Eur. J. Biochem. 192, 633-642 (1990) 0FEBS 1990

In vivo activation of a microtubule-associated protein kinase during meiotic maturation of the Xenopus oocyte Olivier HACCARD

Catherine JESSUS’, Xavier CAYLA’, Jozef GORIS ’, Wilfried MERLEVEDE’ and Rene OZON’

’ ’

Laboratoire de Physiologie de la Reproduction, Institut National de la Recherche Agronomique/Unite Associee du Centre National de la Recherche Scientifique 555, Universite P. et M. Curie, Paris, France Afdeling Biochemie, Faculteit Geneeskunde, Katholieke Universiteit te Leuven, Belgium (Received January 26/May 15, 1990) - EJB 90 0082

We have characterized a serinelthreonine protein kinase from Xenopus metaphase-11-blocked oocytes, which phosphorylates in vitro the microtubule-associated protein 2 (MAP2). The MAP2 kinase activity, undetectable in prophase oocytes, is activated during the progesterone-induced meiotic maturation (G2-M transition of the cell cycle). p-Nitrophenyl phosphate, a phosphatase inhibitor, is required to prevent spontaneous deactivation of the MAP2 kinase in crude preparations; conversely, the partially purified enzyme can be in vitro deactivated by the low-M, polycation-stimulated (PCSL) phosphatase (also termed protein phosphatase 2Az), working as a phosphoserine/phosphothreonine-specificphosphatase and not as a phosphotyrosyl phosphatase indicating that phosphorylation of serine/threonine is necessary for its activity. S6 kinase, a protein kinase activated during oocyte maturation which phosphorylates in vitro ribosomal protein S6 and lamin C, can be deactivated in vitro by PCSL phosphatase. S6 kinase from prophase oocytes can also be activated in vitro in fractions known to contain all the factors necssary to convert pre-M-phase-promoting factor (pre-MPF) to MPF. Active MAP2 kinase can activate in vitro the inactive S6 kinase present in prophase oocytes or reactivate S6 kinase previously inactivated in vitro by PCSL phosphatase. These data are consistent with the hypothesis that the MAP2 kinase is a link of the meiosis signalling pathway and is activated by a serine/threonine kinase. This will lead to the regulation of further steps in the cell cycle, such as microtubular reorganisation and S6 kinase activation.

M-phase promoting factor (MPF) is the almost universal cytoplasmic agent responsible for the initiation of M-phase [l - 31. It has been purified from Xenopus and starfish eggs [4, 51. All the MPF preparations possess an HI kinase activity and contain a 32-kDa subunit which corresponds to the product of the Schizosaccharomyces pombe cdc2 gene [5 - 81. In Xenopus and starfish oocytes, the appearance of MPF activity is coincident with a major burst in total protein phosphorylation. Cicirelli et al. [9] used a number of different protein and peptide substrates to identify three M-phase-activated kinases. They reported that the rise in protein phosphorylation at the time of MPF appearance is due at least in part to the increase of these protein kinase activities. The transient activation of MPF and of protein kinases at M-phase is concomitant with important architectural events : depolymerization of the lamins and breakdown of the nuclear Correspondence to 0. Haccard, Laboratoire de Physiologie de la Reproduction, UA-CNRS 555, Universite P. et M. Curie, 4 Place Jussieu, F-75252 Paris Cedex 05, France Abbreviations: BSA, bovine serum albumin; MAP, microtubuleassociated protein; MBP, myelin basic protein; MPF, M-phase promoting factor; PCSH, PCSMand PCSL phosphatases, high-MF,medium-M, and low-M, polycation-stimulated protein phosphatase; AMD phosphatase, ATP, Mg2+-dependent protein phosphatase; NpP, p-nitrophenyl phosphate; RCM lysozyme, reduced carboxyamidomethylated and maleylated lysozyme; IC5,,, the concentration giving half-maximum inhibition; EGF, epidermal growth factor; XMAP, 21 5-kDa Xenopus microtubule-associated protein. Enzymes. Alkaline phosphatase (EC 3.1.3.1); acid phosphatase (EC 3.1.3.2); casein kinase I1 (EC 2.7.1.-): cyclic-AMP-dependent protein kinase (EC 2.7.3.7).

envelope [lo, 111, drastic reorganization of cytoplasmic microtubules leading to the formation of the metaphase spindles [12,13], reorganization of intermediate filaments [14]. All these cytoskeletal transformations are linked to post-translational modifications, among them phosphorylation/dephosphorylation reactions. Nonetheless the protein kinases as well as the protein phosphatases that catalyze these in vivo posttranslational modifications of cytoskeletal proteins, necessary for the reorganization of the M-phase cytoskeleton, remain unknown. This is particularly true for microtubules although numerous protein kinases have been shown to phosphorylate in vitro microtubule-associated proteins (MAP) [15, 161 and tubulin itself [17, 181 and to influence in vitro the tubulinpolymerization reaction [IS - 201. However, the in vivo phosphorylation reactions as well as their role in the spatial distribution and the stability of microtubules in oocytes are not well known. Gard and Kirshner [21, 221 isolated a Xenopus MAP (XMAP) of 215 kDa that promotes in vitro tubulin polymerization. Preliminary experiments suggest that the level of phosphorylation of XMAP increases at the time of germinal vesicle breakdown [22]. We have also identified numerous different Xenopus MAPS which copolymerize in vitro and in vivo with microtubules [23, 241. One of these, which is recognized by an anti-(rat brain MAP2) antibody, becomes phosphorylated when Xenopus oocytes are induced to mature (Fellous et al., unpublished results). Altogether, these observations favour the view that Xenopus MAP represent potential in vivo substrates of M-phase-activated protein kinases. We therefore decided to investigate whether one of the protein kinases activated upon entering the M-phase, could

634 use MAP2 as a preferential substrate. In a recent series of experiments, Ray and Sturgill [25, 261 identified and characterized in 3T3-Ll cells a kinase (MAP kinase), activated after insulin stimulation and which preferentially phosphorylated MAP2 from rat brain. In this report, we have used a chromatographic purification procedure adapted from the work of Ray and Sturgill [26] to search for the presence of a Xenopus MAP kinase in metaphase extracts from in-vitromatured oocytes. Furthermore, the MAP kinase from 3T3L1 cells was shown to be phosphorylated on tyrosine and threonine after insulin stimulation [27] and to correspond to pp42, a major tyrosine kinase target protein [28]. This mitogen-activated serine/threonine protein kinase can be inactivated by the catalytic subunit of the polycation-stimulated (PCS) phosphatase (also called phosphatase 2Ac) [29]. Since this phosphatase can dephosphorylate serine and threonine as well as phosphotyrosyl residues under proper conditions [30 331, we investigated which of the activities could be responsible for the inactivation of the M-phase-activated MAP2 kinase from Xenopus. This M-phase-activated MAP2 kinase could activate in vitro an s6 kinase, that, in addition to physical and enzymatic properties, suggests a relationship with the insulinstimulated MAP kinase from 3T3-Ll cells.

oocytes were referred as metaphase oocytes. Activated eggs were prepared as in [41]. Extract supernatant preparation

Prophase oocytes or metaphase oocytes were homogenized at 4°C in 1.5 vol. 25 mM Tris, pH 7.4, 2 mM EGTA, 1 mM dithiothreitol, 40 mM NpP, 25 mM NaCl and 0.2 mM p methyl phenylsulfoxide (buffer A) and centrifuged at 2000 rpm for 5 min in a Beckman J6 centrifuge with JA2 rotor. The supernatant was recovered and centrifuged at 4000 rpm for 10 min at 4°C. The supernatant was finally centrifugated at 30000 x g for 10 min at 4°C in a Beckman L3-50 centrifuge. The supernatant was then passed through a 0.22-pm MillexGV filter (Millipore Corp.) and hereafter referred to as an extract supernatant. Ammonium sulfate fractionation was carried out by addition of 0.5 vol. of a saturated solution of ammonium sulfate in buffer A to the extract supernatant, incubation on ice for 30 min, and resuspension of the pellet collected at 30000 x g for 10 min at 4°C in buffer A without NpP. Chromatography

The extract supernatant or the ammonium sulfate precipitate were diluted twice in buffer A containing 250 mM NaCl and applied to a phenyl-Superose HR 5/5 column previously Materials equilibrated with buffer A containing 250 mM NaCl in the Xenopus laevis adult females (Centre de Recherche de Bio- presence or absence of N p P (see text). The column was washed chimie Macromolkculaire du Centre National de la Recherche with buffer A containing 250 mM NaC1, in the absence or Scientifique, Montpellier, France) were bred and maintained presence of NpP, and eluted with a gradient of decreasing under laboratory conditions. p-Nitrophenyl phosphate (NpP) concentrations of NaCl in the range 250 - 25 mM and increasand histone H1 were purchased from Boehringer Mannheim; ing concentrations of ethyleneglycol in the range 0-60%, in the Superose 12 HR 10/30 column and the phenyl-Superose the absence or in the presence of N p P (see text). The flow HR 5/5 column were obtained from Pharmacia. [y-32P]ATP rate was 0.2 ml/min and 0.5-ml fractions were collected and was purchased from Amersham, the S6 peptide (RRLSSLRA) assayed for MAP2 kinase activity. and cyclic-AMP-dependent protein kinase inhibitor peptide Gel-filtration chromatography was carried out using a (TTYADFIASGRTGRRNAIHD) from a multiple peptide Superose 12 HR 10/30 column, previously equilibrated with system. Alkaline phosphatase (type 111-R from Escherichia 20 mM Tris, pH 7.4, 1 mM EGTA, 1 mM EDTA, 0.5 mM coli), sweet potato acid phosphatase (type X) and all other benzamidine, 2% glycerol, 0.1 M NaCl and 1% isopropanol reagents, unless otherwise specified, were obtained from (buffer B). The column was eluted with buffer B with a flow Sigma. rate of 0.4 ml/min. 0.5-ml fractions were collected. Tubulin was purified from Sprague-Dawley rat brain using the method described in [34].Heat-stable MAP2 and zproteins were prepared as described in [35]. High-M,, medium-M, and Kinase assay low-M, polycation-stimulated protein phosphatases (PCS", All MAP2 kinase assays were performed for 20 min at PCSM and PCSJ phosphatases) were purified from rabbit 30°C in a final volume of 40 p1 50 mM Tris, pH 7.5, 2 mM skeletal muscle [36], the PCSL phosphatase also from Xenopus EGTA, 1 mM dithiothreitol, 20 mM MgC12 containing oocytes [30]. Phosphotyrosyl phosphatase activator was 100 pM [y-32P]ATP(0.5 cpm/fmol), 5 pl MAP2 kinase preppurified from rabbit muscle as described in [33,37]. The active aration and either 2 pg MAP2 or 10 pg myelin basic protein catalytic subunit of the ATP, Mg2+-dependent phosphatase (MBP). CAMP-dependent protein kinase inhibitor peptide (AMDc phosphatase) was purified from dog liver as in [38]. (10 pM) was always included for reasons discussed in [9]. To A Xenopus oocyte homologue of the TyrP protein phosphatase assess the substrate specificity of the MAP2 kinase, the assays type 1B [39] was partially purified as described in [40] by were performed under the same conditions in the presence of ACA34, polylysine and mono Q chromatography as ad- 1.56 pg 5 proteins, 4 pg tubulin, 20 pg histone H I , 4 pg histone ditional purification steps. The final preparation had a specific H2A, 4 pg casein, 30 pg S6 peptide or 3.8 pg ribosomal proactivity releasing 40 nmol P, . min- . mg- with reduced car- teins prepared, as described in [42]. The kinase reaction was boxyamidomethylated and maleylated lysozyme as substrate, stopped by adding 40 p1 Laemmli buffer [43] and boiling for exclusively phosphorylated on tyrosyl residues [40]. 3 min. Usually, 40-pl samples were run on electrophoresis according to [43], with appropriate polyacrylamide concentrations: 6.5% for MAP2,8% for zproteins and tubulin, 10% Oocytes prepmation for casein and ribosomal proteins, 12% for histones H I and Isolated oocytes were prepared as described [24]. Fully H2A. After staining, the gels wre dried prior to exposure to grown oocytes (referred as prophase oocytes) were induced X-Omat AR films (Kodak). The protein bands corresponding to mature by addition of 1 pM progesterone. The matured to the different substrates were further excised from the gel

MATERIALS AND METHODS

635 and radioactivity measured in water by Cerenkov effect in an SL 3000 counter (Intertechnique). The level of protein phosphorylation was also quantified by trichloroacetic acid precipitation of 20 p1 of each assay on 3MM Whatman paper. Phosphorylation of S6 peptide was quantified as in [9] using Whatman phosphocellulose P81 paper to separate the incorporated phosphate from the remaining ATP. Phosphoamino acid analysis after partial acid hydrolysis was performed on the [32P]MAP2 band eluted from the electrophoresis gel as described in [44]. Treatment o f t h e MAP2 kinase by phosphatases All incubations of the MAP2 kinase in the presence of phosphatases were performed for 20 min at 30°C in a final volume of 20 pl. MAP2 kinase activity was tested subsequently in a 40-pl volume as described previously. AMD,, PCS and type-IBphosphatasrs. Incubations of the MAP2 kinase with the phosphatases were performed in 50 mM Tris, pH 7.5, 2 mM EGTA, 1 mM dithiothreitol, 125 pM ATP, 6.25 mM MgClz and 1 mg/ml bovine serum albumin (BSA). Both ATP and MgClz were omitted when the incubation was performed with type-1B phosphatase. AMD, and PCS phosphatases were stopped by adding 10 pM okadaic acid, a potent inhibitor of both these activities [45-471, in order to prevent their action in the subsequent kinase assays. Controls were carried out by incubating the AMD, or PCS phosphatases and the MAP2 kinase in the presence of 10 pM okadaic acid. Since the MAP2 kinase was demonstrated to be a serine/threonine kinase, inhibition of the type-I B phosphatase, a specific tyrosine phosphatase, was not required. Acid and alkaline phosphatases. Incubation with acid phosphatase was performed in 100 mM Mes, pH 5.5 and 1 mg/ml BSA, and incubation with alkaline phosphatase in 50mM Tris, pH 9.0, 2 mM EGTA, 1 mM dithiothreitol and 1 mg/ml BSA. Both acid and alkaline phosphatases were stopped by adding 40 mM NpP. Controls were made by incubating the acid or alkaline phosphatases and the MAP2 kinase in the presence of 40 mM NpP. Activation o j PCS, phosphatase by TyrP phosphatase activator. PCSL phosphatase was incubated with TyrP phosphatase activator for 10 min at 30°C in a 10 p1 final volume of 50 mM Tris, pH 7.5, 2 mM EGTA, 1 mM dithiothreitol, 500 pM ATP, 2.5 mM MgClz and 1 mg/ml BSA. MAP2 kinase was further added for 3 min at 30"C, as described. This short incubation time was mandatory in order to obtain maximal stimulation by TyrP phosphatase activator over the basal activity, since it is known that the TyrP protein phosphatase activity is deactivated during the assay [33, 371. All controls were carried out to eliminate a possible direct effect of TyrP phosphatase activator on the MAP2 kinase. Activation oj the PCS, phosphatase by ATP. In order to activate specifically the TyrP protein phosphatase activity of the PCS, phosphatase, the enzyme was incubated for 10 rnin at 30°C in a 10-pl final volume of 50 mM Tris, pH 7.5, 2 mM EGTA, 1 mM dithiothreitol, 1 mM ATP and 1 mg/ml BSA, as described in [30 - 321. MAP2 kinase was further added for 20 min in the same conditions as described. Units of Ser/Thr and phosphotyrosyl phosphatase activities were measured with, respectively, 10 pM phosphorylase a and 1 pM reduced carboxyamidomethylated and maleylated lysozyme as in [40]. Ss kinase Preparation The inactive S6 kinase used in this study was partially purified from a 33% ammonium sulfate fraction, prepared

i

350

-E

-*

n

250

5 E

.-

200

c

-> c

150

;

100

W

O

!

50

t

0 0

20

40

60

80

100

120

140

160

Fractions

Fig. 1. Preparation of the inactive and activated S6 kinase. 2.3 ml dialysed 0-33% (NH4)ZS04 fraction (originating from 20 ml oocytes) were divided into two equal parts and incubated either with (+) or without (0)MgATP and an ATP-regenerating system for 30 min at room temperature as described in [48], applied to two identical DEAE-Sepharose CL-6B columns (14 cm x 2.5 cm) equilibrated in 20 mM Tris, pH 7.4,1 mM EDTA, 1 mM EGTA and 0.5 mM benzamidine, further washed (60 ml) and eluted with a 500-ml linear (0 -0.5 M) NaCl gradient in the same buffer. Fractions of 3.6 ml were collected. 10 p1 of each fraction was assayed for Sb peptide kinase, by adding to 20 yl kinase assay mixture containing 25 mM glycerol 2phosphate, 3.5 mM NaF, 0.15 mM EDTA, 7.5 mM MgCI2, 1 mM dithiothreitol, 100 pM CAMP protein kinase inhibitor, 0.5 mg/ml Ss peptide and 200 pM [y-3zP]ATP(lo6 cpm/assay) for 20 rnin at 30°C. The reaction was stopped as described in [9]. 1 U S6 kinase incorporates 1 nmol phosphate/min. Fractions 85 - 120 were pooled and concentrated as described in Materials and Methods

from prophase oocytes as described in [48]. This fraction is known to contain all the factors necessary to convert pre-MPF into MPF by addition of MgATP and an ATP-regenerating system. As shown in Fig. 1, the S6 kinase present in this fraction is also activated in these conditions. After separation of the 33% ammonium sulfate fraction on a DEAE-Sepharose CL-6B column, the activated S6 kinase fractionsand the corresponding inactive fractions of a parallel column run in otherwise identical conditions, were pooled as indicated in Fig. 1 , concentrated by dialysis against 20% poly(ethy1ene glycol) in the column equilibration buffer and further against 60% glycerol in the same buffer. Both these fractions could be stored at -20°C for several months without loss of activity and are referred to as inactive and activated S6 kinase respectively. RESULTS Preparation of a MAP2 kinase extract In a first series of experiments, we used a phenyl-Superose column to fractionate the supernatant prepared from an extract of Xenopus oocytes arrested at metaphase 11, after being induced to mature in vitro by 1 pM progesterone. We utilized MAP2 as a substrate to screen the presence of MAP2 kinase activites in the presence of [p3'P]ATP in the different fractions. As shown in Fig. 2, these enzymatic activities are detectable in two main fractions of equal importance. We focused our attention on the activity retained on the column, which, when eluted by two simultaneous linear opposite gradients

636

0

2

4

6

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12

Elution volume

8

(ml)

14

16

18

Fig. 2. Phenyl-Superose chromatography of the M A P 2 kinase. Extract supernatants from either 200 prophase oocytes (0)or 200 metaphase oocytes ( 0 )were applied to phenyl-Superose. The column was eluted with a gradient of simultaneously decreasing concentrations of NaCl (250 -25 mM) and increasing concentrations of ethyleneglycol (060%) as described under Materials and Methods. 0.5-ml fractions were collected and assayed for kinase activity as described

94

-

67-

43?

__---9 9.5

10

10.5

11

-Elution

volume (mi)

B

h

.. 1

2

Fig. 3. Phosphorylation of'MAP2 by thephenyl-Superosepeakfractions of the M A P 2 kinase. (A) Autoradiogram of a 6.5% polyacrylamide gel electrophoresis showing the phosphorylation of MAP2 by the phenyl-Superose fractions from either prophase oocyte extract supernatant (p) or metaphase oocytes extract supernatant (m). (B) MAP2 was phosphorylated by the MAP2 kinase as described in Materials and Methods. The band (4pg MAP2) was excised from the gel and processed for phosphoamino acid analysis as described. Lane 1, migration of phosphorylated amino acid standards visualized by reaction with ninhydrin. Lane 2, 32P-labelled amino acids were visualized by autoradiography. @S, phosphoserine; OT, phosphothreonine; B Y , phosphotyrosine

of increasing concentrations (0 - 60%) of ethyleneglycol and decreasing concentrations (250 - 25 mM) of NaC1, resolves in one peak at 21 YOethyleneglycol. The chromatographic behaviour of the Xenopus MAP2 kinase activity compares well with MAP kinase activity from stimulated 3T3 cells [26]. When an identical chromatographic procedure was used to investigate the extract supernatant from prophase oocytes, MAP2 kinase activity was also found in the flow through from

Y,"

~

1.4

1.6

1.8

2.0

2.2

2.4

2.6

2.8

3.O

VeIVo

Fig. 4. Gel-filtration chromatography. 100 p1 of active MAP2 kinase fractions eluted from the phenyl-Superose were applied to a Superose 12 column. 0.5-ml fractions were collected and assayed for MAP2 kinase activity (0) as described in Materials and Methods. The elution position of the marker proteins (aldolase, 158 kDa; BSA, 67 kDa; chymotrypsinogen, 25 kDa; ribonuclease A, 13.7 kDa; vitamin B12 1377 Da) is also indicated ( 0 )

the column; no MAP2 kinase activity was retained on the column (Fig. 2). In a similar way, no MAP2 kinase activity was isolated by elution of an extract supernatant from activated eggs (interphase state) on phenyl-Superose column. The phosphorylation of MAP2 was also analyzed by SDS gel electrophoresis and autoradiography (Fig. 3 A). A strong band of radioactivity was found to be present at the position of MAP2, only when the partially purified enzyme from metaphase oocytes was used. Phosphoamino acid analysis revealed that the phosphorylation of MAP2 by the MAP2 kinase occurs equally on serine and threonine residues (Fig. 3B). The peak fractions from metaphase oocytes were pooled and used further as MAP2 kinase. The presence of 40 mM NpP, an inhibitor of protein phosphatases [30,49], was required during homogenization of the oocytes, the chromatographic purification and for storage of the enzyme. When N p P was omitted from the buffer containing the kinase or removed by gel filtration on a Sephadex G25 column, the MAP2 kinase activity is rapidly lost. This indicates that an inactivating NpP-sensitive factor, probably a protein phosphatase, could co-purify with the MAP2 kinase throughout the purification procedure. In order to eliminate this factor, the extract supernatant of metaphase oocytes was fractionated by 33 % ammonium sulfate precipitation. The 0- 33% ammonium sulfate precipitate was further applied on the phenyl-Superose column and eluted in the absence of NpP. In these conditions, a stable MAP2 kinase activity eluted from the column in an identical manner as in Fig. 2. In further experiments, this fractionation step was always included during the purification procedure. Biochemical properties of the MAP2 kinase

The apparent molecular mass of the MAP2 kinase was estimated by Superose 12 FPLC gel filtration. The kinase activity was recovered in a single peak eluting with an apparent molecular mass of 45 kDa (Fig. 4). The optimal conditions for the kinase assay were further determined. The time course studies showed that the linearity was maintained for at least a 20-min incubation at 30°C. The optimal Mg2+ concen-

637 10

Table 1 . Substrate specificity of the MAP2 kinase Substrate specificity assays were performed as described under Materials and Methods using 2 pg MAP2,lO pg MBP, 1.56 pg z proteins, 4 pg tubulin, 20 pg histone HI, 4 pg histone HzA, 4 pg casein or 30 pg Ss peptide (RRLSSLRA)/assay. n.d., not detectable (less than 0.01 mol phosphate/mol substrate)

I

Substrate

Phosphate bound mol/mol

-0.02

0.04

0.02

0

0.06

MAP2 M BP z Protein Tubulin Histone H I Histone H2A Casein S6 Peptide

0.1

0.08

fltsl (PM-')

-2

0

2

4

6

IltSI

8

10

12

14

(PM-1)

Fig. 5. Kinetic parameters of the Xenopus MAP2 kinasefor ATP and MAP2. Kinase reactions were carried out as described in Materials and Methods except that [Y-~'P]ATPwas present in concentrations of 10-150 pM (A) or MAP2 at concentrations of 71.4-357 nM (B). The radioactivity in MAP2 was determined after 6.5% polyacrylamide gel electrophoresis and escision of the MAP2 band

tration was 20 mM. The kinase activity was stable at a pH range of 7 - 8 and in a temperature range of 20 - 37 "C. When assays were performed at 30°C, pH 7.4, for 20 min in the presence of 20 mM Mg2+,ATP was the preferred phosphate donor. No change in incorporation of [32P]phosphate into MAP2 was observed when 0.5 mM unlabelled GTP was added to the kinase assay. The K , for ATP was estimated to be 8 0 p M (Fig. 5A). As shown in Fig. 5B, the apparent K, for MAP2 was 1 pM with a V,,, of 3.72 pmol phosphate transferred/min. The MAP2 kinase could transfer 5 mol phosphate/mole MAP2. The MAP2 kinase extract could be dialyzed on a 25 nm Millipore filter (VSWP 025000) for 2 h without any loss of activity, indicating that no stabilizing dialyzable compound copurified with the kinase. To differentiate the MAP2 kinase activity from other wellcharacterized kinases, several effectors known to inhibit or to activate certain protein kinases were tested. The MAP2 kinase activity was insensitive to the presence of 0.01 - 10 pM calcium, 12 pM cyclic AMP, 2 mM cAMP protein kinase inhibitor peptide (the specific inhibitor of the CAMP-dependent protein kinase), 2 mM spermine or 30 pg heparin/ml. N,NDimethyl-6-aminopurine which is known to be a non-specific protein kinase inhibitor [50], inhibited the MAP2 kinase activity in a dose-dependent manner with an ICs0 of 0.4 mM. Further distinction of the MAP2 kinase from other types of protein kinases was achieved by assaying the MAP2 kinase on different polypeptide substrates. As shown in Table 1,

4.83 0.5 0.4 0.15 n.d. n.d. 0.033 0.03

MAP2 was a good substrate among the substrates tested. The other microtubular proteins (z and tubulin) could be phosphorylated to a reasonable phosphorylation level. Taking into account that in MAP2 incorporation of up to 40 phosphates has been described [51], the occupancy of the individual phosphorylation sites might be in the same range for the different microtubular proteins. MBP was also easily phosphorylated and could be used as an alternative substrate. The elution profiles of the MAP2 kinase and MBP kinase were very similar in the phenyl-Superose column (not shown). Although it has been reported that other kinases such as the cyclic-AMP-dependent protein kinase or kinase FAcan phosphorylate MBP [52], in the presence of cAMP protein kinase inhibitor, the major MBP kinase activity also comigrates with the M-phase-activated MAP2 kinase in a DEAE-Sepharose column (not shown). The other substrates tested (Table 1, Fig. 6) were poorly phosphorylated or not phosporylated at all. When ribosomal proteins of Xenopus oocytes were incubated with the MAP2 kinase, no proteins were phosphorylated, as jugded by autoradiography (not shown), indicating that in vitro MAP2 kinase does not phosphorylate S1 nor s6 which are known to be highly phosphorylated in vivo [53- 561. Activation of the

s 6

kinase by the MAP2 kinase

It has been previously demonstrated that reactivation and phosphorylation of Xenopus s6 kinase occurs in vitro with the MAP kinase purified from 3T3 cells [29]. Since s6 kinase is in vivo activated during the meiotic maturation of Xenopus oocytes [57, 581, it was important to determine whether the partially purified MAP2 kinase from metaphase oocytes was also able to activate a Xenopus S6 kinase. To answer this question, two different s6 kinase preparations were used. (a) The inactive s6 kinase from prophase oocytes after partial purification by 33 YO (NH4)2S04 precipitation and DEAESepharose chromatography (see Materials and Methods). This preparation was free of endogenous MAP2 kinase activity and free of endogenous PCS phosphatases (b) The same preparation after an activation/inactivation cycle. The s6 kinase was activated in the 33% (NH4)2S04fraction, partially purified as described in the Materials and Methods section, and inactivated by a PCSLphosphatase treatment. Thereafter, the PCSL phosphatase was blocked by okadaic acid and the s6 kinase reactivated by the MAP2 kinase. As shown in Fig. 6,

638 30

A

20

1 r

+MAP2 Kinase

+Phosphalase +MAP2 Kinase

10

1

+Phasphalase I

0

6

12

18

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30

Time (min)

0

6

12

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36

Time (min)

Fig. 6 . Activation ofS, kinase by MAP2 kinase. The inactive (A) or activated (B) Ss kinase (see Fig. 1) was incubated for 20 min at 30°C with (B; a,+) or without 24 Ujml PCSL oocyte phosphatase. After this incubation, the mixtures were fivefold diluted in 50 mM glycerol 2phosphate, pH 7.4, 7 mM NaF, 0.3 mM EDTA, 15 mM MgCI2, 1 mM dithiothreitol and 0.25 pM okadaic acid, and incubated with ( A , m) or without ( A ,0.0 ) MAP2 kinase in the presence of 0.3 mM cold ATP and 1.5 mM MgC12.At the indicated times, 15-pl samples were in the kinase added to 15 p1 containing 1 mg/ml S6 peptide, 100 pM CAMP protein kinase inhibitor and 2.8 x l o 6 cpm carrier free [Y-~’P]ATP buffer, incubated for 10 min at 30°C and 25 pl were stopped as in [9]. The control S6 kinase activity of the MAP2 kinase preparation is indicated by ( x ) in (A). The units of S6 kinase activity were defined in Fig. 1

+,

the MAP2 kinase can activate the inactive S6 kinase up to the same level as the Sb kinase activated in the total 33% (NHJ2S04 fraction and partially purified afterwards. If the activated S6 kinase is inactivated first by PCSI, phosphatase treatment, the MAP2 kinase can reactivate the S6 kinase but only up to an intermediate level (Fig. 6B). Apparently, the PCSL phosphatase did remove phosphates from sites that could not be rephosphorylated by the MAP2 kinase, and that also influenced the S6 kinase activity. Note that the MAP2 kinase could apparently activate both inactive S6 kinase preparations during the S6 kinase assay conditions (10 min), which could explain the already high activity levels at time zero, as compared to controls without MAP2 kinase.

Inactivation qf the M A P 2 kinase by phosphatase treatment Previous studies have shown that in vivo the MAP2 kinase from 3T3 cells is phosphorylated on threonine and tyrosine 1271 and inactivated in vitro by phosphatase 2A [29]. Since the MAP2 kinase isolated from the metaphase oocytes presents strong biochemical similarities to the MAP2 kinase from 3T3 cells, we have also determined whether phosphorylation of the kinase might play a role in the enzyme activity. The MAP2 kinase from metaphase oocytes was therefore incubated with several protein phosphatases and assayed for MAP2 kinase activity. The MgATP-dependent phosphatase (AMD phosphatase or phosphatase I), which is absolutely specific for dephosphorylation of serine and threonine, the oocyte phosphotyrosyl phosphatase homologous to the placental phosphatase 1B [39], which is specific for dephosphorylation of tyrosyl residues, and both alkaline and acid phosphatases, which have a broad specificity, had no effect on the MAP2 kinase activity, using either MBP or MAP2 as substrate

(Table 2). In contrast, incubation of the kinase with the lowM , PCS,, protein phosphatase, also termed phosphatase 2Az, which possesses both serinelthreonine and tyrosyl protein phosphatase activity, resulted in a time- and dose-dependent inactivation of the MAP2 kinase (Fig. 7). The catalytic subunit of the PCS phosphatase lead to the same inactivation of the kinase (Table 2). In contrast, the high- and medium-M, polycation-stimulated PCSH and PCSMprotein phosphatases were unable to inactivate the MAP2 kinase activity (Table 2). PCSFIZphosphatase could also partially inactivate the MAP2 kinase (Table 2). These results suggest that the regulatory subunits of the PCS phosphatases might control the substrate specificity of the enzyme. To determine whether the selective ability of the PCSL protein phosphatase to inactivate the MAP2 kinase activity is due to its selective ability to dephosphorylate either phosphotyrosyl sites or serinelthreonine sites, we tested the effect of free ATP which is known to inhibit the serinelthreonine phosphatase activity and to enhance the tyrosyl phosphatase activity of thre PCSL phosphatase [30, 31, 331. Incubation of PCSL phosphatase in the presence of free ATP clearly prevented is inhibitory action on the MAP2 kinase (Table 2), indicating that the serine/ threonine phosphatase activity of the PCSL phosphatase is absolutely necessary for the effect on the kinase. On the other hand, the TyrP phosphatase activator [33, 371 is kown to strongly enhance the phsophotyrosyl phosphatase activity of the PCSL phosphatase without affecting its serine/threonine phosphdtase activity. When the PCSLphosphatase was treated with TyrP phosphatase activator, its inactivating effect on the MAP2 kinase remained unchanged (Table 2). We therefore concluded from our experiments that the inactivation of the MAP2 kinase by the PCSL phosphatase was triggered by dephosphorylation of serine or threonine rather than by dephosphorylation of tyrosyl residues.

639 Table 2. Deactivation of MAP2 kinuse by phosphatase treatment (A) MAP2 kinase was incubated for 20 rnin at 30°C with several phosphatases as described under Materials and Methods. After arrest of the phosphatease activities, the MAP2 kinase activity was further mcasured by using 12.5 pg MBP/assay. Results are expressed relative to controls incubated for the same period of time with phosphatases treated with their specific inhibitors. (B) PCS, phosphatase (30 Ujml) was incubated without or with a saturating concentration of TyrP phosphatase activator for 10 rnin at 30°C as described under Materials and Methods. The MAP2 kinase was further added for 3 rnin at 30°C (final concentration of PCS, phosphatase, 15 Ujml). After addition of 10 pM okadaic acid, the MAP2 kinase activity was tested as described. Results are expressed as in (A). (C) PCS, phosphatase (30 U/ml) was incubated for 10 min at 30°C in the absence or in the presence of 1 mM ATP and in the absence of M g 2 + as described under Materials and Methods. MAP2 kinase was further added for 20 rnin at 30'C in the same conditions as in (A) (final concentration of PCSL phosphatase, 15 Ujml). After addition of 10 pM okadaic acid, MAP2 kinase activity was measured as previously described. Results are expressed as in (A). n.m., not measured; n.d., not detectable (below 0.5% of the phosphate release from the substrate in the assay) Experiment

Phosphatase type

alkaline (8.4 pg/ml) acid ( 5 pg/ml) type 1B AMD

PCSC PCSHl

PCSHZ

PCSM PCS, PCS, PCSL/PTPA PCS, PCSL/freeATP a

Phosphatase specificity

Inactivation of MAP2 kinase

Ser/Thr

TYr

n.m. n.m. n.d.

0.5 0.4" 2 n. d.

70

29

15

n.d. n.d. 0.18 n.d. 0.12 0.12

15 15 n. d.

0.12 0.9

34 50 23 15

0 0 0 0 40 0 13 0 67 52 50

1.8

61

3

Measured at pH 5.5.

DISCUSSION We have identified a protein kinase indicated as XMAP kinase (Xenopus microtubule-associated protein kinase, or Xenopus metaphdse-activated protein kinase) from metaphase Xenopus oocytes, which utilizes MAP2 as preferential substrate (Table 1). The XMAP kinase can be distinguished by its substrate specificity and its insensitivity to various effectors of several classical protein kinases such as cyclic-AMP-dependent protein kinase, casein kinases or calcium-regulated protein kinases. The partially purified enzyme is inactivated by PCSL phosphatase (also termed protein phosphatase 2A2) working as a serine/threonine phosphatase (Table 2). XMAP kinase activity is absent in prophase extracts, appears in Mphase extracts and disappears after parthenogenetic activation of eggs. We therefore conclude that XMAP kinase belongs to the family of M-phase activated kinases [9]. Histone HI kinase, the protein homolog of the product of the cdc2' gene of the fission yeast S. pombe has been shown to be a component of Xenopus M P F [6, 71. It corresponds to a major M-phase-activated kinase. The preliminary characterization of XMAP kinase indicates that it can be clearly differentiated from histone H1 kinase because (a) their substrate specificities are distinct, e. g. XMAP kinase phosphorylates histone H I with a low efficiency (Table l), (b) whereas histone H1 kinase appears to be activated via a dephosphorylation reaction [59, 601, XMAP kinase is inactivated by dephosphorylation (Table 2, Fig. 7), and (c) although the histone H1 kinase can physically associate with p l 3 sucl protein bound to Sepharose beads [7], it was not possible to bind XMAP kinase to pl3-Sepharose. Altogether, these experiments indicate that histone H1 and XMAP kinases are distinct enzymes.

S6 kinase is another M-phase-activated protein kinase which specifically phosphorylates ribosomal protein s6 [9, 57, 58, 611. However, its substrate specificity and its molecular mass show that it is also a n enzyme different from XMAP kinase. A third M-phase-activated kinase, called MAK-M [9], was found to phosphorylate MBP. Since XMAP kinase also phosphorylates MBP in vitro, it is therefore possible that the XMAP kinase isolated in this report using a different purification procedure might correspond to MAK-M identified in Xerzupus extracts and to the MBP kinase I described in maturing sea star oocytes [62]. Recently, a mitogen-activated MAP2 kinase was isolated from rat 3Y1 cells after stimulation by epidermal growth factor (EGF) or 12-0-tetrddecanoylphorbol 13-acetate [63]. Its Ca2 sensitivity may differentiate it from the XMAP kinase. Several of our observations suggest that XMAP kinase from oocytes might be related to M A P kinase, a recently identified mitogen-activated serine/threonine kinase [25, 291. Both enzymes phosphorylate MAP2 equally on serine and threonine, activate the s6 kinase from Xenupus oocytes and are inactivated in vitro by phosphatase 2A treatment. Furthermore, their chromatographic behaviour and their molecular masses are similar. It has been demonstrated that the MAP kinase from 3T3-Ll cells is phosphorylated in vivo on tyrosine and threonine [27] and that it corresponds to the pp42 protein, a major tyrosine kinase target protein [28]. It is not known however if this kinase is a direct substrate of the intrinsic tyrosine kinase activity of the insulin receptor. Until now we have no direct arguments indicating that XMAP kinase present in metaphase oocytes is a phosphotyrosine protein. However, our results demonstrate unequivocally that removal of phosphate from serine or threonine by PCSL phosphatase +

640

r

loo

0

A

5

10

15

20

25

35

30

40

Time (min) 16000

12000

hB

0 ' 0

3

6

9

12

15

18

PCSL Phosphatase (U/ml)

Fig. I . Deactivation o j the MAP2 kinase by the PCSLphosphutuse. MAP2 kinase was incubated with 17.8 U/ml of PCSL phosphatase at 30°C for 0 to 40 min (A) or with 0.89- 17.8 U/ml PCSL phosphatase for 20 min at 30°C (B) in a final volume of 20 pl. PCSL phosphatase activity was blocked with 0.6 pM okadaic acid (PCSLphosphatase is 100% inhibited by 0.6 pM okadaic acid, as demonstrated in [45--471. Kinase assays were performed using 12.5 pg MBP as substrate in a final volume of 40 pl. Results are expressed in percentage of the inhibition of the MAP2 kinase in (A) and as the radioactivity incorporated into 12.5 pg MBP estimated by trichloroacetic acid precipitation (B). As a control, the MAP2 kinase activity was tested in the presence of 0.6 pM okadaic acid without phosphatase; no kinase inhibition or activation was observed

is sufficient for the complete inactivation of the kinase when MAP2 and MBP are used as substrates, in concordance with the results obtained with MAP kinase from 3T3 cells [29]. Recently, it has been shown [64] that monoclonal antibodies could mimic the insulin activation of S6 kinase in 3T3/HIR cells without activation of the insulin receptor tyrosine kinase, and that rat hepatoma cells, expressing an insulin receptor mutant with three key tyrosine autophosphorylation sites in the p subunit changed to phenylalanines, could still activate the S6 kinase by the monoclonal antibodies, without stimulating either the insulin receptor autophosphorylation or the tyrosine phosphorylation of a cellular protein, normally phosphorylated on tyrosine after insulin treatment. These results suggest that activation of receptor tyrosine kinase and subsequent tyrosine phosphorylation of cellular proteins may

not be crucial for S6 kinase activation by the insulin signalling system. Our results suggest that also in the signalling system of the meiosis induction by progesterone, phosphorylation of MAP2 kinase on tyrosine could not be essential for activation of the S6 kinase. The Xenopus oocyte arrested in prophase contains numerous microtubules located in the cortical region and also around the nucleus [13, 651. During meiotic maturation, the perinuclear microtubules disappear at the time of germinal vesicle breakdown, whereas the formation of a giant cytoplasmic network precedes the meiotic spindles [12, 131. Moreover, it has been shown that the critical level for tubulin assembly decreases [24,66,67]. Therefore the machinery which controls the spatial distribution and stability of microtubules is profoundly modified during the transition from interphase to metaphase in Xenopus oocyte, as in mitotic cells. The possibility that the phosphorylation of endogenous MAP is involved in this process is therefore probable. In fact, we recently identified with a set of specific antibodies directed against rat brain MAP2 a protein of 240 kDa which is associated with the oocyte microtubules and which becomes phosphorylated in vivo at the time of germinal vesicle breakdown (Fellous et al., unpublished results). An important task will be to know if XMAP kinase is the endogenous kinase responsible for its phosphorylation, and if this post-translational modification changes the interaction between MAP and microtubules. In addition, both MAP kinase isolated from 3T3 cells [29] and XMAP kinase from Xenopus metaphase oocytes are capable to activate in vitro Xenopus S6 kinase. In 3T3 cells, MAP kinase and S6 kinase are stimulated following a 1015-min treatment with insulin [29]. In Xenopus oocyte, insulin treatment induces a biphasic activation of S6 kinase [57, 581. The first peak of activation follows insulin application and the second occurs at germinal vesicle breakdown, and remains high as long as the cell remains in M-phase. Only the second peak of activation is abolished by inhibitors of protein synthesis [58]. In contrast, progesterone, the natural inducer of maturation, only activates S6 kinase at the onset of germinal vesicle breakdown, in a cycloheximide-dependent manner [9, 571. Therefore, the mitogens provoke an initial transitory burst of activation of S6 kinase, which takes place immediately following mitogen action, whereas the activation of S6 kinase at metaphase corresponds to a longer-lasting event. In 3T3 cells as well as in fibroblasts, it is believed that MAP kinase, a major tyrosine kinase target [28] could be responsible for the stimulation of S6 kinase following mitogen action. It would be important to know whether a similar mechanism is also operating during the transient activation of S6 kinase in Xenopus oocytes induced by insulin. Since XMAP kinase activates S6 kinase in vitro, it is a good candidate to function in vivo as a S6 kinase kinase. The mechanism of activation of XMAP kinase in the cell entering M-phase implicated serinelthreonine phosphorylations since the dephosphorylation of serinelthreonine is sufficient to inactivate XMAP kinase. The phosphotyrosyl phosphatase activites of PCSLphosphatase or type-1B phosphatase do not affect the XMAP kinase, suggesting that tyrosine phosphorylation may not be implicated. However, it cannot be excluded that a more specific phosphotyrosyl phosphatase could lead to inactivation of XMAP kinase, or that tyrosine phosphorylation is the permissive factor to allow the serine/ threonine residues, crucial for the activity change, to become phosphorylated. A major challenge will be to identify the serinelthreonine kinase responsible for XMAP kinase activation.

641 In conclusion, XMAP kinase, whatever the mechanism of its activation, may participate to the cascade of kinase activation leading to phosphorylation of MAP and spatial microtubular distribution. It could be also involved via the activation of Ss kinase in the control of protein synthesis [68, 691 and the phosphorylation of lamin [70,71] which is known to be correlated with nuclear envelope breakdown [72]. In Anderson et al. [73], the authors show that MAP kinase from 3T3 cells stimulated by EGF, can be activated equally well by the PCS (2A) phosphatase acting as a phosphothreonyl phosphatase, as by the phosphotyrosyl-specific CD45 phosphatase, the soluble phosphatase 1B being much less effective. These data endorse our conclusions, while substantiating the importance of tyrosyl phosphorylation in the regulation of MAP kinase in EGF-stimulated 3T3 cells. However, the role of tyrosyl phosphorylation in the regulation of XMAP kinase during meiotic maturation of the Xenopus oocyte remains an open question. Okadaic acid was a kind gift from Dr J. Tsukitani (Fujisawa Co, Osaka, Japan). We thank Dr A. Fellous for providing the purified MAP2 and for her critical advice. We wish to thank R. Poulhe for stimulating discussion and material support. We are grateful to R. Verbiest, R. Bollen and E. Lefrangois for the expert technical assistance. This research was supported by Institut Nationalde la Recherche Agronomique, Centre National de la Recherche Scientifique, Institut National de la SantC et de la Recherche MCdicale and Ministere de la Recherche et de la Technologie (France) as well as from the Nationaal Fonds Wetenschappelijk Onderzoek, Fonds voor Geneeskundig Wetenschappelijk Onderzoek and Onderzoeksfonds Katholieke Universiteit, Leuven (Belgium).

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