THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1988 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol . 263, No. 25, Issue of September 5, pp. 12721-12727,1988 Printed in U.S.A.

Characterization of Insulin-stimulated Microtubule-associated Protein Kinase RAPID ISOLATION AND STABILIZATION OF A NOVEL SERINE/THREONINE KINASE FROM 3T3-Ll CELLS*

(Received for publication, February 2, 1988)

L. Bryan Ray$ and Thomas W. SturgillQ From the Departments of Znternal Medicine and Pharmacology, Uniuersity of Virginia School of Medicine, Charlottesuille, Virginia 22908

A protein kinase, termed microtubule-associated protein (MAP) kinase, which phosphorylates microtu- bule-associated protein 2 (MAP-2) in vitro and is stim- ulated 1.5-3-fold in extracts from insulin-treated 3T3- L1 cells has been identified (Ray, L. B., and Sturgill, T. W. (1987) Proc. Natl. Acad. Sei. U. S. A. 84,1502- 1506). Here, we describe chromatographic properties of MAP kinase and provide biochemical characteriza- tion of the partially purified enzyme. Isolation of the enzyme is facilitated by its unusually high affinity for hydrophobic interaction chromatography matrices. The molecular weight of the partially purified enzyme was determined to be 35,000 by gel filtration chro- matography and 37,000 by glycerol gradient centrif- ugation. MAP kinase activity of chromatographic frac- tions correlated precisely with the presence of a 40- kDa phosphoprotein detected by sodium dodecyl sul- fate-polyacrylamide gel electrophoresis. MAP kinase has a K,,, of 7 I.~M for ATP and does not utilize GTP. Acetyl-coA carboxylase, ATP citrate-lyase, casein, histones, phosvitin, protamine, and ribosomal protein S6 were all poor substrates relative to MAP-2. The enzyme is inhibited by fluoride and &glycerol phos- phate but not by heparin. These properties of MAP kinase distinguish it from protein kinases previously described in the literature.

One of the consequences of exposure of a variety of cell types to insulin is increased phosphorylation of certain pro- teins. These proteins include the insulin receptor, ribosomal protein S6, ATP citrate-lyase, acetyl-coA carboxylase, and a number of other proteins of unknown function (1). Modifi- cation of the phosphorylation state of enzymes is well docu- mented as an effective regulatory mechanism (2). To under- stand this mechanism of intracellular signaling by insulin, it is necessary to biochemically characterize both the substrates for increased phosphorylation and the protein kinases or phosphatases which mediate these effects. The insulin recep- tor is a protein kinase with specificity for tyrosine residues

* These investigations were supported by Grant BC-546 from the American Cancer Society and by a grant from the Jeffress Founda- tion. Additional support was provided by the University of Virginia Diabetes Research Center (Grant DK-38942) and the Cancer Center (Grant CA-44579). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Supported by a postdoctoral fellowship from the Juvenile Dia- betes Foundation, International.

Q To whom correspondence should be addressed.

(3). Since most of the substrates for increased phosphoryla- tion induced by insulin are found to be phosphorylated on serine or threonine residues, activation of the receptor kinase alone cannot account for these modifications. Thus, it has often been proposed that activation of a cascade of protein kinases may occur, beginning with the insulin receptor and resulting in the activation of one or more serine/threonine kinases (e.g. Refs. 4 and 5).

Many reports have established that insulin can activate at least one serine/threonine kinase which phosphorylates ri- bosomal protein S6 (5-10). S6 kinases have been purified to near homogeneity from chicken embryos ( l l ) , bovine liver (12), and Swiss mouse 3T3 cells (13). These enzymes exhibit properties (e.g. M, = 65,000) similar to those of the insulin- stimulated enzymes from chicken embryo fibroblasts or 3T3- L1 cells. An S6 kinase (S6 kinase 11) has also been purified to homogeneity from Xenopus eggs (14). The amphibian en- zyme (Mr = 90,000) appears to be related to S6 kinase in higher vertebrates since antibodies to the Xenopus enzyme can also immunoprecipitate S6 kinase activity from avian cells. Very recently, it has been reported that S6 kinase I1 and the mitogen-activated S6 kinase from 3T3 cells can be inac- tivated by dephosphorylation with protein phosphatase 2A (13, 15). These findings strongly support the hypothesis that S6 kinases are activated by phosphorylation on serine or threonine residues.

We have identified an insulin-sensitive protein kinase from 3T3-Ll cells that phosphorylates microtubule-associated pro- tein 2 (MAP-2)’ in vitro on serine and threonine residues (16, 17). The enzyme, referred to hereafter as MAP kinase, is fully activated within 10 min of exposure of these cells to insulin. Insulin-stimulated MAP kinase activity is extremely labile unless phosphatase inhibitors are included in the buffer used for its isolation. The phosphatase inhibitor most effective in protecting MAP kinase wasp-nitrophenyl phosphate, a struc- tural analog of phosphotyrosine. This observation, along with our finding that phosphotyrosine also had a protective effect while phosphoserine and phosphothreonine were ineffective, led us to speculate that activation of MAP kinase in cells treated with insulin involved phosphorylation of the enzyme, possibly on tyrosine (17). Most recently, we have presented evidence that MAP kinase is phosphorylated in vivo on tyro- sine and threonine (18). In addition we have demonstrated that MAP kinase phosphorylates and activates ribosomal

The abbreviations used are: MAP, microtubule-associated pro- tein; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid EGTA, [ethylenebis(oxyethylenenitrilo)]tetraacetic acid BSA, bo- vine serum albumin; SDS, sodium dodecyl sulfate; FPLC, fast protein liquid chromatography.

12721

12722 Insulin-stimulated MAP Kinase

protein S6 kinase I1 from Xenopus eggs in uitro.’ These results are consistent with the possibility that MAP kinase may function in a cascade of protein kinases activated by insulin.

To perform the studies described above on the mechanism of activation of MAP kinase and its function in intracellular signaling by insulin, it was necessary to rapidly isolate the activated enzyme from insulin-treated cells. In this report, we describe th chromatographic properties of MAP kinase which we have utilized to separate the activated form of MAP kinase from contaminating kinases and phosphatases and allow its storage in an active form for later use. Also, we present data characterizing other biochemical properties of MAP kinase which establish it as a novel enzyme, distinct from protein kinases previously described in the literature.

EXPERIMENTAL PROCEDURES

Cell Culture and Preparation of Extract Supernatants”BT3-Ll cells were grown and differentiated by the methods of Rubin et al. (20) and used on days 5-8. Plates of cells were washed with Krebs- Ringer bicarbonate-Hepes buffer, treated with insulin, harvested, and disrupted as previously described (17). After homogenization in buffer A (25 mM Tris, pH 7.5 at 5 “C, 25 mM NaC1, 40 mM p-nitrophenyl phosphate, 2 mM EGTA, 1 mM dithiothreitol, 0.2 mM phenylmeth- ylsulfonyl fluoride), the cell extract was centrifuged at 30,000 X g for 5 min, and the supernatant, hereafter referred to as an “extract supernatant,” was removed.

Kinase Assays-MAP-2, purified from bovine brain by the method of Kim et al. (21), was used as substrate. In all assays of MAP kinase other than those to determine substrate specificity described below, 5-pl aliquots of the enzyme were incubated in a final volume of 40 p1 for 10 min in the presence of 50 mM @-glycerol phosphate, pH 7.5, 1 mM dithiothreitol, 10 mM magnesium acetate, 0.1 mg/ml MAP-2, and 40 p~ [y3’P]ATP (5 cpm/fmol). Phosphorylated MAP-2 was re- covered from SDS-polyacrylamide gels as previously reported (17). Assays to determine substrate specificity of the purified fraction were performed at 30 “C for 15 min in a 60-pl reaction volume containing 5 pl of the enzyme fraction, 50 mM @-glycerol phosphate, pH 7.5, 1 mM dithiothreitol, 10 mM magnesium acetate, 40 p M [T-~’P]ATP (1 cpm/fmol), and either 0.1 mg/ml MAP-2 in the presence of 1 mg/ml BSA as carrier or 0.5 mg/ml histone IIA or 111s (Sigma), histone HA2B or HF2B (Worthington), dephosphocasein (Sigma), phosvitin (Sigma), protamine sulfate (Lilly), or 40 S ribosomal subunits from Artemia salina (17). Reactions were terminated by applying 40 p1 to a filter (Whatman GF/C, 2.5 cm) which was immediately immersed in a solution of 20% trichloroacetic acid, 60 mM sodium pyrophos- phate. The filters were washed, dried, and counted as described by Glover et al. (22). For ribosomal protein S6, the reaction was stopped with 3 X Laemmli SDS sample buffer and the phosphorylated protein recovered from SDS gels as described (17). Acetyl-coA carboxylase and ATP citrate-lyase (generously provided by Drs. W. Benjamin, SUNY, Stony Brook and L. Witters, Dartmouth) were assayed at concentrations of 0.2 mg/ml and resolved on 7% SDS gels. The amount of 32P incorporated was quantitated by liquid scintillation spectrometry of the stained excised bands. Gels containing ATP citrate-lyase were washed in 1 N HCl, 10% acetic acid for 4 h to release acid-labile phosphohistidine prior to staining.

Chromatography-All chromatography was performed in a cold room at 5 “C. DEAE-cellulose (Whatman DE52) was equilibrated with buffer A. Extract supernatants from cells treated with or without insulin (80 nM for 10 min) were applied to the column which was then washed with 3 ml of buffer A and eluted with an 8-ml gradient of 25-500 mM NaCI. Fractions were immediately assayed for MAP kinase activity.

A phenyl-Superose HR 5.5 FPLC column (Pharmacia LKB Bio- technology Inc.) was equilibrated with buffer A containing 250 mM NaCl. Extract supernatants from control or insulin-treated cells (in the same buffer) were passed through a 0.2-pm Millex-GV filter (Millipore Corp.) and applied to the column. The column was washed with 6 ml of buffer and eluted with a 12-ml gradient of decreasing concentration of NaCl, 250-25 mM, and simultaneously increasing concentration of ethylene glycol, 0-60%. The flow rate was initially 0.2 ml/min and was gradually reduced to 0.05 ml/min during the

Sturgill, T. W., Ray, L. B., Erikson, E., and Maller, J. L. (1988) Nature, in press.

elution to avoid excessive back pressure induced by the high viscosity of the eluting buffer. All fractions were assayed for MAP kinase activity.

Gel filtration chromatography was carried out using a Superose 12 FPLC column (Pharmacia LKB Biotechnology Inc.). The column was equilibrated with buffer A containing 10% glycerol. Cells (3 plates) were homogenized in 150 pl/plate of the column buffer. Extract supernatants were filtered, as above, and 200 pl was applied to the column. Column fractions were assayed as above.

Storage of Active Enzyme-Peak fractions from phenyl-Superose chromatography were combined and BSA (Pentex, crystallized, Miles Laboratories Inc.) added to yield 0.3 mg of protein/ml. This sample was applied to a Centricon microconcentrator (Amicon), centrifuged as recommended by the manufacturer for 1.5-2 h, collected, and stored at -70 “C. Samples were concentrated 3-4-fold by this proce- dure, and recovery of enzymatic activity was usually 80% or better.

Glycerol Gradient Sedimentation-Peak fractions from phenyl-Su- perose chromatography were concentrated in the presence of BSA (6 mg/ml, final). Aliquots (100 pl) to which 5 p1 of [14C]carbonic anhy- drase (5 pCi/ml, Du Pont-New England Nuclear) had been added were applied to 4 ml of linear 10-40% glycerol gradients prepared in buffer A. After centrifugation at 50,000 rpm for 16 h in an SW 55Ti rotor, six drop fractions were collected from the bottom of the tube. Fractions from the same gradient were assayed for MAP kinase activity and for the presence of the molecular weight standards. The presence of [“Clcarbonic anhydrase (Mr 30,000) was determined by liquid scintillation spectrometry, and the migration of BSA (Mr 68,000) was detected by assay of the fractions for total protein content by the method of Bradford (Bio-Rad (23)), since the BSA added as carrier was the principal protein present.

Expression of Results-All experiments presented under “Results” are representative of two or more independent experiments.

Materials-Bovine insulin and protamine sulfate were gifts from Lilly. [Y-~’P]ATP was synthesized by the method of Johnson and Walseth (24); [Y-~’P]GTP was from Du Pont-New England Nuclear.

RESULTS

Chromatographic Properties of Insulin-stimulated MAP Ki- nase-Extract supernatants (see “Experimental Procedures”) of 3T3-Ll cells which have been treated with insulin (80 nM) for 10 min contain MAP kinase activity that is stimulated 1.5-3-fold above that in extracts of control cells (17). The increased activity persisted after chromatography of such extracts on DEAE-cellulose (Fig. 1). The insulin-stimulated MAP kinase activity was retained on the column and eluted by a linear gradient of NaC1. A single peak of kinase activity was eluted at 0.25 M NaCl and was stimulated approximately 4-fold by prior treatment of the cells with insulin. A minor

FIG. 1. DEAE-cellulose chromatography of MAP kinase. Extract supernatants (2 ml, 0.7 mg/ml protein) from 4 plates of cells treated with 80 nM insulin (0) or diluent (0) for 10 min were applied to a 1.5-ml column equilibrated with buffer A. The column was washed and eluted with a gradient of 25-500 mM NaCl. Fractions (0.5 ml) were collected and assayed for MAP kinase activity. The insulin- treated and control supernatants catalyzed the incorporation of 8620 and 2720 cpm of 32P into MAP-2, respectively. FT, flow-through; W, wash.

Insulin-stimulated MAP Kinase 12723

portion of the MAP kinase activity eluted during washing of the column.

Initial experiments examining the behavior of MAP kinase during hydrophobic interaction chromatography indicated that the insulin-stimulated activity interacted strongly with phenyl-Sepharose and phenyl-Superose, being adsorbed in buffers with low salt concentrations and requiring high con- centrations of ethylene glycol for elution. Anticipating the necessity of a rapid method of preparing partially purified fractions of the kinase, we tested the feasibility of applying peak fractions from DEAE-cellulose chromatography directly to a phenyl-Superose FPLC column. Extract supernatants from insulin-treated and control cells prepared in buffer con- taining 0.25 M NaCl were applied to a phenyl-Superose col- umn. The column was washed and then eluted with a gradient of simultaneously increasing (0-60%) ethylene glycol and decreasing (250-25 mM) NaCl. Under these conditions, a considerable portion of the MAP kinase activity was not adsorbed to the column and eluted during washing (Fig. 2). However, chromatography of extracts from insulin-treated cells yielded a single peak of activity which was retained and subsequently eluted by 30% ethylene glycol. Essentially no kinase activity was detectable in corresponding fractions when extracts of untreated control cells were subjected to identical chromatography. About 40-50% of the insulin-stim- ulated activity phosphorylating MAP-2 recovered from the column was not bound and eluted during washing. This ma- terial was not retained when subsequently applied to a second phenyl-Superose column.

Physical Properties-The apparent molecular weight of the insulin-stimulated MAP kinase was determined by gel filtra- tion chromatography and glycerol gradient ultracentrifuga- tion. Extract supernatants of 3T3-Ll cells were applied to a Superose 12 FPLC column. MAP kinase activity was found primarily in a peak eluting just prior to carbonic anhydrase with an apparent molecular weight of 35,000 (Fig. 3). A smaller peak of activity exhibiting a less pronounced stimu- lation by insulin was usually observed eluting earlier with an apparent molecular weight of 150,000-200,000. Additional experiments (not shown) verified that MAP kinase eluted from phenyl-Superose with ethylene glycol also had a M, of 35,000 on gel filtration. Peak fractions of MAP kinase from phenyl-Superose chromatography of extracts from insulin- treated cells were concentrated (see “Experimental Proce-

*

t t I

FIG. 2. Phenyl-Superose chromatography of MAP kinase. Extract supernatants (3.5 ml, 0.8 mg/ml protein) from 5 plates of insulin-treated (0) or control cells (0) were applied to phenyl-Super- ose as described under “Experimental Procedures.” The column was washed and then eluted with a gradient of simultaneously decreasing concentration of NaCl (250-25 mM) and an increasing concentration of ethylene glycol (0-60%). Fractions (0.5 ml) were collected and assayed as described. The insulin-treated and control supernatants catalyzed the incorporation of 4030 and 1290 cpm of 3zP into MAP- 2, respectively. W, wash.

O’ ’ I b 16 ’ 1’8 ’ 2 b ’ 212 ’ 2; Ve/ V o

FIG. 3. Gel filtration chromatography of MAP kinase. Ex- tract supernatants (200 p1, 0.8 mg/ml protein) from 3 plates of cells exposed to 80 nM insulin (0) or diluent (0) for 10 min were applied to a Superose-12 column. Fractions (0.3 ml) were collected and assayed as described. The positions of elution of marker proteins (ferritin, 470 kDa; alcohol dehydrogenase, 150 kDa; carbonic anhy- drase, 30 kDa; or-chymotrypsinogen, 25 kDa; myoglobin, 17 kDa; vitamin BIZ, 1.3 kDa) are indicated by the squares. The insulin- treated and control supernatants catalyzed the incorporation of 15,910 and 6,060 cpm of 32P into MAP-2, respectively.

FIG. 4. Glycerol gradient sedimentation of MAP kinase. Peak fractions from phenyl-Superose chromatography of extracts of cells treated with insulin for 10 min were concentrated and centri- fuged through a glycerol gradient. Fractions were collected from the bottom of the tube (left) and assayed for MAP kinase activity (0) or the presence of marker proteins, bovine serum albumin, M, = 67,000 (m), ~r,[’~C]carbonic anhydrase, M, = 30,000 (A) as described under “Experimental Procedures.”

dures”) and applied to a glycerol gradient. Upon sedimenta- tion, the MAP kinase was found to migrate with a M, of 37,000 (Fig. 4).

Although the insulin-stimulated MAP kinase activity in extracts of 3T3-Ll cells is unstable (17), the peak fractions from phenyl-Superose chromatography retained activity when frozen at -70 “C in the presence of 1 mg/ml BSA. Such stored fractions have been used to further characterize the kinase. Typically, these have been prepared by applying the crude extract supernatant to DEAE-cellulose which is then washed and eluted with buffer containing 350 mM NaCl. The DEAE eluate is then applied to phenyl-Superose, eluted as described above, and the peak fractions collected and concentrated in the presence of BSA.

In some experiments the concentrated peak fractions from phenyl-Superose chromatography were further purified by gel filtration chromatography. In the experiment shown in Fig. 5, MAP kinase was isolated from cells labeled with 32Pi. Resolution of the proteins in fractions eluting from Superose- 12 by SDS-polyacrylamide gel electrophoresis and subsequent silver staining of the gels revealed that the principal stained band (not attributable to BSA added as carrier) is a 40-kDa protein (Fig. 5A). The amount of the 40-kDa protein present

12724 Insulin-stimulated MAP Kinase

17 18 19 20 21 22 A. FRACTION E 17 18 19 20 21 22

kDa - &m0""66

"-97 - -

\ I 9-43 / \

" 3 6 -

"-3 1 -

a.22 -

FIG. 5. Polyacrylamide gel electrophoresis of the MAP ki- nase preparation after ion exchange, hydrophobic interac- tion, and gel filtration chromatography. MAP kinase was iso- lated from 10 plates of cells which had been labeled with "Pi (2 mCi/ plate in 8 ml) for 2 h and then exposed to insulin (80 nM) for 10 min. Chromatography on DEAE-cellulose, phenyl-Superose, and Super- ose-12 was performed as described under "Experimental Procedures." Fractions eluted from the gel filtration chromatography column were concentrated and applied to a 12% SDS gel. The silver-stained gel is shown in panel A. The heavily stained band is BSA added prior to gel filtration chromatography. The 40-kDa phosphoprotein which co- elutes with MAP kinase is identified by the arrows. An autoradiogram made by exposing the gel to X-Omat AR film (Kodak) for 3 days a t -70 "C with a Lightning Plus intensifying screen (Du Pont) is shown in panel R. The top of the gel and the dye front are indicated by arrows in B. The same fractions, 17-22, were assayed for MAP kinase activity. They catalyzed the incorporation of 80, 1070, 2820, 2650, 1140, and 580 cpm of "P into MAP-2, respectively.

in each fraction correlates precisely with the level of MAP kinase activity in that fraction (Fig. 5). As described in detail elsewhere (18), the 40-kDa protein is phosphorylated in vivo on tyrosine and threonine. After the gel filtration step, the 40-kDa protein is the only phosphorylated band detected (Fig. 5B).

Subcellular Localization-Assays of extract supernatants and the corresponding pellets from insulin-treated or control cells indicate that MAP kinase is primarily cytosolic. In a representative experiment, a resuspended particulate fraction from cells treated with insulin catalyzed the transfer of only 11% (1,163 cpm) of the 32P transferred from [y3'P]ATP to MAP-2 by the supernatant (10,892 cpm). MAP kinase activity in the supernatant fractions was stimulated 2.5-fold by insulin treatment while that in the particulate fraction was stimu- lated only 1.5-fold. To test whether high ATPase activity or other factors in the membrane fraction might interfere with the assay, the pellet and supernatant fractions were mixed and assayed. The kinase activity of the supernatant fraction was only slightly diminished by the addition of the insoluble fraction (9,383 cpm), indicating that the assay did accurately measure the activity of MAP kinase present in the insoluble fraction.

Kinetic Analysis-Kinetic studies were performed on par- tially purified fractions of MAP kinase. In assays using MAP- 2 (0.1 mg/ml) as substrate in the presence of 10 mM magne- sium, ATP was the preferred phosphate donor. The transfer of phosphate from [y-"P]GTP was negligible compared to the rates obtained using ATP (Fig. 6). The K, for ATP was 7 PM under these conditions (Fig. 6, inset).

MAP-2 was dialyzed against assay buffer and assayed under standard conditions at concentrations of 0.06-1 mg/ml. MAP- 2 was utilized as a substrate with apparent K,,, of 4-12 PM (range = K,,, determined by linear regression analysis k S.E., not shown).

Substrate Specificity-Partially purified MAP kinase was assayed for phosphorylation of proteins known to be sub-

.I .9 .8 -

-

1 I [ATP], pM 0

I I t 1 1 1 1 1 1 1 1 1 1 , 1 ~

10 ,,w 15 2 0 25 5

FIG. 6. Comparison of ATP and GTP as phosphate donors for MAP kinase activity. MAP kinase from insulin-treated cells was prepared by phenyl-Superose chromatography and assayed under standard conditions with the indicated concentrations of [y3'P]ATP (0) or [y3*P]GTP (8,000 cpm/pmol) (0). Kinetic analysis of the affinity of MAP kinase for ATP is shown in the inset.

TABLE I Substrate specificity of partially purified MAP kinase

MAP kinase was prepared from insulin-treated cells by DEAE- cellulose and phenyl-Superose chromatography. Stored peak fractions were assayed as described under "Experimental Procedures." Results are expressed as percent of incorporation of 32P into MAP-2.

Substrate Activity

% MAP-2 100 Acetyl-coA carboxylase 0 ATP citrate-lyase 0 Casein 8 Histone IIA 3 Histone HA2B 1 Histone 111s 2 Histone HF2B 4 40 S ribosomal subunits 0

pgmlml 5 50 1oc

0 40 80 120 16C mM

FIG. 7. Effects of KF, &glycerol phosphate, and heparin on the activity of MAP kinase. MAP kinase was prepared from insulin-treated cells by DEAE-cellulose and phenyl-Superose chro- matography and assayed as described in the presence of the indicated concentrations (in mM) of KF (O), &glycerol phosphate (A), or (in pg/ml) of heparin (B). Data are representative of several experiments. Incorporation of "1' into MAP-2 in the absence of added factors ranged from 16,600 to 22,400 cpm.

strates for other protein kinases. MAP-2 was preferentially phosphorylated. Phosphorylation of casein by MAP kinase preparations was detected but resulted in incorporation of less than 10% of the 32P transferred to MAP-2. Phosvitin, protamine, histones, ribosomal protein S6, acetyl-coA car- boxylase, and ATP citrate-lyase were phosphorylated poorly, if a t all, by MAP kinase (Table I).

Other Biochemical Properties of MAP Kinase; Effects of pH,

Insulin-stimulated MAP Kinase 12725

Salts, and Inhibitors-Extract supernatants from insulin- treated and control cells were assayed for MAP kinase activity over a range of pH. Optimal activity was observed in a broad peak around pH 8 (data not shown).

The effects of several compounds known to inhibit certain protein kinases were tested (Fig. 7). Phosphorylation of MAP- 2 by partially purified MAP kinase is inhibited by KF. Activity of the kinase is reduced approximately 50% when 60 mM KF is present in the assay mixture. &Glycerol phosphate is also a potent inhibitor of the enzyme. Half-maximal inhibition was observed with 45 mM @-glycerol phosphate. Heparin had no effect at 0.5-5 pg/ml, but higher concentrations had a slight stimulatory effect on incorporation of 32P into MAP-2.

DISCUSSION

We have identified a soluble protein kinase from 3T3-Ll cells which phosphorylates microtubule-associated protein 2 from bovine brain in vitro (16, 17). The enzyme is present in 3T3-Ll cells, BC3-H1 and 23A2 myocytes, H4 hepatocytes, and freshly isolated rat adipocytes and hepatocytes (25). In each cell type, the kinase is rapidly stimulated by treatment of intact cells with insulin. The partial purification and char- acterization of MAP kinase described here allow the distinc- tion of the enzyme from previously described insulin-stimu- lated protein kinases and also from other kinases which have been isolated from mammalian tissues.

Very recently, a protein kinase which phosphorylates MAP- 2 in vitro has been detected in extracts of human lung fibro- blastic (TIG-3) cells (26). That activity was increased follow- ing treatment of cells with insulin or other growth factors and, like MAP kinase, was not adsorbed by phosphocellulose. Curiously, the enzyme from TIG-3 cells was either inhibited or inactivated when low concentrations ( 4 pM) of calcium were added to cell extracts. Under the conditions employed in our experiments, both MAP kinase activity in extract supernatants and the highly purified enzyme are insensitive to micromolar concentrations of calcium (data not shown). Further study of the TIG-3 cell enzyme will be required to determine its relationship to MAP kinase.

Insulin and other growth factors stimulate one or more protein kinases which phosphorylate the ribosomal protein S6 in vitro (5-10). Although S6 kinase activity elutes from DEAE-cellulose near MAP kinase, no S6 kinase activity is adsorbed to phenyl-Superose, and we have been unsuccessful in detecting S6 kinase after gel filtration chromatography (data not shown). Phosphorylation of S6 was not detected in assays of partially purified MAP-2 kinase. Furthermore, we have shown in collaboration with J. Maller's laboratory' that MAP kinase can phosphorylate and activate S6 kinase 11, the ribosomal protein S6 kinase that group has purified to ho- mogeneity from Xenopus Zaevis eggs. Other kinases, including the insulin receptor kinase, pp60"-"", the tyrosine kinase as- sociated with the Abelson murine leukemia virus, protein kinase C, the catalytic subunit of cyclic AMP-dependent protein kinase, and casein kinases I and 11, failed to activate S6 kinase I1 (data not shown). Thus, the present findings support our previous conclusion (17) that insulin-stimulated phosphorylation of MAP-2 and S6 is mediated by distinct enzymes.

A soluble Mn2+-dependent serine kinase from rat adipocytes which is stimulated by insulin has been characterized (27). The Mn'+-dependent enzyme was much less active when Mg2+ was substituted for Mn2+ and exhibited insulin-stimulated activity only within a narrow concentration range of Mg2+ around 1 mM, about 10-fold less M F than we used in our assays. The apparent molecular weight of that enzyme as

determined by gel filtration chromatography was 50,000- 60,000. Our determinations of the molecular weight of MAP kinase by gel filtration chromatography and glycerol gradient centrifugation indicate that it is considerably smaller (Mr = 35,000-40,000). The Mn'+-dependent enzyme is apparently not inhibited by concentrations of fluoride (60 mM) which strongly inhibit MAP kinase. Also, MAP kinase does not appreciably phosphorylate ATP citrate-lyase and histones which are preferred substrates of the Mn2+-dependent en- zyme. Therefore, the Mn'+-dependent enzyme and MAP ki- nase are likely to be distinct enzymes.

A membrane-bound serine/threonine kinase from rat adi- pocytes which phosphorylates exogenous histone has also been described (28). This activity was increased by treatment of isolated cells with insulin. We have found MAP-2 kinase to be primarily located in the cytosol and to phosphorylate histones poorly, if at all. These properties of these insulin- stimulated kinases are dissimilar, but further characterization of the microsomal enzyme will be required to allow a more definitive comparison of these activities.

We have considered the relationship of MAP kinase to casein kinases I and 11, both of which have some properties in common with MAP kinase. Casein kinase I is known to phosphorylate MAP-2 (29). Recently, casein kinase I1 has been shown to be activated in 3T3-Ll adipocytes upon expo- sure of those cells to insulin (30). Casein kinase I is of similar size to MAP kinase as is the catalytic subunit of casein kinase I1 (31, 32). The most obvious dissimilarity between MAP kinase and these enzymes is that casein is not a good substrate for MAP kinase. While casein kinase I1 utilizes both GTP and ATP, MAP kinase prefers ATP. Heparin, a potent inhib- itor of casein kinase 11, has no inhibitory effect on MAP kinase. We have shown elsewhere that MAP kinase is not adsorbed by phosphocellulose (17), and we have found that highly purified casein kinase I (received from E. Itarte, Uni- versitat Autonoma, Bellaterra, Barcelona, Spain) does adhere to that matrix under the same conditions. These observations clearly distinguish MAP kinase from casein kinases I and 11.

Two protein kinases, protin kinase C and protease-acti- vated kinase 11, have been described which are activated in vitro by limited proteolysis. The smaller form of protease- activated kinase is reported to have a molecular weight larger (45,000-55,000) than that of MAP kinase (33). Unlike MAP kinase, the protease-activated kinase adheres to phosphocel- lulose in buffers containing low salt and readily phosphoryl- ates ribosomal protein S6. MAP kinase is also smaller than protein kinase M (Mr = 64,000), the proteolytically activated form of protein kinase C (34). Furthermore, MAP kinase does not phosphorylate protamine, a preferred substrate of protein kinase M (34). Based on the above analysis, we conclude that MAP kinase is a novel serine/threonine kinase not previously described in the literature.

The procedures described here allow the rapid purification of MAP kinase away from contaminating kinases and phos- phatases. The absence of other kinases is evidenced by the poor phosphorylation of substrates such as histone or casein by the purified fractions. When assays of MAP kinase using MAP-2 as substrate are stopped by the addition of excess ATP and subjected to continued incubation at 30 'C, no loss of 3'P incorporated into MAP-2 is detected (data not shown). Thus, the partially purified fraction is devoid of phosphatases with activity toward the site(s) on MAP-2 phosphorylated by MAP kinase.

For several reasons it is not possible to calculate the -fold purification of MAP kinase achieved by the purification steps described. In the crude extract supernatants, there are several

12726 Insulin-stimulated MAP Kinase

contaminating kinases which are known to phosphorylate MAP-2 in vitro. A significant proportion (40-50%) of the insulin-stimulated kinase activity that phosphorylates MAP- 2 in vitro is not retained on phenyl-Superose. This activity may represent other insulin-regulated kinases (e.g. ribosomal protein S6 kinase(s), casein kinase 11) or a form of MAP kinase with lower affinity for the hydrophobic matrix. It is, therefore, difficult to quantitate the activity in cell extracts which is contributed by MAP kinase. Furthermore, the small amounts of protein obtained from 10 plates of cultured cells are such that the protein concentration of fractions eluted from phenyl-Superose chromatography is nearly undetectable by standard assays. These fractions are supplemented with BSA prior to further purification by gel filtration chromatog- raphy.

In spite of these problems, it is clear that the fractions obtained are highly purified. As mentioned above, the frac- tions are free of contaminating kinases and phosphatases. In partially purified preparations of MAP kinase from 32P-la- beled cells, the 40-kDa phosphoprotein correlated with MAP kinase (see below) is the only remaining phosphoprotein band detected by autoradiography of SDS gels. Silver staining of such gels reveals that the 40-kDa band is a major component of such fractions.

Several observations indicate that the mechanism of acti- vation of MAP kinase may involve covalent modification rather than allosteric regulation. The increased activity of MAP kinase in extracts from insulin-treated cells is main- tained during gel filtration, ion exchange, or hydrophobic interaction chromatography. Even incubation of extract su- pernatants with a high concentration of NaCl(0.5 M) prior to rapid gel filtration by a centrifuge-column procedure, a ma- nipulation which completely reversed the activation of acetyl- CoA carboxylase from insulin-treated cells (35), failed to reduce the insulin-stimulated activity of MAP kinase (data not shown). However, these observations do not exclude the possibility of allosteric regulation of a larger inactive complex to yield an activated 40-kDa subunit. Insulin-induced relocal- ization of the enzyme cannot account for the increased activ- ity in the soluble fraction since we found low levels of MAP kinase activity in pellets containing insoluble material from homogenized 3T3-Ll cells and no evidence of translocation of the activity upon stimulation of cells with insulin.

We have recently obtained additional evidence favoring the view that MAP kinase may be activated by phosphorylation. When MAP kinase is prepared by the methods described in this report from 3T3-Ll cells labeled with 32Pi, a phosphopro- tein which migrates during SDS-polyacrylamide gel electro- phoresis with an apparent molecular mass of 40 kDa is found to co-purify with MAP kinase through each step of the puri- fication ((18) Fig. 5 ) . The 40-kDa phosphoprotein contains phosphotyrosine and phosphothreonine. The apparent molec- ular weight of the phosphoprotein is consistent with the values obtained here by gel filtration chromatography and glycerol gradient sedimentation. Like MAP kinase activity, the 40- kDa protein is detected in eluates from phenyl-Superose, by either autoradiography or silver staining, only when chroma- tography is performed on extracts from cells treated with insulin. Taken together, these observations provide strong evidence, but do not prove, that MAP kinase and the 40-kDa phosphoprotein are identical.

The observation that neither MAP kinase activity nor the 40-kDa phosphoprotein is found in extracts from control cells after phenyl-Superose chromatography is of interest. Since MAP kinase is very rapidly activated by insulin and its activation is not blocked by pretreatment of the cells with

cycloheximide (10 pg/ml) for 30 min (data not shown), de novo synthesis of the enzyme is unlikely to account for the appearance of the protein. The most likely explanation, then, is that MAP kinase undergoes post-translational modification (e.g. phosphorylation, proteolysis) in cells stimulated with insulin. This would result in adsorption of the activated form of the enzyme (presumably the 40-kDa phosphoprotein) to phenyl-Superose while the less active precursor would not be retained. Selective adsorption of the active form of MAP kinase by phenyl-Superose could result from a conformational change which exposed a hydrophobic region(s) of the poly- peptide. Examples of such behavior have been described for calmodulin and calpain which display calcium-dependent binding to phenyl-Sepharose (19, 36).

MAP kinase exhibits unusually high affinity for hydropho- bic interaction chromatography matrices. While most cyto- solic proteins bindphenyl-Superose only in buffers containing high concentrations of salt, MAP kinase is adsorbed in buffer containing relatively low salt concentration and requires high concentrations of ethylene glycol for elution. This character- istic of the kinase results in substantial purification during hydrophobic interaction chromatography. The properties de- scribed above will make hydrophobic interaction chromatog- raphy useful in analyzing the mechanism of activation of MAP kinase by insulin.

Further studies are underway in this laboratory to elucidate the manner in which MAP kinase is activated in cells treated with insulin. It is, of course, possible that other modifications of the enzyme besides or in addition to phosphorylation are important to its activation. The mode of activation of MAP kinase is of particular interest in light of our recent findings that MAP kinase can phosphorylate and activate ribosomal protein S6 kinase I1 from Xenopus eggs i n vitro2 and is itself phosphorylated on tyrosine and threonine (18). Those results suggest that MAP kinase may participate in a cascade of phosphorylation reactions which are important in producing the cellular actions of insulin.

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