Yeast MCKI Protein Kinase Autophosphorylates at Tyrosine and ...

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1993 by The American Society for Biochemistry and Molecular Biology, Inc

Vol. 268, No. 28, Issue of October 5, PP. 21155-21164, 1993 Printed in U.S.A.

Yeast MCKI Protein Kinase Autophosphorylates at Tyrosine and Serine but Phosphorylates Exogenous Substrates at Serine and Threonine*

(Received for publication, January 7, 1993, and in revised form, July 28, 1993)

Moon-Young Lim8, David DaileyO, G. Steven Martin, and Jeremy Thorned From the Department of Molecular and Cell Biology, Division of Biochemistry and Molecular Biology, and The Cancer Research Laboratory, University of California, Berkeley, California 94720

The product of the Saccharomyces cerevisiae MCKl gene is a protein kinase that phosphorylates poly (Glu,Tyr) in vitro and is itself phosphorylated at both tyrosine and serine in vivo. To characterize the substrate specificity of Mckl, the enzyme was purified to apparent homogeneity from the soluble fraction of yeast cell extracts by ammonium sulfate precipitation, followed by ion exchange chromatography (Q- and S-Sepharose), dye-ligand affinity chromatography (Or- ange A-agarose), adsorption chromatography (hydroxylapatite), and ion exchange fast protein liquid chromatography (Mono-s). In the absence of an exogenous substrate, purified Mckl was able to autophosphorylate on tyrosine and serine. A catalytically inactive mutant (K68R in conserved kinase domain 11) expressed in an mcklA strain did not contain detectable phosphotyrosine, confirming that the tyrosine phosphorylation observed in vivo is due to autophosphorylation, but did contain phosphoserine, suggesting that Mckl is a target for other cellular protein kinases. Purified Mckl phosphorylated a variety of proteins in heat-inactivated yeast extracts, primarily on serine (and threonine). The purified enzyme also used a number of mammalian proteins as phosphoacceptors, including myelin basic protein (MBP), microtubule-associated protein 2 (MAP-2), and tau protein. All of these substrates were phosphorylated on either serine or threonine (or both). Mckl isolated from yeast extracts by immunoprecipitation with an anti-Mckl antibody directed against its C terminus also phosphorylated MBP at serine. In the same immune complex kinase assay, the K68R mutant did not detectably phosphorylate MBP, indicating that the serine-specific phosphotransferase activity of Mckl is intrinsic and not due to contamination by an associated kinase. These findings demonstrate that Mckl is a member of a novel class of protein kinases that displays the ability to phosphorylate all three hydroxyamino acids in proteins.

* This investigation was supported by National Institutes of Health Research Grants GM44173 (to G. S. M.) and GM21841 (to J. T.) and by facilities provided by the Berkeley campus Cancer Research Laboratory. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertzsernent” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

?I. Supported by United States Public Health Service Postdoctoral

Award Postdoctoral Fellowship CA09067. Traineeship CA09041 and Recipient of National Research Service

0 Supported by United States Public Health Service Predoctoral Traineeships CA09179, GM07232, and CA09041. Present address: Plant Gene Expression Center, U. S. Dept. of Agriculture, 800 Bu- chanan St., Albany, CA 94710. 1 To whom correspondence should be addressed: Dept. of Molecular

and Cell Biology, Division of Biochemistry and Molecular Biology, Rm. 401, Barker Hall, University of California, Berkeley, CA 94720. Tel.: 510-642-2558; F a : 510-643-5035.

Cell metabolism and proliferation are controlled by processes that involve phosphorylation of proteins on serine, threonine, and tyrosine residues. These reactions are catalyzed by protein kinases (ATP:protein phosphotransferases) and can be re- versed by the action of phosphoprotein phosphatases (phosphoprotein phosphohydrolases), thereby allowing for rapid and re- versible regulation. The majority of protein kinases have been classified into two groups based on their specificity for the hydroxyamino acid phosphorylated in substrate proteins (Hanks et al., 1988). One class, the protein-serinekhreonine kinases, modifies either Ser or Thr, or both; the other class, the protein-tyrosine kinases, phosphorylate only Tyr. When opti- mally aligned, the catalytic domains of all known protein kinases share ll conserved subdomains (designated I-XI) (Hanks and Quinn, 1991). Based on such alignments, it has been suggested that the sequences of subdomains VI and VI11 are predictive of the hydroxyamino acid selectivity of a protein kinase (Hanks et al., 1988).

Recently, however, a number of protein kinases that seem to be able to phosphorylate proteins both at Tyr and at Ser and Thr (so-called “dual specificity” enzymes) have been described. Examples of such protein kinases include: mammalian Clk (Cdc28/cdc2+-!ike kinase), also called Sty (Ben-David et al . , 1991; Howell and Afar, 1991; Johnson and Smith, 1991); mammalian Erkl and Erk2, which are mitogen-activated OL4€“AP1

kinases (Crews et al., 1991; Seger et al . , 1991); a kinase ( M A P kinase kinase) that activates the Erk kinases (Gomez and Co- hen, 1991; Alessandrini et al., 1992; Crews and Erikson, 1992; Kosako et al., 1992; Nakielny et al., 1992); Schizosaccharomy- cespombe weel+ gene product (Featherstone and Russell, 1991; Parker et al., 1992); and Saccharomyces cereuisiae Mckl (Dai- ley et al., 1990) and Spkl (Stern et al., 1991). All of these protein kinases appear to undergo autophosphorylation both at Tyr and at Ser (or Thr). In some cases, the enzymes have been shown to phosphorylate exogenous substrates such as poly- (Glu,Tyr), angiotensin 11, or ~34‘~‘~’ at Tyr in uitro, or to result in phosphorylation of bacterial proteins a t Tyr when expressed in Escherichia coli. The sequences of subdomains VI and VI11 in this set of enzymes resemble those of known protein-serine/ threonine kinases. Hence, no sequence features yet identified can accurately predict whether a protein kinase will have dual specificity (Lindberg et al., 1992). In fact, the human insulin receptor, hitherto regarded as a canonical protein-tyrosine ki-

The abbreviations used are: MAP-2, microtubule-associated protein 2; BSA, bovine serum albumin; CIP, calf intestinal alkaline phosphatase; FPLC, fast protein liquid chromatography; LPSM, low phosphate synthetic medium; MES, 2-(N-morpholino)ethanesulfonic acid; MBP, myelin basic protein; MeP, 1-methyl-2-pyrrolidone; PBS, phosphate- buffered saline; PVDF, polyvinylidene difluoride; RIPA, radioimmune precipitation assay; RSA, rabbit serum albumin; PAGE, polyacrylamide gel electrophoresis; SMBS, rn-maleimidobenzoyl-N-hydroxysulfosuccin- imide ester; kb, kilobase(s).

~ ~~

21155

21156 Yeast Mckl Dual Specificity Protein Kinase

nase, has been reported to undergo autophosphorylation at both Ser and Thr (Baltensperger et al., 1992). Thus, the hydroxyamino acid selectivity of a protein kinase cannot be determined unambiguously from its sequence alone.

We previously described a protein kinase from S. cerevisiae that was purified on the basis of its ability to phosphorylate poly(Glu,Tyr) (Schieven et al., 1986; Dailey et al., 1990). We demonstrated that this enzyme is the product of the MCKl gene, which we initially designated YPKl .2 Subdomains VI and VI11 of the Mckl sequence closely resemble those of the S e d Thr-specific class of protein kinases. Nonetheless, we showed that the Mckl protein isolated from yeast cells is phosphorylated on Tyr and Ser, and that partially purified Mckl undergoes autophosphorylation on Tyr and Serin vitro (with low levels of phosphorylation of Thr).

Some of the physiological roles of the MCKl gene product have been characterized genetically. MCKl function is required in MATdMATa diploids for expression of the IMEl gene, a positive regulator of meiosis and sporulation, and for formation of a mature ascus (Neigeborn and Mitchell, 1991). Further- more, increased MCKl gene dosage accelerates the sporulation program, whereas mckl mutations cause both a delay and a decreased efficiency of sporulation. In addition, overexpression of MCKl in haploid cells suppresses the poor fidelity of chromosome segregation manifested by certain mutations in one subdomain of S. cerevisiae centromeric DNA, the 25-base pair centromere DNA element (or CDE) I11 (Shero and Hieter, 1991). Strains carrying an mckl null mutation (mcklA allele) are viable under standard growth conditions (30 "C) but are cold-sensitive (unable to grow at 11 "C) and are hypersensitive to the growth inhibitory effects of benomyl, a microtubule-de- stabilizing agent. These observations suggest that Mckl may be involved in some aspect of spindle andfor kinetochore me- chanics.

As one approach for further elucidating the function of Mckl, we undertook extensive purification of this enzyme and detailed characterization of its substrate specificity. In support of our previous findings, we demonstrate here that apparently homogenous Mckl undergoes autophosphorylation at both Tyr and Ser, yet phosphorylates exogenous substrates exclusively at Ser and Thr.

EXPERIMENTAL PROCEDURES Growth of Yeast Cells-S. cereuisiae strain YPH500 (MATa u ra352

lys2401amb"' ade2-101°chrc trpl-A63 his3-A200 leu2-Al) and its deriva- tives were grown in YPD medium (Sherman et al., 1986). To maintain selection for plasmids, transformants were grown in synthetic minimal medium supplemented with the appropriate nutrients (Sherman et al., 1986). When cells were labeled with 32P,, cultures were grown in a low phosphate medium (LPSM) (Reneke et al., 1988).

Plasmid and Strain Constructions-A 3.9-kb ClaI-PstI fragment of yeast genomic DNA that contains the MCKl gene (Dailey et nl., 1990) was inserted into the corresponding sites in the polylinker of the vector, pBluescript I1 SK+ (Stratagene), to yield plasmid pDD2 (Dailey, 1992). To generate a plasmid for overexpression of MCKl in yeast, the gene was inserted into a high copy number vector, as follows. The entire MCKl gene and its promoter were excised from pDD2 by digestion with SphI (at -340) and XbaI (a site in the polylinker downstream of the MCKl coding sequence). The resulting 3.0-kb fragment was purified by agarose gel electrophoresis and ligated into the yeast 2-pm DNA vector, YEp352 (Hill et al., 19861, that had been cleaved with SphI and XbaI, generating YEp[MCKlI.

An mckl null allele was constructed in vitro, as follows. pDD2 was digested with Sal1 (a site in the polylinker upstream of the MCKl coding sequence) and with SphI, thereby removing genomic sequences 5' to the SphI site. The overhanging ends so generated were converted to flush ends by incubation with the Klenow fragment of E. coli DNA

* The GenBank accession number for MCKl is M55984. We originally called this sequence, YPK1, but the gene designation was subsequently changed to MCKl (see Dailey et al., 1991).

polymerase I (Boehringer Mannheim) in the presence of deoxyribo- nucleoside triphosphates and religated to generate plasmid pDD5. To substitute a selectable marker for the sequence between the EcoRI (+46) and EcoRV (+491) sites in the MCKl open reading frame, pDD5 was digested with EcoRV, ligated to synthetic EcoRI linkers (New Eng- land Bio-Labs), digested exhaustively with EcoRI, treated with CIP (Promega), and then ligated with a fragment containing the URA3 gene t o which had been attached EcoRI linkers with 5'-phosphoryl ends, yielding plasmid pDD7. To prepare the URA3-containing fragment, the gene was excised from plasmid pNKY51 (Alani et al., 1987) by digestion with BamHI and BglII, and the ends so generated were filled in by incubation with Klenow fragment, ligated to EcoRI linkers, and digested exhaustively with EcoRI.

To produce a genomic mckl null mutation, pDD7 was digested with XhoI (in the upstream polylinker sequence) and XbaI (in the downstream polylinker sequence), and the resulting 6.3-kb fragment containing the mckl::URA3 deletion-substitution allele was used for DNA- mediated transformation of strain YPH500 via the lithium acetate procedure (It0 et al., 1983). To confirm that the genomic MCKl locus had been transplaced with the mutant allele, genomic DNA was prepared from several Ura' transformants and analyzed by restriction enzyme digestion and Southern blot analysis. When digested with SphI and PstI and hybridized to an appropriate probe (BamHI fragment 3' to the MCKl coding sequence), the normal genomic SphI-PstI fragment was absent and was replaced by a band of the expected length; similarly, when digested with ClaI and hybridized to a different probe (ClaI-StuI fragment 5' to the MCKl coding sequence), the same result was obtained (data not shown). One such transformant, DDY1, was used in subsequent experiments; the alteration it carries was designated the mckl-ASrtURA3 mutation.

Site-directed mutagenesis was used to generate a mutant gene en- coding a full-length, but catalytically inactive, Mckl protein, as follows. The ApaI site in pBluescript I1 SK+ was eliminated by cleaving with ApaI, removing the resulting 3"overhangs by digestion with the 3'- to 5'-exonuclease activity of T4 DNA polymerase, and ligating the blunt ends so generated using T4 ligase, yielding plasmid pML1. The 3.9-kb ClaI-PstI fragment from pDD2 was subcloned into pMLl that had been digested with ClaI and PstI to produce pML2. Using the 3.9-kb ClaI- PstI fragment from pDD2 as the template, a 30-base oligonucleotide (Primer A) was extended with VentTM polymerase (New England Bio- Labs) by incubation for 5 cycles in a thermal cycler (Perkin-Elmer Cetus) set to a program of 94 "C (1 min) for denaturation, 48 "C (1 min) for renaturation, and 72 "C (1 min) for extension. Primer A corresponds to nucleotides +184 to +213 ofthe MCKl coding sequence, but with G in place of the A at position +203, converting codon 68 (AAA) for Lys in conserved kinase domain I1 to a codon (AGA) for Arg. After 5 cycles, a second 30-base oligonucleotide (Primer B) complementary to nucleotides +325 to +354 of the MCKl sequence was added, and polymerase chain reaction amplification was continued for an additional 25 cycles. The resulting double-stranded product was converted to flush ends by incubation with Klenow fragment, inserted by blunt-end ligation into the EcoRV site in pML1, and used for transformation of E. coli strain XL1 Blue. Plasmids of the appropriate size and possessing the expected restriction sites were recovered from several of the transformants and were subjected to double-stranded sequencing (Kraft et al., 1988) using Primer B to verify the presence of the A203G mutation. One of these plasmids, pML3, was digested with ApaI and NcoI, and the resulting 150-base pair fragment (corresponding to nucleotides +191 to +341 of the MCKl coding sequence) was ligated into pML2 that had been digested with ApaI and NcoI, purified by agarose gel electrophoresis, and treated with CIP, thereby creating plasmid pML4. TO generate a plasmid for overexpression of the mutant gene in yeast, the SphI-XbaI fragment of pML4 containing the K68R mutant and its promoter were ligated into the 2-pm DNA vector, yEp351 (Hill et al., 19861, that had been digested with SphI and XbaI, yielding pML5.

Immunological Reagents-Preparation, characterization, and affinity purification of rabbit polyclonal anti-Mckl and anti-phosphotyrosine antibodies were described previously (Dailey et al., 1990). To prepare an antibody directed against the C terminus of Mckl, a peptide (CEL- QILGEFADKIKPTKVAE), which corresponds to a cysteine followed by the 19 C-terminal residues of Mckl, was synthesized chemically. TO activate the carrier protein, RSA (2.5 mg) was dissolved in 50 pl of 50 m~ sodium phosphate (pH 7.2), and SMBS (0.8 mg, Pierce Chemical Co.) dissolved in 25 pl of MeP was added. After an additional 25 PI of MeP were added to clarify the solution, the mixture was incubated for 35 min at 22 "C, and then the excess SMBS was removed by filtration of the RSA through a bed (1 ml) of Sephadex G-25 in a disposable plastic pipette. The synthetic C-terminal peptide (2 mg) was then coupled via

Yeast Mck l Dual Specificity Protein Kinase 21157

the sulfhydryl of its N-terminal Cys by incubation with the activated RSA for 3 h a t 22 "C in about 2 ml of 0.1 M sodium phosphate and 2 mM EDTA(pH 6.5). Unreacted peptide was removed by extensive dialysis of the peptide-protein conjugates against PBS. The peptide-RSA antigen was used to immunize a rabbit (200 pghjection) subcutaneously three times at 21-day intervals. Following the third injection, serum was withdrawn three times a t 1-week intervals. To prepare a matrix for affinity purification of the anti-C-terminal peptide antibodies, vinyl sulfone-activated agarose (200 mg, Sigma) was swollen in water, washed sequentially with 1 M sodium phosphate (pH 6.5),0.5 M sodium carbonate (pH lo), water, and 0.2 M N-methylmorpholine and 10 m~ EDTA (adjusted to pH 7.8 with glacial acetic acid), and then incubated with the synthetic C-terminal peptide (2 mg) in 2.5 ml of the latter buffer for 18 h at 22 "C with gentle agitation by rocking. To block unreacted sites, 5 pl of undiluted P-mercaptoethanol was added and incubation continued for 2 h. The peptide-agarose beads were washed thoroughly with PBS and used to purify anti-peptide antibodies from the crude serum by methods described before (Dailey et al., 1990).

Purification of MckI-YPH500 carrying YEp[MCKIl was grown a t 30 "C to late exponential phase in synthetic minimal medium lacking uracil in a 200-liter fermenter. The cells were harvested in an air-driven continuous flow centrifuge (Sharples) and stored at -70 "C. To initiate a purification, frozen cells (100 g) were thawed, washed, and lysed as described before (Dailey et al., 1990). Mckl was enriched from the clarified extracts following the procedures documented previously (Dai- ley et al., 19901, up to and including dye-ligand affinity chromatography on Orange A-agarose. The Mckl content of various fractions was moni- tored by dot-immunoblot analysis using rabbit polyclonal anti-Mckl antibodies. Despite the approximately 10-fold increase over wild-type cells in the level of Mckl in the starting material from the overproducing strain, a significant number of contaminating species were detectable in the Orange A-agarose fraction. To achieve greater purification, the pooled peak fractions from Orange A-agarose chromatography (-10 mg of protein in 50 ml) were diluted 5-fold in 10 mM potassium phosphate (pH 6.8) and loaded onto a bed (20 ml) of hydroxylapatite ( H A - Ultrogel; IBF) in a column (1.5 x 12.5 cm). The column was washed with 40 ml of the same buffer, bound proteins were eluted with a linear gradient (80 ml total) from 10 to 500 mM potassium phosphate (pH 6.81, and fractions (2 ml) were collected. Protein species in each fraction were resolved by SDS-PAGE, transferred to PVDF membranes (Millipore) by electroblotting, and stained either with colloidal gold (AuroDye forte, Amersham Corp.) or with rabbit polyclonal anti-Mckl antibodies followed by alkaline phosphatase-coupled goat anti-rabbit IgG (Bio-Rad). As the final step, three peak fractions (19-21) from hydroxylapatite chromatography were pooled, diluted 5-fold in 0.1% Triton X-100, 1 m~ EDTA, 50 mM MES (pH 6.51, and subjected to FPLC on a Mono-S column (HR5/5; Pharmacia LKE3 Biotechnology Inc.) as described previously (Dailey et al., 19901, and fractions (1 ml) were collected and analyzed by staining with colloidal gold and by immunoblotting with anti-Mckl antibodies. Fractions containing apparently homogeneous Mckl were adjusted to 50% glycerol and stored at -70 "C.

Analysis of Proteins-SDS-PAGE was conducted as described else- where (Laemmli, 1970) using prestained marker proteins (Sigma) as the molecular weight standards. Staining with colloidal gold was carried out according to the manufacturer's instructions. Radiographic and colorimetric detection of bound antibodies on immunoblots was performed as described before (Dailey et al., 1990). 32P-Labeled proteins were visualized by autoradiography enhanced by the use of Cronex Lightning Plus intensifying screens (DuPont). To quantitate the amount of 32P or lZ5I present, the corresponding regions of the PVDF membranes were excised and analyzed by liquid scintillation counting in Filter Count (Packard Instrument Co.). Phosphoamino acid analysis (both one- and two-dimensional) was conducted by methods described by others (Cooper et al., 1983). Protein concentrations were determined, using BSA as the standard, either by a dye binding assay in solution (Bradford, 1976) or by staining proteins blotted to PVDF membranes with colloidal gold.

Assays of Protein Kinase Actiuity-Autophosphorylation was measured as the incorporation of radioactivity from [Y-~~P]ATP into a species that co-migrated with authentic Mckl protein and was immunoprecipi- table with affinity-purified anti-Mckl antibodies. The standard reaction mixture (30 pl final volume) contained 10 m~ MnCl,, 10 mg/ml BSA, 50 m~ MES (pH 6.5), and pure Mckl enzyme (2 ng; pooled Mono-S fractions 20-26). Reactions were initiated by the addition of [Y-~~P]ATP (8.8 x lo5 cpdpmol) to a final concentration of 2.5 p~ and incubated a t 30 "C for 30 min to 1 h. The [Y-~~PIATP used was either a commercial product (Du Pont) or was prepared with a Gamma Prep-A kit (Pro- mega). Reactions were terminated by the addition of an equal volume of

stop buffer (two times concentrated SDS-PAGE sample buffer) containing 100 mM EDTAand 48% (wiv) urea. For analysis, terminated reaction mixtures were loaded directly onto SDS-polyacrylamide gels without boiling (Schieven and Martin, 1988).

The phosphotransferase activity of Mckl in solution was measured as the incorporation of radioactivity from [Y-~~PIATP into a species that co-migrated upon SDS-PAGE with the authentic protein added as the exogenous phosphoacceptor substrate. The standard reaction mixture (30 pl final volume) contained 10 m~ MgCl,, 50 12" Tris-HC1 (pH 7.01, 10 pg of substrate protein, and pure Mckl enzyme (2 ng). Reactions were initiated by the addition of [Y-~~PIATP (7,000 cpdpmol) to a final concentration of 100 p~ and incubated at 30 "C for 10-30 min. Reac- tions were terminated by addition of an equal volume of stop buffer followed by boiling for 5 min prior to analysis by SDS-PAGE. Purified bovine MAP-2 and tau protein mixture was the gift of B. Poschal and R. Vallee (Worcester Foundation, Worcester, MA), and purified bovine MAP-2 and recombinant tau protein were the gifts of D. Drechsel and M. Kirschner (University of California, San Francisco, CAI. Purified bovine MBP was purchased from a commercial supplier (Sigma).

The phosphotransferase activity of Mckl in immune complexes was measured, as follows. YPH5OO carryingYEp[MCKIl and DDYl carrying pML5 [ m ~ k l ~ ~ ~ ~ l were grown in 10 ml of synthetic minimal medium lacking uracil or leucine, respectively, harvested in midexponential phase, and washed once with ice-cold water. The cells were resuspended in 500 pl of lysis buffer (250 m~ NaCl, 10 mM KC1, 1 m~ EDTA, 25 m~ MES, pH 6.5) containing protease inhibitors (1 m~ phenylmethylsulfo- nyl fluoride, 0.01 mM benzamidine HCl, 1 mg/ml o-phenanthroline, and 0.5 mg/ml each antipain, leupeptin, pepstatin A, and aprotinin) and broken by ten 30-s bursts of vigorous vortex mixing (with 30-9 periods of cooling on ice between bursts) with an equal volume of chilled acid- washed glass beads (0.45-0.5-mm diameter, Braun Melsungen AG). To remove glass beads and cell debris, the lysates were subjected to centrifugation a t 14,000 x g for 10 min. Samples of the clarified lysates (100 pg total protein) were precleared by: addition of 2 pl of normal rabbit serum followed by incubation a t 4 "C for 1 h; addition of 30 pl of a 10% suspension of fixed Staphylococcus aureus cells (IgSorb, The Enzyme Center) followed by incubation at 4 "C for 30 min; and centrifugation in a microcentrifuge a t 14,000 x g for 10 min to remove the IgSorb and adsorbed proteins. The precleared lysates were then diluted 2-fold with twice concentrated RIPA buffer (2 x = 300 mM NaCl, 2 mM EDTA, 2% Nonidet P-40, 2% sodium deoxycholate, 40 m~ Tris-HC1, pH 7.2) containing twice the concentrations of the same protease inhibitors as in the lysis buffer and incubated for 1 h with 20 pl of PBS containing an excess of previously titrated affinity-purified anti-C-terminal peptide antibody. An aliquot (30 pl) of a 10% suspension of S. aureus cells (which had been preblocked by incubation overnight at 4 "C in 10% BSA and then washed thoroughly with RIPA buffer) was added and incubation continued for another 30 min. The immune complexes bound to the IgSorb were collected by centrifugation in a microcentrifuge, washed twice with -1 ml of RIPA buffer and once with -1 ml of lysis buffer, resuspended in 30 pl of 10 mM MgC12, 50 mM Tris-HC1 (pH 7.0) 100 p~ [Y-~~PIATP (7,000 cpdpmol) containing 10 pg of MBP, and incubated for 10-30 min at 30 "C. The products were solubilized as described above for the standard phosphotransferase assay.

All radiolabeled phosphoprotein products were resolved by SDS- PAGE, transferred electrophoretically to PVDF membranes, and visualized by autoradiography.

Radiolabeling and Immunoprecipitation of Yeast Proteins-To metabolically label yeast proteins with 32Pi, cultures of YPH500 carrying YEp[MCKIl or DDYl carrying pML5 were pregrown a t 30 "C in minimal medium lacking uracil or leucine, respectively, then diluted 100- fold into LPSM, and grown overnight. The saturated overnight cultures were diluted 10-fold into fresh LPSM and cultured at 30 "C for 2 h. The cells in portions (5 ml) of these fresh cultures were collected by centrifugation in a clinical centrifuge, resuspended in LPSM (0.5 ml) containing 5 mCi of 32Pi, and incubated at 30 "C for 4 h. The labeled cells were harvested by centrifugation in a microcentrifuge, washed twice with ice-cold PBS, lysed in 100 pl of 1% SDS by vortexing with glass beads, and boiled for 3 min. To each sample was added 1 ml of RIPA buffer containing 10 mM NaF and 1 m~ sodium orthovanadate, and the mixtures were boiled again for 3 min. To remove the glass beads and any other debris remaining, the lysates were subjected to centrifugation in a microcentrifuge a t 14,000 x g for 20 min.

Samples (250 pl) of the cleared extracts were incubated with 30 pl of PBS containing excess affinity-purified anti-C-terminal peptide antibody for 2 h at 4 "C. A sample (50 pl) of a 10% suspension of S. aureus cells (which had been preblocked by mixing with a total extract of unlabeled DDYl cells) was added, and incubation continued for 1 h at


4 "C. The immune complexes bound to the IgSorb were collected by centrifugation in a microcentrifuge, washed twice with RIPA buffer, once with RIPA buffer containing 500 mM NaCI, and once with 150 mM NaCl in 20 mM Tris-HC1 (pH 7.0). Proteins in the final washed pellets were solubilized by boiling in SDS-PAGE sample buffer and analyzed by SDS-PAGE and autoradiography.

Phosphatase Deatrnent of Phosphorylated Mckl-Pure Mckl (2 ng) was labeled with n2P in uitro under the conditions described above for autophosphorylation. "P-Mckl was immunoprecipitated by the addition of 20 pl of PBS containing excess affinity-purified anti-C-terminal peptide antibody followed by incubation for 1 h at 4 "C and by addition of 50 pl of a 1 0 1 suspension of S. aureus cells followed by incubation for an additional 30 min. The immune complexes were collected by centrifugation in a microcentrifuge, washed twice with 150 m~ NaCl and 20 mM Tris-HC1 (pH 7.0) containing the same protease inhibitors as in lysis buffer, and then once either with phosphatase buffer A (1 mg/ml BSA, 0.170 P-mercaptoethanol, 25 mM imidazole HCl, pH 7.0) or with phosphatase buffer B (10 mM magnesium acetate, 10 mM Tris acetate, 50 mM potassium acetate, pH 8.0). Immune complexes washed with phosphatase buffer A were resuspended in 20 pl of the same buffer and then treated with either 1 1.11 of a buffer (50% glycerol, 1 mM dithiothreitol, 1 mM EDTA, 50 mM Tris-HCl, pH 7.4) containing 9.8 pg of purified phosphoprotein phosphatase 2A ( g i f t of M. Mumby, University of Texas Southwestern Medical Center, Dallas, " X ) or with 1 pl of a buffer (20% glycerol, 2 m~ EDTA, 0.1% P-mercaptoethanol, 1 mM benzamidine HCI, 50 m~ NaCI, 10 mM imidazole HCl, pH 7.2) containing 0.7 pg (1 unit) of the truncated form (ATC) of the T cell phosphatase (gift of N. Tonks, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). Immune complexes washed with phosphatase buffer B were resuspended in 20 pl of the same buffer and treated with 1 pl of a buffer (0.5 mM MgCl2, 1 M diethanolamine, pH 9.8) containing 5 pg (17 units) of CIP (Pharmacia). Reaction mixtures were incubated for 1 h at 30 "C. After the dephosphorylation reactions, the immune complexes were washed twice with 150 mM NaCl and 20 mM Tris-HC1 (pH 7.0) and once with 10 mM MgClz and 50 mM Tris-HC1 (pH 7.0) containing the protease inhibitors. To assess the extent of dephosphorylation of Mckl following each phosphatase treatment, the protein in a sample (one-halo of each pellet was solubilized by boiling in 50 pl of SDS-PAGE sample buffer, subjected to SDS-PAGE, transferred to a PVDF membrane, and examined by autoradiography and liquid scintillation counting. To assess the effect of the dephosphorylation treatment on the phosphotransferase activity of Mckl, the other half of each pellet was resuspended in 30 pl of 10 mM MgC12 and 50 mM Tris-HC1 (pH 7.0) containing 10 PM okadaic acid, 1 m~ sodium orthovanadate, 50 pg of MBP, and 100 p~ [y-32P]ATP (7,000 cpdpmol), and incubated for 10 min a t 30 "C. The immune complex kinase reactions were terminated by addition of an equal volume of stop buffer followed by boiling for 5 min. The radiolabeled products were separated by SDS-PAGE, transferred to PVDF membranes, and examined by autoradiography and liquid scintillation counting.

RESULTS Improved Purification of Mckl Protein Kinase-Using am-

monium sulfate fractionation, both anion and cation exchange chromatography, and dye-ligand affinity chromatography, we previously achieved an 8,000-10,000-fold purification in rea- sonable yield (25%) of the S. cerevisiae Mckl enzyme from both a protease-deficient laboratory strain (AB1031 and from a commercial stock (Red Star) (Dailey et al., 1990). Even after this large enrichment, however, the final fraction (Orange A-agarose eluate) still contained contaminating protein species, as revealed by SDS-PAGE and silver staining. As one approach to eliminate these contaminants, we previously used FPLC on Mono-S resin. Although additional purification was obtained, Mckl eluted as two separate peaks, and the major peak was still not homogeneous (Dailey et al., 1990).

Two additional strategies, which we describe here, allowed us to obtain apparently homogeneous preparations of Mckl. First, the level of Mckl in the starting material was increased approximately 10-fold, as determined by immunoblotting (see Fig. 4), by expressing the MCKl gene from a multi-copy plasmid as well as from its normal locus on chromosome XIV. Sec- ond, the Orange A-agarose eluate was subjected to adsorption chromatography on hydroxylapatite before proceeding to the last step. When the Orange A-agarose eluate was diluted into

10 mM potassium phosphate (pH 6.8) and applied to hydroxylapatite, nearly all of the Mckl bound to the column and was recovered in a broad peak which eluted from 220 to 400 mM potassium phosphate. The first three Mckl-containing fractions comprised only 25% or so of the total Mckl, but contained very few contaminating species. Therefore, these three fractions were pooled and subjected to FPLC on Mono-S. As observed previously, Mckl eluted from this resin as two incom- pletely resolved peaks; however, as the result of the modifications used in the improved purification scheme, the first peak was virtually free of significant contaminants, and the second peak appeared to be completely free of contaminating proteins, as judged by the highly sensitive colloidal gold staining method (Fig. JA). Immunoblotting with anti-Mckl antibodies confirmed that the 40-kDa species in both peaks was the Mckl protein (Fig. LB 1. The apparently pure material from the second peak (pooled fractions 20-26) was used for the subsequent studies reported here. Using the modified procedure, about 10 pg of pure Mckl protein were obtained from 100 g of the overproducing cells.

Autophosphorylation of Mckl at Drosine and Serine in Vitro -The majority of previously described protein kinases undergo autophosphorylation in uitro. The partially purified preparations of Mckl we obtained before became phosphorylated at both Ser and Tyr when incubated with ATP and a divalent metal ion (Dailey et al., 1990). To confirm that this apparent autophosphorylation activity is intrinsic to Mckl and not due to a contaminating kinase, the ability of the homogeneous Mckl protein to undergo autophosphorylation was examined. Pure Mckl underwent autophosphorylation when incubated with ATP and either Mn2+ or Mg2' (Fig. 2). Addition of 1% SDS (data not shown) or chelation of the divalent metal ion by EDTA (Fig. 2 B ) completely abolished this activity. The efficiency of the autophosphorylation reaction was highly dependent on the reaction conditions. The highest degree of autophosphorylation was observed using Mn2+ as the cofactor in MES buffer at pH 6.5 in the presence of a non-substrate carrier protein (for ex-

A KDa 180 - 116 - 84 - 70 - 52 -

-c 36 - 27 -

f a n 2 4 6 810121416182022242628303234

B -c .Fa"- -

FIG. 1. Final step of yeast Mckl purification. Mckl was purified from an overproducing strain (see "Experimental Procedures") through

al., 1990) and then subjected to adsorption chromatography on hydrox- the Orange A-agarose step by methods presented previously (Dailey et

ylapatite, as described under "Experimental Procedures." Three pooled peak fractions from the hydroxylapatite eluate were applied to a Mono-S FPLC column, and bound proteins were eluted with a linear gradient from 0 to 1 M NaCI. Protein in individual fractions ( frxn) was subjected to SDS-PAGE, transferred to a PVDF membrane, and stained with either colloidal gold (panel A ) or rabbit anti-Mckl antibodies followed by alkaline phosphatase-coupled goat anti-rabbit IgG (panel B) . Migration position of Mckl at an apparent M, of 40 kDa is indicated (arrowhead).

Yeast Mckl Dual Specificity Protein Kinase 21159

A citrate acetate MES BES TEA pH 3 4 5 6 4 5 5.5 6 6.5 6.5 7 7.5 7 7.5

A B

P - s P-T

. I . . .. . , . ..

V

B 5mMMni ' SmMMg++ ~~

EDTA 0 0.1 510 0 0.1 5 10 ( m W

4

FIG. 2. Effect of reaction conditions on Mckl autophosphorylation. Mckl(2 ng; pooled Mono-S fractions 20-26) was incubated for 30 min under standard autophosphorylation conditions, and the product was analyzed, all as described under "Experimental Procedures," except for the modifications indicated. Panel A, reactions carried out in various buffers (at 50 mM) at the pH values indicated;panel B, reactions carried out with either 5 mM MnCI2 (lanes 1-4) or 5 mM MgCI2 (lanes 5-8). in the absence and presence of the concentrations of EDTA indicated. Migration position of Mckl is indicated (arrowhead). BES, N,N" bis(2-hydroxyethyl)-2-aminoethanesulfonic acid; TEA, triethanola- mine.

ample, BSA) (Fig. 2A) . Replacement of Mn2+ with Mg2+, in- creasing the ionic strength (either by raising the concentration of the buffering species or by addition of NaCl or KCI), omission of BSA, and addition of dithiothreitol or Triton X-100 all significantly reduced the extent of autophosphorylation (Fig. 2B and data not shown). In two experiments conducted under op- timum conditions for autophosphorylation, the dependence of the rate of incorporation per molecule on the concentration of Mckl added was more consistent with an intramolecular mechanism than with intermolecular transphosphorylation (data not shown).

Phosphoamino acid analysis revealed that autophosphorylation of Mckl occurred on both Tyr and Ser. The relative content of phosphotyrosine was dramatically dependent on the reaction conditions and was highest under the same conditions that were optimal for maximizing total incorporation (Fig. 3).

Autophosphorylation of Mckl a t l'yrosine in Vivo-We previously found that Mckl isolated by immunoprecipitation and SDS-PAGE from 32P-labeled yeast cells is phosphorylated at both Tyr and Ser (Dailey et al., 1990). To determine whether this phosphorylation depends on the kinase activity of Mckl itself, we examined the in vivo phosphorylation state of a Mckl(K68R) mutant that encodes a catalytically inactive protein. The K68R mutation that was engineered (see "Experimen- tal Procedures") alters a residue that is conserved in every protein kinase examined to date (Hanks and Quinn, 1991) and that appears to be crucial for the formation of a functional ATP binding site (Knighton et al., 1991). The normal MCKl gene on a multi-copy plasmid was introduced into YPH500 (MCKl'), and the mutant mcklK68R gene on a multi-copy plasmid was introduced into DDYl (mckl-A9::URA3). Mckl protein was immunoprecipitated from total extracts of these cells, resolved on SDS-polyacrylamide gels, and transferred to PVDF membranes. The level of expression of Mckl was examined by immunoblotting with anti-Mckl antibodies, and the extent of tyrosine phosphorylation was assessed by immunoblotting with anti-phosphotyrosine antibodies. As expected, no Mckl was detectable in DDYl (or in DDYl transformed with the multi-copy

P-Y

FIG. 3. Autophosphorylation of Mckl at and Ser in vitro. Panel A, Mckl (2 ng) was incubated for 30 min under standard autophosphorylation conditions, except that the buffer was 50 mM Tris-HCI (pH 7.5), and both MgCI2 (5 mM) and MnClz ( 5 mM) were present. Panel B, Mckl (2 ng) was incubated for 30 min under standard autophosphorylation conditions, except that the concentration of MnCI2 was 20 mM. ARer termination of the reaction, the radiolabeled product was subjected to SDS-PAGE, transferred to a PVDF membrane, and located by autoradiography. The position corresponding to "2P-Mckl was excised, subjected to partial acid hydrolysis, and the resulting phosphoamino acids identified by two-dimensional electrophoresis in the presence of internal phosphoamino acid standards, as described previously (Dailey et al., 1990). p-S, phosphoserine; pT, phosphothreonine; p-Y, phosphotyrosine.

A B 1 2 3 4 5 1 2 3 4 5

KD; 180 116 84 70 52

36 27

1

of Mckl in vivo. Extracts (400 pg total protein) of DDYllpML51 (lane FIG. 4. The K68R mutation abolishes tyrosine phosphorylation

I ) , DDYI[YEp3511 (lane 21, YPH500[YEp(MCKI)l (lane 3). DDYl (mckl-A9::URA3) (lane 4 ) , and YPHBOO (lane 5) were immunoprecipi-

jected to SDS-PAGE, transferred to PVDF membranes, and analyzed by tated with anti-Mckl antibodies, and the immunoprecipitates were sub-

immunoblotting either with anti-Mckl antibodies (panel A ) or with anti-phosphotyrosine antibodies (panel B ) , followed by 12sII-Protein A. Migration position of Mckl is indicated (arrowhead).

expression vector lacking an insert), whereas Mckl was readily detectable in extracts from its isogenic MCKl' parent, YPH5OO (Fig. 4A). In cells overexpressing either MCKl or mcklK6", a t least 5-10 times more Mckl was present than in the wild-type strain (Fig. 4A). In agreement with our previous findings, Mckl immunoprecipitated from normal yeast cells contained phosphotyrosine, as judged by the binding of anti-phosphotyrosine antibodies (Fig. 4B ). The absence of any 40-kDa phosphotyrosine-containing protein in immunoprecipitates of the mckl null mutant (or the mutant transformed with the control plasmid) and the pronounced overproduction of such a phosphotyrosine- containing species in the cells expressing MCKl from a multi- copy plasmid confirmed that the 40-kDa phosphotyrosyl protein observed was Mckl (Fig. 4B). Despite its dramatic overproduction, however, the Mckl(K68R) mutant protein contained a barely detectable level of phosphotyrosine (Fig. 4B). These results indicate that tyrosine phosphorylation of Mckl requires that the enzyme be catalytically active and suggest further that the tyrosine phosphorylation of Mckl observed in vivo is due primarily to autophosphorylation.

To determine whether the Mckl(K68R) mutant protein

21160 East Mckl Dual Specificity Protein Kinase might still be phosphorylated at Ser and to confirm that the mutant protein was not phosphorylated at Tyr, cultures of the same cells were metabolically labeled with 32Pi. 32P-Mckl was then recovered from extracts of the cells by immunoprecipitation, subjected to SDS-PAGE, transferred to a PVDF membrane, located by autoradiography, excised, hydrolyzed, and analyzed by two-dimensional phosphoamino acid analysis. As observed before, cells overexpressing mcklK6sR and MCKl both overproduced Mckl protein (Fig. 5A ). Correspondingly, more 32P-labeled Mckl was recovered from the strain overproducing normal Mckl than from wild-type cells and, as expected, no detectable "P-Mckl was recovered from the mckl null mutant carrying the control vector; however, a high level of 32P-Mckl was also recovered from the cells overproducing the Mckl(K68R) mutant protein (Fig. 5B). Mckl from the strain overproducing the normal enzyme contained both phosphotyrosine and phosphoserine (and a trace of phosphothreonine) (Fig. 5C). The ratio of phosphotyrosine to phosphoserine was similar to that found in Mckl isolated from the isogenic parent, YPH500 (data not shown), and similar to that observed previously in Mckl isolated from another wild-type strain (Dailey et al., 1990). In contrast, the Mckl(K68R) mutant protein did not contain any detectable phosphotyrosine but showed little or no reduction in the amount of phosphoserine (or in the trace of phosphothreonine) present (Fig. 5B 1. These findings confirm that tyrosine phosphorylation in vivo requires that Mckl be active and suggest that most of the in vivo serine (and threonine) phosphorylation observed arises from the action of other cellular protein kinases. Hence, although autophosphorylation of purified Mckl at serine was readily detectable in vitro (Fig. 3), autophosphorylation a t serine must contribute only a minor portion of the total serine phosphorylation that occurs in vivo.

Phosphorylation of Exogenous Substrates by Mckl -Although Mckl phosphorylates synthetic poly(Glu,Tyr) poly- mer and itself at tyrosine, we previously found that Mckl was unable to utilize as substrates other peptides and proteins (including angiotensin 11, enolase, casein, and mixed histones) that are typically eficient phosphoacceptors for several well documented protein-tyrosine kinases (Dailey et al., 1990). As one approach for identifying potential Mckl substrates, a heat- inactivated total yeast extract was incubated with either active or heat-inactivated Mckl and [Y-~~PIATP and then examined

1 2 3 4 A 4 Ilr B -

I

1 4

p;Y

rylation of Mckl in vivo. Cultures of DDYl[pML5] (lane I), FIG. 5. The K68R mutation does not abolish serine phospho-

DDYl[YEp3511 (lane 2), YPH5OO (lane 3). and YPH500[YEp(MCKI)] (lane 4 ) were metabolically labeled with OZPi as described under "Ex- perimental Procedures," and Mckl protein in the extracts was recovered and analyzed as described in the legend to Fig. 4. Migration position of Mckl is indicated (arrowhead). After detection, first, by autoradiography (panel B ) the same gel was probed with anti-Mckl antibodies (panel A). The radiolabeled Mckl(K68R) mutant protein ( I ) and the radiolabeled Mckl wild-type protein ( 4 ) were subjected to phosphoamino acid analysis (panel C ) a s described in the legend to Fig. 3. p-s, phosphoserine; pt, phosphothreonine; py, phosphotyrosine.

by SDS-PAGE and autoradiography. Despite heat inactivation of both the extract and the enzyme some residual phosphorylation of yeast proteins was observed; however, many more yeast proteins were phosphorylated when the heated extract was incubated with active Mckl (Fig. 6A). To determine the nature of the Mckl-dependent phosphorylation events, phosphoamino acid analysis was performed on the most prominent individual bands excised from the gels. These phosphoproteins contained primarily phosphoserine andor phosphothreonine (Fig. 6B). The phosphotyrosine detected in the band at 40 kDa presumably arose from autophosphorylation of Mckl itself. Traces of phosphotyrosine were also recovered in the 30-kDa region of the gel; however, this species might represent a product of the proteolysis of Mckl rather than an authentic substrate for protein tyrosine phosphorylation by Mckl. Overall, these results suggested that Mckl phosphorylates exogenous substrates on Ser and Thr.

Given the genetic findings, which implicate Mckl in spindle function andor chromosome segregation, we tested as substrates some purified proteins that are known to be phosphoacceptors for protein kinases that phosphorylate microtubule- associated proteins. Both a mixture of MAP-2 and tau proteins, as well as each of these proteins individually, were phosphorylated when incubated in vitro with active Mckl but not when incubated with heat-inactivated Mckl; similarly, MBP was ef- ficiently phosphorylated in the presence of active Mckl but not in the presence of heat-inactivated Mckl (Fig. 7A). MBP appeared to be the most efficient substrate, based on total incorporation per mole, and was used to define the conditions that are optimal for the phosphotransferase activity of Mckl. Maxi- mal phosphorylation of MBP was observed when Mg2+ was used as the cofactor in Ws-HCI buffer at pH 7.0 (Fig. 8). Fur- thermore, in contrast to the optimal conditions for autophosphorylation, Mckl-dependent phosphorylation of MBP was in- hibited by the addition of Mn2+ (Fig. 8) and by the addition of BSA (data not shown). Detailed kinetic analysis performed under conditions optimal for phosphotransferase activity yielded an apparent K , of 180 PM for MBP3 and an apparent K, of 70 1.1~ for ATP, with a kc,, of -2.51s (data not shown).

As judged by direct phosphoamino acid analysis of the radiolabeled products (Fig. 7B) , purified "-2 protein from two independent sources, both purified and recombinant tau protein and MBP, were all phosphorylated predominantly at Ser. Significant incorporation at Thr was only observed for the recombinant tau protein, which had been treated with SDS. Hence, like the unidentified endogenous yeast substrates, other defined targets of Mckl phosphorylation became modified at Ser and Thr, but not at Tyr.

Other proteins tested, including ~ 3 4 ' ~ ' ~ ' (gift of D. Morgan, University of California, San Francisco), a peptide fragment (residues 2-20) of ~ 3 4 ' ~ ' ~ ' (gift of D. Beach, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY), p60""", a Xenopus laevis homolog of mammalian Erk2 (gift of J. Posada, Fred Hutchison Cancer Institute, Seattle, WA), S. cerevisiae Kssl kinase (gift of D. Ma, this laboratory), and S. cereuisiae Spol5Npsl protein (a yeast homolog of mammalian dynamin, a microtubule-bun- dling protein; gift of K. Bloom, University of North Carolina, Chapel Hill, NC) were not detectably phosphorylated by purified Mckl (data not shown).

As another means to confirm that the phosphotransferase

The concentration of MBP was calculated based on estimates of the purity of the commercial preparation used. The content of MBP in this preparation, as reported by the supplier, was -50%; and we confirmed this value both by staining of a sample separated by SDS-PAGE and by analysis of the major peak resolved by reverse-phase high pressure liquid chromatography using electrospray ionization-mass spectrom- etry.

Yeast Mckl Dual Specificity Protein Kinase

B

1 2 3 4 5 6 7 8

21161

A 1 2

K Da

PS * PT .)

PY - PS .) PT - x -

*

10

9

0

t 11

*

12

9

9

13

0

t

15

s 16

0

i 17

9

0

18

FIG. 6. Mckl-dependent phosphorylation of proteins in a heat-inactivated yeast extract. Panel A, Mckl(2 ng), either active (lane 1 ) or inactivated by treatment a t 75 “C for 10 min (lune 2 ), was incubated at 30 “C for 30 min with 100 pg of total yeast extract protein (prepared by glass bead lysis and heat-inactivated at 75 “C for 10 min) in a final volume of 30 pl containing 5 mM MnCI2, 5 mM MgCI,, 50 mM Tris-HCI (pH 7.51, and 50 p~ [y-:l”PIATP (70,000 cpdpmol). Products were subjected to SDS-PAGE, transferred to PVDF membranes, and examined by autoradiography. Panel B, individual phosphoprotein bands (1-18) were excised, subjected to partial acid hydrolysis, analyzed by one-dimensional electrophoresis in the presence of internal phosphoamino acid standards, and examined by autoradiography. Migration positions of phosphoserine ( P S ) , phosphothreonine ( P T ) , and phosphotyrosine ( P Y ) are indicated. The X designates a product of partial acid hydrolysis that did not migrate at the position of any of the three phosphoamino acid standards.

*MAP 2

4 t a u

*MBP

FIG. 7. Phosphorylation of exogenous protein substrates by Mckl. Panel A , Mckl (2 ng), either active (lanes 2, 3, 5, and 7) or heat-inactivated as described in the legend to Fig. 6 (lunes 1, 4, 6, and 8). was incubated for 20 min in standard phosphotransferase reaction mixtures containing either a MAP-2 and tau protein mixture (-10 pg; lanes 1 and 2) , or purified MAP-2 (-2 pg; lunes 3 and 4 ) , or recombinant tau protein (-1 pg; lunes 5 and 6), or purified MBP (10 pg; lanes 7 and 8). Reactions were terminated and the products analyzed as described under “Experimental Procedures.” Panel B, radiolabeled products (a-f) were excised, and their phosphoamino acid content was analyzed as described in the legend to Fig. 3. p-s , phosphoserine; p-t , phosphothreonine.

activity of Mckl is intrinsic to this protein, we raised an anti- serum directed against the extreme C terminus of Mckl and used the affinity-purified anti-C-terminal peptide antibody to develop an immune complex kinase assay for Mckl, as described under “Experimental Procedures.” To demonstrate the specificity of this assay, Mckl was immunoprecipitated from extracts of cells overexpressing either wild-type Mckl or the Mckl(K68R) mutant. A portion of each immunoprecipitate was analyzed by SDS-PAGE and immunoblotting with polyclonal anti-Mckl antibodies to determine the content of Mckl; another portion was incubated under standard phosphotransfer-

Mn++ Mg++ Mn++ & Mg++

I

ase activity. Mckl(2 ng) was incubated with MBP (IO pg) and 100 p~ FIG. 8. Effect of reaction conditions on Mckl phosphotransfer-

[y-32P1ATP (7,000 cpdpmol) at 30 “C for 30 min in a final volume of 30 pl in the presence of either 10 my MnCI2 or 10 mM MgCI,, or both, and with the buffers (50 mM) and at the pH values indicated. Radiolabeled products were analyzed as described under “Experimental Procedures.” Arrowhead designates the migration position of MBP. TEA, triethanol- amine; DEA, diethanolamine.

ase assay conditions with MBP and [Y-~~PIATP and the products examined by SDS-PAGE and autoradiography. Normal Mckl in the immune complexes was able to phosphorylate MBP and, to a lesser extent, the IgG heavy chain, whereas the Mckl (K68R) protein was completely inactive (Fig. 9A), despite the fact that both normal Mckl and the mutant protein were present at similar levels in the immunoprecipitates (Fig. 9B). As observed for phosphotransferase reactions conducted in solution, MBP was phosphorylated exclusively on Ser, and the IgG heavy chain was phosphorylated at both Ser and Thr (data not shown). The results of this immune complex kinase assay, taken together with our other findings, demonstrate unequivo- cally that Mckl possesses protein-serinekhreonine kinase activity.

Effect of Phosphatase Zkeatment on Mckl Activity -Mammalian MAP kinases (Anderson et al., 1990; Payne et al., 1991) and two S. cerevisiae MAP kinase homologs, Fus3 (Gartner et al., 1992) and Kssl,4 are phosphorylated at a specific Tyr and at a specific Thr, and require phosphorylation at both of these sites for activity. To determine whether the phosphorylation state of Mckl affects its kinase activity, Mckl was prelabeled by autophosphorylation with [Y-~~PIATP in the presence of Mn2+ and then treated with either a phosphoserine- and

D. Ma, J. G. Cook, and J. Thorner, submitted for publication.

21162 Yeast Mckl Dual Specificity Protein

A 1 2

K Da 1so - 116 -

* IgH

A 180 116

% 52

36 27

36 - 27 -

Kinase

1 2 3 4 5 6 7 8 9 1 0

Mckl

4 M B P

4 X I B P

B a 4 v c k 1

FIG. 9. Immune complex kinase assay for Mckl. Panel A, Mckl was immunoprecipitated from cell extracts (100 pg total protein) prepared from either YPH500[YEp(MCKl )I (lane 1) or DDYl[pML5] (lane 2 ) grown in minimal medium lacking uracil or leucine, respectively, using an excess of affinity-purified anti-C-terminal peptide antibody. The immune complexes were collected, washed, and incubated with MBP (10 pg) for 20 min under standard phosphotransferase reaction conditions, as described under "Experimental Procedures." The products were analyzed as described in the legend to Fig. 6. Panel B, the same PVDF membrane as shown in panel A was probed with polyclonal anti-Mckl antibodies followed by '*"I-Protein A.

phosphothreonine-specific phosphatase (phosphoprotein phosphatase 2A) or a phosphotyrosine-specific phosphatase (the 37- kDa truncated form of the T cell phosphotyrosine phosphatase), or a combination of both of these enzymes, or with a nonspecific phosphatase (CIP) purportedly capable of dephosphorylating phosphoserine, phosphothreonine, and phosphotyrosine. The dephosphorylated enzymes were then recovered by immunoprecipitation with the anti-C-terminal peptide antibodies, washed thoroughly, and treated with appropriate specific inhibitors of each phosphatase. The extent of dephosphorylation was then analyzed, and the ability of the dephosphorylated enzymes to phosphorylate MBP was assessed. In agreement with our previous finding that Mckl is autophosphorylated at both Tyr and Ser, treatment with either phosphatase 2A or the T cell phosphatase each removed a significant fraction of the original radioactivity incorporated, whereas a combination of both enzymes removed nearly all of the label. Removal of phosphate from serine by phosphatase 2A only modestly reduced the rate of MBP phosphorylation (to about 65% of the control level), and removal of phosphate from tyrosine by the T cell phosphatase did not significantly change the rate of MBP phosphorylation. Moreover, dephosphorylation of Mckl by treatment with both phosphatase 2A and the T cell phosphatase reduced the rate of MBP phosphorylation only slightly more (to about 40% of the control level) than did treatment with phosphatase 2A alone. These results suggest that modification by autophosphorylation of the Tyr or Ser sites accessible to phosphatase 2A and the T cell phosphatase is not essential for Mckl catalytic activity, although these residues may represent positive regulatory sites. By contrast, treatment with CIP removed less than half of the radioactivity incorporated into Mckl by autophosphorylation yet resulted in a significant (nearly 2-fold) stimulation of the rate of MBP phosphorylation, suggesting that modification of a site accessible to CIP (but not to phosphatase 2A or the T cell phosphatase) may exert a negative regulatory effect on Mckl kinase activity.

Regardless of the degree of dephosphorylation achieved by phosphatase treatment, Mckl was capable of catalyzing a high level of incorporation into MBP (Fig. 1OA 1; yet, under the same conditions, no significant additional incorporation into Mckl itself was detected (Fig. 10, A and B 1.

1 Z 3 . 1 5 6 7 8 9 1 0

FIG. 10. Effects of dephosphorylation on Mckl phosphotransferase activity. Panel A, Mckl (2 ngkeaction) was incubated with [-p3*P]ATP under standard autophosphorylation conditions (see "Ex- perimental Procedures"), collected by immunoprecipitation with excess anti-C-terminal peptide antibody, washed, resuspended in an appropriate buffer, and treated with either buffer alone (lunes 1 and 6), or phosphoprotein phosphatase 2A (lanes 2 and 7), or T cell phosphotyrosine phosphatase (lanes 3 and 8), or both phosphatase 2A and the T cell phosphatase (lanes 4 and 9), or CIP (lanes 5 and IO), as described under "Experimental Procedures." The treated samples were then washed thoroughly and resuspended in phosphotransferase assay buffer containing specific phosphatase inhibitors, as described under "Experimen- tal Procedures." Equal portions of the washed immune complexes were subjected to SDS-PAGE and autoradiography (right side), and the extent of dephosphorylation was quantified by direct scintillation counting (panel B, right side, closed bars), as described under "Experimental Procedures." Identical samples of the washed immune complexes were incubated for 10 min under standard phosphotransferase reaction conditions with MBP (50 pg) (left side), and the amount of radioactivity incorporated into MBP (panel B, left side, hatched burs) and into Mckl (panel B, left side, closed bars) was quantified by direct scintillation counting, as described under "Experimental Procedures." The relative amount of label in Mckl (% control) was normalized to the radioactivity present in Mckl that had not undergone any dephosphorylating treatment (lane 6). The relative amount of label in MBP (% control) was normalized to the radioactivity incorporated into this substrate by Mckl that had not undergone any dephosphorylating treatment (lane I ).

DISCUSSION The results presented here demonstrate that S. cereuisiae

Mckl is a protein kinase that is capable of phosphorylating Ser, Thr, and Tyr residues in proteins. Mckl autophosphorylates at Tyr and Ser. In this respect, Mckl resembles several other "dual specificity" kinases that have been identified, which undergo autophosphorylation both a t Tyr and at Ser (or Thr). However, our data indicate that the most efficient phosphoacceptor proteins are phosphorylated by Mckl exclusively a t Ser and Thr. Moreover, removal of phosphate from the Tyr residue(s) in Mckl that are modified by autophosphorylation by treatment with phosphatase did not detectably alter the phosphotransferase activity of Mckl. Nonetheless, Mckl is a phosphotyrosine-containing protein in vivo (Dailey et al., 1990) and, as we have shown here, Mckl activity is required for this modification, suggesting that the phosphorylation of Mckl at Tyr observed in uiuo is due to autophosphorylation. Thus, tyrosine phosphorylation of Mckl may have some physiological signifi- cance. Although dephosphorylation of Tyr did not affect its activity on the heterologous substrates used in this study, perhaps recognition and/or phosphorylation of authentic targets of Mckl action in yeast are influenced by the state of phosphorylation of Mckl at Tyr. Alternatively, perhaps tyrosine phosphorylation of Mckl affects its subcellular localization or its

Yeast Mckl Dual Specificity Protein Kinase 21163

ability to associate with other cellular components that regu- late Mckl activity. Mapping and mutation of the site(s) of tyrosine phosphorylation in Mckl, which are in progress, should help address these issues.

We originally detected and purified Mckl on the basis of its ability to phosphorylate synthetic poly(Glu,Tyr), which is an effective substrate for many well documented protein-tyrosine kinases (Braun et al., 1984). However, Stern et al. (1991) have reported that two well characterized protein-serinekhreonine kinases, CAMP-dependent protein kinase and type I1 calmodu- lin-dependent protein kinase, are able to phosphorylate poly- (Glu,Tyr), albeit more weakly than Mckl. This observation and our demonstration that Mckl phosphorylates exogenous substrates at Ser and Thr indicate that the capacity to phosphorylate poly(Glu,Tyr) is unlikely to reflect adequately the physiologically relevant substrate preference of a protein kinase. On the other hand, given that Mckl autophosphorylates at Tyr, whereas CAMP-dependent protein kinase and type I1 calmod- ulin-dependent protein kinase do not, it could be argued that the ability of a protein kinase to phosphorylate poly(Glu,Tyr) a t a significant rate reveals some special feature of the catalytic domain and leaves open the possibility that Mckl may phosphorylate some authentic yeast substrate at Tyr. In this regard, it should be recalled that a protein with an apparent molecular mass of 30 kDa in yeast extracts became phosphorylated at Tyr when incubated with purified Mckl; however, the identity of this species and whether it might be derived from Mckl itself are unresolved.

Based on comparative sequence analysis of their catalytic domains (Lindberg et al., 1992), the dual specificity protein kinases appear to be a rather heterogenous group of enzymes, which also differ with respect to their specificity toward exogenous substrates. Some of these enzymes can phosphorylate phosphoacceptor proteins at both Tyr and Ser (or Thr). For example, S. pombe Weel' phosphorylates its major physiologically important substrate, ~ 3 4 ~ ~ " ' + , at a Tyr within a glycine- rich loop (conserved kinase domain I) (Parker et al., 1992) but also phosphorylates angiotensin I1 at Tyr and acid-denatured enolase at both Tyr and Ser (Featherstone and Russell, 1991; Parker et al., 1992). Similarly, MAP kinase kinase phosphorylates its physiologically relevant substrates, Erkl and Erk2, both at Tyr and at Thr (Gomez and Cohen, 1991; Alessandrini et al . , 1992; Crews and Erikson, 1992; Kosako et al., 1992; Nakielny et al., 1992). In contrast, the MAP kinases (Erkl and Erk2) themselves have been reported to undergo autophosphorylation a t both Tyr and Thr, albeit at a low rate (Crews et al., 1991; Seger et al., 1991;Ahn et al . , 1992), yet phosphorylate exogenous substrates only a t Ser and Thr (for review, see Pelech and Sanghera (1992)).

In certain respects, Mckl resembles the MAP kinases. First, Mckl(375 residues) is similar in size to other MAP kinases, for example S. cereuisiae Kssl (Courchesne et al., 1989), which is 368 residues. Second, Mckl shares somewhat greater homology with MAP kinases (for example, 31% identity to Kssl) than it does with other kinases (for example, 25% identity to S. cereuisiae Cdc28 (Lorincz and Reed, 1986)). Third, both Mckl and MAP kinases are dually phosphorylated in uiuo (Mckl at Tyr and Ser, and MAP kinases at Tyr and Thr), and both enzymes display dual autophosphorylation activity, albeit with different efficiencies, on the same residues in uitro. Fourth, both Mckl and MAP kinases phosphorylate MBP, "-2, and tau eff- ciently; however, Mckl modifies Ser in MBP, whereas Erk2 modifies Thr, and the apparent K , for MBP phosphorylation by MAP kinase is 50 PM (Erickson et al., 19901, whereas the apparent K , for MBP phosphorylation by Mckl is about %fold higher.

Mckl appears to differ, however, from the MAP kinase family

in its mode of regulation. MAP kinases, including mammalian Erkl and Erk2 and S. cereuisiae Kssl and Fus3, possess a common sequence motif, -TEY-, 7 residues N-terminal to conserved kinase domain VI11 (-APE-), and phosphorylation of both Thr and Tyr at this site is essential for MAP kinase activity (Anderson et al., 1990; Gartner et al., 1992L4 Further- more, phosphorylation of MAP kinases at the TEY motif cannot depend on autophosphorylation because a catalytically inactive MAP kinase mutant microinjected into progesterone-stimulated frog eggs (Posada and Cooper, 1992) or a catalytically inactive Kssl mutant expressed in a-factor-stimulated MATa cells4 become phosphorylated on both the Tyr and the Thr at this site. Indeed, activation of the MAP kinases by phosphorylation at the TEY site appears to be due to the action of a discrete enzyme, MAP kinase kinase (Crews and Erikson, 1992; Kosako et al., 1992; Nakielny et al . , 1992). In contrast, Mckl contains the sequence -1SY- at the corresponding position, and Tyr phosphorylation of Mckl does not appear to be necessary for its catalytic activity, at least when assayed with MBP as the substrate. Furthermore, our results indicate that phosphorylation of Mckl at Tyr results from autophosphorylation and requires catalytically active Mckl. In agreement with this conclusion, Mckl does not appear to be a substrate for a t least one preparation of MAP kinase kinase (gift of N. Ahn (Seger et al., 1992)h5 In fact, treatment of Mckl with CIP (but not with a phosphotyrosine-specific phosphatase) led to a stimulation of phosphotransferase activity, suggesting that phosphorylation of a Ser (or Thr) site in Mckl exerts a negative effect on its catalytic function. Moreover, heat-inactivated Mckl was not a substrate for purified X . laeuis Erk2 (gift of J. Posada (Posada and Cooper, 1992)) nor was heat-inactivated X . laeuis Erk2 a substrate for purified M ~ k l . ~ These observations suggest that Mckl participates in a cellular regulatory path- way distinct from that involving the MAP kinases. Indeed, of the growing family of dual specificity kinases characterized to date, Mckl shares greatest amino acid sequence identity with mammalian glycogen synthase kinase-3 and its recently cloned homologs in other organisms (Shero and Hieter, 1991; Hughes et al., 1993; Rue1 et al., 1993). Moreover, the differences among Mckl and other dual specificity kinases reinforce the idea that during evolution that capacity to phosphorylate Ser, Thr, and Tyr was independently acquired by a diverse set of enzymes.

Acknowledgments-We thank N. Ahn, D. Morgan, M. Mumby, J. Posada, and N. Tonks for generously providing purified enzyme preparations, D. Beach, K. Bloom, D. Drechsel, M. Kirschner, D. Ma, B. Poschal, and R. Vallee for generously supplying purified peptides and proteins, J. Onuffer for the synthesis of oligonucleotides, and D. King for the synthesis of the Mckl C-terminal peptide and general advice on peptide chemistry.

REFERENCES

Ahn, N. G., Seger, R., and Krebs, E. G. (1992) Cum Biol. 4,992-999 Alani, E., Lao, L., and Kleckner, N. (1987) Genetics 116, 541-545 Alessandrini, A., Crews, C. M., and Erikson, R. L. (1992) Proc. Natl. Acad. Sci.

Anderson, N. G., Maller, J. L., Tonks, N. K., and Sturgill, T. W. (1990) Nature 343,

Baltensperger, K., Lewis, R. E., Woon, C.-W., Vissavaijhala, P., Ross, A. H., and

Ben-David, Y., Letwin, K., Tannock, L.. Bernstein, A., and Pawson, T. ( 1991 j EMBO

Bradford, M. M. (1976) Anal. Biochem. 72, 248-254 Braun, S., Raymond, W. E., and Racker, E. (1984) J. Biol. Chem. 259,2051-2054 Cooper, J. A., Sefton, B. M., and Hunter, T. (1983) Methods Enzymol. 9 9 , 3 8 7 4 0 2 Courchesne, W. E., Kunisawa, R., and Thorner, J. (1989) Cell 58, 1107-1119 Crews, C. M., and Erikson, R. L. (1992) Proc. Natl. Acad. SCL U. S. A. 89, 8205-

8209 Crews, C. M., Alessandrini, A. A., and Erikson, R. L. (1991) Proc. Natl. Acad. Sci.

U. S. A. 88, 8845-8849 Dailey, D. (1992) Purification, Cloning, and Characterization of MCK I , a Protein-

SerinelThreoninel5rosine Kinase from Saccharomyces cereuisiae. Ph.D. dis-

U. S. A. 89, 8200-8204

6 5 1 4 5 3

Czech, M. P. (19921 Proc. Natl. Acad. Scr. U. S . A. 89, 7885-7889

J. 10,317-325

M.-Y. Lim, unpublished results.


Dailey, D., Schieven, G. L., Lim, M. Y., Marquardt, H., Gilmore, T., Thorner, J., and sertation, University of California a t Berkeley

Dailey, D., Schieven, G. L., Lim, M.-Y., Marquardt, H., Gilmore, T., Thorner, J., and Martin, G. S . (1990) Mol. Cell. Biol. 10, 6244-6256

Erickson, A. K., Payne, D. M., Martino, P. A,, Rossomando, A. J., Shabanowitz, J., Martin, G. S . (1991) Mol. Cell. Biol. 11, 2333

Weber, M. J., Hunt D. F., and Sturgill, T. W. (1990) J. Biol. Chern. 266,19728- 19735

Featherstone, C., and Russell, P. (1991) Nature 349, 808411 Gartner, A,, Nasmyth, K., and Ammerer, G. (1992) Genes & Deu. 6, 128LL1292 Gomez, N., and Cohen, P. (1991) Nature 353, 170-173 Hanks, S. K., and Quinn, A. M. (1991) Methods Enzymol. 200, 38-62 Hanks, S. K., Quinn, A. M., and Hunter, T. (1988) Science 241,4242 Hill, J. E., Meyers, A. M., Koerner, T. J., and Tzagoloff, A. (1986) East 2,

Howell, B. W., and Afar, D. E. H. (1991) Mol. Cell. Biol. 11, 56a572 Hughes, K., Nikolakaki, E., Plyte, S . E., 'lbtty, N. F., and Woodgett, J. R. (1993)

Ito, H., Fukuda, Y., Murata, K., and Kimura, A. (1983) J. Bucteriol. 153,

Johnson, K. W., and Smith, K. A. (1991) J. Biol. Chern. 266,3402-3407 Knighton, D. R., Zheng, J., Ten Eyck, L. F., Ashford, V. A., Xuong, N.-H., Taylor, S.

Kosako, H., Gotoh, Y., Matsuda, S., Ishikawa, M., and Nishida, E. (1992) EMBO J.

Kraft, R., Tardiff, J., Krauter, K. S., and Leinwand, L. A. (1988) Bionchniques 6,

Laemmli, U. K. (1970) Nature 227,680-685

163-167

EMBO J. 12,803-808

163-168

S. , and Sowadski, J. M. (1991) Science 253,407-414

11,2903-2908

544-547

Lindberg, R. A,, Quinn, A. M., and Hunter, T. (1992) Z-ends Biochern. Sci. 17,

Lorincz, A. T., and Reed, S . I. (1986) Mol. Cell. Biol. 6, 4099-4103 Nakielny, S . , Cohen, P., Wu, J., and Sturgill, T. (1992) EMBO J. 11,2123-2129 Neigeborn, L., and Mitchell, A. (1991) Genes & Deu. 5,533-548 Parker, L. L., Athertonfessler, S., and Piwnica-Worms, H. (1992) P m . Natl. Acad.

114-119

Payne, D. M., Rnssomando, A. J., Martino, P., Erickson, A. K., Her, J.-H., Sha- Sci. U. S. A. 89, 2917-2921

banowitz, J., Hunt, D. F., Weber, M. J., and Sturgill, T. W. (1991) EMBO J. 10,

Pelech, S . L., and Sanghera, J. S. (1992) fiends Biochern. Sci. 17,233-238 Posada, J., and Cooper, J. A. (1992) Science 265,212-215

Ruel, L., Bourouis, M., Heitzler, P., Pantesco, V., and Simpson, P. (1993) Nature Reneke, J., Blumer, K., Courchesne, W. F., and Thorner, J. (1988) Cell 55,221-234

Schieven, G., and Martin, G. S. (1988) J. Biol. Chem. 263, 1559LL15593 Schieven, G. L., Thorner, J., and Martin, G. S . (1986) Science 231, 390-393 Seger, R., Ahn, N., Boulton, T., Yancopoulos, G., Panayotatos, N., Radziejewska, E.,

Ericsson, L., Bratlien, R., Cobb, M., and Krebs, E. (1991) Proc. Natl. Acad. Sci.

Seger, R., Ahn, N. G., Posada, J., Munar, E. S . , Jensen, A. M., Cooper, J. A,, Cobb, U. S. A. 88,6142-6146

Sherman, F., Fink, G. R., and Hicks, J. B. (1986) in Laboratory Course Manual for M. H., and Krebs, E. G. (1992) J. Biol. Chern. 287, 14373-14381

Methods in Eas t Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY

885-892

362,557-560

Shero, J., and Hieter, P. (1991) Genes & Deu. 5,549-560 Stem, D., Zheng, P., Beidler, D. R., and Zerillo, C. (1991) Mol. Cell. Biol. 11,

987-1001

Yeast MCKI Protein Kinase Autophosphorylates at Tyrosine and ...

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