Cyclin activation of p34cdc2

Cell, Vol. 63, 1013-1024,

November

30, 1990, Copyright

Cyclin Activation

0 1990 by Cell Press

of p34cdc2

Mark J. Solomon,” Michael Glotzer,’ Tina H. Lee: Michel Philippe,t and Marc W. Kirschner* *Department of Biochemistry and Biophysics University of California San Francisco, California 94143-0448 tLaboratoire de Biologie et Genetique du Developpement Universite de Rennes I Campus de Beaulieu 35042 Rennes CEDEX France

Summary The gradual accumulation of cyclin in the frog egg induces an abrupt and concerted activation of ~34~~~~ that initiates mitosis. Activation is delayed even after the accumulation of cyclin to a critical threshold concentration. We have reproduced these unusual kinetic propertles of p34Cdc2 activation in vitro using bacterially expressed cyclin proteins and extracts derived from Xenopus eggs. Abrupt activation follows a lag period, the length of which is independent of the concentration of cyclin. The threshold concentration of cyclin and the length of the lag period are regulated by INH, an inhibitor of MPF activation in oocytes recently identified as a type 2A protein phosphatase. Blnding to cyclin induces both tyrosine and threonine phosphorylation of the previously unphosphorylated p34-, rendering it inactivated. The concerted transition into mitosis involves both a reduction in the rate of p34phosphorylation on tyrosine and an increase in its rate of dephosphorylation. Introduction The transitions of the cell cycle are abrupt, yet most biochemical processes are continuous. During the simple cell cycles of early Xenopus development, the abrupt transition from interphase to mitosis is driven by the continuous accumulation of cyclin. Although cyclin appears to be the only protein whose synthesis is required for the early Xenopus embryonic cell cycles (Minshull et al., 1989; Murray and Kirschner, 1989), its association with ~34~“~ is insufficient to produce active maturation-promoting factor (MPF) and Hi kinase activities (Pines and Hunter, 1989; Pondaven et al., 1990); additional, posttranslational steps are required. For example, tyrosine phosphorylation of ~34~~~ inhibits its kinase activity, and subsequent progress into M phase is accompanied by tyrosine dephosphorylation (Draetta et al., 1988; Dunphy and Newport, 1989; Gould and Nurse, 1989; Morla et al., 1989). Although it is known that the mitotic state is generated by complexes of p34Ck* and cyclin (Booher et al., 1989; Draetta et al., 1989; Labbe et al., 1989a; Meijer et al., 1989; Gautier et al., 1990) and that these activities are controlled posttranslationally, the critical question of how

the oscillations of the cell cycle are generated remains unanswered. Specifically, how does the continuous accumulation of cyclin lead to the abrupt activation of ~34~~~ and the consequent concerted transition from interphase to mitosis? (see Figure 1). Previous experiments have shown that cyclin must accumulate to beyond a critical level in order to trigger mitosis (Evans et al., 1983; Pines and Hunt, 1987; Minshull et al., 1989; Murray and Kirschner, 1989). Biochemically, what is the cyclin threshold and how is it monitored? In addition, a lag has been defined in vivo as the period before ~34~~2 activation occurs during which protein synthesis is no longer required (Wagenaar, 1983; Picard et al., 1985; Karsenti et al., 1987). Thus, even after the threshold concentration of cyclin has been reached, there must be other, slow processes that must be completed before ~34~~~ is.activated. This paper presents an in vitro system designed to facilitate biochemical dissection of the entry into mitosis. Interphase extracts derived from Xenopus eggs that carry out all the major biochemical reactions of the cell cycle (Lohka and Maker, 1985; Hutchison et al., 1987; Newport and Spann, 1987; Murray and Kirschner, 1989; Murray et al., 1989) are combined with exogenous cyclin proteins made in Escherichia coli. We found that even when a large excess of cyclin protein was added all at once, there was a lengthy lag before the abrupt appearance of Hl kinase activity in the extract. The lag period and cyclin threshold necessary for activation could be altered by changing the activity of INH, a negative regulator of MPF recently shown to be a type 2A protein phosphatase (Cyert and Kirschner, 1988; T. H. L. et al., unpublished data). Cyclin binding induced both tyrosine and threonine phosphorylation of the previously unphosphorylated p34&*, preventing premature activation during the lag period. The subsequent abrupt transition into mitosis can be explained by a positive regulatory loop in which active ~34~&* leads to an inhibition of ~34~~2 phosphorylation on tyrosine and a stimulation of its dephosphorylation. These results provide a simplified biochemical explanation for how cyclin accumulation induces the concerted transition into mitosis and for the existence of the G2 phase of the cell cycle. Results Expression and Purification of Cyclin Proteins We expressed derivatives of a sea urchin type B cyclin as fusion proteins in E. coli. Two fusion constructs to residues 13-409 of sea urchin cyclin (Murray et al., 1989) were prepared: cyclin-prA, containing the IgG binding domain of protein A inserted at amino acid 91, and GT-cyclin, containing the enzyme glutathione transferase fused to the N-terminus of cyclin. Each of the two cyclin derivatives has particular advantages. Cyclin-prA binds extremely tightly to IgG-Sepharose and can be quantitatively depleted from solution. GT-cyclin is easily purified under very gentle conditions (glutathione competition provides specific

Cell 1014

1234

5

nM CYCLIN.

9

10

1500 460 150 46 IS 46

6

7

6

15

A

SOLUBLE CYCLIN M

TIME

Figure 1. A Schematic See text for details.

View of Cell Cycle

A Threshold Concentration of Cyclln Protein Ie Required to Initiate Mitosis In Vitro ! Extracts of Xenopus eggs can be released from their physj, iological metaphase arrest by Ca2+ and are blocked in interphase by cycloheximide, which prevents the reaccumulation of cyclins (Lohka and Maker, 1985; Murray and Kirschner, 1989); subsequent addition of cyclin-prA induces mitosis. The initial egg extract, arrested at metaphase, had high levels of Hl kinase and MPF activities (Figure 2A, lane l), whereas the interphase extract derived from it (lane 2) and an interphase extract supplemented with a mock cyclin sample (lane 3) had low levels of both activities. Addition of cyclin-prA to the interphase extract induced very high levels of both MPF and Hl kinase activities (Figure 2A, lane 4). In addition, the nuclear envelopes of added nuclei broke down and chromosomes condensed (not shown), indicating that a true mitotic state had been produced. to cyclin-pd in vitro was essentially all The response or none: over a 30-fold range of cyclin concentration there was only a 2-fold change in MPF and Hl kinase activities, but at lower concentrations of cyclin there was no induction of activity (Figure 2A, lanes 4-10). The induced levels of activity, especially at the highest cyclin concentrations, were significantly higher than those seen in the egg extract

or in vivo.

In fact,

addition

of cyclin

to a metaphase

egg extract consistently

elevated the level of Hl kinase ac-

tivity

3- to 4-fold

to approximately

(not

shown).

The

full

potential activation of p34cdc2 is thus not normally achieved. There was a quantitative association of both the resulting Hl clin species

kinase (Figure

and MPF 28).

activities

with

this

single

cy-

T

S

P

Hl KINASE.

106 15

136

144

MPF.

440 ~30

520

560 <30

Activation

elution), and complexes containing this protein (such as Hl kinase) can be purified and eluted with retention of activity. Moreover, GT-cyclin is not degraded during mitosis, presumably owing to its modified N-terminus (Murray et al., 1989), and is thus an extremely useful reagent for studying the activation process, divorced from subsequent inactivation.

I

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Figure 2. Cyclin-Induced Activation of p&l&2 (A) Threshold activation of Hl kinase and MPF. Lane 1 shows Hl kinase and MPF activities in an egg extract, and lane 2 shows the activities in the interphase extract derived from it. To this interphase extract a control extract (lane 3; see Experimental Procedures) or various concentrations of cyclin-prA (lanes 4-10) were added. Aliquots were taken after 20 min and assayed for Hl kinase and MPF activities. The autoradiograph of the Hl kinase assay is shown. Shown below is quantitation of both activities. Hl kinase activity is expressed as pmollmg per min of phosphate transferred to histone Hl, and MPF activity is expressed as units per microliter of extract (see Experimental Procedures for quantitation). (B) Physical association of Hl kinase and MPF activities with added cyclin. Lanes M and I denote M phase and interphase extracts, respectively. To the interphase extract, cyclin-prA (SOLUBLE CYCLIN, 73 nM final concentration), mock cyclin-prA (lane C), IgG-Sepharose beads with bound cyclin-prA (CYC BEADS, approximately 700 nM cyclin), or IgG beads incubated in a control E. coli extract (CONT. BEADS) were added. Total (T), supernatant (S), and pellet(P) fractions were prepared after a 20 min incubation. Aliquots representing equivalent amounts of extract were assayed for both Hl kinase (gel shown) and MPF activities. The supernatant remaining after removing the cyclin beads could not generate further Hl kinase activity with additional cyclin (not shown). Furthermore, serial transfers of interphase extracts activated with soluble cyclin to fresh interphase extracts only induced mitosis as long as the cyclin concentration remained above the threshold concentration established above (not shown).

A Deflned Lag Period Precedes Cyclln-Induced Activation of ~34~~~~ Cyclin accumulates fraction

slowly in vivo, and it is unclear what

of the interphase

period

is due

to this

accumula-

tion as opposed to subsequent posttranslational reactions. The availability of purified cyclin has enabled us to clearly distinguish the contributions of these two processes to the overall kinetics. At a high concentration of cyclin-prA (1000 nM, Figure 3A), activation of Hl kinase occurred between 10 and 20 min after cyclin addition. Activation required the same time at lower cyclin-prA concentrations, but at the lowest concentrations activity subss quently declined, presumably owing to cyclin degradation. No activation occurred at 10 nM cyclin-pd. A longer lag was observed at a lower temperature (WC) (Figure 38). The constancy of the lag period over at least a 30-fold range of cyclin concentrations was unexpected for a bi-

Cyclin 1015

Activation

Figure 3. Kinetics trations

of p34cdc2

of Hl Kinase

Activation

at Different

Cyclin

Concen-

(A) Interphase extracts were incubated at 23% with the indicated final concentrations of cyclin-prA and assayed for Hl kinase activity at the indicated times. (6) An interphase extract was incubated with 250 nM cyclin-prA at 15% for the indicated times and assayed for Hl kinase activity.

molecular reaction. Presumably, either association of cyclin with ~34~~~ is so rapid that a 30-fold reduction in rate is insignificant compared with the time required for other activation events, or the rate of association is limited by proteins other than cyclin and ~34~~~~. We have examined the rate of complex formation in two ways: by looking at the rate of cyclin binding to ~34~~~~ that was then bound to ~13~~~’ beads (not shown), and by using cyclin beads to remove ~34~~~~ from an extract after various incubation times (see Figure 7). By both methods association was rapid (~5 min at 23%) compared with the lag, but slow compared with the estimated diffusion controlled rate (tllP = ~6 s). The lag also does not represent the time required for the interphase extract to “age” into a cyclin-responsive state since the lag was identical in interphase extracts taken 15, 40, or 120 min after release from M phase arrest (data not shown). The Lag and Threshold Are Controlled by the MPF Inhibitor INH, a vpe 2A Protein Phosphatase An activity termed INH inhibits the generation of MPF activity in Xenopus oocyte extracts (Cyert and Kirschner, 1966). This activity has recently been purified and shown to contain the catalytic subunit of protein phosphatase type 2A and possibly unique regulatory chains (T. H. L. et al., unpublished data). In addition, it has recently been found that okadaic acid, an inhibitor of type 1 and 2A phosphatases, permits certain interphase extracts that were blocked with cycloheximide to enter mitosis (Felix et al., 1990). We examined the possible contribution of protein phosphatases to the lag period by measuring the length of the lag in the presence and absence of okadaic acid. Addition of okadaic acid shortened the lag period from about 20 min to 12.5 min (Figure 4A, 0 U/f11 INH). A sharp transition was observed (not shown), indicating that

a lag still existed, and had not been transformed into a gradual, continuous process. Since addition of a small amount of mitotic extract could also shorten the lag (not shown), okadaic acid might shorten the lag by bypassing the requirement for the accumulation of an intermediate complex to some critical level. Okadaic acid might, then, also be expected to eliminate the cyclin threshold. We therefore tested whether okadaic acid modified the strong dose dependence of p34&* activation on cyclin concentration. Figure 48 shows that there was no threshold in the presence of okadaic acid. Instead, the final level of Hl kinase activity was roughly proportional to the concentration of cyclin, even at cyclin concentrations of less than one-tenth the normal threshold concentration. Increasing INH activity had the opposite effect, lengthening the lag to 60 min and longer (Figure 4A; data not shown). The length of the lag period increased in proportion to the amount of added INH (open circles). Okadaic acid completely eliminated this effect and produced a lag period whose length was independent of INH concentration (closed circles). If the effect of INH can be extrapolated, then the interphase extract contained about 1.5 U/PI INH. (The extract was diluted to 50% for the activation reactions.) This figure is in good agreement with that calculated independently (~0.6 U/f& T. H. L. et al., unpublished data). The effect of increased INH levels on the threshold concentration of cyclin required for activation was also determined (Figures 4C and 4D). As expected, addition of INH greatly increased the threshold (Figure 4C, closed circles and squares), opposite to the effect of okadaic acid. Significantly, the threshold level was directly proportional to the concentration of INH (Figure 4D; see Discussion). Tymslne Phosphorylation of ~34~~~~ Induced by Cyclln Binding We sought an explanation for the delay in ~34~~~~ activation following cyclin addition. We found that 0.3 mM sodium orthovanadate (vanadate) completely inhibited ~34~~ activation and that lower concentrations lengthened the lag period (unpublished data). Although not completely specific, vanadate inhibition can be taken as indicative of the action of a phosphotyrosine phosphatase (Cantley et al., 1977; Gibbons et al., 1978; Klarlund, 1985; Tonks et al., 1988). To assess this, we looked at the modification states of ~34~~~~ (Figure 5). After incubation of the extracts under various conditions, total ~34~~~ (lanes l-4) or cyclinbound ~34~~~~ (lanes 5 and 6) was isolated for analysis. For the samples in lanes 1-4, ~34~~2 was purified on ~13~~~’ beads (Dunphy et al., 1988). Alternatively, for the samples in lanes 5-6, GT-cyclin attached to glutathione beads was added directly to the extract, and the ~34~~~~ was isolated in association with the cyclin. Incubation of the extracts with no additions or after addition of vanadate had no effect on the low basal level of Hl kinase activity (Figure 5A, lanes 1 and 4). Incubation with GT-cyclin induced high kinase activity (lanes 3 and 6) whereas incu-

Cell 1016

A

p13 BEADS CYC BEADS CYC ‘CYC ’ tv CYC v tv CYC

’

HlK

~13 BEADS

B

CYC BEADS CYC ‘CYC ’ +v CYC v +v CYC

’ p32

a-PSTAIR

~13 BEADS

C

’

p32

CYC BEADS CYC “CYC ’ +v CYC v +v CYC

-

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p32

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CYC +v CYC

CYC BEADS “CYC ’ v +VCYC

C&p32 M

Figure

5. Cyclin-Induced

123

Tyrosine

456

Phosphorylation

of p34cdc2

An interphase extract was incubated alone (-, lanes l), with 360 nM GT-cyclin (CYC, lanes 3) with 2 mM vanadate (V, lanes 4) or with GT-cyclin and vanadate (CYC + V, lanes 2) for 30 min. Both GT-cyclin and vanadate were added from concentrated stock solutions to minimize dilution. Small aliquots were taken for Hl kinase assay ([A], HlK). Larger samples were bound to ~13~~’ beads, washed, eluted into SDS-PAGE sample buffer, and analyzed by Western blotting with antisera to the PSTAIR region of p34 c*z ([El, a-PSTAIR), phosphotyrosine ([Cl, a-P-TYR), or p32 (ID], a-p32; antiserum kindly provided by J. Gautier and J. Mailer). Alternatively, GT-cyclin was prebound to glutathione beads that were subsequently added to an interphase extract with (CYC + V, lanes 5) or without (CYC, lanes 6) 2 mM vanadate (final concentration). The final concentration of GT-cyclin was 450 nM. After 30 min, aliquots for Hl kinase measurement and for Western blotting were prepared. Lanes M contained 20 ng of purified p32 expressed in E. coli. See Experimental Procedures for further details.

Figure

4. Regulation

of the Lag and the Threshold

by INH

(A) GT-cyclin (70 nM) was added to an interphase extract containing variousconcentrationsof partially purified INH, atype 2A protein phosphatase. The mixtures also contained either 3% DMSO (- okadaic acid) or 3.7 pM okadaic acid (+ okadaic acid) and were incubated at 15%. Samples were taken every 5 min and assayed for Hl kinase activity. The lag was calculated as the time required for half-maximal kinase activation. (B) Okadaic acid lowers the required threshold concentration of cyclin. An interphase extract was incubated at 23% with different concentrations of GT-cyclin in the presence of 1% DMSO (open circles) or of 1.3 pM okadaic acid (closed circles). Samples were taken at 30 min for Hl kinase determinations. One hundred percent activation in the absence and presence of okadaic acid corresponded to 124 and 167 pmollmg per min, respectively. Activity plateaued at approximately246 pmollmg per min at higher GT-cyclin concentrations (not shown). (C) INH increases the cyclin threshold. GT-cyclin was incubated in an interphase extract at 15% in the presence of 3 U/p1 (closed circles) or 6 Ulul (closed squares) INH. Aliquots were taken at 60 min and assayed for Hl kinase activity. One hundred percent activation cor-

bation with GT-cyclin in the presence of vanadate allowed only a low level of activation (lanes 2 and 5). The modification state of p34ck2 was examined by Western blotting with an antiserum directed to a peptide conserved in all known p34C’% (Figure 58, a-PSTAIR). A major ~34~~~ band was seen in the untreated extract (Figure 58, lane 1) as well as in the extracts that were treated with either GT-cyclin (lane 3) or vanadate (lane 4) alone. Two electrophoretically retarded bands were observed only when both cyclin and vanadate were added

responded to 263, 151, and 246 pmollmg per min in the presence of 0, 3, or 6 U/p1 INH, respectively. (D) The cyclin thresholds determined in (C) and in (9) were plotted versus INH concentration. The endogenous level of INH was extrapolated from (6) (see text).

Cyclin 1017

Activation

of ~34~~~~

M

1

Figure 6. Cyclin-Induced ration into ~34~“’

VM MITOTIC

VANADATE-ARRESTED c

4 PIJ-SOUND

CYCLIN-BOUND

a-PSTAIR

(lane 2). Since these modified forms did not accumulate in the presence of vanadate alone (lane 4), cyclin must induce the modifications of ~34~~~ that can subsequently be trapped by vanadate. Similar results were seen with GT-cyclin beads: electrophoretically retarded forms of ~34~~~ were only produced when cyclin and vanadate were present (lane 5). These modified forms of ~34~~~ bound efficiently to GT-cyclin (see also Figure 8). The same ~34~~ samples were also analyzed by blotting with a monoclonal antibody to phosphotyrosine (Figure 5C, a-P-TYR). The reactivity of ~34~“2 to this antibody correlated extremely well with the presence of electrophoretically detectable modified forms seen with the a-PSTAIR serum. The two bands detected with the antiphosphotyrosine antiserum corresponded to the upper two bands of ~34~~2 detected with the a-PSTAIR serum (data not shown). Although there was a low level of phosphotyrosine in ~34~“2 in the untreated interphase extract (Figure 5C, lane l), high levels were only seen after addition of both Gl-cyclin and vanadate (lane 2). The near absence of ~34~~2 containing phosphotyrosine in interphase contrasts with previous reports; this discrepancy will be addressed in detail in the Discussion. Vanadate alone was insufficient for phosphotyrosine accumulation, and phosphotyrosine was completely removed prior to entry into mitosis (Figure 5C, lane 4; Figure 7C, lane 3). The lower a-PSTAIR reactive band (see also Figure 78) is ~32, a ~34~~~ “look-alike” protein encoded by Egl, a Xenopus clone selected by differential screening (Paris and Philippe, 1990) that is specifically deadenylated and released from polysomes after fertilization (Paris et al., 1990). Although p32 binds to the a-PSTAIR antiserum, and to p13*uC1, it fails to bind to GT-cyclin (Figure 5D, lanes 5 and 6).

Both Phosphorylatlon and Dephosphorylation Events Accompany p34CdC2Activation a2P incorporation ly that GT-cyclin

into p34e2 was used to verify directinduces tyrosine phosphorylation of

Phosphate

Incorpo-

(A) ~34~~ was s2P-labeled in extracts and purified by binding to p13sucr beads followed by immunoprecipitation as described in Experimental Procedures, The lanes contain ~34~~ from an egg extract (M, lane 2) an interphase extract (I, lane 3) or an interphase extract incubated with CT-cyclin in the presence 01, lane 4) or absence (M, lane 5) of 2 mM vanadate. The lower panels show the Hl kinase activities of the various extracts (HlK) and Western blot analysis of the purified forms of ~34~“~ (a-PSTAIR). (B) and (C) Two-dimensional tryptic peptide analyses of the p34=@ in lanes 4 and 5 are shown in (B) and (C), respectively. The letters in (B) and (C) indicate the phosphorylated amino acids. (D) and (E) Two-dimensional tryptic phosphopeptides from cyclin-associated and immunoprecipitated, 32P-labeled ~34~~ prepared from extracts incubated in the presence and absence of 2 mM vanadate, respectively.

~34~~2 and, in addition, to obtain information about the sites of p34cdc2 phosphorylation (Figure 6). 32P was added to an egg extract (Figure 6A, lane 2), which was then released into interphase in the presence of cycloheximide (lane 3). GT-cyclin plus vanadate (lane 4) or GT-cyclin alone (lane 5) was added to aliquots of the interphase extract. p34cdc2 was purified from these extracts by rapid binding to ~13~~~’ beads on ice followed by immunoprecipitation. No 32P was incorporated into ~34~~~ in the egg extract or during the subsequent interphase (Figure 6A, top panel, lanes 2 and 3). In contrast, high levels were incorporated into ~34~~~~ in the vanadate plus GT-cyclin treated extract (lane 4) as well as in the mitotic sample does not occur (lane 5). Thus, p34 cdc2 phosphorylation simply upon exit from M phase, but rather requires association with cyclin. To examine the changes in ~34~~~~ phosphorylation during cyclin-induced activation, the phosphopeptides produced by tryptic digestion of ~34~~~ isolated from a vanadate-arrested extract (Figure 6A, lane 4) and from a mitotic extract (lane 5) were analyzed by two-dimensional electrophoresis/chromatography (Figures 6B and 6C). The vanadate-arrested ~34~dC2 gave rise to four phosphopeptide spots. (The unlabeled spots are not reproducibly observed and the one in Figures 6B and 6D does not appear to be a phosphopeptide.) Spots 3 and 4 were also seen in the mitotic sample (Figure 6C). Spot 2 contained phosphotyrosine, confirming that cyclin induced the tyrosine phosphorylation of p34Cdc2. Spot 1 contained both phosphotyrosine and phosphothreonine. Only one site of tyrosine phosphorylation (at amino acid 15) has been reported in any ~34~~~ (Gould and Nurse, 1989). The adjacent Thr-14 is phosphorylated in mammalian ~34~~~~ (C. Norbury and f? Nurse, personal communication), suggesting that peptide 1 may be a doubly phosphorylated version of peptide 2. The relative mobilities of these spots are consistent with this interpretation (Ward and Kirschner, 1990) as is the variation in their relative intensities between experiments.

Cell 1019

p13 BEADS

’-ivCYC ” 5’

A

II

CYC

BEADS

CYC’ 10’ 1s *iI 30 5’ 10’ 15’ 20‘ 30’ iv

HIK

1 2 3 4 5 6 7 8 9 10 11 12 13 14 BEADS I 5’ I 10WC ’-iv WC” p135’BEADS IO’1sZD’ 30 1s203vCYC’ +”

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4 5 6 7 8 9 10 11 12 13 14 p13BEADS WC BEADS , I WCI 5 IO 15’ 23 30’ 5’ Ill 15 , “.

a-P-TYR

1 2 3 Figure

7. Cyclin-Induced

4 5 6 7 8 9 10 1, 12 13 14 Transient

Modification

of ~34~“~

An interphase extract was diluted with 1 vol of X6 and incubated at 23% for 30 min either alone (lanes l), with GT-cyclin plus 2 mM vanadate (lanes 2) or with 2 mM vanadate alone (lanes 3). Separately, a diluted extract was incubated with GT-cyclin for the indicated times (lanes 4-9). Samples were taken at the appropriate times for Hl kinase assay ([A], HlK) or for analysis by Western blotting following purification on p13sUc’ beads. Alternatively, Gl-cyclin was prebound to glutathione beads and added to an interphase extract in the absence (lanes 9-13) or the presence (lane 14) of 2 mM vanadate. Samples were taken at the indicated times (lanes 9-13) or at 30 min (lanes 14) for HI kinase assay (A) or for analysis by Western blotting. Blots were probed with the a-PSTAIR antiserum ([El, a-PSTAIR) or a monoclonal antibody against phosphotyrosine ([Cl, a-P-TYR). The final concentration of soluble GT-cyclin was 100 nM and of GT-cyclin on glutathione beads was 175 nM.

We have consistently observed mitotic phosphorylation of p34Cdc2 on threonine (spots 3 and 4). Since this result contradicted a previous report that Xenopus ~34~~~~ is unphosphorylated during M phase (Gautier et al., 1989) we sought independent confirmation for this finding. The p34Ck2 shown in Figure 8A was purified by binding to p13SuC1 beads followed by immunoprecipitation with an a-PSTAIR antiserum. Although mitotic ~34~~~~ should be electrophoretically separable from p32 (Figures 58 and 5D, lane 4; see also Figure 6A, lowest panel; Figure 78) we achieved a greater purification of ~34~~~ by taking advantage of the failure of p32 to bind to cyclin (Figure 5D). 32P-labeled ~34~~~~ purified from vanadate-arrested and mitotic extracts by ‘binding to GT-cyclin beads followed by immunoprecipitation, generated phosphopeptide maps (Figures 8D and 8E) that were indistinguishable from those obtained following purification on ~13~~~’ beads (Figures 6B and 6C). Therefore, all of the phosphopeptides, including those present in mitosis, were specific for p34c”p. Tyrosine-Phosphorylated ~34~dC~ Is a Transient Intermediate in Mitotic Actlvatlon A time course of ~34~~~ activation showed that the tyrosine-phosphorylated form was a normal activation in-

termediate and not a consequence of vanadate treatment (Figure 7). As expected, Hl kinase activity appeared after a lag of 10 to 15 min and remained high through 30 min (Figure 7A, lanes 4-8); vanadate blocked this activation (Figure 7A, lane 2). Electrophoretic variants of p34CdC2 appeared at 5 min, peaked at 10 min, and then declined (Figure 7B, lanes 4-8). Paralleling the mobility shifts, phosphotyrosine content also peaked at 10 min and then declined to interphase levels (Figure 7C, lanes 4-8). During the lag prior to Hl kinase activation, all of the cyclin-associated ~34~~~~ was phosphorylated. This was shown by prebinding GT-cyclin to glutathione beads prior to the addition of interphase extract (Figure 7, lanes 914). ~34~~~~ modification and phosphotyrosine content peaked just before 10 min, as Hl kinase activation was beginning. At 5 min (lane 9) or in the presence of vanadate (lane 14) only modified ~34~~~‘was found in association with GT-cyclin. Unshifted ~34~~~~ first appeared at 10 min, during Hl kinase activation. These experiments confirmed that p32 does not bind to cyclin, although it did bind well to ~13~~~’ (lanes 1-8). Modified ~34~~~~ Is Quantitatively Bound to Cyclin The results in Figure 5 demonstrated that cyclin-bound p34c”2 was quantitatively modified, but they did not address the modification state of cyclin-free ~34~~~~. If modified ~34~~~~ could dissociate from cyclin, then cyclin could act catalytically, as has been suggested (Labbe et al., 1989b; Murray et al., 1989). Figure 8 shows that little, if any, modified ~34~~~’ existed free of cyclin. Serial dilutions of cyclin-pd were added to an interphase extract. p34Ck2 was fully activated at the higher cyclin concentrations but not activated at all at the lower, subthreshold concentrations (Figure 8A). As is frequently observed, there was incomplete demodification of activated ~34~“~ (Figure 88, lanes 2 and 3; unpublished data; see also Figure 5). Modification was maximal at 50 nM cyclin (lane 4) and declined in proportion to the concentration of cyclin (lanes 5-7). The cyclin-free pool of ~34~~~~ contained no modified forms (Figure 8C), and there was a noticeable loss of total ~34~~~~ at the higher cyclin concentrations (lanes 2 and 3). Thus, any ~34~~~ that dissociated must have rapidly lost its modifications. This result suggests that modification only occurs in the cyclin-containing complex and that cyclin does not act catalytically to generate free, modified ~34~~~~. Mitotic Regulation of p34Cdc2 Tyrosine Phosphorylation and Dephosphorylation At some point during interphase sufficient cyclin will have accumulated to induce mitosis. The abruptness of the transition into mitosis suggests that the activities that control ~34~~~~ inactivation may change. Two reactions were studied: tyrosine phosphorylation and tyrosine/threonine dephosphorylation. The relative rates of tyrosine phosphorylation of interphase and mitotic ~34~~~~ were determined following addition of vanadate (to block dephosphorylation) to an interphase extract (in the presence of GT-cyclin) and to a mitotic extract (Figures 9A and 9B). As expected, vanadate blocked Hl kinase activation in the in-

Cyclin 1019

Activation

of p34=‘jc2

ure 6A). Half of the ~34~~ was dephosphorylated to the mitotic level by 1 min (lane 7), and a limit state was reached by 5 min (lane 6). The rate of ~34~~ dephosphorylation is thus at least an order of magnitude greater in mitosis than during interphase.

1234567

Discussion

“M C”Cli” ~ I ~

B

‘0

200

100

50

25

12

6

I P32

Cyclln-induced Phoephorylation of p34cdc2

TOTAL P34CDC2

“M cyc,in

C

‘0

200

100

50

25

12

6’

~32

1

2

3

4

5

6

7

M

CYCLIN-FREE P34CDC2

Figure

8. Modified

Forms of ~34~~~

Are Bound

to Cyclin

One-tenth volume of cyclin-prA was added to an interphase extract to the indicated final concentrations. After a 40 min incubation, samples were taken for Hl kinase assay ([A], HlK). p34&* was purified from larger samples on ~13~~’ beads and analyzed by Western blotting with the a-PSTAIR antiserum ([B], TOTAL ~34~9. Alternatively, cyclin-containing samples were first incubated with IgG-Sepharose beads to remove cyclin-associated ~34~~, and the remaining ~34~’ was purified on p133uc1 beads and analyzed by Western blotting ([Cl, CYCLIN-FREE ~34~~3. Lanes labeled M contained 20 ng of purified ~32.

terphase extract (Figure 9A, lanes 2-6). Surprisingly, it also caused a slow inactivation of mitotic activity (lanes 7-11) to nearly the interphase level by 60 min (not shown). Mitotic activity was stable in the absence of vanadate through at least 60 min (lane 12). The rates of tyrosine phosphorylation were determined by Western blotting of pl3S”C’ -purified ~34~~~ (Figure 96). Phosphotyrosine levels plateaued in the interphase extract within 5 min (lanes 2-6). In contrast, phosphotyrosine was undetectable in the mitotic extract until 30 min, at which time it was close to, but less than, the interphase level at 5 min. The rate of tyrosine phosphorylation was thus at least 6-fold higher in interphase than it was during mitosis. The converse reaction that might be modified by the mitotic state is p34=k2 dephosphorylation. To make 32P-labeled ~34~~2 for use as a phosphatase substrate, GT-cyclin and 32P-orthophosphate were added to an interphase extract in the presence of vanadate, and labeled complexes were purified on and eluted from glutathione-agarose beads (see Experimental Procedures). This material was added to interphase and mitotic extracts, and p34cdc2, with associated Gl-cyclin, was recovered on ~13~uC’ beads and analyzed by electrophoresis (Figure 9C). Little dephosphorylation occurred in the interphase extract through 30 min (lanes 2-5). In striking contrast, significant dephosphorylation and an assqciated increase in the electrophoretic mobility of ~34~“* occurred in the mitotic extract. These changes presumably reflect dephosphorylation at the shift-producing tyrosine and threonine sites with retention of phosphorylation at the mitotic sites (see Fig-

We have shown by immunoblotting and by direct 32P analysis that p34&* is unmodified in the absence of cyclin; binding to cyclin induces phosphorylation of specific tyrosine and threonine residues. This result contradicts previous assumptions about the control of ~34~~~ phosphorylation on tyrosine and its possible function during p34C~2 inactivation (Dunphy and Newport, 1989; Gautier et al., 1969; Murray, 1990; Nurse, 1990). In fact, all recent models that address this issue indicate that p34@* is tyrosine phosphorylated prior to binding cyclin and that such phosphorylation is a characteristic of the interphase state that may be responsible for the inactivity of monomeric p34&2 (Gautier el al., 1989; Murray, 1990; Nurse, 1990). We agree that specific threonine and tyrosine residues are rapidly dephosphorylated upon entry into mitosis, but our experiments clearly show that they remain unphosphorylated during the following interphase so long as cycloheximide is present to prevent the reaccumulation of cyclin. It is now well established that significant amounts of cyclin are present in the oocyte interphase state (Westendorf et al., 1989), as well as during interphases of cultured cells (Pines and Hunter, 1989) and yeasts (Booher.et al., 1989), and we consider it likely that cyclin was present whenever tyrosine-phosphorylated ~34~~2 was previously reported. We showed that the unphosphorylated interphase state of ~34~~~ was inactive. Thus, the cyclin-induced transient threonine and tyrosine phosphorylations, even though they block kinase activity, are part of the activation pathway, not the inactivation pathway, of ~34~~~ regulation. When only a small proportion of ~34~~~ molecules becomes tyrosine phosphorylated, all of these molecules are subsequently dephosphorylated (Dunphy and Newport, 1989). Since the same event (cyclin binding) targets a particular ~34~~~ molecule for both activation and for tyrosine phosphorylation and dephosphorylation, only one selective event, cyclin binding, is required. The appearance of modified forms of ~34~~ (Bailly et al., 1989; Morla el al., 1989; Pines and Hunter, 1989) are therefore presumptive evidence for cyclin association. In addition to two inactivating phosphorylations, cyclin also induced mitotic phosphorylation of p34Ce2. The phosphorylated protein was clearly not ~32. We do not fully understand why M phase phosphorylation of Xenopus p34C”* was not observed previously. The absence of electrophoretic retardation may have obscured its presence, but that cannot be the entire explanation. Perhaps, too, it is transient and vanishes by the points in mitosis at which cells can easily be arrested. The mitotic phosphorylations of ~34~~~ could either be obligatory for activity or simply an indirect consequence of a cyclin-induced con-

Cell 1020

tophosphorylatable (Hindley and Phear, 1984; Ldrincz and Reed, 1984; Simon et al., 1988; Toh-e et al., 1988; Courchesne et al., 1989; Elion et al., 1990).

CYCLIN

-

Figure 9. Mitotic phorylation

Regulation

of ~34 cdcz Phosphorylation

and Dephos-

(A) and (6) ~34~~~ phosphorylation. Aliquots were taken from an untreated interphase extract (lanes 2) or from an interphase extract incubated in the presence of 160 nM GT-cyclin and 2 mM vanadate for the indicated times (lanes 3-6) and assayed for HI kinase activity (A) or by blotting pWUCf-purified ~34 cdcz for its phosphotyrosine content (B). A mitotic extract was produced by incubating an interphase extract with 160 nM GT-cyclin for 30 min. Aliquots were taken from the untreated mitotic extract (lanes 7) or at the indicated times following addition of vanadate to 2 mM and processed as for the interphase samples (lanes 6-11). Lane 12 contained mitotic extract incubated for a further 60 min in the absence of vanadate. Lane 1 contained 20 ng of ~32. (C) ~34~~~ dephosphorylation. 32P-labeled GT-cyclin-p34CdC2 complexes were purified from vanadate-arrested extracts as described in Experimental Procedures and were added to interphase and mitotic extracts for the indicated lengths of time. The 0 min samples were mixed with extract on ice in the presence of vanadate to prevent dephosphorylation. GT-cyclin-p34CdC2 complexes were purified on p13sucr beads, eluted with SDS-PAGE sample buffer, and analyzed by gel electrophoresis and autoradiography.

formational change of p34Cdc2. An intriguing possible phosphorylation site is a Gly-Thr-Pro motif that is absolutely conserved in a very large number of ~34~~ homologs and other related proteins and that might be au-

A Model of Cyclin-Induced Activation of ~34~~~~ Figure 10 presents a working model for how cyclin accumulation triggers the activation of ~34~~~. This model contains three key features: First, ~34~~~ is unmodified prior to association with cyclin. Second, cyclin binding induces a conformational change in p34cdc2, rendering it susceptible to phosphorylation by both a tyrosine and one or more threonine kinases. We have no direct kinetic or mechanistic evidence that phosphorylation follows a particular order, either kinetically or mechanistically, although the presence of phosphotyrosine in both electrophoretitally retarded forms of p34Cdc2 suggests that phosphorylation of tyrosine may occur first. Dephosphorylation of tyrosine may be required for the subsequent dephosphorylation of threonine, since vanadate, a phosphotyrosine phosphatase inhibitor, blocks dephosphorylation at both sites. This form of the cyclin-p34”c2 complex may be a conformationally active state, inhibited only by the double phosphorylation within the ATP binding site. As cyclin is synthesized, cyclin-p34cdc2 complexes presumably accumulate in this state until a threshold is reached, leading to the series of reactions that activate the complex. Third, the transition into mitosis is associated with a significant decrease in the rate of ~34~~~~ tyrosine phosphorylation and increase in its rate of dephosphorylation. Although the combined effect of these changes could exceed lOOfold between mitosis and interphase (see Figure 9) far smaller changes in activity may occur just as the cyclin threshold is surpassed. A slow initial rise in Hl kinase activity could produce further changes in these p34cdc2modifying enzymes, leading to ever faster activation. Such a concerted activation of kinase activity could explain the long-recognized autocatalytic nature of MPF activation and the abrupt rise in MPF activity during the cell cycle. This sort of accelerating feedback loop, starting from a very low level of activity, would also predict that a lag might be present even if the threshold were surpassed instantaneously or even eliminated, as in the presence of low levels of mitotic extract or of okadaic acid (see Figure 4).

+

Figure

10. Model for Cyclin-Induced

phosphorylation

ACTIVE INACTIVE

and inactivation.

Activation

Mitosis

is as-

phosphorylation at inhibitory sites and an increased rate of its dephosphorylation. Other sites are specifically phosphorylated in mitosis. The transition to the activating state may begin with a small increase in Hl kinase activity, either of the fi%‘ly active form or inherent in the tyrosine-phosphorylated intermediate. INH directly inactivates ~34~“~. See text for further details.

Cyclin 1021

Activation

of ~34~~~~

The initiation of the transition into mitosis requires that there be a mechanism to “sense” the concentration of the accumulating inactive cyclin-p34Ch2 complexes. The most direct measure would be if these complexes themselves had low but significant kinase activity. Although these complexes probably do not have high activity in view of the double phosphorylation of ~34~~~ within its ATP binding region, it has not been demonstrated that they are completely inactive. A second way to measure complex formation would be if a low concentration of fully active complexes were also formed, either by equilibration with inactive complexes or by a stochastic process whereby some newly formed complexes acquired mitotic phosphorylations to the exclusion of inactivating phosphorylations. In support of either of these general schemes, we have consistently observed a low level of Hl kinase activation in the presence of vanadate (Figures 5A and 7A) as well as at early times during activation (Figure 7A). We have placed INH, a negative regulator of Hl kinase and MPF activation (Cyert and Kirschner, 1988; T H. L. et al., unpublished data), at the final step in the pathway. INH is a type 2A phosphatase that can inactivate Hl kinase in vitro (T. H. L. et al., unpublished data). The proportionality between INH and the cyclin threshold is readily explained by this model: near the threshold concentration of cyclin, the low concentration of the active complex would be directly proportional to the cyclin concentration and inversely proportional to INH activity. The qualitative relationship between INH and the length of the lag is also understandable: as the threshold of active complexes is reached, the slow “autocatalytic” increase in net active complex formation would be highly sensitive to INH levels. The approximately linear responses of the lag and threshold to INH concentration (Figure 4) are most readily explained by modeli such as this in which INH acts directly on cyclin or p34Ce2. Since both cyclin and ~34~~~ are phosphorylated during mitosis (Meijer et al., 1989; Pines and Hunter, 1989; Gautier et al., 1990; T. H. L. et al., unpublished data; this paper), either could be a site of INH action, and further work will be necessary to determine its precise target(s). This model makes a number of testable predictions about p34Cdc2-modifying activities. We expect there to be at least two ~34~~ kinases, one or more specific for threonines and one for tyrosine. These kinases should be specific for p34cdc2 in association with cyclin and should not phosphorylate monomeric ~34~~~. We have partially purified unphosphorylated cyclin-p34cdc2 complexes that activate spontaneously in the presence of ATP, even when vanadate is present. The ability to thus bypass the vanadate-sensitive regulatory system suggests that either unmodified cyclin-p34c@ complexes have an innate ability to activate, or a specific activating enzyme exists. Finally, the changes we observed in the rates of ~34~~~ phosphorylation and dephosphorylation during mitosis should not necessarily be interpreted as changes in kinase and phosphatase activities. It is equally plausible that the association of another protein with the cyclinp3@’ complexes is regulated, and that this protein controls the availability of the substrate. One candidate is

pl3S”C’, which might bind to p34d2 the latter is tyrosine phosphorylated, could be cdc25. ExperImental

only transiently, when for instance; another

Prowdums

Preparation of Extracts Extracts of Xenopus eggs arrested in M phase were prepared as dsscribed (Murray and Kirschner, 1989; Murray et al., 1989). interphase extracts were derived from the M phase extracts by addition of cycleheximide to 100 pg/ml and CaC12 to 0.4 mM, and incubation at 23OC for 40 min. Aliquots were frozen in liquid nitrogen and stored at -8ooC. Antiwra The anti-PSTAIR serum was raised in rabbits by BAbCo (Richmond, CA) against the peptide EGVPSTAIREISLLKEC coupled 10 KLH through the C-terminal cysteine residue (Green et al., 1982). The rabbit antiserum to the C-terminal 16 amino acids of p32 was a generous gift of J. Gautier and J. Mailer. Culture supernatant containing monoclonal antibody 4GlO against phosphotyrosine was a generous gift of D. Morrison and T M. Roberts. Expmssion and Purifkation of p32 A 1.3 kb Ncol-Sspl fragment of plasmid CDC91 containing Egl, the p32 gene, was inserted into the T7 expression vector pET-8c that had been cu1 with Ncoi and Bamlil and that had a filled-in BamHi site. This construct produced p32 beginning at its own initiator methionine (no fusion amino acids) under the control of the T7 promotor. The protein was expressed in E. coli strain BL21(DE3). Cells were induced at an ABoo of 0.4 with 0.4 mM IPTG for 3 hr. The protein was expressed to very high levels but was insoluble. The cell pellet was washed in 0.9% (w/v) NaCI, resuspended in 0.9% NaCI, lysed directly in SDS-PAGE sample buffer, boiled for 5 min, and briefly sonicated to reduce the viscosity. The protein was purified by preparative gel eiectrophoresis on 1.5 mm thick, 5%-15% gradient gels. Proteins were stained with Coomassie blue, and the p32 band was cleanly excised. Protein was eluted into 0.1% SDS, 50 mM NH&lCOa using an lsco model 1750 electroelutor according to the manufacturer’s instructions. Protein recovery was approximately 1 mg from 40 ml of culture. Purification of iNI4 INH was partially purified from Xenopus eggs by DEAE chromatography (T H. L. et al., unpublished data). The AmSO, and heating steps were omitted. A concentrated AmSO precipitate of the peak fractions was resuspended in EB (15 mM MgClp, 20 mM EGTA, 10 mM dithiothreitol, 60 mM K+glycerophosphate [pH 7.31, containing 25 wg/ml aprotinin, 25 pglml leupeptin, 1 mM benzamidine, 0.5 mM PMSF, 10 Kg/ml pepstatin) and dialyzed extensively against XB (100 mM KCI, 50 mM sucrose, 1 mM MgCIP, 0.1 mM CaC12, 10 mM K-HEPES [pH 7.41, with 10 pa/ml each of leupeptin, chymostatin. and pepatatin) containing 1 mM dithiothreitol. The specific activity of the INH was 2800 Ulmg. Pmparation of p13*UC1 Beads ~13~~’ was purified essentially according to published procedures (Brizuela et al., 1987; Labbe et al., 1989a). pRK172kucl was grown in BL21(DE3)LysS and induced by adding IpT(; to 0.4 mM when the & was 0.4. After 3 hr, cells were rinsed and lysed, and a clarified extract was prepared as described above for the preparation of cyclin extracts. A 30%-50% AmSO cut was prepared, resuspended in lysis buffer, heated to 65OC for 3 min, clarified, and 4 ml was applied to a CWB column (18 x 2.5 cm, Pharma’cia) equilibrated in 2 mM EDTA, 50 mM Tris (pli 6.0). The essentially pure fractions of ~13%’ were pooled and dialyzed against 0.5 M NaCI, 0.1 M NaHC03 (pH 8.3), and coupled to CNBr-activated Sepharose 48 (Pharmacia) according to the instructions of the manufacturer. Unreacted groups were quenched with 0.2 M ethanolamine (pH 8.0). The beads were extensively washed according lo the manufacturer’s instructions and stored in 0.5 M NaCI, 5 mM EDTA, 0.1 M Tris (pH 8.0). The concentration of coupled pl3sm’ was approximately 2 mg per ml of gel. Construction To construct

and Purification of Cyciin Fusion Proteina cyclin-pd, a 0.6 kb Sau3A fragment of pRIT2T (containing

Cdl 1022

the IgG binding domain of protein A; Pharmacia [Nilsson et al., 19851) was inserted into the Bglll site of cyclinAl3 (Murray et al., 1989) at amino acid 91. An Nsil-EcoRI fragment was excised. The Nsil end was chewed back, and the EcoRl end was filled in with T4 polymerase. The blunt-ended fragment was inserted into a T4 polymerase-treated BamHl site of PET-3a. Strain BL21(DE3) was used for expression of the fusion protein. In a routine cyclin purification, 2 liters of cells was grown to an As,,, of 0.8 and induced for 30 min with 0.4 mM IPTG. The cells were pelleted, washed twice with 0.9% NaCI, and resuspended in 14 ml of 150 mM NaCI, 5 mM dithiothreitol, 2 mM EDTA, 50 mM Tris (pH 8.0) with 40 uglml each of leupeptin, chymostatin, and pepstatin to which 0.5 mg/ml lysozyme was then added. After 30 min, NP-40 was added to 0.5% (producing buffer A + lysozyme), and the extract was subjected to sonication (30 s at setting #4 on a Branson model 350 Sonifier with a microtip) followed by ultracentrifugation for 30 min at 45,000 rpm in an SW50.1 rotor. The clarified extract was incubated with 1 ml of IgG-Sepharose beads (Pharmacia, #17-0989-01) for 90 min at 4OC with rocking. Prior to incubation, the beads were washed extensively in buffer A, followed by buffer B (3 mM dithiothreitol, 10 mM sodium-HEPES [pti 8.01, with 10 pglml each of leupeptin, chymostatin, and pepstatin), 0.25 M lithium diiodosalicylate (LIS) in buffer B, buffer B again, and finally buffer A. After protein binding, the beads were washed extensively in buffer A, poured into acolumn, and washed with buffer B. Bound protein was eluted in 2 ml of 0.2 M LIS in buffer B and was subsequently desalted into buffer B on a Sephadex G-25 column and concentrated in aCentriconcell (Amicon) to approximately 300 11 (about 0.2 mglml). Protein concentrations were determined on stained gels by comparison with known amounts of bovine serum albumin (determined spectrophotometrically). To construct the GT-cyclin fusion protein, an Ncol-EcoRI fragment of A4OOp27 (Murray et al., 1989) with filled-in ends was inserted into a filled-in EcoRl site of pGEX2 (Smith and Johnson, 1988). The fusion protein was expressed in HBlOl. For purification, 8 liters of culture was grown to an ABoo of 0.4, induced for 3 hr with 0.4 mM IPTG, and a clarified extract was prepared as described above, scaled up to J-fold. Approximately 45 ml of clarified extract was incubated with 3 ml of glutathione-agarose beads (S-linkage, Sigma), previously washed extensively in buffer A, for 30 min at 4OC on a rotating platform. The beads were subsequently washed in buffer A followed by buffer B. Bound protein was eluted by three 2 min rinses of 3 ml each in buffer B containing 5 mM glutathione (reduced, freshly prepared). The released protein was concentrated in Centriconcells and desalted on a Sephadex G-25 column into buffer 8. The typical recovery was 309 ul at 0.2 mglml. Although almost all of the expressed protein was insoluble, when supernatants containing very low levels of the cyclin fusion proteins were passed over affinity columns, proteins of the expected sizes and immunoreactivity could be specifically eluted. This approach may be a useful general method for the purification of proteins that appear to be totally insoluble. Contaminating bands in both preparations are probably bacterial stress-induced proteins bound to small fragments of each fusion protein. Proteinsof about 30 kd in theGT-cyclin preparation contained glutathione transferase fused to small peptides of cyclin; they did not bind to ~34~“~ attached to p13sucr beads. We have consistently found that a ~40-50 nM concentration of either cyclin protein was required to induce near maximal levels of Hl kinase activity in extracts, close to our estimate of 50-100 nM for the endogenous pool of ~34~~~ (see, for example, Figures 5 and 7). This value is also consistent with the estimated concentration of cyclin B during mitosis (7 nM; J. Minshull, personal communication), which does not generate such high levels of Hl kinase activity. Injection of a final concentration of 11 nM of either cyclin into Xenopus oocytes induced maturation, even in the presence of cycloheximide. The time required for maturation (70 min) was independent of cyclin concentration and 20 min shorter than that required for MPF-induced maturation.

Occyte

Maturation

and MPF Assays

Frog care and oocyte procedures were as described (Cyert and Kirschner, 1988). Oocytes from pregnant mare’s serum gonadotropin-primed frogs were “plucked” and injected on the equatorial plane with 50 nl samples. In MPF assays, samples were diluted into EB containing 0.025% NP-40. Germinal vesicle breakdown was scored at 2 hr, following TCA fixation and dissection of the oocyte. A unit of MPF activity is

the amount that, under the above conditions, induces maturation in half of the injected oocytes. Activity is expressed as units per microliter of extract. Thus, if an extract is diluted with cyclin, and then diluted with EB before injection into oocytes, the concentration activity is adjusted to refer to the undiluted extract (before cyclin addition).

Hl Klnaae

Assays

Samples for assay were diluted 50-fold into EB (relative to the extract before additions) and stored in liquid nitrogen until use. Ten microliters of sample was mixed with 8 ul containing 287 W/ml histone HI (Boeringer-Mannheim; stored in 0.2 M NaCI, 0.1 mM EDTA, 1 mM dithiothreitol. 20 mM Tris [pH 7.41, and 10 pglml each of leupeptin. chymostatin, and pep&tin), 1 mM ATP, and 0.25 t&i/p1 [r-*P]ATP (ICN, 4500 Cilmmol, aqueous). (The PKA inhibitory peptide was never observed to have any effect on background activity under these conditions and was not routinely used.) After 15 min, 18 PI of 2x electrophoresis sample buffer was added, and the samples were run on 5%-15% gradient gels (acrylamide:bisacrylamide = 30:0.8). Bands were detected by autoradiography of dried gels without intensifying screens. For quantitation, labeled bands were excised and radioactivity determined by Cerenkov counting. Activity is expressed as pmoles of phosphate incorporated per milligram per minute.

Additlon

of Cyclln

to Interphase

Extracts

Unless specified otherwise, cyclin was diluted into XB (Murray and Kirschner, 1989) and mixed with an equal volume of interphase extract. After incubation, aliquots for Hl kinase assay were diluted 25-fold into EB (50-fold relative to the initial interphase extract) and stored in liquid nitrogen until assay. Aliquots for MPF assay were diluted with an equal volume of EB and frozen in liquid nitrogen. To prepare cyclin-prA beads (Figure 28). IgG-Sepharose beads were rinsed in buffer A and then incubated for 90 min in approximately IO vol of a clarified extract containing cyclin-pd (see above). The beads were subsequently rinsed in buffer A and then in XB. Equal volumes of cyclin beads and interphase extract were mixed. Control cyclin beads contained identically treated beads incubated in a clarified extract derived from strain HBlOl not containing any cyclin-expressing plasmid. After incubation, supernatant and pellet fractions were prepared by transferring 8 ul into 200 PI of EB (to stabilize any activity not associated with the beads) and centrifuging in a microfuge for 3 s. The supernatant was transferred and immediately frozen in liquid nitrogen; the pellet was resuspended in 200 11 of EB and frozen.

Prepsration

of Samples

for Western

Blotting

For ~13~~ bead purification of ~34~~~ prior to blotting, up to 100 91 of extract was added to approximately half that volume of ~13~“~’ beads. The beads were first rinsed extensively in XB containing 0.5% NP-40 and 0.5 M NaCI, in EB without protease inhibitorsor dithiothreitol, and finally in EB. After 5 min on ice with periodic mixing, the beads were pelleted and rinsed once with 0.9 ml of EB containing 0.2% NP40, 0.5 M NaCI, 2 mM vanadate, twice with 0.9 ml of EB containing 2 mM vanadate, and once with 3 mM vanadate, 1 mM dithiothreitol, 1 mM EDTA, containing 10 uglml each of leupeptin, chymostatin, and pepstatin, and 10 mM HEPES (pH 8.0). The final pellet was resuspended in at least an equal volume of 2x sample buffer (lx sample buffer contained 5 mM EMA, 5 mM vanadate, 20 mM sodium pyrophosphate, 30 mM NaF, 10% SDS, bromophenol blue, 80 mM Tris [pH 8.81, 10% glycerol, 0.1 M dithiothreitol). Material bound to GT-cyclin beads was prepared in a similar manner. Glutathione beads (S-linkage, Sigma) were washed extensively in 150 mM NaCI, 0.5% NP-40, 5 mM dithiothreitol, 2 mM EDTA, 50 mM Tris (pH 8.0) containing 10 i@ml each of leupeptin, chymostatin, and pepstatin (buffer A). GT-cyclin was added and incubated with the beads for 20 min, followed by extensive rinsing in buffer A and then in XB. Where appropriate, the beads were rinsed in XB containing 4 mM vanadate prior to addition to the interphase extract, Following incubation with the extract, GT-cyclin beads were pelleted and rinsed according to the procedure for p13syc’ beads. Bead-bound material from 8-10 pi was used for all blots, except a-P-TYR blots, which contained 13-25 nl.

Western

Blotting

Samples were electrophoresed on 8.5% polyacrylamide gels (total acrylamide:bisacrylamide = 3O:l) containing 2 mM EDTA. Slow elec-

Cyclin 1023

Activation

of ~34~~~~

trophoresis (4-5 hr for a 13 cm gel) significantly improved resolution of the modified forms of ~34~“~. Proteins were transferred to ImmobiIon membranes (Millipore) at 0.4 mA for 3-4 hr in a Hoefer model TE42 electrotransfer device in 225 mM glycine, 30 mM Tris base, 20% (VA’) methanol. Except for anti-phosphotyrosine blots, filters were blocked in TEST (0.2 M NaCI, 0.1% Tween, 10 mM Tris [pH 7.41) containing 5% nonfat dry milk (blotto), incubated in antibody in the same buffer, washed with multiple changes for 1 hr in TBST, incubated with a secondary antibody in blotto, and washed for 1 hr in TEST. The antisera directed against the PSTAIR peptide of ~34~*~, ~32, and the C-terminus of p32 were used at dilutions of 1:200. The a-P-TYR culture supernatant was used at a dilution of 1:l. Primary and secondary antibody incubations for anti-phosphotyrosine blots were in TBST containing 5% bovine serum albumin (Miles). Primary and secondaryantibody incubations for Figure 8 contained in addition 10 mg/ml human r-globulin to reduce reactivity with the cyclin-protein A fusion protein. sera Detection in Figure 6 was with lz51-protein A. All other secondary were alkaline phosphatase conjugated. Analysis of ~34~~~~ Phosphoiylstlon Sites Freshly prepared egg extract (400 ul) was added to 10 mCi of dried 32P-orthophosphate (NEN. nNEX-053) and incubated at 23OC for 10 min to allow equilibration; 100 ul was transferred to 60 ul of ~13~~’ beads in EB on ice. Three microliters of 40 mM CaC12 was added to the remaining extract; 100 ul was removed to ~13~“~~ beads after 30 min. The remaining extract was split and the halves incubated with 330 nM GT-cyclin in the presence or absence of 2 mM vanadate for 30 min, and then bound to ~13~’ beads. Aliquots of all extracts were diluted for Hl kinase assay. The p13suc’ beads were incubated with extracts for 10 min and then washed extensively in RIPA+ (0.1% SDS, 1.0% NP-40, 1.0% deoxycholate, 0.15 M NaCI, 1 mM EDTA, 1 mM dithiothreitol, 3 mM vanadate, 50 mM NaF, 30 mM sodium pyrophosphate, 10 uglml each of leupeptin, chymostatin, and pepstatin, and 20 mM HEPES [pH 7.51). After a single rinse in 1 mM dithiothreitol, 3 mM vanadate, 1 mM EDTA, 50 mM NaF, 30 mM sodium pyrophosphate, and 10 mM HEPES (pH 7.5) containing 10 @ml each of leupeptin, chymostatin, and pepstatin, bound protein was eluted by boiling twice in 60 nl of 0.5% SDS, 1 mM dithiothreitol, 1 mM EDTA, 3 mM vanadate, 50 mM NaF, 30 mM sodium pyrophosphate, and 10 pg/ml each of leupeptin, chymostatin, and pepstatin. The pooled eluate was adjusted to 600 ul of RIPA+ and added to 50 ul of protein A-Sepharose CL4B (Sigma) prebound with 100 ul of a-PSTAIR antiserum in RIPA+. After 60 min, the beads were washed extensively with RIPA+ and resuspended in 150 ul of SDS-PAGE sample buffer (see Western Blotting section). To purify labeled, cyclin-associated p34cdc2, 100 pl of interphase extract (preequilibrated with 2.5 mCi of dried 32P-orthophosphate) was incubated with 70 nM GT-cyclin in the presence or absence of 2 mM vanadate for 20 min at 23“C. The extracts were bound to 100 ul of glutathione-agarose beads for 15 min at 23OC and then washed twice in EB containing 0.5 M NaCI, 0.2% NP-40,2 mM vanadate, 50 mM NaF, and 30 mM sodium pyrophosphate, once in EB containing 2 mM vanadate, 50 mM NaF, and 30 mM sodium pyrophosphate, and twice in 150 mM NaCI, 1 mM EDTA, 1 mM dithiothreitol, 3 mM vanadate, 50 mM NaF, 30 mM sodium pyrophosphate, and 20 mM HEPES (pH 7.5) containing a 10 @ml concentration each of leupeptin, chymostatin, and pep&tin. Bound proteins wereeluted into 300 ul of this last buffer supplemented with 5 mM glutathione by three 2 min rinses at 23OC. The eluates were made 0.2% in SDS, boiled, and then adjusted to 600 ul of RIPA+ and immunoprecipitated as described above. Aliquots of immunoprecipitated samples were analyzed by Western blotting to determine the forms of ~34~’ present. 1251-protein A detection was employed since a lot of IgG was present. Thin glass filters (made of coverslips) were used to verify that no 32P contributed to the observed 125l signal. lmmunoprecipitated proteins were electrophoresed on 5%-15% gradient gels. Phosphopeptides in the excised, labeled proteins were analyzed as described (Ward and Kirschner, 1990). Phosphoamino acids were separated by two-dimensional electrophoresis at pH 1.9 and pH 3.5 in the presence of unlabeled phosphoamino acids. 32P-Lsbsled p34cdcz-Cyclln Complexes Interphase extract (100 ul) was desalted on a Sephadex into XB containing 5 mM Mg&. One-half of the protein

G-25 column peak was in-

cubated with 144 nM GT-cyclin for 30 min in the presence of 2 mM vanadate, 1.3 mCi of [T-~P]ATP (ICN. >4ooo Cilmmol, #35001x), and 0.3 mM ATP The extract was then added to 60 ul of glutathione beads previously rinsed in XB containing 0.2% NP40,0.5 M NaCI, and 2 mM vanadate, and then in XB containing 2 mM vanadate. After 20 min with periodic mixing, the beads were pelleted and rinsed twice in XB containing 0.2% NP-4905 M NaCI, and 2 mM vanadate, and three times in XB (no vanadate). Bound complexes were specifically eluted in three 2 min rinses of 50 ul each containing XB with 1.0 mg/ml ovalbumin and 5 mM glutathione. To examine phosphatase activities, 10 ul of these complexes was incubated with 10 nl of an interphase or a mitotic extract. The mitotic extract was prepared by incubating an interphase extract with 260 nM GT-cyclin for 30 min. At various times, samples were added to p1380c1 beads in EB. The material was incubated, washed, and eluted in sample buffer as for Western blotting (see above). Acknowledgments We are indebted to David Morgan for initially helping to show that cyclin-prA could activate HI kinase activity and to Bill Hansen for advice about constructing protein A-containing fusion proteins. Ira Clark and Lucy Godley assisted at early stages of this work. We are grateful to those who read our two manuscripts and confronted us with the need to combine them: Bob Booher, Leslie DeLong, Ray Deshaies, Jeremy Minshull, Andrew Murray, Rob Needleman, and Tim Stearns. This work has been supported by a fellowship from the American Cancer Society (M. J. S.), a predoctoral fellowship from the National Science Foundation (M. G.), an NIH departmental training grant (T H. L.), and by general support from the National Institute of General Medical Sciences (M. W. K). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 16 USC Section 1734 solely to indicate this fact. Received

June

26, 1990; revised

September

11, 1990.

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of

The

In Proof

The paper referred to throughout as T. H. L. et al., unpublished data, is now in press: Lee, T. H., Solomon, M. J., Mumby, M. C., and Kirschner, M. W. (1991). INH, a negative regulator of MPF, is a form of protein phosphatase 2A. Cell 84. It is also shown in this paper that INH acts directly on p34 cdcz by removing a phosphate required for kinase activity.