- and cyclin B-dependent kinases might be due to different catalytic subunits. However, the firm demonstration in this study that cyclin A and cyclin B activate efficiently the same catalytic subunit means that the two cyclins can greatly alter the function of a given catalytic subunit, presumably by modulating its substrate preferences or subcellular targetting. In addition to the A and B-type cyclins, other types of cyclin are being identified (Hunter and Pines, 1991). The existence of multiple cdc2-like catalytic subunits (Lehner and
O'Farrell, 1990a; Elledge and Spottswood, 1991; Fang and Newport, 1991; Paris et al., 1991; Tsai et al., 1991) multiplies the number of possible cyclin-dependen. protein kinases. Control of the activity of these protein kinases is apparently achieved in part by specific temporal patterns of synthesis and degradation of the different cyclins during the cell cycle and also by post-translational regulation of the catalytic subunit. Our finding that at least two of these complexes are regulated differently at the post-translational level suggests that each kinase complex once formed might have distinct regulatory characteristics, allowing different signals to control their activities.
Materials and Methods Materials Triton X-100, ATP, leupeptin, creatine phosphate and creatine kinase were obtained from Boehringer Mannheim. Cytochalasin D, dithiothreitol, Na orthovanadate and PMSF were obtained from Sigma. Other chemicals were obtained from Merck. Human cyclin A was expressed using the pT7F IA vector in E. coli strain BL21(DE3). Cyclin A expression was induced at 25°C by addition of 1 mM IPTG for 6 h. Protein from the soluble fraction was purified to homogeneity by successive ammonium sulphate precipitation, gel filtration and mono-Q anion exchange chromatography (Pagano et al., 1992). Sea urchin cyclin BA90 cloned and expressed in E.coli strain BL(DE3)pLysS was a generous gift of M.Glotzer and M.W.Kirschner (University of California at San Francisco). Cyclin BA90 expression and purification from the inclusion body fraction was as described in Glotzer et al. (1991). Xenopus cyclin A, cloned by J.Minshull and T.Hunt, was expressed as a maltosebinding protein fusion using a pMAL vector in E.coli TBI (New England Biolabs) (Felix,M.A., unpublished). Expression and purification of pl3S" was as described in Felix et al. (1989). Protein concentrations were detennined using a dye-binding assay (Biorad). Cyclin A, cyclin BA90 and p1 3s"u were coupled to CNBr-activated Sepharose (Pharmacia) according to the manufacturers instructions. Cyclins were coupled at 1 mg/ml Sepharose and pl3S`'l at 5 mg/ml. Affinity purified rabbit antibodies raised against a peptide corresponding to the N-terminus of starfish p34cdc2 (MEDYAKIEKIGEC) and against the PSTAIR-containing sequence (EGVPSTAIREISLLKE) were gifts of M.Doree (CNRS, Montpellier). Anti-cdk2 (Xenopus Egl), anti-PSTAIR mouse monoclonal and anti-phosphotyrosine antibodies were donated by J.Gannon and T.Hunt (ICRF, South Mimms), M.Yamashita (Okazaki) and
G.Peaucellier (Roscoff), respectively. Preparation of interphase extracts Extracts of Xenopus eggs were prepared by a modification of a previous method (Felix et al., 1989). Eggs were dejellied with 2% (mass/vol) cysteine-HCI (pH 7.8) and then washed extensively with modified Ringer's MMR/4 (25 mM NaCI, 0.4 mM KCI, 0.25 mM MgSO4, 0.5 mM CaC12, 1.25 mM HEPES and 25 $M EDTA, pH 7.2). Eggs were activated by an electric shock (Karsenti et al., 1984), incubated at 20°C in MMR/4 plus 100 Ag/ml cycloheximide for 90 min and then transferred to centrifuge tubes filled with ice-cold acetate buffer (100 mM K acetate, 2.5 mM Mg acetate, 60 mM EGTA, 5Lg/ml cytochalasin D, 1 mM dithiothreitol, pH 7.2). Subsequent steps were performed at 4°C. Excess buffer was removed and the eggs crushed by centrifugation at 10 000 g for 10 min. The cytoplasmic material between the upper yellow lipid layer and the lower yolk was collected and an ATP-regenerating system (final concentrations of 10 mM creatine phosphate, 80 Ag/ml creatine kinase, 1 mM ATP) added. This cytoplasmic fraction was then centrifuged at 100 000 g, the supernatant collected, aliquoted, frozen and stored in liquid nitrogen. These extracts contained -40 mg protein/ml. In some cases further centrifugation at 200 000 g for 45 min in a Beckman TL100 centrifuge was performed and the subsequent supernatant and particulate fractions were separated, frozen and stored in liquid nitrogen (Leiss et al., 1992). Incubation of cyclins in extracts Cyclins diluted to the appropriate concentration in XB [10 mM K-HEPES (pH7.4), 100 mM KCI, 50 mM sucrose, 1 mM MgCI2, 0.1 mM CaCl2, 1 mM dithiothreitol and 10 pg/ml leupeptin] were added in 1/10 vol to interphasic extracts devoid of endogenous cyclins. Incubations were carried out at 20°C and samples, typically 1 1l, were removed into 20 vol of EB
1759
P.R.Clarke et al. [80 mM ,B-glycerophosphate (pH7.3), 15 mM MgCl2, 20 mM EGTA, 1 mM dithiothreitol, 25 jug/ml aprotinin, 25 yg/ml leupeptin, 1 mM benzamidine and 0.5 mM PMSF) plus 4 mM Na vanadate. Histone HI kinase assays were performed essentially as in Felix et al. (1989) using exogenous histone HI (type IIIS from calf thymus, Sigma).
Isolation of kinase molecules bound to cyclin - Sepharose beads Cyclin A- or cyclin BA90-Sepharose beads were washed in XB prior to use. Beads were added to extracts at 1 vol packed beads to 3 vol of extract and incubated at 20°C for 30 min. Beads were recovered by centrifugation at 5000 g in a microtube centrifuge, washed once with 1 ml of EB plus 4 mM Na vanadate, 1% (by volume) Triton X-100 and 250 mM NaCI, followed by three washes of 1 ml of EB plus 4 mM Na vanadate. Finally, the bound proteins were solubilized in gel sample buffer (80 mM Tris-HCI [pH6.8], 5% (mass/vol) SDS, 50 mM Na fluoride, 5 mM EDTA, 4 mM Na vanadate, 10% (by volume) 2-mercaptoethanol) before analysis. Alternatively, washed beads were used to assay the associated histone H 1 kinase activity after dilution in 60 vol of EB.
Isolation of kinase molecules bound to p 13suc1- Sepharose beads p1 3s"l -Sepharose beads were washed with EB plus 4 mM Na vanadate before use. After incubation of extracts with or without added cyclins, 2 vol of packed p13U"'C beads together with 2 vol of EB plus 4 mM vanadate was added the extract, such that the extract was diluted by 5-fold. After shaking at 4°C for 1 h, p13S"UC beads were recovered by low speed centrifugation, washed and the bound proteins solubiized as for cyclin beads. Alternatively, washed beads were used to assay the associated histone H 1 kinase activity after dilution in 10 vol of EB.
Gel electrophoresis and Western blotting All procedures were carried at room temperature. Samples were analysed by SDS gel electrophoresis using 12% acrylamide gels with prestained molecular mass marker proteins (Biorad, low molecular mass range). Electrophoresis was continued until the 18 kDa marker was almost at the bottom of the gel. Proteins were transferred at 300 mA for 1 h onto nitrocellulose membranes (Schleyer and Schull) in 48 mM Tris base, 39 mM glycine, 0.037% (by mass) SDS and 20% (by volume) methanol using a semi-dry transfer apparatus (Biorad). After transfer, membranes were blocked for 1 h with 5% (mass/vol) dried milk powder or 10% (mass/vol) BSA (in the case of detection using anti-phosphotyrosine antibodies) in Tween-Tris-buffered saline (TTBS) [25 mM Tris/HCI (pH 7.6), 150 mM NaCI, 0.05% (by volume) Tween-20]. After washing with TTBS, blots were incubated for 1 h with anti N-terminal antibody, anti-PSTAIR antibody or anti-cdk2/Egl antibody (all 1/600 dilution in TTBS plus 1% (mass/vol) dried milk powder), or anti-phosphotyrosine antibody (1/1500 dilution in 1% (mass/vol) BSA). After washing in TTBS, detection was performed using biotinylated anti-rabbit IgG, followed by biotinylated horseradish peroxidase-streptavidin complex (Amersham, both 1/400 dilution in TTBS plus 1% (mass/vol) dried milk powder), or protein A coupled to horseradish peroxidase [Amersham, 1/2000 dilution in TTBS plus 1% (mass/vol] BSA) for anti-phosphotyrosine antibodies. When a monoclonal antibody against the PSTAIR sequence was used (1/1000 dilution in TTBS plus 1% (mass/vol) dried milk powder), detection was performed as for the rabbit anti-PSTAIR antibody, except that biotinylated anti-mouse IgG 1 (Amersham) was used. Between each incubation step, blots were washed for 30 min with multiple changes of TTBS. Development was performed using a chemiluminescent method (Amersham ECL) according to the manufacturers instructions.
Acknowledgements We are indebted to Marcel Doree, Gerard Peaucellier, Tim Hunt and Masakane Yamashita for their gifts of antibodies, Michael Glotzer and Marc Kirschner for the cyclin BA90 expression system, and Jacqueline Hayles and Paul Nurse for sucl+ E. coli. We thank Julia Coleman for purifying plj3Sl". We are particularly grateful to Marie-Anne Felix, who performed some of the experiments preliminary to this study. We also thank Giulio Draetta and Tim Hunt for helpful discussions. This study was funded, in part, by the Human Frontier Science Programme. M.P. is supported by a fellowship from Associazione Italiana per la Ricerca sul Cancro. P.R.C. is the recipient of a research fellowship of The Wellcome Trust.
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