Eur. J. Riochem. 203, 353-360 (1992) f3 FEBS 1992

Interaction between the cell-cycle-control proteins ~3 4 '~"and ' p9CKShsZ Evidence for two cooperative binding domains in p9cKShsa Lyarninc AZZI', Laurcnt MEIJER', Steven I. REED', Ravidernath PIDIKITI3 and H. Y . Lim T U N G 3 CNRS, Station Biologiquc, Roscoff, France Department of Molecular Biology, Research Institute of Scripps Clinic, La Jolla, CA, USA Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, USA (Received July 29, 1991)

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EJB 91 1013

A universal intracellular factor, the 'M-phase-promoting factor' (MPF), displaying histone [I1 kinase activity and constituted of at least two subunits, ~ 3 4 ' ~ ' " a ncyclin d Bed'", triggers the G2 --t M transition of the cell cycle in all organisms. The yeast pl3"''and pl 8CKS'subunits and their functiondly interchangeable human homologues, pgCKShsl and p9CKSh"Z, directly interact with ~34'~''and may actually be part of the MPF complex. We have chemically synthesized p9CKShdand several of its peptide domains in order to investigate the binding of p9CKS"S2and ~34'~''.Several arguments support the hypothesis that the N-terminal half (peptide B) and the C-terminal half (peptide E) each contain a ~34'~''-binding site and that these two binding domains cooperate in establishing a stable p9CKSh"p34'd'Z complex: (a) only the combination of peptides B + E, and not B or E alone, is able to elute the cdc2 kinase from p9CKSh"'-Sepharosebeads; (b) only immobilized peptides B E, and not immobilized B or E, bind the cdc2 kinase; (c) only the peptides B + E combination, and not B or E alone, can compete with p9CKShs' for cdc2 kinase binding; (d) only when supplemented with E or B free peptide does the cdc2 kinase bind to B- or E-Sepharose beads, respectively. No binding occurs in the absence of free peptide. This additivity cannot be attributed to the formation of a B-E complex mimicking the full-length p9CKSh".The cyclin B subunit is not required for the formation of the p9CKShsZ-p34cdcZ complex through these two binding domains. The implications of the existence of two cooperative p34'd'2-binding domains in p9CKShs2 on the structure of the active M-phase-specific kinase is discussed.

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The G2 -+ M transition of the cell cycle is triggered by a universal factor, the 'M-phase-promoting factor' (MPF) constituted of at least two subunits, ~ 3 4 " ~ and " ' cyclin Bcdc13 (reviews in [l -31). The p34'd'2-cyclin-B complex displays an easily assayable histone H1 kinase activity [4-71 but it also phosphorylates a variety of mitotically relevant substrates such as lamin B, nucleolin, pp60c-.'rc,pp145'-"'", the tumor suppressor genes products Rb and p53, HMG I/Y and P1, etc. (reviews in [8, 91). The formation and activation of the p34cdc2-cyclin-B complex is highly regulated during the cell cycle. Among the regulators, the 13 - 18-kDa proteins encoded by the sucl jCKSl genes (Schizo.sacchuromyces pomhelSaccharomyces cereityiae appear to play a central role [lo, 111. The gene sue1 was first identified as an extragenic suppressor of certain cdc2 mutants [12, 131. Its homologue in S. cerevisiae, CKSI, rescues mutations of CDC28, the cdc2 homologue [14]. The human homologues, CKShs1 and CKShB, recently cloned by Richardson et al. [l I], both rescue a null mutation of the S . cerevisiae CKSI gene. Deletion of the sucl gene produces a cell cycle arrest, apparently in late Correspondence to L. Mcijer, CNRS, Station Biologique, F-29680 Roscoff, Francc Ahhrevzutions. CKS, CDC28 kinase subunit; CNBr, cyanogen bromide; H I , histone H I ; IMeAde, I-methyladenine; MPF, Mphase-promoting factor; sucl, suppressor of cell cyclc block: Tris/ NaCI, Tris-buffcrcd saline.

anaphase [12, 15, 161. Over-expression of sue1 ' is not lethal but leads to a division delay [12, 151. It has been originally suggested that pl 3'"'l directly interacts with ~ 3 4 ~ [12]. ~ ' ' The physical association between the two proteins and the presence ofpl3"""- pl gCKS' in the active cdc2 - CDC28-kinase complex have been demonstrated in yeast [14, 171. Although it does not seem to interfere with H I kinase activity, ~ 1 3 ~ "directly '' binds the ~3 4 " ~" -cyclin-Bkinase complex from M-phase extracts of mammalian cells [I 8, 191, starfish oocytes [6, 7, 201, sea urchin eggs [21,22] and Physarum [23]. Immobilized on Sepharose beads, it depletes M-phase extracts from MPF activity [24] and it allows fastaffinity chromatography purification of the p34'd'2- cyclinB c d d 3 complex [7, 201. When microinjected, purified pl 3sucf inhibits entry of Xenopus oocytes in meiosis [24]; it blocks ~ 3 4 ' ~ 'tyrosine ' dephosphorylation and subsequent mitotic activation in Xenopus oocyte extracts [25]. Altogether, these data suggest that the pl3""'' protein directly binds to ~34'"'" and acts as a regulator of its activation and inactivation. In an attempt to investigate the interaction between these two cell-cycle-regulatory proteins, we have chemically synthesized p9CKSh"Zand several of its peptide domains. The interaction between these peptides and the M-phase p34'd'Zcyclin-B kinase provides evidence for the existence of two cooperative p34'd'z-binding domains in p9CKShsZ. The implications of this finding on the structure of the active M-phasespecific kinase will be discussed.

354 MATERIALS AND METHODS

Preparation of gametes, maturation and fertilization

All experiments presented in this paper were performed on the starfish Asterias; rubens and the sea urchin Sphaevechinus Sodium vanadate, glycine, 1-methyladenine (1 MeAde), granularis. These animals were collected by diving in Brittany EGTA, EDTA, Mops, glycerol 2-phosphate, sodium fluoride, and kept under running sea water until use. Starfish gonads p-nitrophenyl phosphate, emetine, leupeptin, aprotinin, were dissected out of the animal, gently torn open in ice-cold soybean trypsin inhibitor, benzamidine, histone H1 (type IIIcalcium-free artificial sea water and filtered through cheese S), CNBr-activated Sepharose 4B, 6-aminohexanoic-acid-acticloth. Oocytes were then washed three or four times in calvated Sepharose-4B and Nonidet P40 were obtained from cium-free artificial sea water to remove the l MeAde-producSigma Chemicals; bovine serum albumin (fraction V) was ing follicle cells. They were resuspended, as a 10'/0 (by vol.) obtained from Boehringer Mannheim. suspension in Millipore-filtered natural sea water, until use. ["J-~~PIATP (PB 168) and '251-protein A (IM 144) were Oocyte maturation was triggered by addition of 1MeAde to obtained from Aniersham. Anti-p34"d'zantibodies were kindly a final concentration of 1 pM (for further experimental details provided by Dr G. Draetta (Heidelberg), anti-(cyclin Bcdc'S) on oocyte preparation and induction of maturation, see [27]). antibodies were generously donated by Dr T. Kishimoto Shedding of gametes in sea urchins was induced by injec(Tokyo). tion of 0.2 ml 0.2 M acetylcholine through the peribuccal membrane. Sperm was collected 'dry' and kept undiluted at 4°C. Eggs were collected in Millipore-filtered natural sea water. They were washed once with Millipore-filtered natural Synthesis, purification and characterization of p9CKSksZ sea water and used as a 10% (by vol.) suspension. To facilitate and related peptides elevation of the fertilization membrane in S. granularis, glycine p9CKS'1nZ and related peptides were synthesized from 9- (final concentration 0.1 %, mass/vol.) was added to the eggs. fluorenylmethoxycarbonyl amino acids by the solid-phase Sperm was diluted just before insemination (1 drop 'dry' method on an automated Milligen 9050 peptide synthesizer sperm/5 ml Millipore-filtered natural sea water; 1 drop of as recommended by the manufacturer (Milligen, Burlington, this dilution/lO ml egg suspension). At 2-3 min after sperm MA). Following synthesis, p9CKShSZ was cleaved from the resin addition, the eggs were checked for successful fertilization by treatment with 90% (by vol.) trifluoroacetic acid contain- (100% in all experiments) and the excess sperm was removed ing 5% (by vol.) thioanisole, 3% (by vol.) ethanedithiol and by washing the eggs once with Millipore-filtered natural sea 2% (by vol.) anisole. Following cleavage, p9CKShsZ was purified water. All experiments were performed at + 20°C. by reverse-phase HPLC on a CI8 column and dried on a rotatory evaporator. Following drying, p9CKSh'Z was dissolved in 10 mM imidazole/Cl pH 7.3at about2 mg/ml and dialyzed Preparation of oocyte and egg extracts overnight against the same buffer. The sequence of p9CKShs2 At various times after 1MeAde (starfish oocytes) or sperm was confirmed by sequencing the protein on a Milligen 6600 solid-phase sequencer after it was coupled to an arylamine- (sea urchin eggs) addition, 2-ml aliquots of the oocyte/egg derivatized poly(viny1idene difluoride) membrane as rec- suspension were rapidly centrifuged ( 5 s full speed in an asommended by the manufacturer (Milligen, Burlington, MA), Eppendorf centrifuge). The supernatant was removed piration and 400 p1 homogenization buffer was added to the cell pellet. The tube was immediately frozen in liquid nitrogen and kept at -80°C until further processing (within 24 h). Eggs or oocytes were homogenized by a 10-s sonication. After Buffers centrifugation for 10 min at 14000 g at 4"C, the supernatant Calcium-free artificial sea water contained 452.2 mM was recovered, frozen in liquid nitrogen and stored at NaCI, 10.08 mM KCI, 29.8 mM MgCI2, 17.2 mM MgS04, - 80 'C until loaded on p13'"C'-Sepharose or p9CKShS'i25 mM Tris/HC1 pH 8.0. Sepharose beads. Homogenization buffer contained 60 mM glycerol 2-phosphate, 15 mMp-nitrophenyl phosphate, 25 mM Mops pH 7.2, 15 mM EGTA, 15 mM MgC12, 1 mM dithiothreitol, 0.1 mM Preparation and use of plJ""Bepharose, p9CKSks'-Sepharose sodium vanadate, 0.1 mM sodium fluoride, 10 pg leupeptin/ and p9cKsh"z-Sepharose ml, 3 0 pg aprotinin/ml, 10 pg soybean trypsin inhibitor/ml, p l 3sucJand p9CKShs1 were purified from over-producing 100 pM benzamidine. This buffer had previously been shown strains of Escherichia coli by gel filtration on Sephacryl Sto stabilize the starfish meiotic oocyte [4] and the sea urchin 200 (Pharmacia) as described previously [ l l , 171. p9CKSh"Zwas mitotic egg [26] M-phase-specific histone HI kinase. obtained by total chemical synthesis. These proteins were Buffer C is homogenization buffer with 5 mM EGTA, no conjugated to Sepharose 4B activated by CNBr or 6NaF and no protease inhibitors. aminohexanoic acid according to the instructions of the manuBead buffer contained 50 mM Tris pH 7.4, 5 mM NaF, facturer. Unreacted groups on the resin were quenched with 250 mM NaCI, 5 mM EDTA, 5 mM EGTA, 0.1% Nonidet 1 M ethanolamine pH 8.0. The amount of coupled proteins/ P40, 10 pg leupeptin/nil, 10 pg aprotinin/ml, 10 pg soybean ml gel was 5 mg for pl 3suL1 and 3.9 mg for ~ 9 ~ ~ ' ~ " 'p9cKsh"2, and trypsin inhibitor/ml and 100 pM benzainidine. i.e. 0.5 pmol/ml gel. Synthetic peptides were couTransfer buffer contained 39 mM glycine, 48 mM Tris, pled similarly to CNBr or 6-aminohexanoic-acid-activated 0.37% SDS, 20% methanol. Sepharose 4B at a final concentration of 0.5 pmol/ml packed Tris-buffered saline (Tris/NaCl) contained 50 mM Tris beads. pH 7.4, 150 mM NaCl. The protein-beads were kept at 4°C as a 20% (by vol.) Sodium-bicarbonate-buffered saline contained 200 mM suspension in bead buffer. Just before use, 10 pl packed proNaHCO, pH 8.2,200 mM NaCl. tein-beads was washed with 1 ml bead buffer and resuspended Chemicals

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355 the presence of 1 mg histoue Hl/ml (Sigma, type 111-S) and buffer C in a final volume of 50 pl. Assays were terminated by transferring the tube onto ice. After a brief centrifugation at 10000 g, 30-pl aliquots of supernatant were spotted onto pieces (2.5 x 3 em) of Whatman P81 phosphocellulose paper and, after 20 s, the filters were washed five times (for at least 5 inin each time) in a solution of 10 ml phosphoric acid/l water. The wet filters were transferred into 6-ml plastic scintillation vials, 5 in1 ACS (Amersham) scintillation fluid was added and the radioactivity of the samples measured in a Packard counter. Electrophoresis and Western blotting

B

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CKShs-2

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Time after fertilization (min.)

Proteins bound to p9C"Sh", p13"""', pgCKShsf,or peptides-Sepharose beads were recovered with 50 p1 2 x Laemmli sample buffer. Typically, samples were run in 10% SDS/polyacrylamide gels. Proteins were transJ'erred from the 10% acrylamide gels to 0.1-pm nitrocellulose sheets (Schleicher & Schiill) in a Milliblot/SDS system (Millipore) for 30 min at 2.5 mA/cm2 in transfer buffer. The filters were subsequently blocked with Tris-buffered saline (Tris/NaCl) containing 3% bovine serum albumin for 1 h at 37°C. The filters were then incubated overnight at 4°C with G I antip34'd'2antibodies (1 : 1000 dilution) or anti-cyclin-B antibodies (1 :500 dilution). After four washes of 15 min each with Tris/ NaCl containing 0.2% Nonidet P40, the filters were treated with 1 pCi 1251-protein A (30mCi/mg, Amersham) in 3% bovine serum albumin in Tris/NaCl for 2 h at room temperature. After four washes of 15 min each with 0.2% Nonidet P40 in Tris/NaCl, the filters were exposed overnight to Hyperfilm MP.

RESULTS ~ 9 ' ~ and ~ ~ p9CKShs2 " ' both bind the ~34'~''- cyclin-B kinase

~ 9 ' ~ ~and ~ "p9CKSh"Z ' have been recently cloned and described as the human homologues of the budding yeast pl XCKS' 3: 0 5 10 15 20 25 30 and the fission yeast p13""" [I 11. Both proteins bind active HI Time after lMeAde addition (min.) kinase from M-phase extracts [Ill. To compare these two Fig. 1. ~ 9 ~and~p9CKSh" ~ ~ bind " equally ' the M-phase-specific HI kinase proteins further we have investigated their binding properties ' a complete cell (A, C) and ~ 3 4 ' ~(B). ' ~ (A, B) Mitotic sea urchin eggs. Eggs were towards H1 kinase and ~ 3 4 ' ~ ' throughout fertilized at time 0 and exlracts prepared throughout the first mitotic cycle (sea urchin egg mitosis; Fig. 1A, B) and throughout a division wcre loaded on p9CKSh"-or p9"Ksh"ZSepharose beads. Thc highly synchroneous G 2+ M transition (starfish oocyte meibound material was then assayed for HI kinase (HlK) activity (A) or osis; Fig. 1C). Extracts prepared during these time-courses rcsolved by SDS/PAGE prior to immunoblotting with ar1ti-p34'~'' were loaded on p9CKShs'-Sepharoseand p9"KSh"2-Sepharose antibodies; the autoradiographs at the level of ~34'~''are presented beads. The H1 kinase activity (Fig. 1 A, C ) and p34'd'2pr~tein here (B). (C) Meiotic starfish oocytes. Oocytes ( A . ruhens) were treated amount (Fig. 1B) bound to ~ 9 ' ~ ~or~p9CKShs2 " ' were indiswith 1 pM 1 MeAde at time 0, and extracts prepared throughout the tinguishable. p9CXShsf and p9CKSh", under these criteria, appear G 2+ M transition were loaded on p9CKShS'-or p9CKSh3-Sepharose to be functionally interchangeable. We have thus selected one beads. The bound material was then assaycd for F11 kinase activity. to study its affinity for ~34'~". of these two proteins, p9CKShSZ, in 400 pl bead buffer. The egg extract supernatant (390 pl) was added to the beads and the tubes were kept under constant rotation at 4°C for 30 min. After a brief centrifugation at 10000g and removal of the supernatant, the beads were washed three times with bead buffer and used either for HI kinasc assay or for immunoblotting. Histone H1 kinase assay

Samples (10 pl) of packed beads were incubated 10 min at 30'C with 15 pM [jj-32P]ATP(3000 Ci/mmol, 1 mCi/ml) in

Elution assay identifies two potential p34cd'z-binding domains

Full-length p9CKs"s2,as well as several peptides (A- J) covering various domains of the protein, were synthesized by the 9-fluorenylmethoxycarbonyl amino acid methodology (Fig. 2). An elution assay was set up t o investigate p9CKshh"2/ P34""2 interaction: . starfish M-phase oocyte extracts were loaded on p9CKSh"'-Sepharose beads; after extensive washing, the beads were exposed for 30 min under constant rotation to various peptides, alone or in combination; the beads were washed again and the HI kinase activity remaining on the

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Fig. 2. Sequence of p9CKShsZ 1111 and the related peptides used in this study. Boxed amino acids are identical in p13""cJ(Schizosaccharomyces pomhe) [15], pl sCKsJ (Saccharomyces cerevisiue) [I 41, ~ 9 ' "(Gullus ~ ~ gallus) ~ (Maridor, Gallant and Nigg, personal communication), p9CKShs1 (Homosupiens) [l I]. and p9CKShsZ

Table 1. Only p9CKShszand the combinationsof peptides A + E or B + E Qclin B elute ~34'~''- cyclin-B HI kinase bound to p9CKSh"'-Sepharosebeads. Starfish oocyte M-phase extracts were loaded on p9CKSh"1-beads as p34cdc2 described under Materials and Methods. The beads were then exposed for 30 min, at 4' C, under constant rotation to 500 pl peptides A - J or A A B B B B C C C D D ECKSvarious combinations thereof (final concentration: 250 pM). ~ 9 ' ~ ~ ~ " + +++ + hs2 and buffer were used as positive and negative controls, respectively. E C D E D E E After two washes with bead buffer, the beads were assayed for remaining HI kinase activity; (-)no release of HI kinase; (+)more than 50% Elution Peptides HI kinase release (representative of two independent experiments Fig. 3. The apparent ~ 3 4 ' ~ " -cyclin-B H1-kinase-releasing effect by performed in triplicate). p9CKShsz, A E and B E is due to effective elution of p34cd'z-cyclinB and not to inhibition of H l kinase. Starfish oocyte M-phase extracts Elution Peptides were loaded on p9CKSh"'-beadsas described under Materials and of H I kinase Methods. The beads were then exposed for 30min, at 4"C, under constant rotation to 500 pl peptides A - E or various combinations A thereof (final concentration: 250 pM). p9CKShsZ and buffer were used B as positivc and negative controls, respectively. After two washes with C bead buffer, the proteins bound to the beads were resolved by SDS/ D PAGE prior to immunoblotting with anti-p34'dczand anti-cyclin B E antibodies; the autoradiograph at the level of p3FdCzand cyclin B is F presented here. -

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but to a true release of p34'dc2-cyclin-B, we have analyzed by immunoblotting the remaining p34'dcZand cyclin B after elution with peptides (Fig. 3). Clearly peptides A + E, B + E and p9CKShr2 elute the ~ 3 4 ' ~ ~ cyclin-B 'complex from the p9CKSh"'-beads.The H1 -kinase-releasing effect of peptides B + E was analysed further: the extent of H1 kinase release by the B + E combination depends on the final B + E concentration (Fig. 4A) but also on the B/E proportion (Fig. 4B).

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beads was measured (Table 1). No H1 kinase releasing effect by a single peptide was observed. Full-length p9CKShs2, however, is able to release more than 50% of the bound HI kinase. When tested in combination, only peptides A + E and peptides B + E release H1 kinase from the p9CKSh"'-Sepharosebeads (Table 1). To confirm that this apparent H1-kinase-releasing effect was not due to an inhibition of the bound HI kinase

Additivity of B and E domains in ~34'~''binding We next investigated the binding of ~ 3 4 ~ ~ "cyclin-B 'H1 kinase to various peptides immobilized on Sepharose beads (Fig. 5). Although the M-phase H1 kinase binds to immobilized p136uc1, ~ 9 ' ~ ~(bacterially ~ " ' produced) and ~ 9 ' ~ ~ "(chemically ' synthesized), it does not bind at all to immobilized peptides A, B, C, D, E (Fig. 5A). This lack of binding is also observed when peptides A- E are immobilized through an eight-carbon spacer to the Sepharose beads (Fig. 5B). However, when peptides B E are immobilized together on Sepharose beads, H1 kinase binds with a similar dose/response curve as p9CKSh"-Sepharosebeads (Fig. 6A). The mixture of B-Sepharose and E-Sepharose beads is unable to bind HI kinase (Fig. 6B). The additivity of B and E peptides in ~ 3 4 ' ~ binding '' is also observed in competition experiments (Fig. 7). Addition of B or E peptide to the M-phase extract does not prevent H1 kinase fixation to p9CKSh"'-Sepharose beads. However, when both B and E peptides, or p9CKShS2, are added to the M-phase extract, they reduce HI kinase binding

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Fig. 4. Elution of p34'*''- cyclin-B H1 kinase from p9CKShs'-Sepharosebeads by B + E: doselresponse curve (A) and B/E ratio curve (B). (A) HI kinase (HIK) bound to p9"KSh"-Sepharose beads was exposed to increasing concentrations of peptides B, E, B E or p9CKSh3(500 PI) for 30 min, at 4"C, undcr constant rotation. After two washes with bead buffer, the beads were assayed in duplicates for remaining HI kinase activity, expressed as a percentage of the activity recovered on p9CK"h"'-beadsexposed to control buffer. (B) H I kinase bound to p9CK"hs2Sepharose beads was exposed to 50 or 100 pM of B E in various proportions for 30 min, at 4"C, under constant rotation. After two washes with bead buffer, the beads were assayed in duplicates for remaining HI kinase activity, expressed as a percentage of the activity recovered on p9CKShs'-beadsexposed to E alone.

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Fig. 5. Immobilized pW"", ~ 9 ' ~ ~or~p9CKShs2 " ' but not peptides A, B, C, D, E bind the p34'dcZ-cyclin-B H1 kinase. p13""", p9CK"h"',p9CK"h'2or peptides A - E were covalently linked to CNBr-activated Sepharose 4B (1-atom spacer; A) or to CNBr-activated CH-Sepharose 4B (%atom spacer; B) to a final concentration of 0.5 pmol/ml packed beads. Increasing volumes of starfish meiotic oocytes extracts were loaded on the beads and the bound H I kinase (HIK) was assayed as described in Materials and Methods.

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Fig. 6. p34'd"-cyclin-B H1 kinase binds to (B + E)-Sepharose beads but not to B- or E-Sepharose beads. p9CKShs2, B, E and (B E) were covalently linked to CNBr-activated Sepharose 4B at a final concentration of 0.5 pmol/ml packed beads. In (A) increasing volumes of starfish meiotic oocytes extracts were loaded on the beads and the bound HI kinase (HIK) was assayed in duplicate as described in Materials and Methods. In (B), the H1 kinase activity bound to (R + E)-Sepharose beads or to an equivalent mixture of B-beads and E-beads was compared (duplicate assays).

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Fig. 7. Binding of p34'd'2- cyclin-B H1 kinase to p9CKShs'-Sepharose beads is compcted by peptides B E together but not by B or E alone. Starfish meiotic oocytes extracts, supplemented with peptides B, E, B + E or p9

Interaction between the cell-cycle-control proteins p34cdc2 and p9CKShs2. Evidence for two cooperative binding domains in p9CKShs2.

A universal intracellular factor, the 'M-phase-promoting factor' (MPF), displaying histone H1 kinase activity and constituted of at least two subunits...
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