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Nerve growth factor-induced phosphorylation cascade in PC12 pheochromocytoma cells. Association of S6 kinase II with the microtubule-associated protein kinase, ERK1.

Nerve growth factor-induced phosphorylation cascade in PC12 pheochromocytoma cells. Association... Nerve growth factor-induced phosphorylation cascade in PC12 pheochromocytoma cells. Association of S6 kinase II with the microtubule-associated protein kinase, ERK1. E Scimeca, R Ballotti, C Scimeca, T. Nguyen, C. Filloux, E. van Obberghen To cite this version: E Scimeca, R Ballotti, C Scimeca, T. Nguyen, C. Filloux, et al.. Nervegrowth factor-induced phospho- rylation cascade in PC12 pheochromocytoma cells. Association of S6 kinase II with the microtubule- associated protein kinase, ERK1.. Journal of Biological Chemistry, 1992, 267 (24), pp.17369-74. hal-02108969 HAL Id: hal-02108969 https://hal.science/hal-02108969 Submitted on 18 Nov 2021 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Vol. 267, No. 24, Issue of August 17369-17374,1992 25, PP. THE JOURNAL OF BIOLOGICAL CHEMISTRY Printed in U.S.A. 0 1992 by The American Society for Biochemistry and Molecular Biology, Inc. Nerve Growth Factor-induced Phosphorylation Cascade in PC12 Pheochromocytoma Cells ASSOCIATION OF S6 KINASE I1 WITH THE MICROTUBULE-ASSOCIATED PROTEIN KINASE, ERK1’ (Received for publication, February 7, 1992) Jean-Claude ScimecaS, Tien T. NguyenQ, Chantal Filloux, and Emmanuel Van Obberghen From the Znstitut National de la Sante et de la Recherche Medicale U145, Facult6 de Medecine, 06107 Nice, Cedex 2, France erful tool to search for additional physiologically rel- Microtubule-associated protein (MAP) kinases form evant substrates for MAP kinase, a key integrator a group of serinelthreonine kinases stimulated by var- enzyme for growth factors and hormones. ious growth factors such as nerve growth factor (NGF) and hormones such as insulin. Interestingly, MAP kinases are thought to participate in a protein kinase cascade leading to cell growth as they have been shown to phosphorylate and activate ribosomal protein S6 During the last 5 years much research in the area of cell kinase. To further evaluate the interactions between signaling has focussed on the serine/threonine MAP kinases, the different components of this cascade, we looked at which are thought to play a central role in metabolic and the possible coprecipitation of MAP kinase activator(s) mitogenic effects induced by various extracellular stimuli. In or MAP kinase substrate(s) with MAP kinase. Using response to most growth-promoting and mitogenic factors antipeptides to the C terminus of the M, 44,000 MAP tested, as well as to many other signals such as phorbol esters kinase, ERK1, and cell extracts from unstimulated or or phosphatase inhibitors, MAP’ kinase phosphorylation is NGF-treated PC12 cells, we obtained in addition to increased on both threonine and tyrosine residues, leading to MAP kinase itself coprecipitation of a protein with a the stimulation of its kinase activity (for review see Ref. 1). M, in the 90,000 range. We further show that this While tyrosine protein kinase receptors have been implicated protein is a protein kinase since it becomes phosphoryl- in a wide variety of physiological functions, the understanding ated on serine residues, after sodium dodecyl sulfate- of the molecular mechanisms of their actions continues to polyacrylamide gel electrophoresis and transfer to a polyvinylidene difluoride membrane. In vitro phos- represent a major challenge. Dissection of the transductional phorylation performed before sodium dodecyl sulfate- cascades induced by insulin (2), EGF (3-5), and NGF (6, 7) polyacrylamide gel electrophoresis demonstrates NGF- has identified MAP kinases as a link between the tyrosine sensitive phosphorylation of this 90-kDa protein on kinase receptors of these polypeptides, and serine/threonine both serine and threonine; the serine phosphorylation phosphorylations. Although the precise role(s) of MAP is likely to be due to autophosphorylation, and the kinases remain(s) to be clarified, they have emerged as key threonine phosphorylation due to phosphorylation by “switch kinases,” i.e. serine/threonine kinases which are ca- the copurifying MAP kinase. Furthermore, immuno- pable of converting a tyrosine phosphorylation signal coming precipitation of this 90-kDa protein was obtained with from a tyrosine kinase receptor into a serine/threonine phos- antibodies to S6 kinase 11. Finally, using in situ chem- phorylation. In 1990 Cobb et al. (8, 9) obtained an almost ical cross-linking, we were able to demonstrate in in- complete sequence of an “extracellular signal-regulated kinase tact cells the occurrence of an anti-ERK1 immunoreac- 1” (ERKl), which was found to be closely related to two yeast tive species with a molecular mass of approximately kinases involved in cell cycle control, and whose properties 125,000 compatible with a complex between ERKl strongly indicated identity with previously identified MAP a 90-kDa S6 kinase. Taken together, our obser- and kinases. Moreover, their study presented evidence for the vations demonstrate that the 44-kDa MAP kinase is existence of a family of at least 4 ERK proteins with molecular associated, in intact PC12 cells, with a protein kinase weights ranging from 41,000 to 62,000 (10, 11). which is very likely to be S6 kinase 11. In conclusion, Using immunoprecipitation with antipeptides to the car- our data represent strong evidence for a physiological boxyl terminus of ERK1, we have shown that mouse fibroblast role of the MAP kinase436 kinase cascade in PC12 cells. Finally, our antipeptides provide us with a pow- ERKl was phosphorylated in vitro on both threonine and tyrosine. Moreover, this dual phosphorylation was concomi- tant with an enhanced ERKl kinase activity as measured by * This work was supported in part by funds from the Institut myelin basic protein phosphorylation (12). However, this National de la Santi et de la Recherche Midicale; the Universite de activation remained small compared to the level of ERKl Nice-Sophia-Antipolis; Grant 6760 from the Association pour la Recherche contre le Cancer; and the Ligue Nationale Fran~aise contre le Cancer, Fediration des Comites Dipartementaux, Comiti Dipar- The abbreviations used are: MAP, microtubule-associated pro- temental du Var. The costs of publication of this article were defrayed tein; BSA, bovine serum albumin; C peptide, ERKl peptide/sequence in part by the payment of page charges. This article must therefore 356-367; DTT, dithiothreitol; EGF, epidermal growth factor; ERK, be hereby marked “aduertisement” in accordance with 18 U.S.C. extracellular signal-regulated kinase; Hepes, N-2-hydroxyethylpiper- Section 1734 solely to indicate this fact. azine-N’-2-ethanesulfonic acid ISPK, insulin-stimulated protein ki- $ Recipient of a fellowship from the Ligue Nationale Francaise nase; NGF, nerve growth factor; PBS, phosphate-buffered saline; Contre le Cancer, Federation des Comitis Dipartementaux, Cornit6 PMSF, phenylmethylsulfonyl fluoride; PVDF, polyvinylidene difluo- Departemental du Var. ride; Rsk, ribosomal protein S6 kinase; SDS-PAGE, sodium dodecyl § Recipient of a postdoctoral fellowship from the Medical Research sulfate-polyacrylamide gel electrophoresis; TLC, thin-layer chroma- Council of Canada. tography; DSS, disuccinimidyl suberate. 17369 17370 NGF-induced Phosphorylation of ERKl activity observed after in uiuo treatment of cells with growth leupeptin, and 0.18 mg/ml PMSF). Cells were scraped, and proteins were solubilized for 15 min in buffer A supplemented with 1% Triton factors or hormones such as insulin. At the same time three X-100 (solubilization buffer). Cell extracts were then submitted to groups reported independently that ERKl and ERK2 ob- centrifugation (18,000 X g for 15 min), and samples were incubated tained by expression in Escherichia coli was phosphorylated for 90 min at 4 "C with anti-ERK1 antibodies adsorbed on protein in vitro on both tyrosine and threonine and that this was A-Sepharose pellets. concurrent with a slow and moderate activation of the enzyme 32P Labeling in Intact PC12 Ceh-Confluent PC12 cells, growing (13-15). To summarize, the available data suggest that, al- in 145-mm culture dishes, were starved overnight in RPMI, 0.2% BSA medium (BSA 7030 from Sigma). Cells were then washed twice though ERKl/ERK2 dual self-phosphorylation can occur and with Dulbecco's modified Eagle's medium without phosphate and might be physiologically relevant, the mechanism of ERKl incubated for 3.5 h in this medium containing 500 pCi of ["PI activation is likely to be more complex and probably involves orthophosphate (1.7 mCi/ml). At the end of labeling, NGF (6 X lo-@ other proteins such as activator(s). The latter could be protein M) was added for 5 min. After three washes with ice-cold PBS, kinases or allosteric modulators. In an attempt to address the proteins were solubilized for 15 min on ice in 50 mM Hepes, pH 7.5, phosphorylation cascade issue, we investigated here the pos- containing 150 mM NaCl, 10 mM EDTA, 10 mM Na4P,07, 2 mM sibility of a coprecipitation of protein(s) with ERK1. Using sodium orthovanadate, 100 mM NaF, 1% Triton X-100,100 units/ml aprotinin, 20 pM leupeptin, and 0.18 mg/ml PMSF. Cell extracts were PC12 cells and an antipeptide to the carboxyl terminus of then incubated for 90 min with antibodies to ERKl adsorbed on a ERK1, we immunopurified ERKl from unstimulated or NGF- protein A-Sepharose pellet. After five washes with solubilization treated cells. After elution from antipeptide precipitates, we buffer, Laemmli buffer (3% sodium dodecyl sulfate) was added to performed in vitro phosphorylation experiments. The follow- dried pellets, and proteins were submitted to SDS-PAGE under ing key observations were made: (i) NGF induced a stimula- reducing conditions. tion of 32P incorporation into a protein in the 90-kDa range; In Vitro Phosphorylation of Immunopurified ERKl-At the end of incubation with anti-ERK1 antipeptides, protein A-Sepharose pellets (ii) this phosphorylation occurred to the same extent on both were washed five times in solubilization buffer. After an additional threonine and serine residues. We next addressed the question wash in HNTG buffer (50 mM Hepes, pH 7.5, 150 mM NaCI, 0.1% of a possible autophosphorylation of this protein. To this end Triton X-100, 10% glycerol, 20 PM leupeptin, 100 units/ml aprotinin, we performed a denaturation/renaturation procedure after 0.18 mg/ml PMSF), ERKl was eluted by a 30-min incubation at transfer to a PVDF membrane, followed by a kinase assay room temperature in HNTG buffer containing 10 p~ C peptide and reaction. We observed phosphorylation of both ERKl and the 0.2 mM sodium orthovanadate. In vitro phosphorylation was per- formed for 1 h at room temperature in the presence of 5 mM MnC12, 90-kDa protein, and phosphoamino acid analysis of the 90- 10 mM MgAc, and [y-32P]ATP (5 pM, 33 Ci/mmol). The reaction was kDa phosphoprotein identified solely serine residues. Fur- terminated by addition of 4-fold concentrated Laemmli buffer and thermore, using anti-S6 kinase I1 antibodies, we obtained samples were submitted to SDS-PAGE under reducing conditions on specific precipitation of a 90-kDa protein from anti-ERK1 a 10% acrylamide resolving gel. antipeptide eluates. Finally, using in situ chemical cross- Phosphoamimacid Analysis-For phosphoamino acid analysis, linking, we observed by immunoblotting with anti-ERK1 phosphorylation was performed as described above, and samples were submitted to SDS-PAGE under reducing conditions. After electro- antipeptides, a protein with an electrophoretic mobility con- phoresis, 32P-labeled proteins were localized by autoradiography, and sistent with a complex between ERKl and S6 kinase 11. As a gel pieces corresponding to the phosphoproteins of interest were whole, our data indicate that ERKl is associated in living excised. Labeled proteins were then eluted from the gel by an over- PC12 cells with a phosphoprotein of 90 kDa, which corre- night incubation at 37 "C in 50 mM NH4HC03, pH 8,0.1% SDS, and sponds very likely to the mammalian homologue of the frog 5% 0-mercaptoethanol. Eluted proteins were precipitated for 30 min S6 kinase 11. on ice in the presence of 10% trichloroacetic acid and 25 pg of bovine y-globulin as a carrier. Pellets were washed once with 100% EtOH and once with EtOH/ethylic ether (1:l). Proteins were hydrolyzed for EXPERIMENTAL PROCEDURES 90 min at 110 "C in 6 N HCl. Phosphoamino acids were then separated Materials-C peptide (356-367: TARFQPGAPEAP), correspond- on cellulose thin-layer plates by electrophoresis at pH 3.5 for 2 h at ing to the C terminus of ERKl (8), was produced by Neosystem 1000 V and analyzed by autoradiography as previously described (17). (Strasbourg, France). Antipeptides against this region of ERKl were Protein Transfer, Renaturation Procedure, and Kinase Reaction- obtained as previously described (12). Immobilon PVDF transfer After immunopurification and elution from anti-ERK1 antibodies, membrane was from Millipore. Bovine serum albumin (BSA; type eluates were submitted to SDS-PAGE under reducing conditions on 7030) for cell culture, and dimethyl sulfoxide were from Sigma. a 10% acrylamide resolving gel. Proteins were transferred to a PVDF Disuccinimidyl suberate (DSS) was from Pierce. BSA for immunoblot membrane, and the membrane was incubated for 60 min at 4 "C in a experiments was from Intergen Company (Providence, RI). Anti-Rsk denaturation solution (6 M guanidinium chloride, 50 mM Tris-HC1, serum (Rsk: ribosomal protein S6 kinase), from a rabbit injected with pH 8.3, 50 mM DTT, 2 mM EDTA). The PVDF membrane was bacterially produced Rsk protein, was a generous gift of Drs. E. washed twice with renaturation buffer (20 mM Tris-HC1, pH 7.5, 150 Erikson and J. Maller (Department of Pharmacology, University of mM NaC1,2 mM DTT, 2 mM EDTA, 0.1% Nonidet P-40,2% glycerol), Colorado Health Sciences Center, Denver, CO). NGF was kindly and incubated overnight at 4 "C in renaturation buffer. The mem- provided to us by Dr. P. Kitabgi (Nice-Sophia-Antipolis, France). brane was saturated by incubation for 60 min at room temperature Cell Culture-PC12 cells were cultured in RPMI medium contain- in renaturation buffer containing 0.2% polyvinylpyrrolidone, 0.2% ing 10% horse serum and 5% fetal calf serum. Cells were plated at ficoll, and 20 mM sodium pyrophosphate. The kinase assay was then 2.5 X lo4 cells/cm* and grown to confluence. Before incubation with performed in HNTG buffer containing 10 mM magnesium acetate, 5 the effectors, cells were starved overnight in RPMI/O.2% BSA. NIH mM manganese chloride, and 5 p~ [y-32P]ATP (50 &i/ml). After 1 3T3 cells, clone HIR 3.5 (HIR cells), transfected with a human insulin h of phosphorylation at room temperature, the blot was washed receptor cDNA construct and expressing lo6 receptors/cell, were extensively with PBS containing 1% Triton X-100 prior to autora- provided to us by Dr. J. Whittaker (Stony Brook, New York, NY) diography. Concerning the ERKl immunoblot after kinase assay, the (16). HIR cells were cultured to confluence in H21/10% fetal calf experiment was performed as previously described (121, except for a serum and starved overnight in H21/0.2% BSA. step of rehydration of the PVDF membrane (after autoradiography) In Vivo Stimulation of ERKl Actiuity and ERKl Immunopurifica- in the saturating buffer. tion-After the starvation period, HIR or PC12 cells were incubated In Situ Cross-linking-After a 5-min incubation in the absence or for 5 min in presence of effectors: insulin M) for HIR cells, and presence of NGF, PC12 cells were washed twice with ice-cold PBS, NGF (6 x lo-' M) or EGF M) for PC12 cells. All the following and incubated for 20 min at 4 "C in PBS/1.2% (v/v) dimethyl sulf- steps were performed at 4 "C. Cells were washed twice with ice-cold oxide as a control or in PBS/l.2% (v/v) dimethyl sulfoxide containing PBS (phosphate-buffered saline; 140 mM NaC1, 3 mM KC1, 6 mM 1.2 mM DSS as previously described (18). Cells were then washed NaZHP04, 1 mM KH2P04, pH 7.41, and once with buffer A (50 mM with ice-cold PBS, and cellular proteins were solubilized before im- Hepes, pH 7.5, 150 mM NaCl, 10 mM EDTA, 10 mM Na4P207, 2 mM sodium orthovanadate, 100 mM NaF, 100 units/ml aprotinin, 20 pM munoprecipitation as described above. 17371 NGF-induced Phosphorylation of ERKl NGF ADDED RESULTS TO PC12 CELLS: NO YES In Vivo and in Vitro Phosphorylation of a 90-kDa Protein OR Coprecipitating with ERK1-To search for coprecipitation with ERKl of cellular activators and/or substrates, confluent - HIR and PC12 cells were incubated for 5 min with buffer or with insulin M) for HIR cells, and with buffer, NGF (6 X lo-* M) or EGF ( M) for PC12 cells. After solubilization, proteins were submitted to precipitation by antipeptides to ERK1, and the washed pellets were incubated with C peptide (10 pM). In vitro phosphorylation was then performed on 66 - eluates, and samples were analyzed by SDS-PAGE under reducing conditions (Fig. 1). In both cell types, we observed two major phosphoproteins, one in the range of 90 kDa, and 45 - - ERKl another one at 44 kDa corresponding to ERK1. Furthermore, we found that insulin, NGF or EGF strongly stimulated the Mr x 10” :IpP incorporation into the 90-kDa species. The band with an estimated molecular weight of 46,000, and whose phosphoryl- ation is significantly stimulated by insulin in HIR cells, could be also detected on shorter exposures of phosphoproteins in eluates from NGF- and EGF-treated PC12 cells. In additional experiments, we performed a 32P labeling of intact PC12 cells, followed by NGF treatment for 5 min. After solubilization and immunoprecipitation with anti-ERK1 an- FIG. 2. Immunoprecipitation of a 90-kDa phosphoprotein tipeptides, the phosphoproteins adsorbed on the pellet were by antibodies to ERKl after “P labeling of living PC12 cells. eluted by incubation with C peptide, and submitted to SDS- Confluent PC12 cells growing in 145-mm Petri dishes were starved PAGE analysis under reducing conditions. As shown on Fig. overnight, and the ‘”P labeling was performed as described under “Experimental Procedures.” NGF (6 X lo-’ M) was added for 5 min, 2, in addition to ERKl which showed increased phosphoryl- and solubilized proteins were submitted to precipitation by antipep- ation after NGF treatment, coprecipitation of an in vivo tides to ERK1. After washes, phosphoproteins adsorbed on the pellet labeled 90-kDa phosphoprotein was revealed (upper arrow). were eluted by incubation with C peptide, and samples were analyzed Moreover, NGF induced the stimulation of 82P incorporation by SDS-PAGE on a 10% acrylamide resolving gel under reducing into this coprecipitating protein. Taken together, we can conditions. The upper arrow indicates the position of the 90-kDa conclude that in living PC12 cells ERKl was associated with phosphoprotein. a NGF-sensitive 90-kDa phosphoprotein. Phosphoarnino Acid Analysis of the in Vitro Phosphorylated IN VmO PHOSPHORYLATION PHOSPHOAMINOACIO ANALYSIS OF - 9OkD PROTEINS 90-kDa Protein-We next determined the phosphoamino acid content of the 90-kDa phosphoprotein obtained from PC12 cells incubated with NGF. After a 5-min treatment of PC12 ADDED70 cells with NGF, precipitation, elution, and in vitro phos- PC12 CELLS T phorylation were performed as described above. The 90-kDa phosphoprotein was eluted from the gel, and after acidic hydrolysis, phosphoamino acids were analyzed by TLC. As shown in Fig. 3, a basal ”P incorporation into threonine and CELLS: HIR PC12 FIG. 3. Phosphoamino acid analysis of the in vitro phos- ” phorylated 90-kDa phosphoprotein. PC12 cells were incubated OR - in presence of NGF (6 X lo-” M) for 5 min and in vitro phosphoryl- ?Mi- ation of the coprecipitating 90-kDa protein was performed as de- scribed in Fig. l (left panel). The phosphorylated 90-kDa protein was 6 N HCI, and phos- 111 - eluted from the gel, submitted to hydrolysis in phoamino acids were analyzed by TLC (right panel). 71 - serine residues was detected. Furthermore, NGF induced a 48.5 - strong stimulation of the labeling on the same 2 residues. Antipeptides to ERKl Coprecipitate an Autophosphorylating Protein Kinase with M, 90,000-We were interested in deter- mining whether this 90-kDa protein was a substrate protein 29- for ERKl and/or whether it was a protein kinase endowed MrxlO‘ with the capacity to undergo autophosphorylation. To do so, ADDEDTOCELLS: BUFFER INSULIN BUFFER NGF EGF a kinase renaturation assay was performed with solubilized proteins from unstimulated PC12 cells, which were submitted FIG. 1. In vitro phosphorylation of a 90-kDa phosphopro- to precipitation with preimmune or antibodies to ERKl pro- tein coprecipitating with ERK1. Serum-depleted HIR or PC12 cells were incubated for 5 min in presence of insulin (lo-’ M), NGF tein. After transfer to a PVDF membrane, renaturation and (6 X lo-” M), or EGF (lo-’ M). Solubilized proteins were submitted incubation with [T-~~PIATP, the blot was extensively washed to precipitation by anti-ERK1 antibodies, and after elution by C and exposed to an autoradiographic film (Fig. 4, right panel). peptide eluates were phosphorylated in uitro in presence of [-p”P] In these conditions, we observed with anti-ERK1 antipeptides ATP as described under “Experimental Procedures.” Phosphorylated a specific immunoprecipitation of two phosphoproteins: (i) proteins were analyzed by SDS-PAGE on a 10% acrylamide resolving ERKl at 44,000, as shown by the Western blot experiment gel under reducing conditions. 17372 NGF-induced Phosphorylation of ERKl WSTERN BLOT WITH KINASE RMANRATK)H contrast to the eluate phosphorylation experiments, this "P ANnERKI AHnPEPTlDES ASSAY incorporation occurred exclusively on serine residues, indicat- ing that the 90-kDa coprecipitating protein displayed the OR - , _" F capacity to undergo autophosphorylation with a specificity 2" for serine residues. .. Precipitation of the in Vitro Phosphorylated 90-kDa Protein 111 - by Antibodies to S6 Kinuse ZI-The experiments described above indicated to us that an autophosphorylating 90-kDa 71 - protein kinase coprecipitated with ERK1. When in vitro phosphorylation was performed in eluates containing ERKl 48.5 - and the 90-kDa protein kinase, phosphoamino acid analysis showed that the 90-kDa protein was phosphorylated on both serine and threonine residues. In contrast, this phosphoryla- tion occurred exclusively on serine residues when it was 29- performed on the membrane after transfer and renaturation. Mr x 10' in As it has been previously described that MAP kinase in uitro reconstitution experiments is able to phosphorylate and SERVY. activate the 90-kDa S6 kinase I1 from Xenopus oocyte, we next tested the hypothesis that the 90-kDa phosphoprotein FIG. 4. Kinase renaturation assay of the 90-kDa coprecip- we described here could in fact correspond to the mammalian itating protein. Proteins from unstimulated PC12 cells were sub- equivalent of the frog protein. To do so, after coprecipitation mitted to precipitation by preimmune and immune antibodies to ERK1. After SDS-PAGE under reducing conditions and tranfer to a of the 90-kDa protein and in vitro phosphorylation, samples PVDF membrane, a kinase renaturation assay was performed (right from unstimulated or NGF-treated PC12 cells were submitted panel), followed by a Western blot with anti-ERK1 antibodies (left to precipitation by nonimmune antibodies or antibodies di- panel). The upper arrow indicates the position of the 90-kDa phos- rected against bacterially produced Xenopus Rsk protein. As phoprotein. shown in Fig. 6, we observed a specific immunoprecipitation of a 90-kDa phosphoprotein, while no signal was detected KINASE RENATURATION ASSAY PHOSPHOAMINOACID ANALYSIS using the nonimmune antibodies. We interpret these data to mean that the 90-kDa coprecipitating protein kinase is very likely to be the mammalian equivalent of the frog S6 kinase 11. More important, our data indicate that this S6 kinase I1 is associated with ERKl in living PC12 cells, as shown by the coprecipitation of the two proteins. In Situ Cross-linking of ERKl and a 90-kDa Phosphopro- tein-In order to establish that ERKl and the 90-kDa copre- 18.5 - cipitating protein were associated in intact cells before solu- bilization, we performed a 5-min NGF treatment of PC12 cells followed by an incubation with DSS in the presence of dimethyl sulfoxide which permeabilizes cells. Then cellular proteins were solubilized and submitted to immunoprecipita- OR - NON-IMMUNE ANTIBODY TO PRECIPITATION 56 KINASE II WITH: ANTIBODY ADDED TO PC12 CELLS BUFFER NGF BUFFER NGF OR - FIG. 5. Phosphoamino acid analysis of the 90-kDa protein zoo- phosphorylated on transfer membrane. Kinase renaturation as- say was performed with the coprecipitating 90-kDa protein from 116 - (buffer) or NGF-treated PC12 cells (6 X lo-* M for 5 unstimulated 97 - min) (left panel). After autoradiography, membrane pieces corre- c1 in sponding to phosphorylated proteins were submitted to hydrolysis 6 N HCI, and phosphoamino acids were analyzed by TLC (right ) . panel 45 - performed on the same membrane with anti-ERK1 antibodies (Fig. 4, left panel); (ii) a protein in the range of 90 kDa (upper arrow). Note that in some experiments this 90-kDa protein appeared as a doublet after short exposure of the autoradi- NOFADDED ographic film. "+ TO PC12 CELLS: "+ As these data supported the notion that the 90-kDa copre- FIG. 6. Immunoprecipitation with antibodies to S6 kinase cipitating protein was an autophosphorylating protein kinase, I1 of in vitro phosphorylated eluates from anti-ERK1 antibody the same kinase renaturation assay was performed using pellets. In vitro phosphorylation of the coprecipitating 90-kDa pro- tein was performed with eluates from unstimulated or NGF-treated extracts from unstimulated and NGF-treated PC12 cells (Fig. PC12 cells (6 X M for 5 min). The phosphorylation reaction was 5, left panel), and phosphoamino acid content of this 90-kDa stopped by the addition of NaF/EDTA (100 mM and 20 mM, respec- protein was determined by TLC (Fig. 5, right panel). Similar tively), and samples were submitted to precipitation by nonimmune to the observations made in the eluate phosphorylation ex- antibodies and antibodies to S6 kinase 11. Phosphorylated proteins periments, NGF was found to induce a small, but reproducible, adsorbed on the washed pellets were analyzed by SDS-PAGE on a stimulation of "P incorporation into the 90-kDa protein. In 10% acrylamide resolving gel under reducing conditions. NGF-induced Phosphorylation of ERKl 17373 tion by anti-ERK1 serum. After SDS-PAGE, samples were ERKl activity also promoted the 32P incorporation into this transferred to a PVDF membrane and finally blotting with 90-kDa protein; (iii) in vitro phosphorylation of this 90-kDa anti-ERK1 serum was used to search for ERKl alone, and protein, in presence of ERK1, occurred on both serine and ERKl cross-linked to other protein(s). As shown in Fig. 7, we threonine residues, while phosphorylation on transfer mem- found that anti-ERK1 antibodies detect in solubilisates from branes revealed an incorporation into serine residues exclu- cells incubated in the presence of DSS, ERK1, and in addition, sively; (iv) specific immunoprecipitation of this 90-kDa pro- a molecular species with a molecular weight of approximately tein could be obtained with antibodies to the frog S6 kinase 125,000. This is compatible with a complex between ERKl 11. In additional experiments, we performed in vivo 32P-label- and a 90-kDa protein. Moreover, NGF-treatment resulted in ing of PC12 cells followed by a 5-min incubation with NGF. an enhanced appearance of the 125-kDa species. In a control After immunoprecipitation by anti-ERK1 antibodies and elu- experiment, we have performed cell lysate precipitation using tion with the C peptide, coprecipitation of the in vivo labeled preimmune serum before SDS-PAGE and immunoblotting 90-kDa protein could be revealed. Moreover, phosphorylation with anti-ERK1 serum (data not shown). No signal was of this protein was stimulated by exposure of PC12 cells to detected compatible with ERKl or in the range of 125 kDa. NGF. Our in situ cross-linking experiments show that in In summary, our in situ cross-linking experiments strongly living PC12 cells, ERKl is associated with a protein with a suggest that a complex between ERKl and the 90-kDa phos- molecular weight in the range of 90,000, and that this complex phoprotein occurs in living PC12 cells and that this associa- formation is stimulated by NGF. These experiments also tion is increased upon stimulation of ERKl by NGF. indicate that this association is not generated by the cell solubilization procedure itself. As a whole, our data strongly DISCUSSION support the notion of the physiological relevance of an asso- ciation in living PC12 cells between ERKl and a mammalian Of great importance for the idea of a ligand-activated phos- form of S6 kinase 11. Concerning the specificity of this inter- phorylation cascade involved in hormone and growth factor action, it would be interesting to determine which domains of receptor tyrosine kinase action, was the original demonstra- the two proteins are involved in this molecular association. tion by Sturgill et al. (19) that 42 kDa/MAP kinase purified Note that the coprecipitation we describe here is reminiscent from insulin-stimulated 3T3-Ll cells was able to phosphoryl- of recent reports by several groups describing association and ate and to activate, in vitro, the S6 kinase I1 from Xenopus coprecipitation of tyrosine kinase receptors and some of their oocytes. Similar results were thereafter obtained by other primary targets, such as phospholipase C-y and phosphatidyl- groups using progesterone-stimulated Xenopus oocyte ex- inositol-3-kinase (21-23). tracts (20), and EGF-stimulated Swiss 3T3 fibroblast extracts In vitro phosphorylation experiments shown in Figs. 1 and (3, 4). Therefore, our finding that ERKl is associated with a 3 suggest that the activated ERKl is capable of in vitro 90-kDa phosphoprotein under various conditions and in dif- interaction with the 90-kDa protein. According to this view ferent cell types led 1:s to test the hypothesis that this protein the activated ERKl leads to threonine phosphorylation of the could correspond to a S6 kinase I1 or to a related protein. Several lines of evidence are in fact in favor of this view, and 90-kDa protein, which then becomes competent to undergo are as follows: (i) antipeptides to ERKl coprecipitated a 90- autophosphorylation on serine residues. The high level of 32P incorporation seen in cell-free systems after immunopurifi- kDa phosphoprotein; (ii) effectors capable of stimulating cation might be explained by the loss, during immunopurifi- cation, of endogenous protein phosphatase(s) involved in reg- NGF ulation of these protein kinases. This issue is particularly r- 4- - +I OR - intriguing, since we observed in our previous work (12) that in vitro ERKl phosphorylation is enhanced by sodium ortho- 221 - vanadate addition to the phosphorylation mixture. While we cannot exclude at present that this effect could be accounted for by a direct interaction between orthovanadate and ERK1, we favor the idea that these data might reflect the copurifi- 106 - cation of one or several sodium orthovanadate-sensitive pro- tein phosphatase(s). 75 - It should be noted that the antibodies to Xenopus oocyte S6 kinase I1 precipitate specifically only a small fraction of the 90-kDa protein when compared to the total amount sub- mitted to immunoprecipitation (data not shown). This could 46 - be explained by the specificity of this antibody raised against bacterially produced Rsk protein and by the possible limited conservation of the amino acid sequence between frog and mammalian proteins. However, we have also observed, in NO YES some renaturation experiments, two phosphoproteins in the molecular species corresponding to the 90-kDa band. Hence, DSS it is possible that one protein can be recognized as a genuine FIG. 7. Immunoblot of anti-ERK1 immunoprecipitates after S6 kinase, while the other one could in fact be a proteolytic in situ cross-linking with DSS. After a 5-min incubation of PC12 degradation product. If the protein with the fastest electro- cells with NGF, in situ cross-linking by DSS was performed as described under “Experimental Procedures.” Samples were then sol- phoretic mobility represents a proteolytic degradation prod- ubilized, submitted to immunoprecipitation by anti-ERK1 serum, uct, the enzyme renaturation experiments indicate that this and separated by SDS-PAGE on a 10% acrylamide resolving gel proteolysis does not impair in a significant way the ability of under reducing conditions. Immunoprecipitated proteins were trans- the proteolytic fragment to undergo autophosphorylation. ferred to a PVDF membrane, incubated in presence of anti-ERK1 Recently, purification and characterization of an insulin- antibodies, and revealed with ’251-protein A. The arrow indicates the position of ERKl/-gO-kDa complex. stimulated protein kinase (ISPK1) from rabbit skeletal mus- 17374 NGF-induced Phosphorylation of ERKl cle was reported by the laboratory of P. Cohen (Department kinase and S6 kinase form functional complexes in intact of Biochemistry, University of Dundee, Dundee, UK) (24). cells and are therefore solid arguments in favor of an impor- The authors demonstrated that ISPKl was closely related, if tant physiological role of these two kinases in growth factor not identical, to the frog S6 kinase 11. ISPKl appears to play and hormone signaling pathways. a major role in glycogen metabolism as it phosphorylates the Acknowledgments-We express our sincere thanks to Dr. Y. Le G subunit of phosphatase 1, that in turn leads to dephospho- Marchand-Brustel, Dr. E. Van Obberghen-Schilling, Dr. V. Baron, rylation and activation of glycogen synthase (25). Together and Dr. R. Ballotti for reviewing the manuscript and constructive these data demonstrate that besides its effect on protein comments. G. Visciano is acknowledged for illustration work. We synthesis via the S6 kinase II/S6 protein pathway, MAP thank Dr. E. Erikson and Dr. J. Maller (Department of Pharmacol- kinase is a key kinase in the regulation of major metabolic ogy, University of Colorado Health Sciences Center, Denver, CO) for the gift of anti-Rsk antibody, Dr. J. Whittaker (Stony Brook, New responses such as glycogen synthesis. It remains to be shown York, NY) for the HIR cells, and Dr. P. Kitabgi (Nice-Sophia- whether such an ISPKl exists in PC12 cells, and whether Antipolis, France) for the gift of NGF. NGF plays a role in glycogen metabolism in these cells as well. REFERENCES The nature of the insulin-, EGF-, and NGF-sensitive 46- 1. Thomas, G. (1992) Cell 68,3-6 Ray, L. B., and Sturgill, T. W. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, kDa phosphoprotein we observed in Fig. 1 is unknown. It 1502-1506 3. Ahn, N. G., and Krebs, E. G. (1990) J. Biol. Chem. 265,11495-11501 might correspond to a copurifying ERKl substrate or to an 4. Ahn, N. G., Weiel, J. E., Chan, C. P., and Krebs, E. G. (1990) J. Biol. Chem. activator. Recently, Ahn et al. (26) and Gomez et al. (27) have 265,11487-11494 5. Hoshi, M., Nishida, E., and Sakai, H. (1988) J. Biol. Chem. 263, 5396- reported the first steps of purification and characterization of MAP kinase activators. Using extracts from EGF-treated 6. Gomez N Tonks N. K. Morrison, C., Harmar, T., and Cohen, P. (1990) FEB'S iktt. 27i, 119-i22 Swiss 3T3 cells or from NGF-treated PC12 cells, both groups 7. Mi asaka, T., Chao, M. V., Sherline, P., and Saltiel, A. R. (1990) J. Biol. found two MAP kinase activators with a molecular weight in $hem. 265,4730-4735 8. Boulton, T. G., Yancopoulos, G. D., Gregory, J. S., Slaughter, C., Moomaw, the range of 50,000-60,000. At the present time, it is not C., Hsu, J., and Cobb, M. H. (1990) Science 249,64-67 known whether these MAP kinase activators are themselves 9. Boulton, T. G., Gregory, J. S., and Cobb, M. H. (1991) Biochemistry 30, 97Q-9QC -." -" protein kinases. Ahn et al. (26) are leaning toward the idea 10. Boulton, T. G., and Cobb, M. H. (1991) Cell Regul. 2,357-371 that these activators are not kinases mainly based on the fact 11. Boulton. T. G.. Nve. S. H.. Robbins. D. J.. ID. N. Y.. Radzieiewska. E.. Morgenbesser, S: D., DePinho, R. A., Panay&tos, N:, Cobb,". H.,'and that neither proteins nor peptides representing a broad range Yancopoulos, G. D. (1991) Cell 65,663-675 12. Scimeca, J.-C., Ballotti, R., Nguyen, T. T., Filloux, C., and Van Obberghen, of known specificities for phosphorylation could be phos- E. (1991) Biochemistry 30,9313-9319 phorylated. Hence the action of these activating molecules 13. Crews, C. M., Alessandrini, A. A,, and Erikson, R. L. (1991) Proc. Natl. Acad. Sci. U. S. A. 88,8845-8849 could be limited to the induction of a conformational change 14. Seger, R., Ahn, N. G., Boulton, T. G., Yancopoulos, G. D., Panayotatos, in MAP kinase, with a resulting increase in the rate and N., Radzie'ewska, E., Ericsson, L., Bratlien, R. L., Cohb, M. H., and Krebs, E. d. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 6142-6146 extent of the autophosphorylation. However, it remains pos- 15. Wu, J., Rossomando, A. J., Her, J.-H., Vecchio, R. D., Weber, M. J., and sible that they are protein kinases with a virtually unique Sturgill, T. W. (1991) Proc. Natl. Acad. Sci. U. S. A. 88,9508-9512 16. Whittaker, J., Okamoto, A. K., Thys, R., Bell, G. I., Steiner, D. F., and specificity for MAP kinases. Hofmann, C. A. (1987) Proc. Natl. Acad. Sci. U. S. A. 84,5237-5241 In summary, we have shown that antipeptides to ERKl 17. Cooper, J. A., Sefton, B. A,, and Hunter, T. (1983) Methods Enzymol. 99, 387-402 precipitate a functional ERKl/SO-kDa phosphoprotein com- 18. Hari, J., Shii, K., and Roth, R. A. (1987) Endocrinology 120,829-831 plex, in which the 90-kDa species undergoes in vitro phos- 19. Sturgill, T. W., Ray, L. B., Erikson, E., and Maller, J. L. (1988) Nature 334,715-718 phorylation on serine and on threonine residues; the latter 20. Haccard, O., Jessus, C., Cayla, X., Goris, J., Merlevede, W., and Ozon, R. (1990) Eur. J. Biochem. 192,633-642 phosphorylation likely to be directly mediated by ERK1. 21. Margolis, B., Rhee, S. G., Felder, S., Mervic, M., Lyall, R., Levitzki, A., Moreover, this 90-kDa phosphoprotein is specifically precip- Ullrich, A,, Zilberstein, A., and Schlessinger, J. (1989) Cell 67, 1101- 11n7 itated by antibodies to Xenopus oocyte S6 kinase 11. As a 22. Morrison, D. K., Kaplan, D. R., Rhee, S. G., and Williams, L. T. (1990) whole, our results provide strong evidence for the idea that in Mol. Cell Biol. 10, 2359-2366 Kaplan, D. R., Morrisson, D. K., Wong, G., McCormick, F., and Williams, intact PC12 cells a mammalian equivalent of the frog S6 L. T. (1990) Cell 61, 125-133 kinase I1 is associated with the MAP kinase/ERKl. The 24. Lavoinne, A., Erikson, E., Maller, J. L., Price, D. J., Avruch, J., and Cohen, P. (1991) Eur. J. Biochem. 199, 723-728 essence of the whole concept of a kinase cascade triggered by 25. Dent, P., Lavoinne, A,, Nakielny, S., Caudwell, F. B., Watt, P., and Cohen, a ligand-activated receptor tyrosine kinase and involving P. (1990) Nature 348,302-308 26. Ahn, N. G., Seger, R., Bratlien, R. L., Diltz, C. D., Tonks, N. K., and Krebs, MAP kinase and S6 kinase rests for the major part on clever E. G. (1991) J. Biol. Chem. 266,4220-4227 reconstitution experiments. Our data clearly show that MAP 27. Gomez, N., and Cohen, P. (1991) Nature 353,170-173 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Biological Chemistry Unpaywall

Nerve growth factor-induced phosphorylation cascade in PC12 pheochromocytoma cells. Association of S6 kinase II with the microtubule-associated protein kinase, ERK1.

Scimeca, J.C.; Nguyen, T.T.; Filloux, C; Van Obberghen, E
Journal of Biological Chemistry – Oct 1, 1992
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Abstract

Nerve growth factor-induced phosphorylation cascade in PC12 pheochromocytoma cells. Association of S6 kinase II with the microtubule-associated protein kinase, ERK1. E Scimeca, R Ballotti, C Scimeca, T. Nguyen, C. Filloux, E. van Obberghen To cite this version: E Scimeca, R Ballotti, C Scimeca, T. Nguyen, C. Filloux, et al.. Nervegrowth factor-induced phospho- rylation cascade in PC12 pheochromocytoma cells. Association of S6 kinase II with the microtubule- associated protein kinase, ERK1.. Journal of Biological Chemistry, 1992, 267 (24), pp.17369-74. hal-02108969 HAL Id: hal-02108969 https://hal.science/hal-02108969 Submitted on 18 Nov 2021 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Vol. 267, No. 24, Issue of August 17369-17374,1992 25, PP. THE JOURNAL OF BIOLOGICAL CHEMISTRY Printed in U.S.A. 0 1992 by The American Society for Biochemistry and Molecular Biology, Inc. Nerve Growth Factor-induced Phosphorylation Cascade in PC12 Pheochromocytoma Cells ASSOCIATION OF S6 KINASE I1 WITH THE MICROTUBULE-ASSOCIATED PROTEIN KINASE, ERK1’ (Received for publication, February 7, 1992) Jean-Claude ScimecaS, Tien T. NguyenQ, Chantal Filloux, and Emmanuel Van Obberghen From the Znstitut National de la Sante et de la Recherche Medicale U145, Facult6 de Medecine, 06107 Nice, Cedex 2, France erful tool to search for additional physiologically rel- Microtubule-associated protein (MAP) kinases form evant substrates for MAP kinase, a key integrator a group of serinelthreonine kinases stimulated by var- enzyme for growth factors and hormones. ious growth factors such as nerve growth factor (NGF) and hormones such as insulin. Interestingly, MAP kinases are thought to participate in a protein kinase cascade leading to cell growth as they have been shown to phosphorylate and activate ribosomal protein S6 During the last 5 years much research in the area of cell kinase. To further evaluate the interactions between signaling has focussed on the serine/threonine MAP kinases, the different components of this cascade, we looked at which are thought to play a central role in metabolic and the possible coprecipitation of MAP kinase activator(s) mitogenic effects induced by various extracellular stimuli. In or MAP kinase substrate(s) with MAP kinase. Using response to most growth-promoting and mitogenic factors antipeptides to the C terminus of the M, 44,000 MAP tested, as well as to many other signals such as phorbol esters kinase, ERK1, and cell extracts from unstimulated or or phosphatase inhibitors, MAP’ kinase phosphorylation is NGF-treated PC12 cells, we obtained in addition to increased on both threonine and tyrosine residues, leading to MAP kinase itself coprecipitation of a protein with a the stimulation of its kinase activity (for review see Ref. 1). M, in the 90,000 range. We further show that this While tyrosine protein kinase receptors have been implicated protein is a protein kinase since it becomes phosphoryl- in a wide variety of physiological functions, the understanding ated on serine residues, after sodium dodecyl sulfate- of the molecular mechanisms of their actions continues to polyacrylamide gel electrophoresis and transfer to a polyvinylidene difluoride membrane. In vitro phos- represent a major challenge. Dissection of the transductional phorylation performed before sodium dodecyl sulfate- cascades induced by insulin (2), EGF (3-5), and NGF (6, 7) polyacrylamide gel electrophoresis demonstrates NGF- has identified MAP kinases as a link between the tyrosine sensitive phosphorylation of this 90-kDa protein on kinase receptors of these polypeptides, and serine/threonine both serine and threonine; the serine phosphorylation phosphorylations. Although the precise role(s) of MAP is likely to be due to autophosphorylation, and the kinases remain(s) to be clarified, they have emerged as key threonine phosphorylation due to phosphorylation by “switch kinases,” i.e. serine/threonine kinases which are ca- the copurifying MAP kinase. Furthermore, immuno- pable of converting a tyrosine phosphorylation signal coming precipitation of this 90-kDa protein was obtained with from a tyrosine kinase receptor into a serine/threonine phos- antibodies to S6 kinase 11. Finally, using in situ chem- phorylation. In 1990 Cobb et al. (8, 9) obtained an almost ical cross-linking, we were able to demonstrate in in- complete sequence of an “extracellular signal-regulated kinase tact cells the occurrence of an anti-ERK1 immunoreac- 1” (ERKl), which was found to be closely related to two yeast tive species with a molecular mass of approximately kinases involved in cell cycle control, and whose properties 125,000 compatible with a complex between ERKl strongly indicated identity with previously identified MAP a 90-kDa S6 kinase. Taken together, our obser- and kinases. Moreover, their study presented evidence for the vations demonstrate that the 44-kDa MAP kinase is existence of a family of at least 4 ERK proteins with molecular associated, in intact PC12 cells, with a protein kinase weights ranging from 41,000 to 62,000 (10, 11). which is very likely to be S6 kinase 11. In conclusion, Using immunoprecipitation with antipeptides to the car- our data represent strong evidence for a physiological boxyl terminus of ERK1, we have shown that mouse fibroblast role of the MAP kinase436 kinase cascade in PC12 cells. Finally, our antipeptides provide us with a pow- ERKl was phosphorylated in vitro on both threonine and tyrosine. Moreover, this dual phosphorylation was concomi- tant with an enhanced ERKl kinase activity as measured by * This work was supported in part by funds from the Institut myelin basic protein phosphorylation (12). However, this National de la Santi et de la Recherche Midicale; the Universite de activation remained small compared to the level of ERKl Nice-Sophia-Antipolis; Grant 6760 from the Association pour la Recherche contre le Cancer; and the Ligue Nationale Fran~aise contre le Cancer, Fediration des Comites Dipartementaux, Comiti Dipar- The abbreviations used are: MAP, microtubule-associated pro- temental du Var. The costs of publication of this article were defrayed tein; BSA, bovine serum albumin; C peptide, ERKl peptide/sequence in part by the payment of page charges. This article must therefore 356-367; DTT, dithiothreitol; EGF, epidermal growth factor; ERK, be hereby marked “aduertisement” in accordance with 18 U.S.C. extracellular signal-regulated kinase; Hepes, N-2-hydroxyethylpiper- Section 1734 solely to indicate this fact. azine-N’-2-ethanesulfonic acid ISPK, insulin-stimulated protein ki- $ Recipient of a fellowship from the Ligue Nationale Francaise nase; NGF, nerve growth factor; PBS, phosphate-buffered saline; Contre le Cancer, Federation des Comitis Dipartementaux, Cornit6 PMSF, phenylmethylsulfonyl fluoride; PVDF, polyvinylidene difluo- Departemental du Var. ride; Rsk, ribosomal protein S6 kinase; SDS-PAGE, sodium dodecyl § Recipient of a postdoctoral fellowship from the Medical Research sulfate-polyacrylamide gel electrophoresis; TLC, thin-layer chroma- Council of Canada. tography; DSS, disuccinimidyl suberate. 17369 17370 NGF-induced Phosphorylation of ERKl activity observed after in uiuo treatment of cells with growth leupeptin, and 0.18 mg/ml PMSF). Cells were scraped, and proteins were solubilized for 15 min in buffer A supplemented with 1% Triton factors or hormones such as insulin. At the same time three X-100 (solubilization buffer). Cell extracts were then submitted to groups reported independently that ERKl and ERK2 ob- centrifugation (18,000 X g for 15 min), and samples were incubated tained by expression in Escherichia coli was phosphorylated for 90 min at 4 "C with anti-ERK1 antibodies adsorbed on protein in vitro on both tyrosine and threonine and that this was A-Sepharose pellets. concurrent with a slow and moderate activation of the enzyme 32P Labeling in Intact PC12 Ceh-Confluent PC12 cells, growing (13-15). To summarize, the available data suggest that, al- in 145-mm culture dishes, were starved overnight in RPMI, 0.2% BSA medium (BSA 7030 from Sigma). Cells were then washed twice though ERKl/ERK2 dual self-phosphorylation can occur and with Dulbecco's modified Eagle's medium without phosphate and might be physiologically relevant, the mechanism of ERKl incubated for 3.5 h in this medium containing 500 pCi of ["PI activation is likely to be more complex and probably involves orthophosphate (1.7 mCi/ml). At the end of labeling, NGF (6 X lo-@ other proteins such as activator(s). The latter could be protein M) was added for 5 min. After three washes with ice-cold PBS, kinases or allosteric modulators. In an attempt to address the proteins were solubilized for 15 min on ice in 50 mM Hepes, pH 7.5, phosphorylation cascade issue, we investigated here the pos- containing 150 mM NaCl, 10 mM EDTA, 10 mM Na4P,07, 2 mM sibility of a coprecipitation of protein(s) with ERK1. Using sodium orthovanadate, 100 mM NaF, 1% Triton X-100,100 units/ml aprotinin, 20 pM leupeptin, and 0.18 mg/ml PMSF. Cell extracts were PC12 cells and an antipeptide to the carboxyl terminus of then incubated for 90 min with antibodies to ERKl adsorbed on a ERK1, we immunopurified ERKl from unstimulated or NGF- protein A-Sepharose pellet. After five washes with solubilization treated cells. After elution from antipeptide precipitates, we buffer, Laemmli buffer (3% sodium dodecyl sulfate) was added to performed in vitro phosphorylation experiments. The follow- dried pellets, and proteins were submitted to SDS-PAGE under ing key observations were made: (i) NGF induced a stimula- reducing conditions. tion of 32P incorporation into a protein in the 90-kDa range; In Vitro Phosphorylation of Immunopurified ERKl-At the end of incubation with anti-ERK1 antipeptides, protein A-Sepharose pellets (ii) this phosphorylation occurred to the same extent on both were washed five times in solubilization buffer. After an additional threonine and serine residues. We next addressed the question wash in HNTG buffer (50 mM Hepes, pH 7.5, 150 mM NaCI, 0.1% of a possible autophosphorylation of this protein. To this end Triton X-100, 10% glycerol, 20 PM leupeptin, 100 units/ml aprotinin, we performed a denaturation/renaturation procedure after 0.18 mg/ml PMSF), ERKl was eluted by a 30-min incubation at transfer to a PVDF membrane, followed by a kinase assay room temperature in HNTG buffer containing 10 p~ C peptide and reaction. We observed phosphorylation of both ERKl and the 0.2 mM sodium orthovanadate. In vitro phosphorylation was per- formed for 1 h at room temperature in the presence of 5 mM MnC12, 90-kDa protein, and phosphoamino acid analysis of the 90- 10 mM MgAc, and [y-32P]ATP (5 pM, 33 Ci/mmol). The reaction was kDa phosphoprotein identified solely serine residues. Fur- terminated by addition of 4-fold concentrated Laemmli buffer and thermore, using anti-S6 kinase I1 antibodies, we obtained samples were submitted to SDS-PAGE under reducing conditions on specific precipitation of a 90-kDa protein from anti-ERK1 a 10% acrylamide resolving gel. antipeptide eluates. Finally, using in situ chemical cross- Phosphoamimacid Analysis-For phosphoamino acid analysis, linking, we observed by immunoblotting with anti-ERK1 phosphorylation was performed as described above, and samples were submitted to SDS-PAGE under reducing conditions. After electro- antipeptides, a protein with an electrophoretic mobility con- phoresis, 32P-labeled proteins were localized by autoradiography, and sistent with a complex between ERKl and S6 kinase 11. As a gel pieces corresponding to the phosphoproteins of interest were whole, our data indicate that ERKl is associated in living excised. Labeled proteins were then eluted from the gel by an over- PC12 cells with a phosphoprotein of 90 kDa, which corre- night incubation at 37 "C in 50 mM NH4HC03, pH 8,0.1% SDS, and sponds very likely to the mammalian homologue of the frog 5% 0-mercaptoethanol. Eluted proteins were precipitated for 30 min S6 kinase 11. on ice in the presence of 10% trichloroacetic acid and 25 pg of bovine y-globulin as a carrier. Pellets were washed once with 100% EtOH and once with EtOH/ethylic ether (1:l). Proteins were hydrolyzed for EXPERIMENTAL PROCEDURES 90 min at 110 "C in 6 N HCl. Phosphoamino acids were then separated Materials-C peptide (356-367: TARFQPGAPEAP), correspond- on cellulose thin-layer plates by electrophoresis at pH 3.5 for 2 h at ing to the C terminus of ERKl (8), was produced by Neosystem 1000 V and analyzed by autoradiography as previously described (17). (Strasbourg, France). Antipeptides against this region of ERKl were Protein Transfer, Renaturation Procedure, and Kinase Reaction- obtained as previously described (12). Immobilon PVDF transfer After immunopurification and elution from anti-ERK1 antibodies, membrane was from Millipore. Bovine serum albumin (BSA; type eluates were submitted to SDS-PAGE under reducing conditions on 7030) for cell culture, and dimethyl sulfoxide were from Sigma. a 10% acrylamide resolving gel. Proteins were transferred to a PVDF Disuccinimidyl suberate (DSS) was from Pierce. BSA for immunoblot membrane, and the membrane was incubated for 60 min at 4 "C in a experiments was from Intergen Company (Providence, RI). Anti-Rsk denaturation solution (6 M guanidinium chloride, 50 mM Tris-HC1, serum (Rsk: ribosomal protein S6 kinase), from a rabbit injected with pH 8.3, 50 mM DTT, 2 mM EDTA). The PVDF membrane was bacterially produced Rsk protein, was a generous gift of Drs. E. washed twice with renaturation buffer (20 mM Tris-HC1, pH 7.5, 150 Erikson and J. Maller (Department of Pharmacology, University of mM NaC1,2 mM DTT, 2 mM EDTA, 0.1% Nonidet P-40,2% glycerol), Colorado Health Sciences Center, Denver, CO). NGF was kindly and incubated overnight at 4 "C in renaturation buffer. The mem- provided to us by Dr. P. Kitabgi (Nice-Sophia-Antipolis, France). brane was saturated by incubation for 60 min at room temperature Cell Culture-PC12 cells were cultured in RPMI medium contain- in renaturation buffer containing 0.2% polyvinylpyrrolidone, 0.2% ing 10% horse serum and 5% fetal calf serum. Cells were plated at ficoll, and 20 mM sodium pyrophosphate. The kinase assay was then 2.5 X lo4 cells/cm* and grown to confluence. Before incubation with performed in HNTG buffer containing 10 mM magnesium acetate, 5 the effectors, cells were starved overnight in RPMI/O.2% BSA. NIH mM manganese chloride, and 5 p~ [y-32P]ATP (50 &i/ml). After 1 3T3 cells, clone HIR 3.5 (HIR cells), transfected with a human insulin h of phosphorylation at room temperature, the blot was washed receptor cDNA construct and expressing lo6 receptors/cell, were extensively with PBS containing 1% Triton X-100 prior to autora- provided to us by Dr. J. Whittaker (Stony Brook, New York, NY) diography. Concerning the ERKl immunoblot after kinase assay, the (16). HIR cells were cultured to confluence in H21/10% fetal calf experiment was performed as previously described (121, except for a serum and starved overnight in H21/0.2% BSA. step of rehydration of the PVDF membrane (after autoradiography) In Vivo Stimulation of ERKl Actiuity and ERKl Immunopurifica- in the saturating buffer. tion-After the starvation period, HIR or PC12 cells were incubated In Situ Cross-linking-After a 5-min incubation in the absence or for 5 min in presence of effectors: insulin M) for HIR cells, and presence of NGF, PC12 cells were washed twice with ice-cold PBS, NGF (6 x lo-' M) or EGF M) for PC12 cells. All the following and incubated for 20 min at 4 "C in PBS/1.2% (v/v) dimethyl sulf- steps were performed at 4 "C. Cells were washed twice with ice-cold oxide as a control or in PBS/l.2% (v/v) dimethyl sulfoxide containing PBS (phosphate-buffered saline; 140 mM NaC1, 3 mM KC1, 6 mM 1.2 mM DSS as previously described (18). Cells were then washed NaZHP04, 1 mM KH2P04, pH 7.41, and once with buffer A (50 mM with ice-cold PBS, and cellular proteins were solubilized before im- Hepes, pH 7.5, 150 mM NaCl, 10 mM EDTA, 10 mM Na4P207, 2 mM sodium orthovanadate, 100 mM NaF, 100 units/ml aprotinin, 20 pM munoprecipitation as described above. 17371 NGF-induced Phosphorylation of ERKl NGF ADDED RESULTS TO PC12 CELLS: NO YES In Vivo and in Vitro Phosphorylation of a 90-kDa Protein OR Coprecipitating with ERK1-To search for coprecipitation with ERKl of cellular activators and/or substrates, confluent - HIR and PC12 cells were incubated for 5 min with buffer or with insulin M) for HIR cells, and with buffer, NGF (6 X lo-* M) or EGF ( M) for PC12 cells. After solubilization, proteins were submitted to precipitation by antipeptides to ERK1, and the washed pellets were incubated with C peptide (10 pM). In vitro phosphorylation was then performed on 66 - eluates, and samples were analyzed by SDS-PAGE under reducing conditions (Fig. 1). In both cell types, we observed two major phosphoproteins, one in the range of 90 kDa, and 45 - - ERKl another one at 44 kDa corresponding to ERK1. Furthermore, we found that insulin, NGF or EGF strongly stimulated the Mr x 10” :IpP incorporation into the 90-kDa species. The band with an estimated molecular weight of 46,000, and whose phosphoryl- ation is significantly stimulated by insulin in HIR cells, could be also detected on shorter exposures of phosphoproteins in eluates from NGF- and EGF-treated PC12 cells. In additional experiments, we performed a 32P labeling of intact PC12 cells, followed by NGF treatment for 5 min. After solubilization and immunoprecipitation with anti-ERK1 an- FIG. 2. Immunoprecipitation of a 90-kDa phosphoprotein tipeptides, the phosphoproteins adsorbed on the pellet were by antibodies to ERKl after “P labeling of living PC12 cells. eluted by incubation with C peptide, and submitted to SDS- Confluent PC12 cells growing in 145-mm Petri dishes were starved PAGE analysis under reducing conditions. As shown on Fig. overnight, and the ‘”P labeling was performed as described under “Experimental Procedures.” NGF (6 X lo-’ M) was added for 5 min, 2, in addition to ERKl which showed increased phosphoryl- and solubilized proteins were submitted to precipitation by antipep- ation after NGF treatment, coprecipitation of an in vivo tides to ERK1. After washes, phosphoproteins adsorbed on the pellet labeled 90-kDa phosphoprotein was revealed (upper arrow). were eluted by incubation with C peptide, and samples were analyzed Moreover, NGF induced the stimulation of 82P incorporation by SDS-PAGE on a 10% acrylamide resolving gel under reducing into this coprecipitating protein. Taken together, we can conditions. The upper arrow indicates the position of the 90-kDa conclude that in living PC12 cells ERKl was associated with phosphoprotein. a NGF-sensitive 90-kDa phosphoprotein. Phosphoarnino Acid Analysis of the in Vitro Phosphorylated IN VmO PHOSPHORYLATION PHOSPHOAMINOACIO ANALYSIS OF - 9OkD PROTEINS 90-kDa Protein-We next determined the phosphoamino acid content of the 90-kDa phosphoprotein obtained from PC12 cells incubated with NGF. After a 5-min treatment of PC12 ADDED70 cells with NGF, precipitation, elution, and in vitro phos- PC12 CELLS T phorylation were performed as described above. The 90-kDa phosphoprotein was eluted from the gel, and after acidic hydrolysis, phosphoamino acids were analyzed by TLC. As shown in Fig. 3, a basal ”P incorporation into threonine and CELLS: HIR PC12 FIG. 3. Phosphoamino acid analysis of the in vitro phos- ” phorylated 90-kDa phosphoprotein. PC12 cells were incubated OR - in presence of NGF (6 X lo-” M) for 5 min and in vitro phosphoryl- ?Mi- ation of the coprecipitating 90-kDa protein was performed as de- scribed in Fig. l (left panel). The phosphorylated 90-kDa protein was 6 N HCI, and phos- 111 - eluted from the gel, submitted to hydrolysis in phoamino acids were analyzed by TLC (right panel). 71 - serine residues was detected. Furthermore, NGF induced a 48.5 - strong stimulation of the labeling on the same 2 residues. Antipeptides to ERKl Coprecipitate an Autophosphorylating Protein Kinase with M, 90,000-We were interested in deter- mining whether this 90-kDa protein was a substrate protein 29- for ERKl and/or whether it was a protein kinase endowed MrxlO‘ with the capacity to undergo autophosphorylation. To do so, ADDEDTOCELLS: BUFFER INSULIN BUFFER NGF EGF a kinase renaturation assay was performed with solubilized proteins from unstimulated PC12 cells, which were submitted FIG. 1. In vitro phosphorylation of a 90-kDa phosphopro- to precipitation with preimmune or antibodies to ERKl pro- tein coprecipitating with ERK1. Serum-depleted HIR or PC12 cells were incubated for 5 min in presence of insulin (lo-’ M), NGF tein. After transfer to a PVDF membrane, renaturation and (6 X lo-” M), or EGF (lo-’ M). Solubilized proteins were submitted incubation with [T-~~PIATP, the blot was extensively washed to precipitation by anti-ERK1 antibodies, and after elution by C and exposed to an autoradiographic film (Fig. 4, right panel). peptide eluates were phosphorylated in uitro in presence of [-p”P] In these conditions, we observed with anti-ERK1 antipeptides ATP as described under “Experimental Procedures.” Phosphorylated a specific immunoprecipitation of two phosphoproteins: (i) proteins were analyzed by SDS-PAGE on a 10% acrylamide resolving ERKl at 44,000, as shown by the Western blot experiment gel under reducing conditions. 17372 NGF-induced Phosphorylation of ERKl WSTERN BLOT WITH KINASE RMANRATK)H contrast to the eluate phosphorylation experiments, this "P ANnERKI AHnPEPTlDES ASSAY incorporation occurred exclusively on serine residues, indicat- ing that the 90-kDa coprecipitating protein displayed the OR - , _" F capacity to undergo autophosphorylation with a specificity 2" for serine residues. .. Precipitation of the in Vitro Phosphorylated 90-kDa Protein 111 - by Antibodies to S6 Kinuse ZI-The experiments described above indicated to us that an autophosphorylating 90-kDa 71 - protein kinase coprecipitated with ERK1. When in vitro phosphorylation was performed in eluates containing ERKl 48.5 - and the 90-kDa protein kinase, phosphoamino acid analysis showed that the 90-kDa protein was phosphorylated on both serine and threonine residues. In contrast, this phosphoryla- tion occurred exclusively on serine residues when it was 29- performed on the membrane after transfer and renaturation. Mr x 10' in As it has been previously described that MAP kinase in uitro reconstitution experiments is able to phosphorylate and SERVY. activate the 90-kDa S6 kinase I1 from Xenopus oocyte, we next tested the hypothesis that the 90-kDa phosphoprotein FIG. 4. Kinase renaturation assay of the 90-kDa coprecip- we described here could in fact correspond to the mammalian itating protein. Proteins from unstimulated PC12 cells were sub- equivalent of the frog protein. To do so, after coprecipitation mitted to precipitation by preimmune and immune antibodies to ERK1. After SDS-PAGE under reducing conditions and tranfer to a of the 90-kDa protein and in vitro phosphorylation, samples PVDF membrane, a kinase renaturation assay was performed (right from unstimulated or NGF-treated PC12 cells were submitted panel), followed by a Western blot with anti-ERK1 antibodies (left to precipitation by nonimmune antibodies or antibodies di- panel). The upper arrow indicates the position of the 90-kDa phos- rected against bacterially produced Xenopus Rsk protein. As phoprotein. shown in Fig. 6, we observed a specific immunoprecipitation of a 90-kDa phosphoprotein, while no signal was detected KINASE RENATURATION ASSAY PHOSPHOAMINOACID ANALYSIS using the nonimmune antibodies. We interpret these data to mean that the 90-kDa coprecipitating protein kinase is very likely to be the mammalian equivalent of the frog S6 kinase 11. More important, our data indicate that this S6 kinase I1 is associated with ERKl in living PC12 cells, as shown by the coprecipitation of the two proteins. In Situ Cross-linking of ERKl and a 90-kDa Phosphopro- tein-In order to establish that ERKl and the 90-kDa copre- 18.5 - cipitating protein were associated in intact cells before solu- bilization, we performed a 5-min NGF treatment of PC12 cells followed by an incubation with DSS in the presence of dimethyl sulfoxide which permeabilizes cells. Then cellular proteins were solubilized and submitted to immunoprecipita- OR - NON-IMMUNE ANTIBODY TO PRECIPITATION 56 KINASE II WITH: ANTIBODY ADDED TO PC12 CELLS BUFFER NGF BUFFER NGF OR - FIG. 5. Phosphoamino acid analysis of the 90-kDa protein zoo- phosphorylated on transfer membrane. Kinase renaturation as- say was performed with the coprecipitating 90-kDa protein from 116 - (buffer) or NGF-treated PC12 cells (6 X lo-* M for 5 unstimulated 97 - min) (left panel). After autoradiography, membrane pieces corre- c1 in sponding to phosphorylated proteins were submitted to hydrolysis 6 N HCI, and phosphoamino acids were analyzed by TLC (right ) . panel 45 - performed on the same membrane with anti-ERK1 antibodies (Fig. 4, left panel); (ii) a protein in the range of 90 kDa (upper arrow). Note that in some experiments this 90-kDa protein appeared as a doublet after short exposure of the autoradi- NOFADDED ographic film. "+ TO PC12 CELLS: "+ As these data supported the notion that the 90-kDa copre- FIG. 6. Immunoprecipitation with antibodies to S6 kinase cipitating protein was an autophosphorylating protein kinase, I1 of in vitro phosphorylated eluates from anti-ERK1 antibody the same kinase renaturation assay was performed using pellets. In vitro phosphorylation of the coprecipitating 90-kDa pro- tein was performed with eluates from unstimulated or NGF-treated extracts from unstimulated and NGF-treated PC12 cells (Fig. PC12 cells (6 X M for 5 min). The phosphorylation reaction was 5, left panel), and phosphoamino acid content of this 90-kDa stopped by the addition of NaF/EDTA (100 mM and 20 mM, respec- protein was determined by TLC (Fig. 5, right panel). Similar tively), and samples were submitted to precipitation by nonimmune to the observations made in the eluate phosphorylation ex- antibodies and antibodies to S6 kinase 11. Phosphorylated proteins periments, NGF was found to induce a small, but reproducible, adsorbed on the washed pellets were analyzed by SDS-PAGE on a stimulation of "P incorporation into the 90-kDa protein. In 10% acrylamide resolving gel under reducing conditions. NGF-induced Phosphorylation of ERKl 17373 tion by anti-ERK1 serum. After SDS-PAGE, samples were ERKl activity also promoted the 32P incorporation into this transferred to a PVDF membrane and finally blotting with 90-kDa protein; (iii) in vitro phosphorylation of this 90-kDa anti-ERK1 serum was used to search for ERKl alone, and protein, in presence of ERK1, occurred on both serine and ERKl cross-linked to other protein(s). As shown in Fig. 7, we threonine residues, while phosphorylation on transfer mem- found that anti-ERK1 antibodies detect in solubilisates from branes revealed an incorporation into serine residues exclu- cells incubated in the presence of DSS, ERK1, and in addition, sively; (iv) specific immunoprecipitation of this 90-kDa pro- a molecular species with a molecular weight of approximately tein could be obtained with antibodies to the frog S6 kinase 125,000. This is compatible with a complex between ERKl 11. In additional experiments, we performed in vivo 32P-label- and a 90-kDa protein. Moreover, NGF-treatment resulted in ing of PC12 cells followed by a 5-min incubation with NGF. an enhanced appearance of the 125-kDa species. In a control After immunoprecipitation by anti-ERK1 antibodies and elu- experiment, we have performed cell lysate precipitation using tion with the C peptide, coprecipitation of the in vivo labeled preimmune serum before SDS-PAGE and immunoblotting 90-kDa protein could be revealed. Moreover, phosphorylation with anti-ERK1 serum (data not shown). No signal was of this protein was stimulated by exposure of PC12 cells to detected compatible with ERKl or in the range of 125 kDa. NGF. Our in situ cross-linking experiments show that in In summary, our in situ cross-linking experiments strongly living PC12 cells, ERKl is associated with a protein with a suggest that a complex between ERKl and the 90-kDa phos- molecular weight in the range of 90,000, and that this complex phoprotein occurs in living PC12 cells and that this associa- formation is stimulated by NGF. These experiments also tion is increased upon stimulation of ERKl by NGF. indicate that this association is not generated by the cell solubilization procedure itself. As a whole, our data strongly DISCUSSION support the notion of the physiological relevance of an asso- ciation in living PC12 cells between ERKl and a mammalian Of great importance for the idea of a ligand-activated phos- form of S6 kinase 11. Concerning the specificity of this inter- phorylation cascade involved in hormone and growth factor action, it would be interesting to determine which domains of receptor tyrosine kinase action, was the original demonstra- the two proteins are involved in this molecular association. tion by Sturgill et al. (19) that 42 kDa/MAP kinase purified Note that the coprecipitation we describe here is reminiscent from insulin-stimulated 3T3-Ll cells was able to phosphoryl- of recent reports by several groups describing association and ate and to activate, in vitro, the S6 kinase I1 from Xenopus coprecipitation of tyrosine kinase receptors and some of their oocytes. Similar results were thereafter obtained by other primary targets, such as phospholipase C-y and phosphatidyl- groups using progesterone-stimulated Xenopus oocyte ex- inositol-3-kinase (21-23). tracts (20), and EGF-stimulated Swiss 3T3 fibroblast extracts In vitro phosphorylation experiments shown in Figs. 1 and (3, 4). Therefore, our finding that ERKl is associated with a 3 suggest that the activated ERKl is capable of in vitro 90-kDa phosphoprotein under various conditions and in dif- interaction with the 90-kDa protein. According to this view ferent cell types led 1:s to test the hypothesis that this protein the activated ERKl leads to threonine phosphorylation of the could correspond to a S6 kinase I1 or to a related protein. Several lines of evidence are in fact in favor of this view, and 90-kDa protein, which then becomes competent to undergo are as follows: (i) antipeptides to ERKl coprecipitated a 90- autophosphorylation on serine residues. The high level of 32P incorporation seen in cell-free systems after immunopurifi- kDa phosphoprotein; (ii) effectors capable of stimulating cation might be explained by the loss, during immunopurifi- cation, of endogenous protein phosphatase(s) involved in reg- NGF ulation of these protein kinases. This issue is particularly r- 4- - +I OR - intriguing, since we observed in our previous work (12) that in vitro ERKl phosphorylation is enhanced by sodium ortho- 221 - vanadate addition to the phosphorylation mixture. While we cannot exclude at present that this effect could be accounted for by a direct interaction between orthovanadate and ERK1, we favor the idea that these data might reflect the copurifi- 106 - cation of one or several sodium orthovanadate-sensitive pro- tein phosphatase(s). 75 - It should be noted that the antibodies to Xenopus oocyte S6 kinase I1 precipitate specifically only a small fraction of the 90-kDa protein when compared to the total amount sub- mitted to immunoprecipitation (data not shown). This could 46 - be explained by the specificity of this antibody raised against bacterially produced Rsk protein and by the possible limited conservation of the amino acid sequence between frog and mammalian proteins. However, we have also observed, in NO YES some renaturation experiments, two phosphoproteins in the molecular species corresponding to the 90-kDa band. Hence, DSS it is possible that one protein can be recognized as a genuine FIG. 7. Immunoblot of anti-ERK1 immunoprecipitates after S6 kinase, while the other one could in fact be a proteolytic in situ cross-linking with DSS. After a 5-min incubation of PC12 degradation product. If the protein with the fastest electro- cells with NGF, in situ cross-linking by DSS was performed as described under “Experimental Procedures.” Samples were then sol- phoretic mobility represents a proteolytic degradation prod- ubilized, submitted to immunoprecipitation by anti-ERK1 serum, uct, the enzyme renaturation experiments indicate that this and separated by SDS-PAGE on a 10% acrylamide resolving gel proteolysis does not impair in a significant way the ability of under reducing conditions. Immunoprecipitated proteins were trans- the proteolytic fragment to undergo autophosphorylation. ferred to a PVDF membrane, incubated in presence of anti-ERK1 Recently, purification and characterization of an insulin- antibodies, and revealed with ’251-protein A. The arrow indicates the position of ERKl/-gO-kDa complex. stimulated protein kinase (ISPK1) from rabbit skeletal mus- 17374 NGF-induced Phosphorylation of ERKl cle was reported by the laboratory of P. Cohen (Department kinase and S6 kinase form functional complexes in intact of Biochemistry, University of Dundee, Dundee, UK) (24). cells and are therefore solid arguments in favor of an impor- The authors demonstrated that ISPKl was closely related, if tant physiological role of these two kinases in growth factor not identical, to the frog S6 kinase 11. ISPKl appears to play and hormone signaling pathways. a major role in glycogen metabolism as it phosphorylates the Acknowledgments-We express our sincere thanks to Dr. Y. Le G subunit of phosphatase 1, that in turn leads to dephospho- Marchand-Brustel, Dr. E. Van Obberghen-Schilling, Dr. V. Baron, rylation and activation of glycogen synthase (25). Together and Dr. R. Ballotti for reviewing the manuscript and constructive these data demonstrate that besides its effect on protein comments. G. Visciano is acknowledged for illustration work. We synthesis via the S6 kinase II/S6 protein pathway, MAP thank Dr. E. Erikson and Dr. J. Maller (Department of Pharmacol- kinase is a key kinase in the regulation of major metabolic ogy, University of Colorado Health Sciences Center, Denver, CO) for the gift of anti-Rsk antibody, Dr. J. Whittaker (Stony Brook, New responses such as glycogen synthesis. It remains to be shown York, NY) for the HIR cells, and Dr. P. Kitabgi (Nice-Sophia- whether such an ISPKl exists in PC12 cells, and whether Antipolis, France) for the gift of NGF. NGF plays a role in glycogen metabolism in these cells as well. 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Biol. found two MAP kinase activators with a molecular weight in $hem. 265,4730-4735 8. Boulton, T. G., Yancopoulos, G. D., Gregory, J. S., Slaughter, C., Moomaw, the range of 50,000-60,000. At the present time, it is not C., Hsu, J., and Cobb, M. H. (1990) Science 249,64-67 known whether these MAP kinase activators are themselves 9. Boulton, T. G., Gregory, J. S., and Cobb, M. H. (1991) Biochemistry 30, 97Q-9QC -." -" protein kinases. Ahn et al. (26) are leaning toward the idea 10. Boulton, T. G., and Cobb, M. H. (1991) Cell Regul. 2,357-371 that these activators are not kinases mainly based on the fact 11. Boulton. T. G.. Nve. S. H.. Robbins. D. J.. ID. N. Y.. Radzieiewska. E.. Morgenbesser, S: D., DePinho, R. A., Panay&tos, N:, Cobb,". H.,'and that neither proteins nor peptides representing a broad range Yancopoulos, G. D. (1991) Cell 65,663-675 12. Scimeca, J.-C., Ballotti, R., Nguyen, T. T., Filloux, C., and Van Obberghen, of known specificities for phosphorylation could be phos- E. (1991) Biochemistry 30,9313-9319 phorylated. Hence the action of these activating molecules 13. Crews, C. M., Alessandrini, A. A,, and Erikson, R. L. (1991) Proc. Natl. Acad. Sci. U. S. A. 88,8845-8849 could be limited to the induction of a conformational change 14. Seger, R., Ahn, N. G., Boulton, T. G., Yancopoulos, G. D., Panayotatos, in MAP kinase, with a resulting increase in the rate and N., Radzie'ewska, E., Ericsson, L., Bratlien, R. L., Cohb, M. H., and Krebs, E. d. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 6142-6146 extent of the autophosphorylation. However, it remains pos- 15. Wu, J., Rossomando, A. J., Her, J.-H., Vecchio, R. D., Weber, M. J., and sible that they are protein kinases with a virtually unique Sturgill, T. W. (1991) Proc. Natl. Acad. Sci. U. S. A. 88,9508-9512 16. Whittaker, J., Okamoto, A. K., Thys, R., Bell, G. I., Steiner, D. F., and specificity for MAP kinases. Hofmann, C. A. (1987) Proc. Natl. Acad. Sci. U. S. A. 84,5237-5241 In summary, we have shown that antipeptides to ERKl 17. Cooper, J. A., Sefton, B. A,, and Hunter, T. (1983) Methods Enzymol. 99, 387-402 precipitate a functional ERKl/SO-kDa phosphoprotein com- 18. Hari, J., Shii, K., and Roth, R. A. (1987) Endocrinology 120,829-831 plex, in which the 90-kDa species undergoes in vitro phos- 19. Sturgill, T. W., Ray, L. B., Erikson, E., and Maller, J. L. (1988) Nature 334,715-718 phorylation on serine and on threonine residues; the latter 20. Haccard, O., Jessus, C., Cayla, X., Goris, J., Merlevede, W., and Ozon, R. (1990) Eur. J. Biochem. 192,633-642 phosphorylation likely to be directly mediated by ERK1. 21. Margolis, B., Rhee, S. G., Felder, S., Mervic, M., Lyall, R., Levitzki, A., Moreover, this 90-kDa phosphoprotein is specifically precip- Ullrich, A,, Zilberstein, A., and Schlessinger, J. 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Published: Oct 1, 1992

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