From INSERM U-385, Faculté de médecine, Avenue de
Valombrose, 06107 Nice Cedex 2, France, and Centre de
Biochimie, CNRS-UMR 134, Faculté des Sciences, 06018 Nice, France
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ABSTRACT |
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In B16 melanoma cells, mitogen-activated protein (MAP) kinases are activated during cAMP-induced melanogenesis (Englaro, W., Rezzonico, R., Durand-Clément, M., Lallemand, D., Ortonne, J. P., and Ballotti, R. (1995) J. Biol. Chem. 270, 24315-24320). To establish the role of the MAP kinases in melanogenesis, we studied the effects of a specific MAP kinase kinase (MEK) inhibitor PD 98059 on different melanogenic parameters. We showed that PD 98059 inhibits the activation of MAP kinase extracellular signal-regulated kinase 1 by cAMP, but does not impair the effects of cAMP either on the morphological differentiation, characterized by an increase in dendrite outgrowth, or on the up-regulation of tyrosinase that is the key enzyme in melanogenesis. On the contrary, PD 98059 promotes by itself cell dendricity and increases the tyrosinase amount and activity. Moreover, down-regulation of the MAP kinase pathway by PD 98059, or with dominant negative mutants of p21ras and MEK, triggers a stimulation of the tyrosinase promoter activity and enhances the effect of cAMP on this parameter. Conversely, activation of the MAP kinase pathway, using constitutive active mutants of p21ras and MEK, leads to an inhibition of basal and cAMP-induced tyrosinase gene transcription. These results demonstrate that the MAP kinase pathway activation is not required for cAMP-induced melanogenesis. Furthermore, the inhibition of this pathway induces B16 melanoma cell differentiation, while a sustained activation impairs the melanogenic effect of cAMP-elevating agents.
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INTRODUCTION |
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Melanocytes are specialized cells located at the basal layer of
the epidermis that synthesize and transfer melanin pigments to
surrounding keratinocytes leading thereby to a uniform skin pigmentation. In vivo, melanin pigments play a key
photoprotective role against the carcinogenic effects of solar
ultraviolet light, which is in other respects the physiologic stimulus
of melanogenesis (1, 2). UV radiation can act directly on melanocytes
or indirectly through the release of keratinocyte-derived factors that
regulate melanogenesis (3, 4). Among the agents secreted by
keratinocytes upon UV-B treatment, -melanocyte-stimulating hormone
(
-MSH)1 is one of the most
potent activators of melanogenesis. Indeed, addition of
-MSH in
cultured human melanocytes (5) or in melanoma cells (6) stimulates
melanization. Further, subcutaneous injection of this hormone causes a
strong stimulation of the local pigmentation in humans (7).
-MSH
binds to a G protein-coupled heptahelical receptor leading to the
activation of G
s protein and to an increase in
intracellular cAMP content. In cultured melanoma cells, the melanogenic
effect of
-MSH can be mimicked by other cAMP-elevating agents such
as cholera toxin, forskolin, and isobutylmethylxanthine (8-10). These
observations emphasize the pivotal role of cAMP in the regulation of
melanogenesis, but the cellular signaling events connecting the rise in
cAMP to the stimulation of melanin synthesis are still incompletely
clarified.
Melanin biosynthesis or melanogenesis consists in a cascade of
enzymatic and spontaneous reactions that converts tyrosine to melanin
pigments. The initial and rate-limiting step in melanin synthesis, the
hydroxylation of tyrosine to L-DOPA, is controlled by
tyrosinase that is the key enzyme in this process. Stimulation of
melanogenesis by cAMP-elevating agents, as well as by other melanogenic
agents, implies an increase in tyrosinase protein amount as the
consequence of the stimulation of the tyrosinase gene transcription
(11). Concerning the early events induced by cAMP increase, we have
recently demonstrated that cAMP-elevating agents inhibit the
phosphatidylinositol 3-kinase and p70S6 kinase activities
(12). Further, the inhibition of these activities by pharmacological
inhibitors mimics the melanogenic effect of -MSH or forskolin,
suggesting that phosphatidylinositol 3-kinase and p70S6
kinase inhibition by cAMP-elevating agents is a key event in the
regulation of melanogenesis by these agents.
The MAP kinases ERK1 and ERK2 are serine/threonine kinases that are activated upon phosphorylation by the dual specificity MAP kinase kinase or MEK. MEK is phosphorylated and activated by Raf-1, which is itself activated by p21ras (13). Upon activation, MAP kinases translocate to the nucleus where they phosphorylate and activate many transcription factors of which ternary complex factor/serum response factor and activator protein-1 are the most studied (14, 15). Although MAP kinases have been clearly shown to play a crucial role in growth control (16), they are also involved in the differentiation process of several cell systems (17, 18).
In a previous report, we have shown the activation of ERK1 during cAMP-induced melanogenesis in B16 melanoma cells (10). We have also observed a translocation of ERK1 to the nucleus, with a concomitant stimulation of activator protein-1 DNA binding activity. Further, stimulation of melanogenesis, which is associated with morphological changes characterized by an increased cell dendricity, reflects the differentiation of melanocytes and melanoma cells. These observations led us to hypothesize that the MAP kinase pathway could be involved in the regulation of melanoma cell differentiation and, more precisely, that the activation of ERKs would be a required event in the induction of melanogenesis by cAMP-elevating agents in B16 melanoma cells.
In the present report, using a pharmacological approach with a specific inhibitor of MEK (PD 98059) (19) and a molecular approach with constitutively active or dominant negative mutants of MEK and p21ras, we clearly demonstrated that cAMP-elevating agents can stimulate melanogenesis in the absence of MAP kinase activation, thereby invalidating our former hypothesis. Further, we showed that a sustained activation of the Ras/MAP kinase pathway led to a down-regulation of melanogenesis, demonstrating the involvement of this pathway in the control of B16 melanoma cell differentiation.
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EXPERIMENTAL PROCEDURES |
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Materials-- Forskolin, TPA, L-DOPA, 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF), aprotinin, and leupeptin were purchased from Sigma. Dulbecco's modified Eagle's medium, LipofectAMINE reagent, and Optimem medium were from Life Technologies, Inc. PD 98059 was from Santa Cruz Biotechnology. Polyclonal rabbit antiserum to human tyrosinase (PEP-7) was provided by Dr. V. Hearing (Bethesda, MD). Antiserum to ERK1 was a generous gift from Dr. E. Van Obberghen (Nice, France). Expression vectors coding for the dominant positive and negative mutants of p21ras, respectively Val-12 p21 and Asn-17 p21 (20, 21), were provided by Dr. J.C. Chambard (Center de biochimie, Nice, France). Expression vector coding for constitutively active MEK (S222D) and constitutively inactive MEK (S222A) mutants were previously described (22).
Cell Cultures-- B16/F10 murine melanoma cells were cultured in Dulbecco's modified Eagle's medium with 7% fetal calf serum (HyClone Laboratories) and penicillin/streptomycin (100 IU/50 µg/ml) in a humidified atmosphere containing 5% CO2 in air at 37 °C.
Kinase Assay--
Serum-starved cells were treated as indicated,
rinsed, and solubilized in ice-cold lysis buffer (50 mM
Hepes, pH 7.4, 150 mM NaCl, 100 mM NaF, 10 mM EDTA, 10 mM
Na4P2O7, 2 mM
Na3VO4, 1% Triton X-100, supplemented with
protease inhibitors, aprotinin (2 µg/ml), leupeptin (10 µM), and AEBSF (1 mM)). Then extracts were
clarified by centrifugation and incubated for 2 h at 4 °C with
ERK1 antibody preadsorbed to protein A-Sepharose. Immune complexes were
washed twice with lysis buffer and twice with HNTG buffer (50 mM Hepes, pH 7.4, 150 mM NaCl, 0.1% Triton
X-100, 10% glycerol, and 0.2 mM
Na3VO4). Pellets were resuspended in 50 µl of
HNTG buffer supplemented with 30 mM magnesium acetate, 15 µM cold ATP, and 0.2 mg/ml myelin basic protein (MBP)
final concentrations. Reactions were initiated at room temperature by
the addition of 3 µCi of [-32P]ATP (3000 Ci/mmol)
and stopped after 45 min by adding Laemmli sample buffer. Samples were
then boiled for 5 min, and proteins were separated by
SDS-polyacrylamide gel electrophoresis on a 12.5% acrylamide gel. The
incorporation of 32P was visualized by autoradiography.
Then film was scanned and bands were quantitated by densitometry using
MacBas software.
Determination of Tyrosinase Activity--
Tyrosinase activity
was estimated by measuring the rate of oxidation of L-DOPA
(23). Cells grown in 6-well dishes were treated as indicated for
48 h in Dulbecco's modified Eagle's medium, 2% fetal calf
serum. Then cells were washed in ice-cold phosphate-buffered saline and
lysed in 100 µl of phosphate buffer (0.1 M), pH 6.8, containing 1% (w/v) Triton X-100, 2 µg/ml aprotinin, 10 µM leupeptin, and 1 mM AEBSF. Cellular
extracts were clarified by centrifugation at 13,000 × g for 5 min. The tyrosinase substrate L-DOPA
(2 mg/ml) was prepared in the same lysis phosphate buffer (without
Triton). 40 µl of each extract were put in a 96-well plate, and the
enzymatic assay was started by adding 100 µl of L-DOPA
solution at 37 °C. Control wells contained 40 µl of lysis buffer.
Absorbance at 570 nm was read every 10 min for at least 1 h at
37 °C using a microplate reader (Dynatech Laboratories). The blank
was removed from each absorbance value, and a plot of absorbance
against time was represented for each condition. The final
activity was expressed in OD/min/µg of protein for each
condition.
Western Blot Analysis-- Cellular extracts were prepared as described above, and an equal amount of protein was separated by SDS-polyacrylamide gel electrophoresis in a 10% acrylamide gel. Proteins were transferred to a Hybond-C extra membrane (Amersham Corp.), and the blot was probed with the PEP-7 polyclonal antibody (directed against tyrosinase) and with an anti-ERK1 polyclonal antibody (to verify that each lane was evenly loaded). Then proteins were visualized by the Amersham ECL system.
Transfection and Luciferase Assays--
B16 melanoma cells,
plated in 24-well dishes, were transfected using the LipofectAMINE
system according to the recommendations of the manufacturer (Life
Technologies, Inc.). The reporter plasmid used consists in a
2.2-kilobase pair fragment of the mouse tyrosinase gene promoter cloned
upstream of the luciferase gene and named 2.2 pMT-Luc (11). For
determination of the PD 98059 effect, 0.25 µg/well of the reporter
plasmid was transfected with 0.05 µg of pCMVGal (Promega) to
control the variability in transfection efficiency. In the experiments
with mutants of MEK and Ras, the transfection was performed in the same
conditions using, 0.25 µg of the reporter plasmid, 0.1 µg of the
expression vector, empty or containing the coding sequence of the
different mutants, and 0.05 µg of pCMV
Gal. Forty-eight hours after
transfection, cells were washed with a saline phosphate buffer and
lysed with 25 mM Tris-phosphate (pH 7.8) buffer containing
1% Triton X-100, 2 mM EDTA, and 2 mM
dithiothreitol. Soluble extracts were harvested and assayed for
luciferase and
-galactosidase activity. All transfections were
repeated at least five times using different plasmid preparations and
gave similar results.
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RESULTS |
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PD 98059 Inhibits cAMP-induced Activation of ERK1-- PD 98059 has been shown to be a specific inhibitor of MEK activity, the kinase functioning directly upstream of MAP kinases (19). We first verified whether this inhibitor was effectively able to inhibit ERK1 activity in B16 melanoma cells. ERK1 was immunoprecipitated from cells treated with TPA or forskolin and preincubated or not with PD 98059. The kinase activity was monitored with MBP as substrate (Fig. 1). Forskolin and TPA caused, respectively, about 2- and 8-fold induction of ERK1 activity. Preincubation of cells with 10 µM PD 98059 resulted in an almost complete inhibition of this induced kinase activity (respectively, 0.7- and 1.2-fold). Identical results were obtained on ERK2 (data not shown). Thus, PD 98059 inhibits the activation of MAP kinases in B16 melanoma cells.
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PD 98059 Induces B16 Melanoma Cell Morphological
Differentiation--
Then, we analyzed the effect of PD 98059 on B16
cell dendricity, which is the first observable parameter of melanoma
cell differentiation (Fig. 2). Forskolin,
which is a potent activator of melanogenesis, strongly stimulated
dendritogenesis as compared with untreated cells. Addition of 10 µM PD 98059 in the culture medium was followed by an
acquisition of the dendritic phenotype. Further, when cells were
treated with forskolin plus PD 98059, dendricity was more pronounced
than with forskolin or PD 98059 alone. It is worth remarking that
-MSH, at the concentration of 10
6 M,
exerted the same effect of forskolin by inducing dendritogenesis in B16
cells (data not shown). Hence, in our cell system, inhibition of MAP
kinases by PD 98059 induces dendrite outgrowth and potentiates the
morphological changes induced by cAMP-elevating agents.
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Inhibition of MAP Kinases by PD98059 Induces Melanogenesis-- Induction of melanogenesis in B16 melanoma cells is characterized by the stimulation of tyrosinase activity resulting from an increase in the tyrosinase protein expression. We therefore studied the effect of PD 98059 on these parameters. First, we measured tyrosinase activity in response to PD 98059. Cells were treated for 48 h with the MEK inhibitor and with forskolin. We then measured, in a cell-free system, the DOPA-oxidase activity that is the second specific activity of tyrosinase. As shown in Fig. 3A, forskolin stimulated about 8-fold the tyrosinase activity, and we observed a 6-fold stimulation with PD 98059. Addition of forskolin together with PD 98059 allowed us to achieve almost a 10-fold stimulation of tyrosinase activity, suggesting that inhibition of MAP kinases potentiates the effect of forskolin on tyrosinase activity.
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Inhibition of MAP Kinases by PD98059 Stimulates the Tyrosinase Promoter Activity-- In a previous report we have shown that the effect of cAMP on melanogenesis is mediated through a stimulation of tyrosinase promoter activity, reflecting an activation of the tyrosinase gene transcription (11). Using a plasmid containing a 2.2-kilobase pair fragment of the tyrosinase promoter cloned upstream of the luciferase coding sequence as a reporter gene, we investigated whether the inhibition of MAP kinase led also to a stimulation of tyrosinase gene expression (Fig. 4). After transfection with the reporter plasmid, cells were incubated with forskolin, PD 98059, or forskolin plus PD 98059 for 48 h. We observed that PD 98059 alone stimulated luciferase activity 4-fold above the basal level. When forskolin was added to PD 98059, the luciferase activity reached a 9-10-fold stimulation, while forskolin alone induced a 8-fold stimulation. Hence, inhibition of the MAP kinase pathway by PD 98059 triggered an increase in tyrosinase gene expression and potentiate the effect of forskolin. It should be noted that the maximal effect on cell dendricity, melanogenesis, and tyrosinase promoter activity was observed with 10 µM PD 98059.
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Expression of Dominant Negative Mutants of MEK and
p21ras Increases Basal and cAMP-induced Tyrosinase Promoter
Activities--
To confirm the results obtained with the
pharmacological inhibitor of MEK we have investigated the effect of
dominant negative mutants of MEK and p21ras on the tyrosinase
gene expression. p21ras is a small GTPase that activates Raf-1,
the kinase that function upstream of MEK. These two mutants, that have
respectively the ability to repress the endogenous activity of MEK and
Ras, were cotransfected with the reporter plasmid (Fig.
5). In cells expressing the dominant
negative mutants of MEK (MEK), both basal and
forskolin-stimulated tyrosinase promoter activities were increased
about 3-fold compared with control conditions. The dominant negative
mutant of Ras (Ras
) induced a 2-fold augmentation of the
tyrosinase gene expression in basal and forskolin-treated cells. Thus,
inhibition of the Ras/MAP kinase pathway by dominant negative mutants
of Ras and MEK stimulates basal and cAMP-induced tyrosinase promoter
activities.
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Expression of Dominant Positive Mutants of MEK and Ras Inhibits Basal and cAMP-induced Tyrosinase Promoter Activities-- Since the inhibition of MAP kinases induces an augmentation of the tyrosinase gene expression, we wondered whether the expression of dominant positive mutants of p21ras (Ras+) and MEK (MEK+) would lead to an inhibition the tyrosinase gene promoter activity (Fig. 6). In cells co-expressing MEK+ and the reporter plasmid, we observed about a 2-fold decrease in luciferase activity in both basal and forskolin conditions. Expression of the dominant positive mutant of Ras (Ras+) appeared to be more potent to inhibit the forskolin-induced tyrosinase promoter activity. Thus, constitutively activation of the Ras/MAP kinase pathway leads to an inhibition of basal and cAMP-induced tyrosinase gene transcription.
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DISCUSSION |
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The different approaches used in this report clearly demonstrate that the inhibition of the MAP kinase pathway by a pharmacological inhibitor of MEK or by dominant negative mutants of Ras and MEK does not prevent the effects of forskolin on different parameters reflecting the stimulation of melanogenesis. On the contrary, inhibition of the MAP kinase pathway is sufficient to induce dendrite outgrowth and to increase tyrosinase expression and activity. Further, sustained activation of this pathway, by over expression of constitutively active Ras and MEK, inhibits basal and cAMP-induced tyrosinase promoter activities. These findings invalidated our initial hypothesis suggesting that the activation of ERK1 by forskolin in B16 melanoma cells was a key event in cAMP-induced melanogenesis. However, our present results disclose the link between the MAP kinase pathway and the regulation of melanoma cell differentiation and demonstrate that the activation of the MAP kinase pathway leads to the inhibition of melanogenesis and differentiation.
At variance with our results, previous works have demonstrated that MAP kinases activation is required for the megakaryocytic differentiation of K562 cells (24, 25), the neuronal differentiation of PC12 (17) and the adipogenic differentiation of 3T3-L1 cells (18). However, concerning the rat pheochromocytoma cells PC12, that share with melanocytes and melanoma cells the same embryonic origin (neural crest), some other reports have shown that MAP kinases activation is dispensable for neurite outgrowth and differentiation (26, 27). Interestingly, infection by the v-Ha-ras oncogene was reported to inhibited melanogenesis in murine melanocytes (28). Additionally, neurofibromin that stimulates the intrinsic GTPase activity of Ras, thereby leading to a sustained inactivation of Ras, was previously shown to increase the tyrosinase gene expression (29). Thus, consistently with our results, the inhibition of Ras would lead to a stimulation of melanogenesis while its activation would suppress melanogenesis.
It is generally accepted that growth and differentiation are antinomic effects; cells cannot proliferate and differentiate at the same time. This general notion was supported by a recent report demonstrating that the differentiation of the neuroblastoma cell line NE1-115 characterized by the induction of neurite outgrowth is allowed when cells are arrested at the G1 phase of the cell cycle (30). In addition, Callus et al. (31) have reported that treatment of J2E cells with amiloride suppressed the erythropoietin-induced proliferation and MAP kinase activity, and favored the differentiation of these cells. Thus inhibition of cell growth and interruption of cell cycle would commit the cell in a differentiation program. Our findings are more consistent with this notion. Keeping in mind the mitogenic role of MAP kinases that are required for cell proliferation and reentry in cell cycle after serum starvation (16), we could expect that the inhibition of MAP kinase pathway would lead to the stimulation of B16 melanoma cell differentiation and of melanogenesis. Conversely, activation of the MAP kinase pathway and stimulation of cell growth would inhibit melanogenesis. This hypothesis is supported by the inverse correlation between cell growth and melanogenesis observed in numerous reports. Indeed, UV-B irradiation that is a potent melanogenic stimulus inhibits growth of cultured human melanocytes (32). Growth factors such as basic fibroblast growth factor or TPA inhibit melanogenesis and stimulate growth of melanocytes (33). However, it should be noted that the stimulation of melanogenesis, by the inhibition of MAP kinases, cannot be solely explained by an inhibition of cell growth. Indeed, 10 µM PD98059 has only a very moderate inhibitory effect on growth of B16 cells, while inhibition of cell growth by serum depletion leads to faint increase in melanogenesis.
Additionally, it should be noted that down-regulation of the MAP kinase pathway is thought to trigger cell cycle arrest in the G1 phase (16). During this phase, a number of cyclin-dependent kinases including Cdk2, 4, 5, and 6 are accumulated (34). Interestingly, it has been recently shown that Cdk5 and its associated regulator protein p39 plays a critical role in neurite outgrowth during neuronal differentiation (35). We can therefore envision that the inhibition of the MAP kinase pathway would result in the accumulation of Cdk5, thereby explaining the stimulation of dendrite outgrowth by PD98059. Consistently, UV-B (32) and cAMP (36), two potent melanogenic agents that stimulate dendricity, were also shown to block cells in G1 phase. The involvement of Cdk5 in the formation of dendrites induced by cAMP-elevating agents or by the MAP kinase pathway inhibition is an appealing hypothesis that needs to be confirmed.
It seems surprising that the activation of MAP kinase leads to the
inhibition of melanogenesis, since forskolin and -MSH, two strong
stimulators of melanogenesis, have been shown to activate MAP kinases
(10). Hence, cAMP-elevating agents seem to trigger at least two
different cascades of molecular events, one ending in the stimulation
of melanin synthesis, and a second one decreasing melanogenesis through
the activation of the MAP kinase pathway. Induction of antagonistic
effects, by a single agent, has already been described in the
literature. For instance, EGF activates MAP kinases which phosphorylate
the EGF receptor, leading thereby to a decrease in the EGF binding
(37). Further, tumor necrosis factor-
induces apoptosis and the
activation of NF
B that plays a key protective role against apoptosis
(38). We can envision that these opposite effects elicited by one agent
can be involved in a fine tuning of a final biological effect. This
retrocontrol would avoid overgrowth, overapoptosis, and over-melanin
production that could be noxious for the cell.
In summary, we clearly establish in this report that activation of the MAP kinase pathway is not a required event in the induction of melanogenesis. Furthermore, the activation of this pathway leads to an inhibition of melanoma cell differentiation demonstrating that the up-regulation of melanogenesis by cAMP-elevating agents is the consequence of the activation of two distinct and antagonistic pathways that might allow a precise control of melanocyte and melanoma cell differentiation.
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ACKNOWLEDGEMENTS |
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We thank A. Grima and C. Minghelli for illustration work, and E. Aberdam for critical discussion of the manuscript. We are grateful to Drs. J. Pouysségur and J. C. Chambard for their help to this work.
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FOOTNOTES |
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* This work was supported by the Association pour la Recherche sur le Cancer Grant 6760, Ligue Contre le Cancer, INSERM, and Université de Nice-Sophia Antipolis.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed. Tel.: (33) 04 93 37 77 90; Fax: (33) 04 93 81 14 04; E-mail: ballotti{at}unice.fr.
1 The abbreviations used are: MSH, melanocyte-stimulating hormone; MAP, mitogen-activated protein; ERK, extracellular signal-regulated kinase; MEK, mitogen-activated/extracellular signal-regulated protein kinase; DOPA, dihydroxyphenylalanine; TPA, 12-O-tetradecanoylphorbol-13-acetate; AEBSF, 4-(2-aminoethyl)benzenesulfonyl fluoride; Cdk, cyclin-dependent kinase; EGF, epidermal growth factor; MBP, myelin basic protein.
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REFERENCES |
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