A Purified S6 Kinase Kinase from Xenopus Eggs Activates S6 Kinase I1 and Autophosphorylates on Serine, Threonine, and Tyrosine Residues*

56 kinases I and I1 have been purified previously from Xenopus eggs and shown to be activated by phosphorylation on serine and threonine residues. An S6 kinase clone, closely related to S6 kinase 11, was sub-sequently identified and the protein product was ex- pressed in a baculovirus system. Using this protein, termed “rsk” for Ribosomal Protein S6 Kinase, as a substrate, we have purified to homogeneity from unfertilized Xenopus eggs a 41-kDa serine/threonine kinase termed rsk kinase. Both microtubule-associated protein-2 and myelin basic protein are good substrates for rsk kinase, whereas a-casein, histone H1, prota- mine, and phosvitin are not. rsk kinase is inhibited by low concentrations of heparin as well as by B-glycero- phosphate and calcium. Activation of rsk kinase during Xenopus oocyte maturation is correlated with phosphorylation on threonine and tyrosine residues. How- ever, in vitro, rsk kinase undergoes autophosphorylation on serine, threonine, and tyrosine residues, iden- tifying it as a “dual specificity” enzyme. Purified rsk kinase can be inactivated in vitro by either a 37-kDa T-cell protein-tyrosine phosphatase or the serinelthreonine protein phosphatase 2A. Phosphatase-treated


6 kinases I and I1
have been purified previously from Xenopus eggs and shown to be activated by phosphorylation on serine and threonine residues. An S6 kinase clone, closely related to S6 kinase 11, was subsequently identified and the protein product was expressed in a baculovirus system. Using this protein, termed "rsk" for Ribosomal Protein S6 Kinase, as a substrate, we have purified to homogeneity from unfertilized Xenopus eggs a 41-kDa serine/threonine kinase termed rsk kinase. Both microtubule-associated protein-2 and myelin basic protein are good substrates for rsk kinase, whereas a-casein, histone H1, protamine, and phosvitin are not. rsk kinase is inhibited by low concentrations of heparin as well as by B-glycerophosphate and calcium. Activation of rsk kinase during Xenopus oocyte maturation is correlated with phosphorylation on threonine and tyrosine residues. However, in vitro, rsk kinase undergoes autophosphorylation on serine, threonine, and tyrosine residues, identifying it as a "dual specificity" enzyme. Purified rsk kinase can be inactivated in vitro by either a 37-kDa T-cell protein-tyrosine phosphatase or the serinelthreonine protein phosphatase 2A. Phosphatase-treated S6KII can be reactivated by rsk kinase, and S6 kinase activity in resting oocyte extracts increases significantly when purified rsk kinase is added. The availability of purified rsk kinase will enhance study of the signal transduction pathway(s) regulating phosphorylation of ribosomal protein S6 in Xenopus oocytes.
Changes in the phosphorylation state of various proteins are a conserved response of cells to mitogenic stimuli. Phosphorylation of the 40 S ribosomal subunit protein S6 is an ubiquitous example of this phenomenon in higher eucaryotes (1)(2)(3)(4)(5). We have studied this event in Xenopus laeuis oocytes, where S6 is phosphorylated during mitogen-induced meiotic maturation as part of a large burst of phosphorylation events that accompany activation of cdc2 kinase (6).
Two chromatographically and immunologically distinct kinases specific for S6, termed S6 kinase I and S6 kinase 11, account for S6 phosphorylation in the maturing oocyte, and each has been purified and characterized from Xenopus eggs (1, 7). Both activated kinases contain phosphoserine and phosphothreonine residues when labeled in uiuo (1,8), and phosphorylation coincides with increased kinase activity during maturation (8). That direct phosphorylation underlies the mechanism of S6 kinase activation is also evident in the subsequent dephosphorylation and deactivation of S6 kinase upon fertilization or parthenogenetic activation of eggs (8). An S6 kinase clone, isolated using oligonucleotide probes based on peptide sequences in purified S6 kinase 11, has been expressed in both Escherichia coli and Sf9 cells infected with recombinant baculovirus (9,10). The predicted amino acid sequence, however, does not contain the exact sequence of some of the peptides isolated from S6 kinase 11, and therefore the recombinant protein product is termed rsk' (Ribosomal protein 5'6 Kinase). When rsk is expressed in Sf9 cells it has very little S6 kinase activity, but rsk can be activated when Sf9 cells are coinfected with baculovirus expressing both rsk and pp60v-*m (10). This increased activity appears to be dependent on phosphorylation of rsk (10).
The regulation of purified S6 kinase I and I1 by phosphorylation and dephosphorylation has also been studied in uitro.
Treatment with phosphatase 2A results in a decrease in S6 kinase activity with either enzyme (1,11). Significantly, S6 kinase I1 activity can be partially restored by addition of a 42-kDa protein kinase partially purified from 3T3-Ll cells (11). This kinase, termed MAP kinase for its original substrate (MAP-2) or for Mitogen-Activated Protein kinase, is activated just prior to the increase in S6 kinase activity when 3T3-Ll cells are treated with insulin (12). One form of MAP kinase has also been shown to become activated and phosphorylated in Xenopus oocytes and eggs in a cell cycle-related manner (13). Moreover, MAP kinase appears to be related to pp42, a phosphotyrosyl protein whose phosphorylation is correlated with the growth-promoting actions of many growth factors and transforming gene products (14, 15).
In addition to S6 kinase I and I1 and MAP-2, the 3T3 cell MAP kinase phosphorylates myelin basic protein (16)  cascade regulating S6 phosphorylation and is the best current example of a serine/threonine kinase activated by direct phosphorylation on tyrosine residues. Recently, a number of kinases related to MAP kinase have been identified in a variety of cell types including: Swiss 3T3 cells (20), sea star oocytes (21), rat 1 HIRc B cells (22), rat liver cells (23), PC-12 cells (24,25), and Xenopus eggs (26)(27)(28). The M, of all of these kinases, with one exception (23), varies between 40,000 and 45,000. All share similar substrate specificity with the 3T3-Ll cell MAP kinase and, of those examined, all are inactivated by treatment with phosphatases. In addition, several partially purified MAP kinases have been shown to activate S6 kinases (29)(30)(31). A 70-kDa S6 kinase from rabbit liver, which is distinct from the rsk family of S6 kinases, has been reported to be activated by a fibroblast MAP kinase (5), but purified 70-kDa S6 kinase cannot be activated by MAP kinase preparations from several other laboratories (32,33).
We are interested in elucidating the mechanism of S6 kinase activation by mitogens using the Xenopus oocyte maturation system as a model. Using rsk isolated from baculovirus-infected Sf9 cells as a substrate, we have purified a kinase that phosphorylates and activates purified Xenopus S6 kinase I1 in vitro. rsk kinase is phosphorylated on threonine and tyrosine residues and autophosphorylates in vitro on serine, threonine, and tyrosine residues, identifying it as a member of the newly emerging class of "dual specificity" protein kinases.

EXPERIMENTAL PROCEDURES
Materials-Female X. laevis were obtained from Xenopus I (Ann Arbor, MI). The RSK-a baculovirus constructs were a gift from Dr. R. L. Erikson (Harvard University), and the Sf9 cells were grown and infected in the cell culture facilities at the University of Colorado Cancer Center. [y3*P]ATP was either prepared by the method of Johnson and Walseth (34) or obtained from ICN (Irvine, CA). Bovine serum albumin (Pentex, fraction V) used in densitometry was from Miles. Fast flow S-Sepharose, DEAE-Sephacel, and G-25 PD-10 disposable columns were obtained from Pharmacia LKB Biotechnology Inc., and hydroxylapatite was from Bio-Rad. The heat stable inhibitor protein of the CAMP-dependent protein kinase was prepared by the method of Whitehouse and Walsh (35). Protein phosphatases 1 and 2A, 40 S ribosomal subunits, and histone H1 were all prepared in this laboratory as described (36)(37)(38). The protein tyrosine phosphatases, CD45 and the spontaneously active, COOH terminally truncated, 37-kDa T-cell form (39) were gifts of Dr. N. K. Tonks (Cold Spring Harbor Laboratory). Bovine brain MAP-2 and 3T3-Ll cell MAP kinase were gifts of Dr. T. W. Sturgill (University of Virginia). Myelin basic protein, phosvitin, heparin (grade 1 from porcine intestinal mucosa), p-nitrophenyl phosphate, a-casein, and human chorionic gonadotropin were purchased from Sigma, and protamine and pregnant mare's serum gonadotropin were purchased from Calbiochem (La Jolla, CA).
rsk Purification-rsk protein was partially purified from insect cells infected with recombinant baculovirus. Sf9 cells were seeded at 8 X lo6 cells/lOO-mm dish (six dishes total) and allowed to attach for 1 h. Recombinant baculovirus constructs containing the RSK-a clone were added to a multiplicity of infection of 10, and the cells were harvested after a 40-h incubation. The cells were washed off the dishes with phosphate-buffered saline, centrifuged at 1000 rpm for 10 min, resuspended in phosphate-buffered saline, pelleted again, and frozen at -70 "C. The cells were lysed in 15 ml of lysis buffer (10 mM potassium phosphate, 1 mM EDTA, 5 mM EGTA, 10 mM MgC12, 50 mM &glycerophosphate, 1 mM Na3V04, 2 mM dithiothreitol, 40 pg of phenylmethylsulfonyl fluoride/ml, 10 pg of leupeptin/ml, 10 pg of pepstatin/ml, pH 7.05) and clarified by centrifugation at 90,000 X g for 30 min at 4 "C. The supernatant was loaded onto a 5-ml Fast Flow S column pre-equilibrated in buffer C1 (10 mM potassium phosphate, 1 mM EDTA, 5 mM EGTA, 5 mM MgCl,, 10 mM pglycerophosphate, 0.1 mM Na3V04, 2 mM dithiothreitol, 0.05% Brij 35, 10% glycerol, pH 7.2) and eluted with 120 ml of 0-1 M NaCl in buffer C1. Aliquots of selected fractions were electrophoresed in a Laemmli polyacrylamide gel (40), stained with Coomassie Blue, and fractions containing rsk were pooled and loaded onto a 4-ml hydroxylapatite (Bio-Gel HTP) column pre-equilibrated in buffer C1. Flowthrough and wash fractions with protein were pooled and dialyzed overnight against C1 buffer. The sample was concentrated using a 1ml Fast S column and eluting with a 0.4 M NaCl step gradient. The fractions were analyzed by polyacrylamide gel electrophoresis and Coomassie Blue staining, and fractions with rsk were pooled and dialyzed overnight against C1 with 50% ethylene glycol. This protocol yielded 110 pg of rsk that had an apparent M, of 83,000. The rsk could be phosphorylated by purified rsk kinase to a stoichiometry of approximately 0.72 mol of phosphate/mol of rsk. Phosphorylation of rsk by rsk kinase increased the apparent molecular weight of rsk in a polyacrylamide gel when compared with autophosphorylated rsk. This shift in apparent molecular weight has been noted previously (10,30). Kinase Assays-Unless otherwise indicated, all kinase assays were performed in: 20 mM HEPES, pH 7.0, 5 mM @-mercaptoethanol, 5 mM MgC12, 0.1 mg/ml BSA, 100 p~ [-p3'P]ATP (1-5 cpm/fmol), 1.4 pg/ml rsk. Reactions were usually incubated for 10-15 min at 30 "C, terminated by the addition of 0.25 volume of 5 X sample buffer (1 X sample buffer contains 70 mM Tris-HC1, pH 6.8, 11% glycerol, 3% SDS, 0.01% bromphenol blue, 5% 2-mercaptoethanol), immersed in boiling water for 2-3 min, and electrophoresed in a 10% SDSpolyacrylamide gel. The labeled bands were identified by autoradiography, excised, and quantified by liquid scintillation spectrometry.
rsk Kinase Purification-Unfertilized eggs were collected from X .
laevis females induced to ovulate with injections of 75 units of pregnant mare's serum gonadotropin and 550 units of human chorionic gonadotropin 3 days and 12-24 h, respectively, prior to collection. The eggs were collected in 0.1 M NaCl, dejellied in 2% cysteine, pH 8.0, and washed five to seven times in 50 mM Tris, pH 7.0, 0.1 M NaCl. Six ml of eggs were homogenized in 6 volumes of buffer consisting of 25 mM Tris, pH 7.5, 25 mM NaCl, 50 mM NaF, 2 mM EGTA, 2 mM dithiothreitol, 100 p~ phenylmethylsulfonyl fluoride, 10 pg of leupeptin/ml, 1 mM Na3V04. The homogenate was centrifuged at 17,000 X g for 15 min, the lipid layer removed, and the buffer A (25 mM Tris, pH 7.5, 2 mM EGTA, 2 mM dithiothreitol) supernatant loaded onto a 15-ml DEAE column pre-equilibrated in containing 25 mM NaCl. The column was eluted with a 200-ml gradient from 25-300 mM NaCl in buffer A. 400 mM p-nitrophenyl phosphate (40 mM final concentration) was added to the collection tubes, and 2.5-ml fractions were collected. The fractions were frozen overnight, and samples were assayed for rsk kinase activity. Peak fractions were pooled, precipitated with 40% ammonium sulfate, and then resuspended in 2 ml of buffer B: 25 mM HEPES, pH 7.0, 250 mM NaCl, 2 mM EGTA, 2 mM dithiothreitol, 0.01% Brij 35, 10% ethylene glycol. Two ml of resuspended sample were loaded and eluted from two tandemly joined TSK G3000SW columns in separate runs of 1 ml in buffer B. The eluates from each half were collected into the same tubes in 1-ml fractions and assayed for rsk kinase activity.
Peak fractions were pooled and exchanged into buffer C (buffer B without Brij 35) using disposable G-25 columns. The eluted peak was divided in half again, and the halves were separately loaded onto a TSK-phenyl5PW column and eluted with a 10-80% ethylene glycol, 250-25 mM NaCl gradient. The eluates from each half were collected into the same tubes in 1-ml fractions and assayed for rsk kinase activity. Protein concentrations were determined by Bradford analysis as described previously (41), except that 2 X concentrated Bradford reagent was used to determine the protein concentration in the gel filtration pool (1). To estimate the amount of rsk kinase in the pooled peak fractions from the phenyl column, a silver-stained SDS-polyacrylamide gel (42) was analyzed by densitometry with a Molecuiar Dynamics densitometer, and the density of the sample was compared with the densities of known amounts of BSA stained on the same gel.
Phosphatase Inactivation of rsk Kinase-rsk kinase was incuaated with the indicated concentration of phosphatase for an anpropriate time, then the reaction was stopped by the addition of 10 illM NaF, 1 mM EDTA (final concentrations) for phosphatases 1 and 2A or 200 p M Na3V04 (final concentration) for the protein tyrosine phosphatases. The buffer was then adjusted for the kinase reaction, rsk and [Y-~*P]ATP were added, and the samples were incubated for 20 min. The reaction was stopped by the addition of 5 X sample buffer, and counts incorporated into rsk were determined by SDS-polyacrylamide gel electrophoresis as described above. The buffer for phosphatases 1 and 2A consisted of 50 mM Tris, pH 7.0, 10 mM P-mercaptoethanol, 0.03% Brij 35, and was adjusted for the kinase reaction to 5 mM MgCl,, 0.1 mg/ml BSA, 100 p~ [T-~'P]ATP (5 cpm/fmol), 1.4 pg/ml rsk. The buffer for the protein tyrosine phosphatases comprised 25 mM imidazole HCl, pH 7.2,l mg/ml BSA, 15 mM 8-mercaptoethanol, and was adjusted for the kinase reaction as above. In control samples, the phosphatases were inactivated by incubation for 15 min in the presence of NaF/EDTA or N&VO4 prior to addition to rsk kinase.

RESULTS
rsk Kinase Purification-Unfertilized Xenopus eggs arrested at metaphase of meiosis I1 exhibit maximal S6 kinase activity (8) and were used as a source of rsk kinase. Following homogenization and a low speed centrifugation, the homogenate was loaded onto a DEAE-Sephacel column and eluted with increasing NaCl (Fig. 1). As found for 3T3 cell MAP kinase previously (17), it is important to inhibit phosphatase activity at this stage of the purification by addition of pnitrophenyl phosphate to the collection tubes prior to elution (final concentration 40 mM). When the phosphatase inhibitor was not used, the yield of rsk kinase activity obtained was decreased about 4-fold. In all preparations to date, one peak of activity eluting at 190 mM NaCl was consistently present, and a second, much smaller, peak of activity at 120 mM NaCl was occasionally seen. The shoulder on the 190 mM peak does not appear to represent a distinct kinase since the activity elutes as one peak on all subsequent columns.
Peak fractions of the 190 mM DEAE-Sephacel peak (usually six to seven 2.5-ml fractions) were pooled and concentrated by precipitation with 40% ammonium sulfate prior to gel filtration chromatography on a TSK G3000SW column. The sample was gel filtered in two consecutive runs due to the amount of protein present. The activity eluted as a very broad peak, well after the protein markers and the p-nitrophenyl phosphate (Fig. 2). The unusual retention of the rsk kinase activity was probably due to adsorption to the silica gel matrix.
The sample was then chromatographed on a phenyl-TSK column (Fig. 3) with a gradient of increasing ethylene glycol and decreasing NaCl(l7). The activity eluted in a single peak at 60% ethylene glycol. A silver-stained polyacrylamide gel of the pooled peak fractions (Fig. 4) shows that the TSK G3000SW column resolved most contaminating proteins from the rsk kinase, and only a 41-42-kDa doublet was observed following chromatography on the phenyl column. This doublet probably represents two isoforms of the same protein since    (Table I) shows that rsk kinase was purified approximately 800-fold with a 4% recovery.
Inhibitors and Substrate Specificity-In order to characterize the purified rsk kinase, the assay conditions were optimized, and the effect of various inhibitors on phosphotransferase activity was examined. The optimal MgC12 concentration was 5 mM, and changes in pH between 6 and 8 had little effect on kinase activity (data not shown). rsk kinase was inhibited by NaC1, NaF, several divalent cations, and by pglycerophosphate and heparin (Table 11)

Inhibitors of rsk kinase activity
All of the inhibitors were examined at five different concentrations. Inhibitors were added to kinase assays so that the lowest concentrations tested did not markedly inhibit kinase activity and the highest concentrations inhibited activity to the maximal extent. The ICso is the extrapolated concentration at which 50% of control kinase activitv is inhibited. inhibitor was heparin, which had an ICso value of 0.18 pg/ml. Since it has been shown that heparin is a competitive inhibitor with respect to the substrate in the case of casein kinase I1 (43) and since rsk is present in the kinase assay at a relatively low concentration (1.4 pg/ml), 100 pg/ml of myelin basic protein and 200 pg/ml MAP-2 were also used as substrates for rsk kinase in the presence and absence of heparin. The ICso for heparin with myelin basic protein as a substrate was 4.9 f 0.4 pg/ml, while up to 100 pg/ml of heparin had no effect on phosphorylation of MAP-2. Protamine (5 mg/ml) increased rsk kinase activity slightly to 117% of control (data not shown).
The substrate specificity of purified rsk kinase was also examined (Table 111). Both MAP-2 and myelin basic protein were phosphorylated at 30-40 times the rate of rsk, but this difference may be due in part to the higher concentration of MAP-2 and myelin basic protein in the assays. a-Casein and histone H1 were phosphorylated at a lower rate than rsk, and protamine, phosvitin, and 40 S ribosomal subunits were poor substrates.
Phosphorylation and Activation of rsk Kinase-Based on previous studies with MAP kinases in other systems, it was important to determine whether rsk kinase was phosphorylated when activated and whether it could be inactivated with phosphatases. rsk kinase activity was assayed during oocyte maturation and compared with the activation of S6 kinase (Fig. 5). Both S6 and rsk kinase activities increased at the same time during the burst of protein phosphorylation just prior to GVBD.
To investigate the phosphorylation state of rsk kinase, oocytes were labeled with H3:"P04, stimulated to mature by the addition of progesterone, and activated rsk kinase was purified from oocytes that had undergone GVBD. Fig. 6A TABLE I11

Substrate specificity of rsk kinase
The substrates were incubated with purified rsk kinase for 3-10 min using standard kinase assay conditions described under "Experimental Procedures." Incorporation of R2P was linear with time with 0.04 pmol/min of phosphate incorporated into rsk. An asterisk (*) indicates no detectable DhosDhorvlation.   (8), labeled for 4 h with H?*PO,, matured with 10 p~ progesterone, and homogenized when 50% of the oocytes had matured. rsk kinase was purified, and the peak fraction from the phenyl column was silver-stained ( l u n e 1 ) and analyzed by autoradiography ( l a n e 2). B, phosphoamino acid analysis of in vivo labeled rsk kinase. The peak fractions were concentrated on a 0.25ml DEAE column, electrophoresed in an SDS-polyacrylamide gel, excised, eluted, precipitated with trichloroacetic acid, washed with NH40H, dried, and hydrolyzed. Two-dimensional electrophoresis was performed at pH 1.9 and 3.5 on a Hunter thin layer electrophoresis system. C, phosphoamino acid analysis of autophosphorylated rsk kinase. Purified rsk kinase was incubated in kinase reaction buffer in the presence of 5 p~ [y3'P]ATP (1000 cpm/fmol). The sample was electrophoresed and analyzed as above for phosphoamino acid content.
shows a silver stain (lune 1 ) of the peak fraction of purified rsk kinase activity and an autoradiogram of the gel (lune 2). Only the upper band was labeled, consistent with the idea that the upper band is a phosphorylated form of the kinase. Phosphoamino acid analysis of the labeled band yielded phosphothreonine and phosphotyrosine residues (Fig. 6B). In contrast, purified rsk kinase autophosphorylated in vitro was labeled on serine, threonine, and tyrosine residues (Fig. 6C). This is the first demonstration of autophosphorylation of a MAP kinase on serine, threonine, and tyrosine residues.
The effects of the serine/threonine protein phosphatases 1 and 2A, the protein tyrosine phosphatase CD45, and the truncated 37-kDa T-cell phosphatase on rsk kinase activity were then examined. rsk kinase activity was not reduced in the presence of 10 units/ml of phosphatase 1, whereas the same concentration of phosphatase 1 decreased S6 kinase I1 activity by more than 50% (data not shown). Phosphatase 2A (10 units/ml), however, effectively inactivated rsk kinase (Fig.  7). Only 5% of rsk kinase activity remained after 60 min, whereas almost 80% of the activity remained when rsk kinase was treated with phosphatase 2A inactivated with NaF. Both CD45 (data not shown) and the T-cell protein tyrosine phosphatase (Fig. 7) also inactivated rsk kinase. After 20 min of incubation in the presence of the T-cell protein tyrosine phosphatase (50 units/ml), only 10% of the original activity remained. Since all of the protein tyrosine phosphatases examined thus far have had an absolute specificity for phosphotyrosine (44), it is likely that the phosphotyrosine is essential for activity. In contrast, phosphatase 2A can remove phosphate groups from phosphotyrosine as well as phosphoserine and phosphothreonine (45); therefore, it cannot be determined from the data shown here whether phosphothreonine is essential for activity. In a similar experiment, however, with 3T3 L1 cell MAP kinase phosphorylated on both serine/ threonine and tyrosine residues, Anderson et al. (19)  Oocyte extracts were prepared as described in Fig. 5, and kinase assays were performed and quantitated as described under "Experimental Procedures" except that 0.2 pg of the heat stable inhibitor of the CAMPdependent protein kinase was added to the reaction. The kinase reaction mixture also contained purified rsk kinase (1.5 pl) or rsk kinase buffer, oocyte extracts (0.3 pl) and 40 S ribosomal subunits (667 pg/ml final concentration). The reactivation experiments were carried out by incubating purified S6KII with 25 units/ml of phosphatase 1 in a buffer with or without 2400 units/ml of Inhibitor-2. After 15 min, MgC12, BSA, cold ATP, and Inhibitor-2 (final concentration, 5 mM, 0.2 mg/ml, 100 pM, and 2400 units/ml, respectively) and rsk kinase (0.1 pl) or rsk kinase buffer were added. After incubation for 15 min, 667 pg/ml of 40 S ribosomal subunits and [y-"P] ATP (5 cpm/fmol) were added. After 15 min the reaction was terminated with the addition of 5 X sample buffer, and counts incorporated into S6 were analyzed as described. Each experiment was performed a t least three times with similar results.
and not the phosphotyrosine in MAP kinase. Phosphorylation of rsk and SGKII-MAP kinase from 3T3 L1 cells has been shown to phosphorylate both S6 kinase I and S6 kinase I1 in vitro and to activate S6KII (11). rsk kinase also phosphorylated both S6 kinases (data not shown), and in addition, caused an 11-fold increase in the activity of phosphatase-treated SGKII (Fig. 8). Consistent with this result, rsk kinase activated S6 kinase activity 15-fold when added to unstimulated oocyte extracts (Fig. 8).
3T3-Ll cell MAP kinase has already been shown to phos-phorylate and activate S6KII. In order to compare the phosphorylation sites of rsk kinase and MAP kinase, two-dimensional-phosphopeptide maps of rsk phosphorylated by each kinase were prepared (Fig. 9). rsk phosphorylated by rsk kinase yielded five phosphopeptides whereas phosphorylation by MAP kinase produced seven phosphopeptides. Based on the results of the mixing experiment, it appears the five phosphopeptides resulting from phosphorylation by rsk kinase are the same as five of the seven phosphopeptides resulting from phosphorylation by MAP kinase. Both kinases phosphorylated rsk on serine and threonine residues (Fig.  9D).

DISCUSSION
In this paper we report the purification of a 41-kDa Xenopus rsk kinase to apparent homogeneity and characterize the kinase with respect to inhibitors, substrate specificity, phosphorylation, in vitro autophosphorylation, and inactivation by phosphatases. Active rsk kinase is a monomeric phosphoprotein inactivated by both a serinelthreonine phosphatase and a protein-tyrosine phosphatase, and it phosphorylates MAP-2 and myelin basic protein in addition to rsk and S6 kinase 11.
In comparing the characteristics of rsk kinase with other kinases, rsk kinase is most similar to the MAP kinases. The both tyrosine and serine or threonine residues. Based on these criteria, rsk kinase appears to be a member of this "family" of enzymes, but a number of important differences distinguish it from previously described members of the family.
While this work was in progress, Gotoh et al. (26) reported purification of a 42-kDa MAP kinase from unfertilized Xenopus eggs. Several differences between this enzyme and rsk kinase are apparent, however. Both CaC12 and P-glycerophosphate inhibit rsk kinase (ICso = 2.5 and 28 mM, respectively) whereas the Xenopus egg MAP kinase purified by Gotoh et al. is not inhibited by CaC12 at concentrations up to 10 mM, and the ICso for P-glycerophosphate is 150 mM. More importantly, the MAP kinase purified by Gotoh et al. (26) is phosphorylated during activation in oocytes on both serine and tyrosine residues, whereas rsk kinase is phosphorylated on threonine and tyrosine residues (Fig. 5).
rsk kinase autophosphorylates in vitro on serine, threonine, and tyrosine residues. In contrast to this result, the MAP kinase studied by Sturgill does not undergo autophosphorylation in vitro and MAP kinase from sea star oocytes autophosphorylates only on serine residues (46). Recently, two mammalian MAP kinases have been cloned (designated ERKl (47) and ERK2 (48)) and it has been reported that both ERKl and recombinant ERK2 autophosphorylate on threonine and tyrosine residues (49). This autophosphorylation correlates with an increase in recombinant ERK2 kinase activity. In contrast to ERKl and ERK2, rsk kinase is extensively autophosphorylated on serine residue(s) as well as on tyrosine and threonine sites (Fig. 6). Seger et al. (49) have suggested that a partially purified activator of MAP kinase may act by stimulating autophosphorylation of MAP kinase. Since the rate of autophosphorylation of purified rsk kinase is very low and since rsk kinase is not phosphorylated on serine during activation in vivo, the effect of autophosphorylation on rsk kinase activity in vivo is unknown. We have observed no activation after autophosphorylation in vitro. However, the results here clearly establish that rsk kinase, a member of the newly emerging ERK/MAP kinase, family, exhibits dual specificity for autophosphorylation in vitro.
Whether rsk kinase can phosphorylate exogenous substrate proteins on tyrosine residues remains to be seen, although we detected no phosphorylation by purified rsk kinase of enolase, lactate dehydrogenase, or poly (Glu, Tyr, 1:4) in vitro. In addition, phosphoamino acid analysis of rsk and MBP phosphorylated by rsk kinase showed no incorporation into phosphotyrosine residues. Recently, several newly cloned yeast and mammalian cell protein kinases distinct from MAP kinase have also been reported to autophosphorylate on serine, threonine, and tyrosine residues (50-53).
There have been two reports subsequent to that of Sturgill et al. (11) showing that partially purified MAP kinases can activate pp90'Sk S6 kinases. Ahn and Krebs (29) have shown that in epidermal growth factor-treated Swiss 3T3 cells, two peaks of MAP kinase activity can be isolated using a Mono Q column, and both activities can activate an S6 peptide kinase activity which may be related to pp90'Sk. In addition, Chung et al. (30,31) have identified two serinelthreonine rsk kinase activities in Swiss 3T3 cells that coelute with MAP kinase activity. These activities also phosphorylate myelin basic protein and MAP-2 and cross-react with antibodies against sea star oocyte MAP kinase and phosphotyrosine antibodies. In the work of both Ahn and Krebs and Chung et al., the preparations of MAP kinase used for activation were relatively crude and may contain more than one enzyme. In contrast, in this paper we have reconstituted reactivation of purified S6KII with a single purified rsk kinase in an homologous system.
The ability of purified rsk kinase to reactivate phosphatasetreated S6KII in uitro supports the supposition that rsk kinase phosphorylates and activates S6KII in vivo. Since fewer phosphopeptides are evident in rsk phosphorylated by rsk kinase as compared to MAP kinase (Fig. 9), rsk kinase may have a more restricted substrate specificity than the 3T3 cell kinase from Sturgill's laboratory. This is additional evidence that rsk kinase is a distinct member of the MAP kinase family.
At present little is known about the sites in rsk phosphorylated by MAP kinase or rsk kinase. Two groups (16,54) have reported that the MAP kinase phosphorylation site in myelin basic protein has the sequence . . .TPRT*PPP. . . and Takishima et al. (55) have shown that rsk kinase phosphorylates a synthetic peptide corresponding to the sequence in the epidermal growth factor receptor around T669 (. . .QPLT*PSG. . .). Recently, we found that Xenopus cyclin B1 was an excellent substrate for rsk kinase at either 594 or S96 (. . .EPSS*PS*PME.. .) (56). Serine 94 has a sequence motif similar to that found in myelin basic protein and the epidermal growth factor receptor peptide, and therefore it seems likely that MAP kinase prefers serine or threonine residues NHz-terminal to a proline with a consensus PXS/ TP. However, examination of the sequence of rsk (9) does not reveal the presence of a site with this consensus. Selectivity for sites NHz-terminal to proline is also evident in the specificity of cdc2 kinase (57), although in that case basic residues are usually NH,-or COOH-terminal to the phosphorylated residue, unlike the known MAP kinase phosphorylation sites. In Xenopus, MAP kinase and rsk kinase appear to be involved in several physiologically important steps. Gotoh et al. (26) have shown that MAP kinase promotes an interphaseto-metaphase transition in microtubule arrays in Xenopus egg extracts. We have shown that rsk kinase phosphorylates and activates purified S6 kinase I1 in vitro as well as in oocyte extracts, and Izumi and Maller (56) have shown that rsk kinase phosphorylates cyclin B1. Considerable evidence supports the view that these events occur in vivo, and the MAP kinase family is clearly a large and important group of regulatory enzymes that mediate transduction of signals from the cell membrane to internal biochemical systems.