Proc. Natl. Acad. Sci. USA Vol. 92, pp. 9303-9307, September 1995 Biochemistry

Reversible phosphorylation controls the activity of cyclosome-associated cyclin-ubiquitin ligase (cyclin degradation/cell cycle/mitosis) SHIRLY LAHAV-BARATZ*t, VALERY SUDAKIN*t, JOAN V. RUDERMANtt, AND AVRAM HERSHKO*t *Unit of Biochemistry, B. Rappaport Faculty of Medicine and the Rappaport Institute for Research in the Medical Sciences, Technion-Israel Institute of Technology, Haifa 31096, Israel; tMarine Biological Laboratory, Woods Hole, MA 02543; and tDepartment of Cell Biology, Harvard Medical School, Boston, MA 02115 Communicated by Elizabeth D. Hay, Harvard Medical School, Boston, MA, June 11, 1995 (received for review April 18, 1995)

ABSTRACT Cyclin B/cdc2 is responsible both for driving a few E3 proteins or activities have been characterized so far cells into mitosis and for activating the ubiquitin-dependent (9, 12). Multiply ubiquitinated proteins are then recognized degradation ofmitotic cyclins near the end ofmitosis, an event and degraded by the 26S protease (13). required for the completion of mitosis and entry into inter- Work with cell-free systems that reproduce cycles of cyclin phase of the next cell cycle. Previous work with cell-free accumulation and destruction (2, 14) has led to identification extracts of rapidly dividing clam embryos has identified two of some parts of the cyclin destruction machinery. Cyclin- specific components required for the ubiquitination of mitotic selective E2 and E3 activities, (E2-C and E3-C, respectively) cyclins: E2-C, a cyclin-selective ubiquitin carrier protein that have been identified and partially purified from clam oocytes is constitutively active during the cell cycle, and E3-C, a (8, 15). These act on the mitotic cyclins A and B but not on cyclin-selective ubiquitin ligase that purifies as part of a most other cellular proteins; cyclin ubiquitination by E2-C and -1500-kDa complex, termed the cyclosome, and which is E3-C depends on the presence of an intact, unscrambled active only near the end ofmitosis. Here, we have separated the N-terminal destruction box domain (15). The E3-C activity is cyclosome from its ultimate upstream activator, cdc2. The part of a large, '1500-kDa complex termed the cyclosome mitotic, active form of the cyclosome can be inactivated by (15). incubation with a partially purified, endogenous okadaic How is the activity of these components regulated? Cdc2 acid-sensitive phosphatase; addition of cdc2 restores activity activation at the beginning of mitosis somehow leads to to the cyclosome after a lag that reproduces that seen previ- activation of the cyclin destruction machinery near the end of ously in intact cells and in crude extracts. These results mitosis and, after a lag, to its subsequent inactivation (for demonstrate that activity of cyclin-ubiquitin ligase is con- reviews, see refs. 1 and 15). Reconstitution experiments using trolled by reversible phosphorylation of the cyclosome com- partially purified E2-C and E3-C from interphase extracts plex. (which do not ubiquitinate cyclins) or from late M-phase extracts (which contain strong cyclin ubiquitination activity) The early embryonic cell cycles of many organisms are driven indicate that E2-C is constitutively active throughout the cell by periodic oscillations in the levels of cyclin B, a positive cycle. By contrast, E3-C is inactive during interphase and early regulatory subunit required for activation of the protein kinase M phase and becomes activated only in late M phase (8, 15). cdc2 (for recent review, see ref. 1). The rise in cyclin B/cdc2 Addition of active cdc2 to interphase extracts leads to E3-C activity at the end of interphase initiates entry into mitosis and, activation. Incubation of early M-phase extracts (which already after a lag, activation of the cyclin destruction machinery near contain high levels of cdc2 activity) with MgATP to allow the end of mitosis. Cyclin degradation is required for cdc2 continuing kinase activity also leads to activation of the cyclin inactivation and exit from mitosis (1-4). The cyclin destruction destruction machinery (15). When these extracts are further machinery is active for only a short time, and the rate of cell incubated, the cell cycle advances further, and the cyclin cycle progression is set primarily by controlling stability of the destruction is inactivated. Inclusion of okadaic acid, an inhib- mitotic cyclin proteins (2, 5). itor of type 1 and 2A phosphatases (16), prevents inactivation Mitotic cyclins become ubiquitinated near the end of mitosis of the cyclin destruction machinery (17). (6-8), a modification that marks them for rapid proteolysis. Cdc2 clearly initiates the pathway leading to activation of the Ubiquitin is usually ligated to target proteins by the sequential cyclosome-associated cyclin-ubiquitin ligase E3-C, but neither action of three enzymes: a ubiquitin-activating enzyme (E1), a the components of that pathway nor the molecular modifica- ubiquitin-carrier protein [termed E2 or ubiquitin conjugating tions directly responsible for cyclosome activation are known. enzyme (UBC)] and a ubiquitin-protein ligase (E3), (for Similarly, an okadaic acid-sensitive phosphatase activity has review, see ref. 9). Numerous E2/UBC proteins have been been implicated in the process of inactivation of this pathway identified by a variety of biochemical and genetic approaches; (17), but the relevant target has not yet been identified. some of these are involved in the ligation of ubiquitin to Because cdc2, the initiator of the whole pathway, is inactivated specific proteins (10). In the budding yeast Saccharomyces by dephosphorylation at Thr-161 by an okadaic acid-sensitive cerevisiae, genetic experiments show that the E2 encoded by phosphatase (18), it is important to know whether activity of UBC9 is required for destruction of both S-phase and mitotic the cyclosome itself is directly regulated by phosphorylation or cyclins, although it is not known whether UBC9 participates indirectly regulated by phosphorylation of other elements in directly in the ubiquitination of these cyclins or is used to the pathway. To answer this question, we first developed a ubiquitinate other proteins required for progression to the method for obtaining an active cyclosome preparation that cyclin destruction point (11). E3 proteins function in the important recognition of specific protein substrates, but only Abbreviations: ATP[y-S], adenosine 5'-[y-thio]triphosphate; PNPP, p-nitrophenylphosphate; rcm-BSA, reduced-carboxymethylated bo- vine serum albumin; El, ubiquitin-activating enzyme; E2, ubiquitin- The publication costs of this article were defrayed in part by page charge carrier protein; E3, ubiquitin-protein ligase; 125I-cyclin, 1251-labeled payment. This article must therefore be hereby marked "advertisement" in cyclin B-(13-91); E2-C and E3-C, cyclin-selective E2 and E3, respec- accordance with 18 U.S.C. §1734 solely to indicate this fact. tively; UBC, ubiquitin conjugating enzyme. 9303 Downloaded by guest on September 28, 2021 9304 Biochemistry: Lahav-Baratz et al. Proc. Natl. Acad. Sci. USA 92 (1995) lacked detectable cdc2 activity. We next identified and par- Proteins adsorbed to the column were eluted with a gradient tially purified an endogenous, okadaic acid-sensitive phos- of 200-440 mM KCl at a flow rate of 1 ml/min. Fractions of phatase from clam oocytes. Using these components, we show 1 ml were collected and concentrated by ultrafiltration with that E3-C cyclin-ubiquitin ligase activity of the cdc2-free Centricon-30 concentrators (Amicon). A single peak of oka- cyclosome can be inactivated by the okadaic acid-sensitive daic acid-sensitive phosphatase eluted at -320 mM KCl phosphatase and that its activity can be restored by the (fractions 10-13). This material was subjected to hydrophobic subsequent addition of cdc2. These results demonstrate that chromatography, as described in Results. The final preparation the activity of the cyclosome itself is regulated by reversible was essentially free of okadaic acid-insensitive phosphatase phosphorylation. activity and was -25-fold purified, with an 8% yield. Assay of Cyclin-Ubiquitin Ligation. Reaction mixtures con- tained in a 10-jil vol 20 mM Hepes-KOH (pH 7.2), rcm-BSA MATERIALS AND METHODS at 1 mg/ml, 1 mM dithiothreitol, 5 mM MgCl2, 0.5 mM ATP, Materials. Ubiquitin, reduced-carboxymethylated bovine 10 mM phosphocreatine, creatine phosphokinase at 50 ,ug/ml, serum albumin (rcm-BSA), and p-nitrophenylphosphate 50 ,tM ubiquitin, 1 ,uM ubiquitin aldehyde, 1 pmol of E1, 1-2 (PNPP, Tris salt) were obtained from Sigma. Okadaic acid and pmol of 125I-labeled cyclin B-(13-91)/protein A (referred to as adenosine 5'-[,y-thio]triphosphate (ATP[,y-S]) were obtained 1251-cyclin, 1-2) x 105 cpm), 1 ,uM okadaic acid, and enzymes, from Boehringer Mannheim. Ubiquitin aldehyde was prepared as specified. After incubation at 18°C for 60 min, the samples as described (19). E1 was purified from human erythrocytes were separated by electrophoresis on an SDS/12.5% poly- (20). Sea urchin (Arbaciapunctulata) cyclin B-(13-91)/protein A acrylamide gel. Results were quantified with a Fuji phosphor was expressed in Escherichia coli BL21(DE3), purified, and imager. The amount of radioactivity in all cyclin-ubiquitin radioiodinated, as described (6). Active cdc2 was purified from conjugates was expressed as percentage of the total radioac- extracts of M-phase clam (Spisula solidissima) oocytes by tivity in each lane (15). affinity chromatography on p13-Sepharose (21) followed by Assay of Inactivation of E3-C by the Phosphatase. Reaction gel filtration chromatography, as described (8). conditions resembled those described for the assay of cyclin- Fractionation of Clam Oocyte Extracts. Extracts of early ubiquitin ligation, except that okadaic acid, ATP, phospho- M-phase or interphase clam oocytes were prepared (8) and creatine, and creatine phosphokinase were omitted and 3 mM fractionated (15) as described. Briefly, extracts were first ATP[,y-S] was added. ATP[,y-S] can efficiently replace ATP in separated on DEAE-cellulose into two fractions: fraction 1, the E1 reaction (9) but not in the activation of E3-C by cdc2 in the flow-through, and fraction 2, which consists of proteins fraction 1A (unpublished observations). ATP[-y-S] was thus that bind to the resin and are eluted with high ionic strength. used as a precaution, to prevent possible reactivation of Fraction 1 was extracted with high salt, to solubilize compo- inactive E3-C during the assay. Incubation was in two stages: nents bound to particulate material. The salt extract was (i) phosphatase was incubated with E3-C or other components, fractionated with ammonium sulfate to fraction 1A (precipi- as specified in the figure legends, in a reaction mixture lacking tated at 0-45% saturation) and fraction 1B (precipitated at 1251-cyclin, for 60 min at 18°C. (ii) 1251-cyclin was added, a 55-85% saturation of ammonium sulfate). Fraction 1A con- second incubation was carried out for 80 min, and then tains latent E3-C, and fraction 1B contains E2-C (15). E3-C was cyclin-ubiquitin ligation was determined as described above. activated and partially purified as described under Results, and E2-C was further purified by gel filtration on Superose-12, as RESULTS described (8). Assay of Phosphatase Activity. This was determined by the Activated Cyclin-Ubiquitin Ligase Does Not Require the okadaic acid-sensitive hydrolysis of the general phosphatase Continued Presence of cdc2. We have reported (15) a proce- substrate p-PNPP, as follows: Reaction mixtures contained in dure for the fractionation of the components of a cyclin- a 10-,u vol: 20 mM Hepes-KOH (pH 7.2), rcm-BSA at 1 ubiquitination ligation system from clam oocytes. The inactive mg/ml, 1 mM dithiothreitol, 25 mM PNPP, and enzyme as form of E3-C was in a 0-45% saturation ammonium sulfate specified. Assays were done with and without 1 pgM okadaic fraction designated fraction 1A. E3-C could be activated by acid. After incubation at 18°C for 60 min, the reaction was incubation of interphase fraction 1A with cdc2 or of early stopped with 1 ml of 0.02 M NaOH, and absorbance at 410 nm M-phase fraction 1A with MgATP (15). In those experiments, was determined. Okadaic acid-sensitive phosphatase activity E3-C activity was found to be part of a large, -1500-kDa was calculated by the difference between values obtained with complex, the cyclosome, that was partially purified by glycerol and without okadaic acid. A unit of enzyme activity was density-gradient centrifugation. However, this preparation was defined as that hydrolyzing 1 nmol of PNPP under the con- not completely separated from cdc2. To prepare active, cyclo- ditions described above; this corresponds to an A410 value of some/E3-C complexes free from cdc2, M-phase fraction 1A 0.016, as determined with p-nitrophenol standard. was preincubated with MgATP and then separated on a Partial Purification of an Okadaic Acid-Sensitive Phos- Superose 6 gel filtration column, in a buffer containing 0.25 M phatase from Clam Oocytes. In preliminary experiments we KCl. The presence of salt in gel filtration was essential to found that almost all okadaic acid-sensitive activity of clam completely separate E3-C from cdc2 because at low ionic oocyte extracts was contained in fraction 2; therefore, we used strength cdc2 aggregates as high-molecular-weight forms (un- this fraction as the source for enzyme purification. Fraction 2 published observations). As shown in Fig. 1, under these from M-phase clam oocytes (60 mg of protein) was applied to conditions, cdc2 migrated at - 100 kDa and was essentially a 1.5 x 90 cm column of Sepharose-6B equilibrated with 50 completely separated from the -1500-kDa cyclosome- mM Tris HCl (pH 7.6)/200 mM KCl/1 mM dithiothreitol. associated E3-C activity, activated by preincubation with Fractions of 3 ml were collected. Okadaic acid-sensitive phos- MgATP. When the gel-filtration column fractions correspond- phatase activity eluted as a single peak with an apparent ing to cdc2-free, inactive cyclosome (Fig. 1, Untreated) were molecular mass of -200 kDa. This phosphatase was partially further incubated with MgATP, the activity of cyclin-ubiquitin separated from okadaic acid-insensitive phosphatases that ligation was not restored unless cdc2 was also supplemented eluted at molecular-mass region <200 kDa. The peak of (data not shown). These results indicate that after the activa- okadaic acid-sensitive phosphatase from the Sepharose-6B tion process the continued presence of cdc2 is not further column (fractions 25-29, -9 mg of protein) was applied to a required to sustain E3-C activity. Mono Q HR 5/5 column (Pharmacia) equilibrated with 50 Active Cyclosome/E3-C Complexes Are Inactivated by an mM Tris-HCl (pH 7.6)/200 mM KCl/1 mM dithiothreitol. Okadaic Acid-Sensitive Phosphatase. Although cdc2 initiates Downloaded by guest on September 28, 2021 Biochemistry: Lahav-Baratz et al. Proc. Natl. Acad. Sci. USA 92 (1995) 9305

C" 0 C x .0 0 0 C_ 70 en E > OW 0cs Cu CO) 0- z XI 0 0 .-C - 0~ Co0 ._ C.) 0. C-o IT-a .2 4. _ 0 C.) 0 _7 o ICo c5 15 20 25 30 35 Fraction number C) FIG. 1. Separation of active E3-C from protein kinase cdc2 by gel filtration chromatography. Fraction 1A from early M-phase cells (1 mg Fraction number of protein) was either untreated (0) or preincubated with MgATP (0) for 30 min at 18°C in a reaction mixture containing in a 200-,ul vol: 50 FIG. 2. Purification of phosphatase by hydrophobic chromatogra- mM Hepes-KOH (pH 7.2), rcm-BSA at 0.5 mg/ml, 1 mM dithiothre- phy. Peak fractions from the Mono Q column (1.2 mg of protein) were itol, 5 mM MgCl2, 0.5 mM ATP, 10 mM phosphocreatine, creatine applied to a 5-ml methyl-HIC (hydrophobic interaction chromatogra- phosphokinase at 50 Ag/ml, and 1 ,uM okadaic acid. Both samples phy) column (Bio-Rad) equilibrated with 1 M ammonium sulfate in a were separated on a Superose 6 HR 10/30 column (Pharmacia) buffer containing 100 mM sodium phosphate (pH 7.0) and 1 mM equilibrated with 50 mM Tris HCl (pH 7.2)/250 mM KCl/rcm-BSA at dithiothreitol. The column was washed with 10 ml of the same buffer 0.2 mg/ml/1 mM dithiothreitol. Fractions of 0.5 ml were collected at and then eluted with a 40-ml decreasing gradient of ammonium sulfate a flow rate of 0.4 ml/min. The fractions were concentrated by (1 M-0 M) in the above buffer. Fractions of 2 ml were collected at a centrifuge ultrafiltration with Centricon-30 concentrators (Amicon), flow rate of 1 ml/min. Fractions were concentrated by ultrafiltration diluted 20-fold with a buffer consisting of 50 mM Tris-HCl (pH 7.2), with Centricon-30 concentrators. Salt was removed by dilution with 20 20% (vol/vol) glycerol, and 1 mM dithiothreitol and concentrated mM Tris-HCl (pH 7.2)/1 mM dithiothreitol, followed by another again to a 50-,l vol. The ligation of 1251-cyclin to ubiquitin (0, *) was concentration to a 50-,ul vol. (A) Phosphatase activity was assayed in assayed as described in 5-,ul samples of column fractions in the 4.5-,ld samples of column fractions without (o) or with (-) okadaic presence of fraction 1B (10 ,ug of protein) as the E2-C source. Activity acid (Ok. ac.) as described, except that incubation was done for 2 hr. of protein kinase cdc2 was assayed in 5-,ul samples of column fractions (B) Inactivation of E3-C (2 ,ul) was assayed as described with 0.12-,l by histone Hi kinase activity after absorption to p13-Sepharose, as samples of column fractions. Results are expressed relative to the described (21). Fractions 18-21 of the sample preincubated with activity of a control incubated without phosphatase, in which 28.4% of MgATP were collected and used as the preparation of active E3-C for 1251-cyclin was converted to ubiquitin conjugates. further experiments. Molecular mass markers (arrows): TG, thyro- globulin; 670 kDa; AF, apoferritin, 440 kDa; CAT, catalase, 200 kDa; purified E2-C (8). Control experiments showed that activity of Vo, void volume. these partially purified preparations in cyclin-ubiquitin liga- tion was unaffected significantly by okadaic acid (Fig. 3A, the pathway leading to formation of an active cyclosome/E3-C 2 complex, it is not known whether this is the direct result of an compare lanes and 3). This result indicates that the partially activating phosphorylation catalyzed by either cdc2 itself or a purified preparations of E2-C and the cyclosome are them- downstream kinase or is an indirect result of some other selves essentially free of contaminating phosphatase activity. posttranslational modification. Similarly, the observation that Incubation of E2-C and the cyclosome complexes with the okadaic acid, an inhibitor of protein phosphatases 1 and 2A phosphatase almost completely abolished cyclin-ubiquitin li- (16), can prevent inactivation of the cyclin destruction ma- gation (Fig. 3A, lane 4). This inhibition could be completely chinery (17) is consistent with the idea that phosphorylation of prevented by okadaic acid (Fig. 3A, lane 5). the cyclosome itself positively regulates its activity but does not To ask whether E2-C or E3-C, or both, are targets for rule out other, indirect mechanisms. Previously, it was not phosphatase action, each component alone or in combination possible to distinguish between direct and indirect activating was first preincubated with the phosphatase, and then phos- phosphorylations because crude extracts and partially purified phatase action was terminated by the addition of okadaic acid. cyclosome preparations also contained some cdc2, which is Subsequently, the missing untreated component(s) (E2-C, itself inactivated by an okadaic acid-sensitive phosphatase E3-C, or both) were added, and the ligation of 1251-cyclin to (18). The availability of the cdc2-free cyclosome preparation ubiquitin was determined. The results are shown in Fig. 3B. A described above allowed examination of this question. control incubation (Fig. 3B, lane 4) showed that phosphatase To ask whether the activity of cdc2-free cyclosome com- action was effectively quenched by okadaic acid because plexes depends on their being phosphorylated, we sought an subsequently added untreated E2-C and E3-C had full activity. endogenous okadaic acid-sensitive phosphatase activity from Another control (Fig. 3B, lane 1) showed that preincubation of clam oocytes that would inactivate the cyclin destruction both E2-C and E3-C with the phosphatase before the addition system. As outlined in Materials and Methods, such a phos- of okadaic acid inhibited cyclin-ubiquitin ligation almost phatase activity was identified and partially purified from completely, indicating that the preparation of phosphatase fraction 2 by gel filtration, ion exchange, and hydrophobic used was active. Preincubation of E3-C with the phosphatase chromatography. By the last fractionation step, this enzyme abolished cyclin-ubiquitin ligation almost completely (Fig. 3B, activity was well-separated from okadaic acid-insensitive phos- lane 3), whereas preincubation of phosphatase with E2-C had phatase activities (Fig. 2A). The effect of this phosphatase on no effect (Fig. 3B, lane 2). These data indicate that E3-C is the the action of the components of the cyclin-ubiquitin ligation target of phosphatase action, and E2-C is not. system is shown in Fig. 3. For these experiments, we used the Although the phosphatase preparation used in these exper- preparation of activated, cdc2-free E3-C (Fig. 1) and partially iments was only partially purified, the following observations Downloaded by guest on September 28, 2021 9306 Biochemistry: Lahav-Baratz et al. Proc. Natl. Acad. Sci. USA 92 (1995) A B Phosphatase preincubated E2C+E3C - + + + + with Phosphatase - - + + E2C + + _ O.A. - + - + E3C + + 1 2 3 4 5 1 2 3 4

- 80 20 sY 20] < ~~~~~~~+Phosphatase,| then cdc2 - 49.5

MM 32.5 - Cyc. +Phosphatase FIG. 3. Cyclosome-associated E3-C is inactivated by okadaic acid- sensitive phosphatase. Experimental conditions were as described for the two-stage assay of the inactivation of E3-C by the phosphatase. (A) 30 60 90 120 150 Effects of phosphatase and okadaic acid on cyclin-ubiquitin ligation Time, min by E2-C and E3-C. Where indicated, 1 Al of E2-C, 2 ,ul of activated E3-C (see Fig. 1), 0.6 unit of phosphatase, and 1 ,uM of okadaic acid (O.A.) FIG. 4. Phosphatase-inactivated E3-C is reactivated by protein were added. After preincubation for 60 min, 125I-cyclin was added, and kinase cdc2. Samples of 4 p.l of activated E3-C (see Fig. 1) were incubation continued for a further 80 min. (B) Phosphatase treatment preincubated at 180C for 60 min without (0) or with (o, i) phos- inactivates E3-C but does not inactivate E2-C. Phosphatase (0.6 unit) phatase (0.6 unit) in a reaction mixture similar to that described for the was preincubated with E2-C (1 p.l), E3-C (2 p.l), both, or neither, as assay of cyclin-ubiquitin ligation, except that okadaic acid and 125k- indicated. Preincubation was terminated by addition of okadaic acid (1 cyclin were omitted and the buffer pH was 7.6. Subsequently, 1 p.M p.M) to all samples. Subsequently, the missing untreated components okadaic acid was added to all samples. Where indicated (U), 15 units (E2-C, E3-C, or both) were added along with 125I-cyclin, and incuba- ofprotein kinase cdc2 were added. After the addition of 125I-cyclin and tion continued for a further 80 min. Cyc, free 1251-cyclin. Numbers at E2-C (1 p.l), samples were incubated at 180C for the periods indicated, right indicate position of molecular-mass markers (kDa). All bands of and cyclin-ubiquitin ligation was determined as described. higher molecular-mass than free 1251-cyclin are cyclin-ubiquitin con- jugates. phosphatase acts is not constitutive but is regulatory. Further- more, the observed lag in the reactivation of phosphatase- indicate that its effects on the activity of E3-C were indeed due treated E3-C by cdc2, which resembles that seen previously to phosphatase action: (i) The inactivation of E3-C by the with crude preparations (8, 15), raises the possibility that the phosphatase preparation was completely prevented by 1 AM mechanisms responsible for the lag are intrinsically built into okadaic acid (Fig. 3). Inhibition of 50% of E3-C inactivating the cyclosome particle (see Discussion). activity of the phosphatase was attained with - 10 nM okadaic acid (data not shown). (ii) The E3-C-inactivating activity of the phosphatase copurified with its PNPP-hydrolyzing activity in DISCUSSION different chromatographic procedures, such as in hydrophobic We have used an okadaic acid-sensitive phosphatase to gain chromatography (Fig. 2B) or in chromatography on Mono Q some insight into the mechanisms that regulate activity of the columns (data not shown). cyclin-ubiquitin ligase E3-C and, thus, activity of the cyclin Phosphatase-Treated E3-C Can Be Reactivated by Protein degradation system in the cell cycle. Purification of the phos- Kinase cdc2. These results demonstrate that one or more phatase was undertaken with the ultimate aim (not yet ad- phosphorylations are required for the activity of the cyclosome dressed in the present study) of defining the role ofthis enzyme complex, but they do not distinguish between constitutive, in the inactivation of the cyclin destruction machinery after nonregulatory phosphorylation and cell cycle stage-specific mitosis. Although the phosphatase was only partially purified, regulatory phosphorylation. To examine this problem, we quite certainly it is the activity of the phosphatase (and not of asked whether phosphatase-treated cyclosome complexes can some impurity in the phosphatase preparation) that causes the be reactivated by incubation with cdc2. Active, cdc2-free inactivation of E3-C. This conclusion is based on the finding cyclosome complexes were preincubated with or without the that inactivation of E3-C by the phosphatase preparation is phosphatase, and then phosphatase action was terminated by completely prevented by okadaic acid (Fig. 3) and on the adding okadaic acid. Subsequently, cdc2 was added to a coincidence of phosphatase and E3-C-inactivating activities in phosphatase-treated sample, and the time course of the liga- enzyme purification (Fig. 2). Also the loss of activity of the tion of 1251-cyclin was determined in the presence of MgATP. cyclin-ubiquitin ligation system seems not to be due to the In the control sample of E3-C preincubated without phos- phosphatase action on the upstream activator cdc2 (18), a phatase (Fig. 4, No additions) the time course of cyclin- possibility consistent with earlier observations on the effects of ubiquitin ligation was nearly linear. As expected, preincuba- okadaic acid in crude extracts (17) because in the present tion with phosphatase markedly inhibited E3-C activity. When study, a cdc2-free preparation of activated E3-C (Fig. 1) was phosphatase-treated E3-C was incubated with protein kinase used as the substrate for the phosphatase. Still, the phos- cdc2, there was a prolonged lag in the initial 30-60 min, phatase could act on another activator of E3-C, present in the following which the rate of cyclin-ubiquitin ligation increased partially purified preparation of E3-C/cyclosome. However, in markedly. The lag was consistently observed in several exper- such a case the putative activator would have to be assumed to iments. These results, showing that E3-C can be reactivated by have a molecular mass similar to that of the cyclosome (- 1500 cdc2, establish that reversible phosphorylation controls the kDa); in complementation experiments between different activity of the cyclosome-associated E3-C cyclin-ubiquitin fractions of the separation of E3-C on Superose 6, we found no ligase. Because cdc2 is the upstream cell cycle regulator of evidence for the existence of an activator of smaller or larger E3-C, it appears likely that the phosphorylation on which the size than the E3-C/cyclosome complex (data not shown). More Downloaded by guest on September 28, 2021 Biochemistry: Lahav-Baratz et al. Proc. Natl. Acad. Sci. USA 92 (1995) 9307 likely the E3-C/cyclosome complex itself is the direct target for 1. King, R. W., Jackson, P. K. & Kirschner, M. W. (1994) Cell 79, phosphatase action. Whether the E3-C moiety of the cyclo- 563-571. some is phosphorylated or the phosphorylation of another 2. Murray, A. W., Solomon, M. J. & Kirschner, M. W. (1989) Nature protein of the cyclosome complex turns on E3-C activity (London) 339, 280-286. 3. Luca, F. C., Shibuya, E. K., Dohrmann, C. D. & Ruderman, J. V. remains to be seen. (1991) EMBO J. 10, 4311-4320. Further purification of the cyclosome complex is required to 4. Ghiara, J. B., Richardson, H. E., Sugimoto, K., Henze, M., Lew, define its subunit composition, to identify the subunits phos- D. J., Wittenberg, C. & Reed, S. I. (1991) Cell 65, 163-174. phorylated, and to obtain quantitative information on the 5. Hunt, T., Luca, F. C. & Ruderman, J. V. (1992) J. Cell Biol. 116, extent of phosphorylations relevant to the regulation of its 707-724. activity. A similar complex containing cyclin-ubiquitin ligase 6. Glotzer, M., Murray, A. W. & Kirschner, M. W. (1991) Nature activity was identified recently in Xenopus egg extracts (22). (London) 349, 132-138. 7. Hershko, A., Ganoth, D., Pehrson, I., Palazzo, R. E. & Cohen, This particle contains homologs of S. cerevisiae proteins L. H. (1991) J. Biol. Chem. 266, 16376-16379. CDC16 and CDC27, proteins that are essential for destruction 8. Hershko, A., Ganoth, D., Sudakin, V., Cohen, L. H., Luca, F. C., of B-type cyclins and the metaphase-anaphase transition (23, Ruderman, J. V. & Eytan, E. (1994) J. Biol. Chem. 269, 4940- 24). Both proteins become phosphorylated in M-phase ex- 4946. tracts, but the functional significance of those modifications 9. Hershko, A. & Ciechanover, A. (1992) Annu. Rev. Biochem. 61, was not tested. 761-807. Our finding that phosphatase-treated E3-C/cyclosome com- 10. Jentsch, S. (1992) Annu. Rev. Genet. 26, 179-207. 11. Seufert, W., Futcher, B. & Jentsch, S. (1995) Nature (London) plex can be reactivated by incubation with cdc2 (Fig. 4) 373, 78-81. suggests that the phosphorylation removed by the phosphatase 12. Scheffner, M., Huigbretze, J. M., Vierstra, R. D. & Howley, P. is regulatory and not constitutive. Our result does not indicate, (1993) Cell 75, 495-505. however, that the E3-C/cyclosome complex is directly phos- 13. Rechsteiner, M., Hoffman, L. & Dubiel, W. (1993) J. Biol. Chem. phorylated by protein kinase cdc2. The lag in the activation of 268, 6065-6068. interphase E3-C by cdc2 (8, 15) suggests an indirect mecha- 14. Luca, F. C. & Ruderman, J. V. (1989)J. CellBiol. 109, 1895-1909. nism, such as a cascade of protein kinase reactions initiated by 15. Sudakin, V., Ganoth, D., Dahan, A., Heller, H., Hershko, J., Luca, F. C., Ruderman, J. V. & Hershko, A. (1995) Mol. Biol. cdc2 and culminated by an activating phosphorylation of the Cell 6, 185-198. cyclosome by a downstream kinase. Because the kinetics are 16. Cohen, P., Holmes, C. F. B. & Tsukitani, Y. (1990) Trends preserved in the reactivation of phosphatase-treated E3-C Biochem. Sci. 15, 98-102. (Fig. 4), components of the cascade are probably integral parts 17. Lorca, T., Fesquet, D., Zindy, F., LeBouffant, F., Cerutti, M., of the cyclosome complex. Further purification and charac- Brechot, C., Devauchelle, G. & Doree, M. (1991) Mol. Cell Biol. terization of the E3-C/cyclosome complex should reveal the 11, 1171-1175. sequence of events by which protein kinase cdc2 activates E3-C 18. Lorca, T., Labbe, J.-C., Devault, A., Fesquet, D., Capony, J.-P., and of the mechanisms that Cavadore, J.-C., LeBouffant, F. & Doree, M. (1992) EMBO J. 11, delay prevent premature activation 2381-2390. of the cyclin degradation machinery in the cell cycle. 19. Mayer, A. N. & Wilkinson, K. D. (1989) Biochemistry 28,66-192. 20. Hershko, A., Heller, H., Elias, S. & Ciechanover, A. (1983) J. We thank Dvorah Ganoth and Michal Blumenfeld for participating Biol. Chem. 258, 8206-8214. in some initial experiments of this work. The skillful technical assis- 21. Labbe, J.-C., Cavadore, J.-C. & Doree, M. (1991) Methods tance of Judith Hershko and Clara Segal is gratefully acknowledged as Enzymol. 200, 293-301. is the help of the staff of the Marine Resources Department at the 22. King, R. W., Peters, J.-M., Tugendreich, S., Rolfe, M., Hieter, P. Marine Biological Laboratory in the excellent collection and mainte- & Kirschner, M. W. (1995) Cell 81, 279-288. nance of clams. This work was supported by National Institutes of 23. Irniger, S., Piatti, S., Michaelis, C. & Nasmyth, K. (1995) Cell 81, Health Grant DK-25614 and a grant from the U.S.-Israel Binational 269-277. Science Foundation (to A.H.) and by National Institutes of Health 24. Tugendrich, S., Tomkiel, J., Earnshaw, W. & Hieter, P. (1995) Grant HD-23696 (to J.V.R.). Cell 81, 261-268. Downloaded by guest on September 28, 2021