THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 269, No. 18, Issue of May 6, pp. 13279-13288, 1994 Printed in U.S.A.

Purification of a 15-kDa cdk4- and cdk5-binding Protein* (Received for publication, November 12, 1993, and in revised form, January 21, 1994)

Lyamine Azzi%$, Laurent MeijerSn, Anne-Carine Ostvoldll, John Lew**, and Jerry H. Wang** From SCNRS, Station Biologique, BP 74, 29682 Roscoff cedex, France, the IkVeurochemical Laboratory, University of Oslo, P. 0. Box 1115, Blindern, 0317 Oslo 3, Norway, and the **Medical Research Council Group in Signal l'bansduction, Department of Medical Biochemistry, Faculty of Medicine, University of Calgary, Calgary, Alberta T2N 4N1, Canada

Yeasts p13"""/p18CKS and their human homologues, p9CKSh"11p9CKShs2, strongly interact with ~ 3 4 " ~ ' and ~34"~". While attempting to purify the starfish oocyte p13nUc1 homologue, we discovered a 15-kDa protein cross- reactive with anti-p9CKSh"2/anti-p13*uc1 antibodies. p15cdk"P-Sepharose binds an anti-PSTAIRE cross-reac- tive protein of 33 kDa when loaded with starfish oocyte extracts. The p15edk-BP-bo~nd "PSTAIRE signal" is part of a 250-kDa complex distinct from p34c&2/cyclin B. p15cdk'BP-Sepharose beads retain a kinase phosphorylat- ing HMG IN, P1, and myelin basic protein (among 24 substrates tested). Major cdc2 kinase substrates are not phosphorylated by the ~ 1 5 ~ ~ - ~ ~ - b o u n d kinase. Phos- phopeptide maps of P1 phosphorylated by the p15cdk-BP- bound kinase, p34'h2/cyclin B, ~ 3 3 " ~ ~ / p 2 5 , and casein kinase 2 showed that these kinases phosphorylate P1 on different sites. Phosphopeptide maps of P1 phos- phorylated by the p15c"'BP-bound starfish kinase and p15cdk.BP-bo~nd human p34cdk4/cyclin D are largely coin- cident. To investigate the nature of the p15Cdk'BP-bound kinase, extracts of mammalian tissues and cells were loaded on pSCKSh"'- and p15cdk-BP-Sepharose and the bound proteins were analyzed using specific anti-cdk antibodies. cdc2 and cdk2 bind to p9CKSh"'-Sepharose, but not to p15cdk-BP. cdk4 and cdk5 bind to p15cdk'BP-Sepha- rose, but not to p9CKSh'1-Sepharose. We conclude that p15cdk-Bp specifically binds the cdk4cyclin D and cdk5 kinases and, along with p13""" and p9CKShs, may be part of a larger family of cdk-binding proteins.

Fission yeast ~ 3 4 ' ~ ' ~ , as well as its budding yeast homologue p34cDc28, has been identified as a major cell cycle regulator (reviewed by Forsburg and Nurse (1991) and Reed (1992)). The discovery of a ~ 3 4 ' ~ " ~ homologue in human suggested the uni- versality of cell cycle control mechanisms (Lee and Nurse, 1987). This was fully demonstrated by the cloning of p3QdC2 homologues from plants to mammals. With the identification of Eg-1 in Xenopus (Paris et al., 1990) and its human homologue cdk2l (Elledge et al., 1991; Ninomiya-Tsuji et al., 1991; Tsai et

Cancer (ARC) Grant 6268 and INSERM Grant 910110 (both to L. M,). * This work was supported by Association pour la Recherche sur le

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisernent" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

8 Supported by a Franco-Algerian fellowship (Programme franco-al- g6rien).

Tel.: 33-98-29-23-39; Fax: 33-98-29-23-42. 7 To whom correspondence and reprint requests should be addressed.

cyclin-dependent Kinase-binding protein; CKS, cdc28 kinase subunit; ' The abbreviations used are: cdk, cyclin-dependent kinase; cdk-BP,

HMG, high mobility group; MBP, myelin basic protein; lMeAde, 1-methyladenine; MPF, M phase-promoting factor; sucl, suppressor of cdc2 mutation; M A P , mitogen-activated protein; ERK, extracellular regulated kinase; PAGE, polyacrylamide gel electrophoresis; TPA, 12-

al., 1991), it was realized that ~34'~' ' was only one member of a large family of related kinases. The cloning of a dozen of p3QdC2-related kinases confirmed this presumption (Bunnell et al., 1990; Hellmich et al., 1992; Johnson and Smith, 1991; Kidd, 1992; Lapidot-Lifson et al., 1992; Lew et al., 199213; Matsu- shime et al., 1992; Meyerson et al., 1992; Okuda et al., 1992). cdc2-related kinases are usually classified as cyclin-dependent kinases (cdk) or named after the sequence of the conserved PSTAIRE motif: cdkl (cdc2, PSTAIRE), cdk2 (PSTAIRE), cdk3 (PSTAIRE), cdk4 (P(W)STVRE), cdk5 (PSSALRE), PCTAIRE-1, -2, -3, PITSLRE-1, -2, ..., etc. Although several of these kinases are clearly involved in cell cycle control, the pres- ence of cdc2-related kinases in non-dividing cells, such as plate- lets (Samiei et al., 1991) and nervous tissue (Hellmich et al., 1992; Lew et al., 1992a, 1992b), implies other major cellular functions.

cdcl3 was initially identified as another cell cycle regulator in fission yeast. Strong homology with the independently dis- covered sea urchin cyclin B and further cloning of cyclin B homologues from plants to mammals demonstrated its univer- sality. Furthermore ""phase promoting factor" (MPF) was identified as p34cdc2/cyclin B (Arion et al., 1988; Draetta et al., 1989; Dunphy et al., 1988; Gautier et al., 1988, 1990; Labbe et al., 1989; Meijer et al., 1989, 1991a; Pondaven et al., 1990; Solomon et al., 1990; Yamashita et al., 1992). I t was soon real- ized that cyclin B was only one member of a large family of cyclins comprising cyclins A, B1, B2, C, Dl , D2, D3, and E (Lew et al., 1992~) (reviewed by Xiong and Beach (1991)). Cyclins transiently associate with some cdc2-related kinases to regu- late various cell cycle steps.

Fission yeast ~ 1 3 ~ ~ " ' , and its budding yeast homologue p18CKS1, were identified as extragenic suppressors of certain cdc2 mutations (Hayles et al., 1986a, 1986b). The human ho- mologues, CKShsl and CKShs2, rescue a null mutation of the Saccharomyces cerevisiae CKSl gene (Richardson et al., 1990). Deletion of the sucl gene arrests cells in late anaphase (Hayles et al., 1986a; Hindley et al., 1987; Moreno et al., 1989). CKSl mutants arrest both at the G,/S and GJM transition (Tang and Reed, 1993). Astriking property of p13"""lp9CKSh" is its ability to establish a very stable association with ~34'~"' and ~ 3 3 ' ~ ~ ' ( A r i -

on et al., 1988; Azzi et al., 1992; Brizuela et al., 1987; Draetta et al., 1987; Ducommun et al., 1991; Hadwiger et al., 1989; Hayles et al., 1986a; Meijer et al., 1989). pl3"""-Sepharose beads de- plete M phase extracts of MPF activity (Dunphy et al., 1988) and allow the fast, affinity chromatography-based, purification of p34'dc2/cyclin B complex (Labbe et al., 1989; Pondaven et al., 1990). Microinjected p13""" inhibits entry of Xenopus (Dunphy et al., 1988) and mammalian (Gavin et al., 1992) oocytes in meiosis. In addition, p13suc' blocks ~34'~' ' tyrosine 15 dephos- phorylation and subsequent mitotic activation in Xenopus

0-tetradecanoylphorbol-13-acetate; MOPS, 3-(N-morpholino)propane- sulfonic acid; TBST, Tris-buffered saline-Tween 20; CaFASW, calcium- free artificial sea water; SBBS, sodium bicarbonate-buffered saline.

13279

13280 A 15-kDa cdk4- and cdk5-binding Protein

oocyte extracts (Dunphy and Newport, 1989). Altogether these data suggest that p13"""/p9CKSh" plays an essential role in the regulation of ~34'~''. This role is largely unknown however.

All experimentations with p13s""/p9CKShs have been per- formed with recombinant proteins. We therefore decided to pu- rify the native pSCKSh8 homologue from starfish oocytes. Unex- pectedly, we discovered a new protein, p15cdk.BP, which does not interact with ~34"~'' or ~33'~'', but strongly binds ( a ) the prod- uct of cdk4, a protein kinase gene initially identified as PSK-J3 using mixed oligonucleotide probes derived from conserved re- gions of serinekhreonine kinases (Hanks, 1987), and later found to associate with cyclin Dl , D2, or D3 (Matsushime et al., 1992; Xiong et al., 1992), ( b ) a starfish oocyte kinase, cross- reactive with anti-PSTAIRE antibodies, which is likely to be the cdk4 homologue, and ( c ) the product of cdk5, a gene pref- erentially expressed in neuronal tissues (Hellmich et aZ., 1992; Lew et al., 1992a, 1992b). Along with pSCKsh", p15'dk-BP may be part of a family of cdk-binding proteins.

MATERIALS AND METHODS

Chemicals Sodium orthovanadate, glycine, 1-methyladenine (lMeAde), EGTA,

EDTA, MOPS, p-glycerophosphate, dithiothreitol, NaF, p-nitrophenyl phosphate, heparin, staurosporine, protein kinase inhibitor, TPA, leu- peptin, aprotinin, soybean trypsin inhibitor, benzamidine, histone H1 (type 111-S), myelin basic protein, casein, protamine, enolase, isopropyl- 1-thio-p-D-galactopyranoside, CNBr-activated Sepharose 4B, octyl-aga- rose, potato acid phosphatase (type X), Tween 20, and LB broth base were all obtained from Sigma Chemicals. Protein A-agarose, Sephacryl S-BOOHR, and Sephacryl S-300 were purchased from Pharmacia LKB Biotechnology Inc. Matrex gel Green A was from Amicon, and Nonidet P-40 from Fluka. The following cDNAs, proteins, and antibodies were generously donated by the persons listed under "Acknowledgments": cdkl, cdk2, cdk3, cdk4, cdk5, PCTAIRE-1 and -3, PITSLRE-1 and -2 cDNAs, histones H1, H2A, H2B, H3, H4, HMG 17, HMG I, HMGY, P1, inhibitor 1, calmodulin, pp6PCM, the peptides AKAKKTPKKAK and SPMRSRSPSRSK, G, anti-~34'~'', anti-p13""", anti-cyclin F d c l 3 (star- fish), anti-rodent cdc2 and cdk2 (C terminal antibodies), anti-human cdk3, anti-human cdk4 (raised against a fusion protein of glutathione S-transferase and a C-terminal portion of cdk4) (Xionget al., 19921, and anti-human cdk5 (directed against the C-terminal peptide YFSDFCPP) (Xiong et al., 1992), anti-phosphotyrosine antibodies, monoclonal (Ya- mashita et al., 1991) and polyclonal antibodies against NH,-EGV- STAIRESLLKEGGC-COOH ("PSTAIRE" peptide), antibodies against NH,-VEKIGEG~GVVYKARHKLS-COOH (a ~ 3 4 " ~ " peptide contain- ing the regulatory threonine 14 and tyrosine 15 residues; this antibody does not recognize tyrosine-phosphorylated p3FdC2 but only tyrosine- dephosphorylated ~34'~'') (Baratte et al., 1992), polyclonal anti-ERKU MAP kinase antibodies (Shibuya et al., 1992). Antibodies against puri- fied pSCKShS2 were raised by Neosystem Laboratoire. [Y-~~PIATP (PB 168), [35S]methionine (SJ 50501, ACS, Amplify, Hyperfilm pmax, and MP were obtained from Amersham Corp.

Buffers

Homogenization buffer-Homogenization buffer consisted of 60 mM p-glycerophosphate, 15 mM p-nitrophenyl phosphate, 25 m~ MOPS, pH 7.2, 15 mM EGTA, 15 mM MgCI,, 1 mM dithiothreitol, 1 mM sodium vanadate, 1 mM NaF, 1 mM phenylphosphate, 10 pg/ml leupeptin, 10 pg/ml aprotinin, 10 pg/ml soybean trypsin inhibitor, and 100 1.1~ benza- midine. This buffer is known to stabilize the starfish meiotic oocyte (Pelech et al., 1987) and the sea urchin mitotic egg (Meijer and Pon- daven, 1988) M-phase-specific histone H1 kinase.

Buffer C-Buffer C consisted of homogenization buffer, but with 5 mM EGTA, no NaF, and no protease inhibitors.

Bead Buffer-Bead buffer consisted of 50 m~ Tris, pH 7.4,5 mM NaF, 250 mM NaCl, 5 mM EDTA, 5 m~ EGTA, 0.1% Nonidet P-40, 10 pg/ml leupeptin, 10 pg/ml aprotinin, 10 pg/ml soybean trypsin inhibitor, and 100 p~ benzamidine.

Dansfer Buffer-'IYansfer buffer consisted of 39 m~ glycine, 48 m~ Tris, 0.37% SDS, 20% methanol.

Dis-buffered Saline-neen 20 (TBSTI-TBST consisted of 50 mM Tris, pH 7.4, 150 m~ NaC1, 0.1% Tween 20.

Calcium-free Artificial Sea Water (CaFASW)-CaFASW consisted of

452.2 mM NaCl, 10.08 mM KCl, 29.8 mM MpCI,, 17.2 mM MgSO,, 5 mM %s-HCl, pH 8.0.

- I - .

Sodium Bicarbonate-buffered Saline (SBBSk-SBBS consisted of 200 mM NaHCO,, pH 8.2, 2 0 0 ' m ~ NaCI.

Gel Filtration Buffer-Gel filtration buffer consisted of 200 mM NaC1, 0.1% Brij-35, 12.5 m~ P-glycerophosphate, 12.5 mM MOPS, pH 7.2, 0.5 mM EGTA, 7.5 m~ MgCl,, 1 mM dithiothreitol, 0.1 mM NaF.

Material Preparation of Gametes

Starfish Oocyte Maturation-Asterias rubens and Marthasterias gla- cialis were kept under running sea water until use. The gonads were dissected out of the animals, gently torn open in ice-cold CaFASW, and filtered through cheese cloth. Oocytes were then washed three to four times in CaFASW to remove the 1MeAde-producing follicle cells. They were resuspended as a 10% (v/v) suspension in Millipore-filtered natu- ral sea water until use. Oocyte maturation was triggered by addition of 0.2 PM lMeAde (final concentration) (see Meijer et al. (1986)).

Preparation of G, and M Phase Oocytes-For large scale oocyte ex- tracts preparations, gonads were removed from ripe M. glacialis, and either directly frozen in liquid nitrogen and kept at -80 "C (G, phase oocytes) or incubated with 10 p~ lMeAde in natural sea water for 30 min (M phase oocytes). By that time all the oocytes had entered the M phase although they were still in the gonads. These were then removed from the incubation medium, rapidly blotted on filter paper, directly frozen in liquid nitrogen, and kept at -80 "C (Baratte et al., 1992).

Purification of p15cdk.BP Frozen starfish gonads were homogenized in homogenization buffer

(1 g/Z ml). After 30 min of centrifugation at 100,000 x g, the supernatant (160 ml) was diluted with ice-cold MilliQ water (640 ml) and mixed with 40 ml of packed S-Sepharose (prewashed with 15 diluted homogeniza- tion buffer) for 30 min at 4 "C. The slurry was then thoroughly washed, on a glass filter, with ice-cold MilliQ water followed by diluted homog- enization buffer (1:5). Proteins were eluted with a 0-1.5 M NaCl gradi- ent in 1 5 homogenization buffer. Eight ml of the p15'dk-BP-containing fractions were loaded on a Sephacryl S-300 column (100 x 2.6 cm) equilibrated with SBBS. Nine-ml fractions were collected at a flow rate of 1 mumin. The elution pattern was monitored by SDS-PAGE and

pSCKShS2 antibodies. "Late p15'dk-BB' fractions (see "Results") were pooled Coomassie Blue staining or immunoblotting using anti-pl3""" or anti-

and run through a 1-ml octyl-agarose column equilibrated with SBBS; p15'dk-BP was recovered in the flow-through fractions, while a minor, high molecular weight contaminant remained bound to the resin. These fractions (.9 ml) were pooled, diluted 1:l with MilliQ water, and loaded on a 2-ml Matrex gel Green A column equilibrated with 50% SBBS. The resin was washed, and p15'dk-BP was eluted by a 60-ml salt gradient (from 0.1 to 1 M NaCl in 0.1 M sodium bicarbonate); this resolved the p15'dk.BP doublet observed under some electrophoretic conditions.

Preparation and Use of pSCKSh"'- a n d p15'd"BP-Sepharose Beads p9cmhs1 was purified from an overproducing strain of Escherichia coli

(Ami et al., 1992; Richardson et al., 1990). The bacterial extract was diluted to a protein concentration of 1 mg/ml with distilled water prior to mixing for 30 min a t 4 "C with a slurry of S-Sepharose beads; these were then packed in a column, washed with 20 m~ sodium bicarbonate. pSCKShS1 was eluted with SBBS and further purified on a 100 x 2.6-cm Sephacryl S-200 column as described for p15'dk-BP purification. ~ 9 ~ " " and p15'dk-BP were conjugated to CNBr-activated Sepharose 4B, accord- ing to the manufacturer's instructions, Unreacted groups on the resin were quenched with 1 M ethanolamine, pH 8.0. The concentration of coupled proteins per milligram of gel was 3.9 mg for pSCKShS1 and 5.75 mg for p15'dk-BP, i.e. 0.5 pmoVml gel. pSCKShB1- and p15'dk-Bp-Sepharose beads were kept at 4 "C as a 20% (v/v) suspension in bead buffer. Just before use, 10 p1 of packed protein beads were washed with 1 ml of bead buffer and resuspended in 400 pl of bead buffer. The oocyte extract supernatant (400 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 10,000 x g and removal of the supernatant, the beads were washed three times with bead buffer and used either for kinase assay or for immunoblotting analysis of the bound proteins. In most cases extracts were loaded three to four successive times on p9CKSh"1-Sepharose beads prior to loading on p15'dk-BP-Sepharose beads.

Purification of cdk5fp25 Complex from Bovine Brain Bovine brain cdk5/p25 was purified as described by Lew et al.

(1992a), excluding the Mono S chromatographic step. The active frac-

A 15-kDa cdkl- and cdk5-binding Protein 13281

tions from the Superose 12 column were pooled and concentrated to a final concentration of approximately 25 pg/ml enzyme.

Gel Filtration of Oocyte Extracts A 100 x 2.6-cm column was packed with Sepharose S-2OOHR and

equilibrated with gel filtration buffer. It was calibrated with Bio-Rad gel filtration molecular weight markers before and after the oocyte extract run. 3.5 ml of oocyte M phase extract were loaded on the column, and 6 ml/lO-min fractions were collected. One-ml aliquots of each fraction were loaded on 10 pl of p9CKSh"'- and p15'dk-Bp-Sepharose beads as de- scribed above. The bound proteins were assayed for histone H1 kinase activity, or resolved by SDS-PAGE prior to immunoblotting analysis.

Protein Kinase Assays Kinase assays were performed by incubation of 10 or 40 p1 of packed

pSCKSha'-or p15'dk.BP-Sepharose beads (loaded with G, or M phase ex- tracts, or column fractions) or protein A-agarose immunoprecipitates, for 10 min a t 30 "C with 15 p~ [y-32PlATP (3,000 Cdmmol; 1 mCi/ml) in the presence of 1 mg of histone Hl/ml of buffer C in a final volume of 40 pl. Assays were terminated by transferring the tube onto ice.

Peptide Substrates and Histone HI-Peptide substrates and histone H1 were processed as follows. After a brief centrifugation at 10,000 x g , 30-pl aliquots of supernatant were spotted onto 2.5 x 3-cm pieces of Whatman P-81 phosphocellulose paper, and, after 20 s, the filters were washed five times (for at least 5 min each time) in a solution of 10 ml of phosphoric acid/liter of water. The wet filters were transferred into 6-ml plastic scintillation vials, and 5 ml ofACS scintillation fluid were added prior to counting in a Packard counter. Kinase activities were expressed in picomoles of phosphate incorporated in histone H1 or peptideholume of beadsflo-min incubation.

Protein Substrates-Protein substrates were processed as follows. After a brief centrifugation at 10,000 x g , 30-1.11 aliquots of supernatant were mixed with 2 x Laemmli sample buffer. [32PlPhosphate incorpo- ration was estimated by autoradiography (pmax) after SDS-PAGE on the appropriate gel system.

Immunoprecipitations Immunoprecipitations were performed with anti-cyclin B antibodies

(5 pVl00 p1 extract; 60 min a t 0 "C); 80 pl of protein A-agarose (50% (v/v) suspension) were added, and the mixture was rotated at 4 "C for 30 min; the beads were then washed twice with 1 ml of bead buffer and resus- pended in 50 pl of 2 x Laemmli sample buffer for analysis on SDS- PAGE. In some instances histone H1 kinase activity was measured on the immunoprecipitates.

PI Phosphopeptide Mapping 3ZP-Labeled P1 was purified by SDS-PAGE. The labeled P1 was de-

tected by autoradiography and the band excised from the gel. P1 was subjected to complete digestion with thermolysin as described by Wa- laas et al. (1989). The phosphopeptides were separated by electrophore- sis at pH 3.5 and chromatography on silica plates. Chromatography was performed twice to improve the resolution of the peptides.

I n Vitro Dunscription 1 Dunslation In vitro transcriptions and translations were performed using the

Promega TNT coupled reticulocyte lysate system. Briefly, 4 pg of DNA were mixed with 1 pl of RNasin (ribonuclease inhibitor, 40 unitdpl), 2 pl of TNT buffer, 1 p1 1 m~ amino acid mixture minus methionine, 25 pl of lysate, 4 pl of [35Slmethionine (1000 Ci/mmol; 10 mCi/ml), and 1 p1 of RNA polymerase T3 in a final volume of 50 pl. After a 90-min incubation a t 30 "C, 5 p1 of the mixture were analyzed by SDS-PAGE and autora- diography to check for the eficiency of the transcriptiodtranslation. The remaining 45 pl were frozen until use. 20 pl were loaded on 20 pl of pSCKSh"'- and 20 pl of p15'dk-Bp-Sepharose beads for 30 min. The beads were washed 4 times with bead buffer before analysis of the bound proteins by SDS-PAGE and autoradiography.

Electrophoresis and Western BZotting-Column fractions and pro- teins bound to pSCKsh"'- or p15dk-BP-Sepharose beads were recovered with 2 x Laemmli sample buffer. Typically, samples were run in 10 or 12.5% polyacrylamide gels and stained with Coomassie Blue. For a better resolution of low molecular weight proteins, the gel system of Schagger and Von Jagow (1987) was used. Gels containing [35S]methi- onine-labeled proteins were exposed for 60 min to Amplify prior to drying and overnight exposure to pmax. Gels containing [32P]phos- phate-labeled proteins were dried prior to overnight exposure to pmax.

For immunoblotting, proteins were transferred from the polyacryl- amide gels to 0.1-pm nitrocellulose sheets (Schleicher & Schuell) in a Milliblot-SDE system (Millipore) for 30 min a t 2.5 mA/cm2 in transfer

buffer. Subsequently the filters were blocked with 5% lowfat milk in TBST for 1 h. The filters were then washed with TBST and incubated for 2 h with G, anti-p34'd'2 antibodies (1:1000), monoclonal anti- PSTAIRE antibodies (1:2000 dilution), affinity-purified polyclonal anti- phosphotyrosine antibodies (1 pg/ml), anti-cdc2 tyrosine 15 peptide (1:1000), anti-cyclin B antibodies (1:1000), anti-p9CKSh'z (1:1000), or anti- pl3'"'' (1:lOOO) antibodies. After four washes of 15 min each with TBST, the filters were treated with horseradish peroxidase-coupled secondary antibody diluted in TBST for 1 h a t room temperature. After four washes of 15 min each with TBST, filters were analyzed by enhanced chemilu- minescence (ECL, Amersham Corp.) and exposed to Hyperfilm MP.

RESULTS

p15cdk-BP, a Protein Recognized by Anti-pl3""" and Anti-pgCKShS2 Antibodies

The early rationale for this work was the purification of a starfish oocytes homologue of yeast p13s""' and human pSCKSh". We thus used purification gel matrices known to interact with bacterially expressed pSCKsh" and used cross-reactivity with an- tibodies directed against p13'"'' or pSCKSha2 as a detection method.

Chromatography on four successive resins led to the purifi- cation of a 15-kDa, cyclin-dependent kinase-binding protein, p15'dk-BP. The first resin, S-Sepharose, was used batch-wise. The second resin, Sephacryl S-300, allowed the separation of a low molecular weight anti-p9CKSh"2-reactive protein ("late ~ 1 5 " ~ ~ - ~ " ' ) (Fig. l A , fractions 70-72) from a high molecular weight protein mixture also containing anti-pSCKshSZ cross-re- activity ("early p15cdk-BP*) (Fig. lA, fractions 3840). This early p15'dk-BP copurified with anti-PSTAIRE reactive proteins (not shown). Although both early and late p15"dk-BP fractions cross- reacted with anti-pl3""" and anti-p9cKshs* antibodies (Fig. lB ), only late p15cdk.BP fractions were further purified. The third resin, octyl-agarose, allowed the elimination of a minor, high molecular weight contaminant. At this stage p15'dk-BP was es- sentially homogeneous (Fig. 1C). The fourth resin, Matrex gel Green A, allowed the separation of the doublet of p15cdk-Bp, which was observed under favorable electrophoretic conditions (not shown). Both forms were recognized by anti-pSCKShs2 anti- bodies, both bound a PSTAIRE-containing protein (see below) from oocyte extracts (not shown). The electrophoretic mobility of both forms was not altered by pretreatment with potato acid phosphatase (not shown). The reason for the existence of two forms of p15'dk-BP remains unclear.

p15"dk-Bp Binds an Anti-PSTMRE Cross-reactive Protein Extracts from starfish G, and M phase oocytes were loaded

on p9CKSh"'-Sepharose and p15'dk-BP-Sepharose beads. The beads were extensively washed with a salt- and detergent-containing buffer. The bound proteins were then resolved by SDS-PAGE and analyzed by various criteria (Fig. 2).

pScKshS'-Sepharose-Three bands were detected in the 34- kDa range after immunoblotting with anti-PSTAIRE antibod- ies, a major upper band in G,, a major middle band in M phase, and a constant lower band. Polyclonal antibodies directed against yeast ~34"'" detected the typical shift of ~34"'" elec- trophoretic mobility associated with G f l transition (Pondaven e t al., 1990). This shift originates from tyrosine dephospho- rylation as shown with anti-phosphotyrosine antibodies and anti-tyrosine 15 peptide antibodies (which only recognize the tyrosine-dephosphorylated form of ~34'~"' (Baratte et al., 1992)). Anti-cyclin B antibodies detected the typical electro- phoretic mobility shift due to cyclin B phosphorylation as fur- ther shown by radioactive phosphate incorporation. ~ 3 4 ' ~ ' ~ de- phosphorylation and cyclin B phosphorylation coincided with activation of histone H1 kinase (Fig. 2, right pane l ).

p15'dk-EP-Sepharose-No signals were detected with anti- p3QdC2, anti-tyrosine 15 peptide, anti-phosphotyrosine, or anti-

13282 A 15-kDa cdk4- and cdk5-binding Protein

A Sephacryl S-300 B kDa "

24- - 0

17- 14"

8-

3 8 4 0 70 72 Fraction number

- - 2.5 111\ l-"l-

G 2 M G2 M p15 p15 p9 M p15 p15 p9

Coomassie Blue mti-p9 blot

~ , , I I I , , I I I I I I I I I I I I I

3 4 3 6 3 8 4 0 4 2 4 4 4 6 4 8 5 0 5 2 5456586062 6 4 6 6 6 8 7 0 7 2 7 4

Fraction number FIG. 1. Purification of p15'dk.RP. G, or M phase oocyte extracts were first loaded on S-Sepharose resin. After elution with a salt gradient, the

p15cdk'RP-containing fractions were loaded on a Sephacryl S300 gel filtration column. Fractions were collected and analyzed by SDS-PAGE and Coomassie Blue staining (A). Early p15'"~1'1' (fractions 38 and 40; left arrow) and late p15'dk"'P (fractions 70-74, right arrow) were detected by anti-p9'K5hs2 immunoblotting ( B ) . p15'".RP from G, and M phase oocytes were further purified on octyl-agarose and Matrex gel Green A and compared with pure p9"Kqh" by Coomassie Blue staining and anti-pSCKShS2 immunoblotting (1'2).

FIG. 2. p15cdk-RP-Sepharose binds a 33-kDa protein cross-reacting with anti-PSTAIRE antibodies. Starfish G, and M phase oocyte extracts were loaded

and p15'nk~R1'-Sepharose beads. The bound proteins were then ana- lyzed for histone H1 kinase activity (right panel) or resolved by SDS-PAGE prior to immunoblotting with antibodies directed against cyclin B, phosphotyrosine, the cdc2 tyrosine 15 peptide, full-length p34"", and the PSTAIRE peptide (left panel, blots are only presented at the level of interest). ["'PlPhosphate incorporation at the level of cyclin B was also recorded (left panel, top).

on pg''"sl"l-

"I" - - P32- cyclin B -

50 l"---

P-Tyr - 0."

Tyr 15 peptide-

cdc2 F= " "

G 2 M G 2 M G2 M G2 M

p9-sepharose pl5-sepharose p9-sepharose p 15-sepharose

cyclin B antibodies; no radioactive phosphate incorporation at the level of cyclin B was observed. No significant histone H1 kinase activity was measured. However p15edk-RP-Sepharose beads bound a protein, in the 33-kDa range, which cross-re- acted with anti-PSTAIRE antibodies (Fig. 2, left panel).

The possibility that the PSTAIRE signal observed on p15'"k'BP-Sepharose was due to a contamination by ~ 3 4 ~ ~ ~ ~ 1 cyclin B was examined by successive depletions on pSCKSh"'- Sepharose (Fig. 3A) or anti-cyclin B immunoprecipitations (Fig. 3B 1. An oocyte extract was loaded four times successively on pSCKShS1-Sepharose beads. The bound proteins were analyzed for histone H1 kinase activity (Fig. 3A, left panel) or probed, after SDS-PAGE, with antibodies directed against cyclin B, the tyrosine 15 peptide, and the PSTAIRE peptide (Fig. 3A, right panel ). These parameters monitored the efficiency of the deple- tion. The supernatants obtained after each depletion were loaded on p15c"k-"P-Sepharose. After a modest decrease, the

PSTAIRE signal bound to p15dk'Rp-Sepharose remained con- stant despite extensive depletion on pScKshS'-Sepharose (Fig. 3A, right panel ). Similarly, the PSTAIRE signal remained es- sentially constant on p15'dk-BP-Sepharose despite four succes- sive immunoprecipitations with anti-cyclin B antibodies (Fig. 3B). These results, along with results shown below, are not in favor of a residual p34cdr2/cyclin B bound to p15cdk-"P-Sepharose beads.

We next fractionated an oocyte extract on a Sephacryl S-2OOHR column (Fig. 4). Fractions were loaded on p15'dk-"- Sepharose and pSCKSh"'-Sepharose. p15cdk-RP-bo~nd proteins were analyzed by anti-PSTAIRE immunoblotting. pSCKShh"'- bound proteins were analyzed by anti-PSTAIRE and anti-cyclin B immunoblotting and for histone H1 kinase activity. Results show t h a t ( a ) the p15edk-BP-bo~nd PSTAIRE-containing protein is not monomeric but part of a high molecular weight complex (-250 kDa); no PSTAIRE signal was detected on p15cdk-Bp beads

A 15-kDa cdk4- and cdk5-binding Protein

A

13283

FIG. 3. The PSTAIRE signal on p15'dk~"p-Sepharose is insensitive to depletion on p9"KSh"'-Sepharose (A) and to immunodepletion with anti- cyclin B antibodies ( B ) . InA, a starfish M phase oocyte extract was loaded on

were then successively loaded three times on p9rKshs'-Sepharose. The bound pro- teins were analyzed for histone HI kinase activity ( lef t panel) or immunoblotted with anti-cyclin B, anti-cdc2-tyrosine 15 peptide and anti-PSTAIRE antibodies (upper right panel ). After each depletion

was also loaded on p15"'k"~"-Sepharose beads. The p15"lk~""-bound material was then immunoblotted with anti-PSTMRE antibodies (lower right panel) . In B, a starfkh M phase oocyte extract was im- munoprecipitated with anti-cyclin B anti- bodies. The non-precipitated proteins were then successively immunoprecipi- tated three times with anti-cyclin B anti- bodies. The precipitated proteins were analyzed for histone HI kinase activity (left panel) or immunoblotted with anti- PSTAIRE antibodies (upper right panel ). After each immunoprecipitation, the su- pernatant was also loaded on p15'dk."1'- Sepharose beads. The bound proteins were then immunoblotted with anti- PSTAIRE antibodies (lower right panel ).

pgCKShsl- Sepharose. The unbound proteins

on pgCKSl>-l Sepharose, the supernatant

250 ' I T cyclin B

ml5 peptide

PSTAIRE

PSTAIRE

1 2 3 4 Depletion on p9-sepharose

B

p9-sepharose 4 .

12.7

-0 - -33

. 0 .- ..- -2 13

1 2 3 4

p 15-se~harose

- 0 - - "33 1 2 3 4

Anti-cvclin B immunoDreciDitates

."~ - .. -".. kDa - PSTAIRE -33

1 2 3 4

p 15-se~harose

PSTAIRE - I -33

1 2 3 4

1 2 3 4 Immunodepletion by anti-cyclin B antibodies

in the 33-kDa range (not shown); ( b ) the peak of p34rd'2/cyclin B (bound to p9"KShs'-Sepharose) (-120 kDa) and the peak of p15cdk~"'-bound PSTAIRE signal do not coincide (Fig. 4).

plFdk-" Binds a Protein Kinase The 15cdk-RP-bo~nd proteins were next tested for kinase ac-

tivity toward a variety of protein and peptide substrates (Table I, Fig. 5).

( a ) substrate specificities for the p15cdk.131'-bound kinase and the p34'd'2/cyclin B kinase are not identical, although they par- tially overlap.

( b ) among 24 substrates tested, only the chromosomal high mobility group proteins HMG I/Y and P1 and myelin basic pro- tein (MBP) were phosphorylated by the pl5'"~"'-bound kinase. All these are substrates for the cdc2 kinase (Meijer et a l . , 1991b).

In all cases the p15'dkk-BP-bo~nd kinase activity was assayed after three successive p34cdr2/cyclin B depletions on pSCKSh"'- Sepharose beads to avoid a possible residual contamination by the cdc2 kinase. This is illustrated in Fig. 5B, where the ~34"''~/ cyclin B depletion was monitored by the decreased autophos- phorylation on cyclin B; despite this extensive depletion, the

p15cdk-"P-bo~nd kinase retained full P1 phosphorylating activity. We further investigated P1 phosphorylation by phosphopep-

tide mapping (Fig. 6) . Clearly the p34"dr2/cyclin B kinase and the p15'dk~1'P-bo~nd kinase phosphorylate P1 on different sites (Fig. 6, B-D). The p15cdk-""-bound kinase seems to phospho- rylate rather acidic domains. Most of these are different from the sites phosphorylated by casein kinase 2 (Fig. 6, A and B ) . P1 was also phosphorylated by purified ~33'~~'/p25, but on sites different from those phosphorylated by the starfish p15'"k'1'"- bound kinase (Fig. 6, B and E). The phosphopeptide maps of P1 phosphorylated by the starfish oocyte p15cdkk'1i1'-bound kinase or by the human p15cdk."'-bound cdk4/cyclin D (see below) were largely coincident (Fig. 6, F-H). Finally we observed an addi- tional spot when P1 was phosphorylated by starfish p15'dk.1'"- bound kinase from M phase oocytes (Fig. 6B) versus p15'"kBP- bound kinase from G, phase oocytes (Fig. 6, compare p a n e l s B and F ) .

plFdk.np Binds cdk4 and cdk5 The activity of the P1 kinase retained by p15cdk~1'1'-Sepharose

was not altered by calcium, EGTA, calcium-calmodulin, cyclic

13284 A 15-kDa cdk4- and cdk5-binding Protein

FIG. 4. The p15'dk-RP-bo~nd PSTAIRE signal belongs to a high mo- lecular weight complex distinct from p34"'r2/cyclin B. A starfish M phase oo- cyte extract was loaded on a Sephacryl S-ZOOHR gel filtration column calibrated with 17-, 44-, 158-, and 670-kDa molecu- lar size markers (upper panel). Markers were run before and after the oocyte ex- tract run, with identical results. Frac- tions were collected and loaded on p15'dk'RP-Sepharose and p9"'iqh''-Sepha- rose beads. The plF"-"'-bound proteins were analyzed by immunoblotting with anti-PSTAIRE antibodies (upper and middle panel ). The p9"KSh"'-bound pro- teins were analyzed for histone HI kinase activity (upper panel) and by immunob- lotting with anti-cyclin B and anti- PSTAIRE antibodies (lower panel ).

40 45 50 55 60 65 70 75 80 LL

Fraction number

p 15-se~harose

PSTAIRE

r

0.12

0.09 8 0 00 e4 cd Y

0.06 0 6

0.03

0.00

kDa

13

14

40 42 44 4 6 4 8 50 5 2 5 4 56 58 6 0 6 2 6 4

p9-sepharose '-47

Cyclin B

PSTAIRE

AMP, protein kinase A inhibitor, cyclic GMP, or TPA (not shown). These experiments eliminate calmodulin-dependent and cyclic nucleotides-dependent kinases, as well as protein kinase C. Casein kinase 2 was eliminated on the basis of three results: (a) the pl5-bound kinase does not phosphorylate casein (Table I), ( b ) it is insensitive to heparin (not shown), and ( c ) the phosphopeptide maps of P1 phosphorylated by casein kinase 2 and the p15cdk.""-bo~nd kinase are different (Fig. 6). Acontami- nation by ERWMAP kinase is unlikely for three reasons: ( a ) a specific antibody readily detected the ERWMAP kinase in crude extracts but not on p15'dkk'"P-Sepharose beads loaded with starfish oocyte extracts (not shown); ( b ) in contrast to the p15cdk-BP-bo~nd kinase, ERWMAP kinase activity massively in- creases during the G,/M transition (Pelech et al., 1988); ( c ) purified starfish E R K I " kinase does not phosphorylate P1 (not shown).

We next prepared [35Slmethionine-labeled cdc2 and related kinases by in vitro transcriptiodtranslation of human cDNAs. The proteins, labeled to the same extent (not shown), were loaded on pSCKSh"'- and p15'"-RP-Sepharose beads; after exten- sive washing, the bound proteins were resolved by SDS-PAGE and analyzed by fluorography (Fig. 7). As expected, cdc2 and cdk2 bound well to pSCKSh"' beads (Meyerson et al., 1992) (Fig. 7). cdk3, cdk4, and cdk5 (Fig. 7), PCTAIRE-1, PCTAIRE-3 and PITSLRE-1 (not shown) did not. Although all expressed pro- teins bound to a minor extent to p15cdk"'p-Sepharose, cdk4,

-33

-24 42 44 4 6 4 8 50 5 2 5 4 5 6 5 8 6 0 62 64

followed by cdk5, were most efficiently retained by the beads (Fig. 7).

We then prepared extracts from various mammalian tissues (regenerating rat liver, human liver, mouse liver, mouse nasal epithelium, and porcine brain) and cell lines (HepG2, HBG2, human HepSB, fibroblasts, and KB cells). Extracts were loaded on p15'dk'R"-Sepharose (before and after depletion on pSCKShs'- Sepharose) and on pSCKSh"'-Sepharose. The bound proteins were analyzed with specific antibodies. cdc2 and cdk2 signals were only observed on pSCKsh"l-Sepharose, whatever the tissue or cell line (Fig. 8, A and B; data not shown). No cdk3 signal was observed in any of the investigated extracts (not shown). cdk4 was only found in human Hep3B cells: it bound strongly to p15'dk-H"-Sepharose but not to pSCKSh"'-Sepharose (Fig. 8C). In addition, both cyclins Dl and D3 were detected on p15'"k-"- Sepharose beads loaded with Hep3B cells extracts (not shown). A strong anti-cdk5 cross-reactive protein was observed on p15cdk'RP-Sepharose in porcine brain, mouse nasal epithelium, and human KB cells (Fig. 8, D and E ) , even after three succes- sive depletions on pSCKShS'-Sepharose. cdk5 was retained very poorly or not at all on pSCKShS'-Sepharose.

We have recently purified to near homogeneity a novel cdc2- homologue from bovine brain, present as a complex consisting of two distinct subunits of 33 and 25 kDa, respectively (Lew et al., 1992a). The purified holoenzyme complex displays high histone H1 kinase activity. cDNA cloning has revealed that the

A 15-kDa cdk4- and cdk5-binding Protein 13285

TABLE I Substrate specificity of the p15""'0P-bound kinase

M phase oocyte extracts were loaded on pSCKShS1- and p15'dk'"p-Sepha- rose beads; after extensive washing, the beads were assayed for kinase activities using a variety of protein and peptide substrates as described under "Materials and Methods." The protein substrates were resolved by SDS-PAGE and analyzed by autoradiography while peptide sub- strates were analyzed after binding to P-81-phosphocellulose. +, sub- strate, (+), poor substrate; -, not a substrate.

Substrates p9"""h"-Sepharose p15"'k'""-Sepharose

Histone HI + - Histone H,, - - Histone H,,, - - Histone H, - - Histone H, - - AKAKKTPKKAK + - HMG17 - - HMGI + + HMGY + + P1 + + SPMRSRSPSRSK + - Protamine - - Myelin basic protein + + Casein (+) - Casein kinase 2 + - pp60'" + - Phosphatase inhibitor 1 + - Calmodulin - - Enolase - - PgCKSh"' - - GST-cdc25A + - GST-Rb + - Tubulin - - Tau - -

33-kDa subunit is virtually identical to human cdk5 (Lew et al., 1992b). To further test the idea that p15'dk-"P binds native cdk5, the partially purified enzyme from brain was loaded on p15'dk-RP-Sepharose, pSCmh"'-Sepharose, or on p15Cdk-RP-Sepha- rose after three passages of the enzyme sample over pSCKShS1- Sepharose. Analysis by Western immunoblot using anti-cdk5 antibodies demonstrated that indeed cdk5/p25 from brain bound specifically to p15Cdk'R''-Sepharose, but not to pSCKShs1- Sepharose (Fig. 8F).

DISCUSSION

P15cdk.RP In an attempt to purify the starfish homologue of p 1 3 ' l

pSCKSh", we have discovered a protein with interesting proper- ties, p15'dk-BP. This protein shares several properties with p13"""/p9CKSh": (a) cross-reactivity with a n t i - p l 3 ' and anti- pSCKShS2 antibodies, ( b ) similar chromotographic behavior on several resins, and ( c ) ability to form stable complexes with "cdc2-like" kinases.

The possibility that p15cdkkRP is a p13"""/p9CKsh" homologue, post-translationally modified so that it no longer binds cdc2 or cdk2, is unlikely. We have almost completely sequenced a star- fish homologue of pSCKSh" that displays strong homology with human p9"K"h"(not shown); available partial p15cdk-"Pprotein se- quences (not shown) do not match with this pSCKSh" homologue. We thus favor the idea that p15cdk.BP is the product of a distinct gene. p15'dk*RP is clearly different from the recently discovered cdk4-binding p16 protein (Seranoet al., 1993): (a) the available partial p15'dk"'Psequences do not share any homology with the p16 sequence (not shown); ( b ) p16, but not p15'dk-RP, inhibits cdk4' (not shown); (c) p15cdk."P, but not p16, binds cdk5. By analogy with the expanding families of cyclins (Lew et al., 1992c; Xiong and Beach, 1991) and cdc2-related kinases (Mey-

* M. F. Roussel and D. Quelle, personal communication.

A - kDa ~ -_ -31

HMG I -21.5 -14.4

G2 M G2 M -31

HMG Y 21.5 14.4

G2 M G2 M &D15

B

P1

Cyclin B

FIG. 5. ~ 1 5 ~ ~ ~ . ~ ~ binds a

-66.2

c

rl

,-97.4 '-66.2

-42.7

1 2 3 3 orotein kinase. In A. starfish G, and M

phase oocyte extracts were loaded on p9'"''-Sepharose *and on p15'dk'B"-Sepharose (after three successive depletions on p9'Ksh"1-Sepha- rose beads). The beads were then incubated with 1 pg of HMG I or Y in the presence of I y-"'PIATP. The "P-labeled HMG I and Y were subjected to SDS-PAGE and analyzed by autoradiography (A) . In B, an M phase extract was depleted three times on p9"K"h'1-Sepharose beads. After each depletion step, the supernatant was loaded on p15'dk'""-Sepharose. The beads were assayed for P I kinase activity (SDS-PAGE and auto- radiography) (upper panel ). The efficiency of the p9'"ish"' depletion was monitored by autophosphorylation of cyclin B on p9''KSh"1 beads (lower panel ).

erson et al., 1992), p15'dk-RP may be the second member of a family of plP'-related, cdk-binding proteins.

p15'dk."p Binds cdk4 and cdk5 The use of calcium, calmodulin, EGTA, CAMP, cGMP, protein

kinase inhibitor, TPA, and heparin has allowed us to eliminate calmodulin-dependent kinase, cyclic nucleotide-dependent ki- nases, protein kinase C , and casein kinase 2 as the p15'dk-131'- bound protein kinase. ERWMAP kinase is also an unlikely candidate (see "Results").

The cross-reactivity of the pl5-bound kinase with anti- PSTAIRE antibodies suggests that it belongs to the cdk family. The p15cdk'""-bound protein kinase is very unlikely to be the p34c"'2/cyclin B complex for the following reasons: (a) It has no cross-reactivity with anti-cyclin B antibodies; ( 6 ) it has no cross-reactivity with anti-p34"'r2 antibodies; ( c ) there is no ty- rosine dephosphorylation during the G f l transition; (d) deple- tion of p34c"r2/cyclin B on p9CKshs'-Sepharose beads or by anti- cyclin B immunoprecipitation does not remove the p15'dk-13P- bound kinase; ( e ) the p15cdk~13"-bound kinase and p34'dc2/cyclin B behave as distinct complexes upon gel filtration chromatogra- phy; ( f , the plSCdk-""-bound kinase does not phosphorylate ma- jor cdc2/cyclin B substrates; (g) the p15'dk-BP-bo~nd kinase and p34r''"2/cyclin B phosphorylate P1 on different sites; ( h ) in vitro transcribedtranslated cdc2 binds poorly to p15cdk-1~"-Sepharose.

cdk2 is unlikely to be the p15'dk.13P-bo~nd protein kinase: (a) cdk2-specific antibodies do not detect any signal on p15'dk-BP- Sepharose loaded with various mammalian cell extracts, while they do so on p9CKSh"l-Sepharose loaded with the same extracts; ( b ) in vitro transcribedtranslated cdk2 binds to pSCKShS1-Sepha-

13286 A 15-kDa cdk4- and cdk5-binding Protein

A Casein Kinase B pl5-sepharose c p9-sepharose D mix (B)+(C) . . . "" __.

E cdk5/p25 F pl5-sepharose G cdk4/cyclin D H mix (F)+(G) ;- -__p -.*-..- ~ .. - ,..., .~

* Q * A

FIG. 6. Phosphopeptide maps of P1 phosphorylated in vitro by the p15'dk.RP-bo~nd kinase and other kinases. P1 was phosphorylated in oitro, in the presence of [Y-:'~PIATP. by purified casein kinase 2 (A), the starfish (M phase oocytes) ~15'" '~ I"'-Sepharose-bound kinase (B), the

( F ) , or human Hep3B cdk4/cyclin D, bound to p15"lk"'"-Sepharose ( G ) . "'P-Labeled P1 was purified by SDS-PAGE and extensively digested by Sepharose-hound p34"''2/cyclin B ( C ) , purified brain cdk5/p25 kinase ( E ) , the starfish (G, phase oocytes) plYtk.""-Sepharose-bound kinase

thermolysin. In D, the phosphopeptides derived from P1 phosphorylated by the p15"k''"'-Sepharose-bound kinase and the p9""""*'-Sepharose-bound p34'""2/cyclin B kinase were mixed. In H , the phosphopeptides derived from P1 phosphorylated by the plY"-"l'-Sepharose-bound starfish kinase and by the p15"~k"'P-Scpharose-bound human Hep3B cdk4/cyclin D kinase were mixed.The phosphopeptides were resolved by chromatography and electrophoresis a t pH 3.5 and detected by autoradiography.

p9('KShsl-

p9-sepharose 3

- mmr -33

-24

cdk: 1 2 3 4 5

p 15-sepharose kDa -

.33

.24

A a-cdc2 B a-cdk2 C a-cdk4 . . -~ - kDa

D a-cdk5 E a-cdk5 F a-cdk5

- 33

- 24

cdk: 1 2 3 4 5 FIG. 7. Binding of in vitro transcribedltranslated cdks to

pSCKSh"'-Sepharose and p15'dk~RP-Sepharose. The cDNAs of cdkl-5 were transcribed and translated in oitro in the presence of ["'Slmethi- onine. Twenty pl of each reaction mixture were then loaded on p9""sh'l- Sepharose (upper panel) and on p15cdk'""-Sepharose (lower panel) for 30 min. After extensive washing the proteins bound to the beads were resolved by SDS-PAGE and the "'S-labeled cdks detected by autoradiog- raphy. Identical levels of:"S-labeled cdks were loaded on the beads, and gels were exposed for an identical period.

rose but poorly to pl5'"~"-Sepharose; (c ) associated with cyclin A (Gu et al., 1992; Lees et al., 1992) or with cyclin E (Dulic et al., 1992; Lees et al., 1992), cdk2 displays histone H1 kinase activ- ity. cdk2 also phosphorylates the retinoblastoma protein (Akiyama et al., 1992). The p15cdk-"-bo~nd kinase does not phosphorylate histone H1 or the Rb protein.

We have been unable to detect cdk3 on pSCKSh"'- and p15'dk-R"-Sepharose beads loaded with various mammalian cells extracts. cdk3 is unlikely to be the p15c""3'-bound protein kinase: (a) in vitro expressed cdk3 binds poorly to both pSCKshS'- and p15'dk-RP-Sepharose (Fig. 7); cdk3 has been reported to bind weakly to p13"'" (Meyerson et al., 1992); ( h ) in contrast with the p15C"k'B1'-bound kinase (Fig. 2), cdk3 is strongly recognized by polyclonal antibodies raised against full-length Schizosac-

p15 p9 p15d p15 p9 ~ 1 5 p15 p9 PI% FIG. 8. Binding of mammalian cdks to p15cdk'nP-Sepharose and

pSCKShS'-Sepharose. Extracts of mammalian tissues and cells were loaded on p9""'""'-Sepharose ( p S ) , p15'dk-"P-Sepharose (pZ5) , or p15'dk'"P-Sepharose after three successive depletions on p9"Kshs'-Sepha- rose (p15d) . After extensive washing of the beads the bound proteins were resolved by SDS-PAGE and analyzed by immunoblotting with specific anti-cdc2, -cdk2, -cdk4, or -cdk5 antibodies. The immunoblots at the level of cdks are presented. A, regenerating rat liver, anti-cdc2; B, regenerating rat liver, anti-cdk2; C, human Hep3B cells, anti-cdk4; D, porcine brain, anti-cdk5; E, mouse nasal epithelium, anti-cdk5; F , pu- rified cdk5/p25 from bovine brain, anti-cdk5.

charomyces pomhe ~34'~'' (Meyerson et al., 1992). cdk4 strongly interacts with p15'"k-": ( a ) a strong anti-cdk4

cross-reactive signal was observed on p15cdk-131'-Sepharose loaded with human Hep3B cells extracts (Fig. 8C ); ( h ) the cdk4 kinase does not phosphorylate histone H1 (Matsushime et al., 1992); (c) it does not bind to ~13""" (Matsushime et al., 1992); ( d ) cdk4 is the best p15c"k'RP-binding protein, among the five in vitro expressed cdks (Fig. 7) (weak binding may be due to the lack of post-translational modifications and lack of subunit as- sociations); ( e ) the p15'dk-"P-bo~nd kinases from starfish oocytes and human Hep3B cells phosphorylate P1 on similar sites (Fig. 6); ( f , the cdk4-associated cyclins D l and D3 are detected on

A 15-kDa cdk4- and cdk5-binding Protein 13287

p15'dk-BP-Sepharose beads loaded with Hep3B cells extracts (not shown); (g) olomoucine, a kinase inhibitor specific to cdc2, cdk2, and ~ d k . 5 ~ does not inhibit the p15'dk-BP-bo~nd kinase or cdk4kyclin Dl. Although full demonstration will require fur- ther work, it can be reasonably suggested that the starfish oocyte p15"dk-BP-bound kinase is a cdk4 homologue or a very related kinase.

In contrast to the p15cdk-BP-bo~nd kinase, cdk4/cyclin D3 phosphorylates the retinoblastoma gene product pRb (Matsu- shime et al., 1992). The reason for such discrepancy is un- known. The p15'dk-BP-bo~nd kinase might already be complexed with endogenous pRb. Alternatively, substrate preference may be specified by the presence of p15cdk-BP; by analogy, p13""" suppresses ~34"~""s ability to phosphorylate intermediate fila- ment proteins (Kusubata et al., 1992). We do not know whether anti-PSTAIRE antibodies cross-react with cdk4.

cdk5 strongly interacts with p15"dk-BP: ( a ) a protein recog- nized by anti-cdk5 antibodies is detected on p15cdk-BP-Sepharose loaded with a variety of mammalian tissue extracts, including neuronal tissues (Fig. 8, D and E ) , in which cdk5 is preferen- tially expressed (Hellmich et al., 1992; Lew et al., 1992a, 1992b); ( b ) the cdk5/p25 complex purified to homogeneity from bovine brain binds very efficiently to p15'dk.BP- but not to p9CKSh"1-Sepharose (Fig. 8F); (c) cdk5 does not bind to ~13""' (Lew et al., 1992a, 1992b; Meyerson et al., 1992), or to pSCKShS1- Sepharose (Fig. 8, D-F); ( d ) both cdk5 and the p15cdk-BP-bo~nd kinase form a high molecular weight complex (Lew et al., 1992a) (Fig. 4). There is some discrepancy about the ability of anti-PSTAIRE antibodies to recognize the PSSALRElcdk5 ki- nase; cdk5 cross-reacts with mouse monoclonal anti-PSTAIRE antibodies (Meyerson et al., 1992), as does the p15cdk-BP-bo~nd kinase. However, polyclonal anti-PSTAIRE very weakly recog- nize the bovine brain cdk5/p25 complex (Lew et al., 1992a).

One property discriminates cdk5 and the starfish ~ 1 5 " ~ ~ " ' - bound kinase; in contrast to the p15'dk-BP-bo~nd kinase, the bovine brain cdk5/p25 phosphorylates histone H1 (Lew et al., 1992a) and tau.4 The substrate specificity of cdk5 may be regu- lated by the subunits it associates with. In this context, a t least four different proteins can associate with cdk5 in W138 cells, including cyclin D l and D3 (Xiong et al., 1992).

cdk5 has been first obtained by screening a HeLa cervical carcinoma cDNA library with degenerate oligonucleotides cor- responding to conserved regions of cdc2 (Meyerson et al., 1992). The cdk5 sequence was essentially confirmed by screening a HeLa cell cDNA library with the anti-p3PdC2 G8 antibody (Xiong et al., 1992). Independently, a neuronal cdc2-like kinase (nclk) was identified by screening a rat brain cDNAlibrary with a mouse cdc2 cDNA probe. nclk turned out to be cdk5 (Hellmich et al., 1992). In early 1992, Lew et al. (1992a) presented the purification to homogeneity of a novel proline-directed protein kinase from bovine brain. Upon cloning, this kinase was iden- tified as cdk5 (Lew et al., 1992b).

Substrates of the p16dk.Bp-bound Kinase The substrate specificities of p34'd'2/cyclin B and the

p15'dk-BP-bo~nd kinase differ to a large extent (Table I). Never- theless, all substrates of the latter kinase can be phospho- rylated by the cdc2 kinase.

P1 is a chromosomal protein that is heavily phosphorylated in proliferating cells (Ostvold et al., 1985). Previous studies have shown that the protein is a substrate for casein kinase 2, protein kinase C, cyclic AMP-dependent kinase, calciudcal-

J. Vesely, M. Strnad, J. J. Blow, and L. Meijer, submitted for publi-

Paudel, H., Lew, J., A i , Z., and Wang, J. H. (1993) J. B i d . Chem. cation.

268,23512-23518.

modulin-dependent protein kinase 11, and p34"d"2/cyclin B (Me- landsmo et al., 1989; Meijer et al., 1991b; Ostvold et al., 1985; Walaas et al., 1989). Phosphopeptide map analysis (Fig. 6) dem- onstrates that P1 is phosphorylated on different sites by casein kinase 11, cdk5, cdc2, and the p15"dk-BP-bound kinase.

The high mobility group chromosomal proteins HMG Ih' are derived from the same gene by alternative splicing of the mRNA (Johnson et al., 1989). These proteins are preferentially expressed in undifferentiated, proliferating tissues (Giancotti et al., 1987; Vartiainen et al., 1988). HMG I/Y has been recently identified as a promotor-specific accessory factor for NF-KB- dependent virus induction of the human interferon+ gene (Thanos and Maniatis, 1992); it is implicated in stimulation of mouse ribosomal RNA synthesis (Yang-Yen and Rothblum, 1988) and in regulation of the lymphotoxin (tumor necrosis factor p) gene (Fashena et al., 1992). HMG VY is phospho- rylated at multiple sites (Ferranti et al., 1992) by several ki- nases including casein kinase 2 (Lund et d . , 1987; Palvimo and Linnala-Kankkunen, 1989) and p34cdc2/cyclin B (Lund and La- land, 1990; Meijer et al., 1991b; Nissen et al., 1991; Reeves et al., 1991).

In summary, we have identified a protein, p15cdk-BP, which strongly interacts with the cyclin-dependent kinases cdk4 and cdk5. Determination of p15'dk-Bp's sequence will allow compari- son with the Sucl/CKS proteins. The existence of a family of proteins comprising pSCKSh", p15cdk-BP, and more members, which form stable complexes with cdk-related kinases, is an open possibility. p15"dk-BP-Sepharose beads provide a convenient affinity chromatography media for the specific purification of cdk4, cdk5, and their associated partners or their specific depletions from a cellular extract. This tool may be of interest in view of the importance of cdk4/cyclin D in early cell cycle progression (Matsushime et al., 1992; Xiong et al., 1992) and of the deregulation of cyclin D in human tumors (see review by Motokura and Arnold (1993)). It may also provide an important tool in the studies of brain functions and pathology, as illus- trated by the increased phosphorylation of tau protein in Alz- heimer's disease (reviewed by Lee (1993)). Tau protein can be phosphorylated in vitro by purified ~34'~'' and a related protein kinase from neurofilaments (Ledesma et al., 1992; Lee et al., 1992; Mawal-Dewan et al., 1992; Vulliet et al., 1989). It has been shown recently that tau is a substrate for the purified cdk5 kinase? Neurofilament proteins are also phosphorylated by the cdc2 and cdk5 kinases (Hisanaga et al., 1991; Lew et al., 1992a, 1992b; Liem, 1993).

Acknowledgments-We particularly thank Nicole Guyard for excel- lent secretarial assistance, Jacques Orillon for expert photographic work, and P. Nurse, Lee Vogel, and Steve L. Pelech for reading the manuscript. Many thanks to the fishermen of the Station Biologique de Roscoff for collecting starfish and sea urchins. We are grateful to the following persons for providing reagents: D. Beach (anti-cdk4 and anti- cdk5 antibodies), P. Cohen (inhibitor-I), L. Detivaud and B. Baratte ( in vitro transcriptiodtranslation), J. Downing (PCTAIRE-1 and -3 cD- NAs), G. Draetta (anti-pl3""" and anti-~34'~'' antibodies), \'. Giancotti (HMG V Y , HMG 17), R. Golsteyn and E. Nigg (AKAKKTPKKAK), V. Kidd and J. Lathi (cdkl, cdk2, cdk5, and PITSLRE-1 and -2 cDNAs; SPMRSRSPSRSK), P. Loyer (preparation of anti-~l5'&.~' antibodies; rat liver, various mammalian cell lines), M. Meyerson (cdk3 cDNA and anti-cdk3 antibodies), D. Morgan (pp60""), S. L. Pelech (polyclonal anti- PSTAIRE antibodies), L. Pinna (casein kinase 2). S. I. Reed (pSCmh"- expressing bacteria), M. Roussel and C. Sherr (cdk4 cDNA, anti-cdk5

bodies), H. Y. L. Tung (anti-cdc2 tyrosine 15 peptide antibodies), J. Y. J. antibodies), H. Shibuya and J. Ruderman (anti-ERWMAP kinase anti-

Wang (anti-phosphotyrosine antibodies), and M. Yamashita (mono- clonal anti-PSTAIRE antibodies).

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