©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Inhibition of MAP Kinase Kinase Blocks the Differentiation of PC-12 Cells Induced by Nerve Growth Factor(*)

Long Pang (2) (1)(§), Takeshi Sawada (2) (1), Stuart J. Decker (1) (3), Alan R. Saltiel (2) (1)(¶)

From the (1) Department of Signal Transduction, Parke-Davis Pharmaceutical Research, Warner-Lambert Co., Ann Arbor, Michigan 48105 and Department of (2) Physiology and (3) Microbiology, University of Michigan School of Medicine, Ann Arbor, Michigan 48109

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The mitogen-activated protein kinase (MAP kinase) pathway is thought to play an important role in the actions of neurotrophins. A small molecule inhibitor of the upstream kinase activator of MAP kinase, MAP kinase kinase (MEK) was examined for its effect on the cellular action of nerve growth factor (NGF) in PC-12 pheochromocytoma cells. PD98059 selectively blocks the activity of MEK, inhibiting both the phosphorylation and activation of MAP kinases in vitro. Pretreatment of PC-12 cells with the compound completely blocked the 4-fold increase in MAP kinase activity produced by NGF. Half-maximal inhibition was observed at 2 µM PD98059, with maximal effects at 10-100 µM. The tyrosine phosphorylation of immunoprecipitated MAP kinase was also completely blocked by the compound. In contrast, the compound was without effect on NGF-dependent tyrosine phosphorylation of the pp140 receptor or its substrate Shc and did not block NGF-dependent activation of phosphatidylinositol 3`-kinase. However, PD98059completely blocked NGF-induced neurite formation in these cells without altering cell viability. These data indicate that the MAP kinase pathway is absolutely required for NGF-induced neuronal differentiation in PC-12 cells.


INTRODUCTION

In the past few years, the mechanisms by which neurotrophins, such as nerve growth factor (NGF),() promote the survival and development of sympathetic and sensory neurons have been the focus of much inquiry. Although a wide variety of signaling processes may be involved, protein phosphorylation appears to play a prominent role (1) . The NGF receptor, pp140, is a tyrosine kinase that undergoes autophosphorylation in response to NGF (2, 3, 4) . Activation of this kinase initiates a cascade of molecular interactions, ultimately resulting in stimulation of the activity of a number of serine/threonine kinases, including the mitogen-activated protein (MAP) kinase (5, 6) . The activation of this kinase is catalyzed by MAP kinase kinase or MEK, which phosphorylates MAP kinase on threonine and tyrosine residues (7, 8) . MEK is itself phosphorylated and activated by an upstream kinase or kinases that appear to require activation of the ras protooncogene (9, 10) . One of these MEK kinases may be the product of the raf protooncogene (11, 12, 13) , although other MEK kinases have been identified (14, 15, 16, 17) .

The precise role of the MAP kinase pathway in growth factor action remains unclear. This enzyme is activated by a wide variety of hormones, cytokines, and growth factors and in turn directs the phosphorylation of transcription factors, such as c-Jun and p62(18, 19) , other kinases such as pp90(20) and MAPKAP (21) , and intermediate regulators of protein synthesis, such as PHAS-1 (22, 23) . Studies using constitutively active mutants of MEK (24) have shown that persistent activation of this pathway may be transforming in some fibroblast cell lines. Moreover, transfection of PC-12 cells with such constitutively active mutants induced neuronal differentiation, whereas dominant negative interfering mutations blocked differentiation (24) .

Although these data provide compelling evidence supporting a critical role for the MAP kinase pathway in neuronal differentiation, the possibility remains that transfected cells may adapt inappropriately to overexpression of these mutants. In this report we describe the use of a specific inhibitor of MEK to completely block the biological effect of NGF in PC-12 cells, providing additional pharmacological evidence that the MAP kinase pathway is required for the differentiative effects of neurotrophins.


EXPERIMENTAL PROCEDURES

Materials

I-Labeled anti-mouse IgG (3000 Ci/mmol) was from Amersham Corp., [-P]ATP (3000 Ci/mmol) was from DuPont NEN, Dulbecco's modified Eagle's medium was from ICN (Irvine, CA), NGF 2.5 S was from Bioproducts for Science (Indianapolis, IN), and anti-phosphotyrosine and anti-Shc antibodies were from Upstate Biotechnology (Lake Placid, NY). All other reagents were from Sigma and were the highest quality available. Anti-pp44(16) and anti-trk(4) antisera were developed as described previously. Anti-MEK antisera was the generous gift of Dr. Kunliang Guan (University of Michigan).

Cell Culture

PC-12 cells were grown on collagen-coated plastic culture dishes in Dulbecco's modified Eagle's medium with 10% fetal bovine serum and 5% horse serum, as described (5, 6) . Prior to treatment with NGF, medium was replaced for 1 h with serum-free medium.

Assay of MAP Kinase Activity

This activity was assayed as described (5) . Briefly, 10-µl aliquots of cell lysates were incubated with pig brain microtubule-associated protein-2 (MAP-2) or myelin basic protein at 0.2 mg/ml for 10 min at 30 °C in a final volume of 25 µl containing 50 mM Tris-HCl (pH 7.4), 2 mM EGTA, 10 mM MgCl, and 40 µM [-P]ATP (1 µCi). The reaction was stopped by the addition of Laemmli SDS sample buffer, and phosphorylated MAP-2 was resolved by 7.2% SDS-PAGE. Coomassie Blue-stained phospho-MAP-2 or myelin basic protein was excised from the gels, and incorporated radioactivity was measured by Cerenkov counting.

Anti-phosphotyrosine Immunoblots

For analysis of protein tyrosine phosphorylation in whole cell lysates, PC-12 cells were grown in 60-mm dishes. After treatment, cells were washed once with ice-cold phosphate-buffered saline, followed by the addition of 100 µl of Laemmli SDS sample buffer. Samples were heated at 95 °C for 5 min, and 30-µg protein aliquots were loaded onto 8% SDS-polyacrylamide gels. Proteins were transferred to nitrocellulose paper and immunoblotted with anti-phosphotyrosine antiserum. In some experiments, lysates were subject to immunoprecipitation. PC-12 cells were grown in 150-mm dishes prior to treatment. Cells were washed once with ice-cold phosphate-buffered saline and lysed in 1 ml of 50 mM HEPES, pH 7.5, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl, 1 mM EDTA, 10 mM sodium pyrophosphate, 100 µM sodium orthovanadate, 100 mM NaF, 30 mMp-nitrophenyl phosphate, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 1 mM phenylmethanesulfonyl fluoride (HNTG buffer) according to Margolis et al.(25) . Lysates were centrifuged for 10 min at 10,000 g, and supernatants were incubated for 60 min with antiserum. Immunoprecipitates were mixed for 30 min with protein A-Sepharose beads, and immune complexes bound to the beads were washed 3 times with 1 ml of the same lysis buffer. Immune complexes were solubilized in 25 µl of Laemmli sample buffer and loaded onto 8% polyacrylamide gels.

Assay of MEK

Cells (150-mm dishes) were serum deprived for 16 h. After growth factor treatments, cells were lysed in HNTG buffer, and lysates were immunoprecipitated with anti-MEK antisera (1:200 dilution) for 2 h at 4 °C. This antiserum precipitates both MEK1 and MEK2. Immunoprecipitates were mixed with Protein A-Sepharose beads for 30 min, and beads were washed twice with 1 ml of HNTG buffer. The sample was then resuspended in 100 µl of reaction buffer containing 20 mM Tris-HCl, pH 7.4, 10 mM MgCl, 1 mM MnCl, 1 mM EGTA. Reactions were initiated by the addition of 10 µCi of -P]ATP (50 µM) and 10 µg of a glutathione S-transferase-fusion protein containing p44 with a Lys Arg substitution (MAPK/KA).() This protein can be phosphorylated by MEK but is catalytically inactive and does not autophosphorylate. After a 15-min incubation at 25 °C, reactions were stopped with 20 µl of Laemmli sample buffer, and phospho-MAPK/KA was detected by SDS-PAGE followed by autoradiography.

Assay of Phosphatidylinositol (PI) 3-Kinase

This was assayed essentially as described previously (27) with the following modifications. Lysates were prepared from confluent cells (100-mm plates) and treated with the indicated concentrations of NGF for 5 min. Cells were lysed in 500 µl of Nonidet P-40 lysis buffer, and lysates were precleared with rabbit IgG and Pansorbin, 5 µl of 4G10 were added, and the samples were incubated overnight. Immune complexes were isolated using 50 µl of protein-G/A agarose. PI 3-kinase was assayed by adding to the immunoprecipitate 50 µl of reaction buffer (10 mM Tris (pH 7.5), 0.2 mM EGTA, 100 mM NaCl, and 20 mM MgCl), 0.4 mg of phosphatidylinositol, and 10 µM [-P]ATP (10 µCi), followed by incubation for 20 min at room temperature. The reaction was stopped by extraction with organic solvents; the organic phase was dried down and chromatographed on silica gel TLC. Incorporated radioactivity was determined by autoradiography.


RESULTS

PD98059 was discovered by screening a chemical library for inhibitors of the MAP kinase cascade. Its structure is shown in Fig. 1 . This compound is a selective inhibitor of MAP kinase kinase or MEK. To evaluate its ability to block the stimulation of MAP kinase, PC-12 cells were preincubated with 0-100 µM PD98059 for 10 min prior to the addition of 100 nM NGF for 2 min. Following the treatment, lysates were assayed for MAP kinase activity by evaluating the phosphorylation of myelin basic protein (Fig. 2). As described previously (5) , NGF produced almost a 4-fold increase in MAP kinase activity. Pretreatment of cells with PD98059 alone was without effect, but the compound completely blocked stimulation of the enzyme by NGF, returning the enzyme activity to basal levels. Half-maximal inhibition was observed at approximately 2 µM, and complete inhibition was observed at 10-100 µM. This blockade of MAP kinase activation by NGF was also observed in 3T3 cells expressing the trk protooncogene. Direct addition of PD98059 to the assay incubation had no effect on MAP kinase activity (data not shown).


Figure 1: Structure of PD98059.




Figure 2: PD98059 blocks the activation of MAP kinase by NGF. Serum-deprived PC-12 cells in 100-mm dishes were treated with an indicated concentration of PD98059 for 30 min prior to the addition of 50 ng/ml NGF for 5 min. Cells were lysed and MAP kinase activity was assayed as described under ``Experimental Procedures,'' using myelin basic protein (MBP) as substrate. Following SDS-PAGE, the phosphorylated substrate was excised from the gel and quantitated by Cerenkov counting. Results are expressed as the means of triplicate determinations ± S.D. and are representative of five separate experiments. Activity assayed in the absence of NGF was 1.2 10 cpm.



MAP kinase is thought to be activated by the dual specificity kinase, MEK. To confirm that PD98059 blocks MAP kinase activation via inhibition of MEK, we examined the activity of MEK in an immune complex assay (Fig. 3). PC-12 cells were preincubated with or without 20 µM PD98059 and then exposed to NGF for 5 min. Cells were lysed, and lysates were immunoprecipitated with an anti-MEK antisera that recognizes both MEK1 and MEK2. MEK activity was assayed by evaluating the phosphorylation of a glutathione S-transferase-fusion protein containing a catalytically inactive MAP kinase. Treatment of cells with NGF caused a 4-fold increase in MEK activity. This stimulation was inhibited approximately 70% by preincubation of cells with PD98059. Interestingly, direct addition of PD98059 to the assay inhibits MEK activity by over 90%, although this inhibition can be reversed by extensive washing. It is likely that the reduced inhibition described here results from some dissociation of PD98059 from the enzyme complex during immunoprecipitation and washing.


Figure 3: PD98059 inhibits the activation of MEK by NGF. Serum-deprived PC-12 cells were grown in 150-mm dishes. Cells were preincubated for 30 min with 20 µM PD98059 or dimethyl sulfoxide, followed by a 5-min exposure to NGF. Cell lysates were immunoprecipitated with anti-MEK antisera, and the phosphorylation of glutathione S-transferase-MAPK/KA was assayed as described under ``Experimental Procedures.'' The migration of glutathione S-transferase-MAPK/KA is shown in the autoradiograph. GST-ERK, glutathione S-transferase-extracellular regulated kinase. In B, regions of the gel corresponding to the phosphorylated band were excised and subject to Cerenkov counting. Identical results were obtained in two separate experiments.



To further confirm that PD98059 specifically blocks the activation of MAP kinase, we examined the tyrosine phosphorylation of the enzyme. PC-12 cells were treated with or without PD98059 as described above, followed by exposure to NGF for 2 min. Cells were lysed, and lysates were precipitated with an anti-MAP kinase antisera. Immunoprecipitates were subject to SDS-polyacrylamide gel electrophoresis, followed by immunoblotting with anti-phosphotyrosine antibodies (Fig. 4A). As described previously (6) , NGF caused the tyrosine phosphorylation of MAP kinase, due to the activation of its upstream kinase, MEK. Preincubation with PD98059 completely blocked this effect. To confirm the specificity of this agent, whole cell lysates from the same cells were immunoprecipitated with anti-trk and anti-Shc antibodies, followed by anti-phosphotyrosine Western blotting (Fig. 4B). Neither autophosphorylation of the receptor nor the phosphorylation of its substrate Shc (28) was affected by PD98059.


Figure 4: PD98059 blocks the tyrosine phosphorylation of MAP kinase but not early NGF-receptor tyrosine phosphorylations. PC-12 cells were cultured as described in Fig. 2. Cells were treated with or without 20 µM PD98059 for 30 min, followed by a 5-min exposure to NGF, as indicated. In A, cell lysates were immunoprecipitated with anti-MAP kinase antisera, followed by SDS-PAGE. The resulting gels were Western blotted with anti-phosphotyrosine antibody. In B, lysates were precipitated with anti-trk or anti-shc antisera, and precipitates were subject to SDS-PAGE and anti-phosphotyrosine immunoblotting as described above. IP, immunoprecipitate.



The binding of NGF to its receptor and the subsequent stimulation of its tyrosine kinase activity lead to an increase in PI 3-kinase activity in PC-12 cells (27) , by virtue of the association of the p85 regulatory subunit of the enzyme with a 100-kDa substrate of the receptor (27) . Recent studies with wortmannin (29) suggest that the activation of PI 3-kinase may be necessary for the biological effects of NGF. To evaluate whether PD98059 had any effect on the stimulation of this enzyme, cells were pretreated with the compound as described above, followed by exposure to NGF (Fig. 5). As described previously (27) , NGF caused the appearance of PI 3-kinase activity in anti-phosphotyrosine immunoprecipitates, correlating with activation of the enzyme and generation of PI-3,4,5-P in PC-12 cells (30) . Treatment of cells with PD98059 was without effect on this stimulation. Moreover, direct addition of the compound to the assay incubation did not affect enzyme activity (data not shown).


Figure 5: PD98059 does not block the activation of PI 3-kinase by NGF. PC-12 cells were preincubated with 20 µM PD98059 and treated with NGF for 5 min as described above. Cell lysates were immunoprecipitated with anti-phosphotyrosine antisera, and PI 3-kinase activity was assayed in the immunoprecipitate. An autoradiograph of the thin layer chromatograph is shown. Identical results were obtained in three separate experiments.



Inhibition of the tyrosine kinase activity of the NGF receptor by staurosporine and related compounds (31) or by tyrphostins (32) can block NGF-dependent neurite outgrowth. To evaluate the specific role of the MEK-MAP kinase pathway in the differentiative action of NGF, PC-12 cells were treated with 10 µM PD98059 for 30 min before the addition of NGF for 2 days (Fig. 6). The compound completely blocked NGF-induced neurite formation, without significantly altering cell viability.


Figure 6: PD98059 inhibits NGF-induced neurite outgrowth. PC-12 cells were treated with 10 µM PD98059 for 30 min prior to addition of 50 nM NGF for 3 days. Cells were photographed using a phase-contrast light microscope.




DISCUSSION

The stimulation by hormones of the MAP kinase pathway is thought to be involved in a number of cellular changes, including both growth and differentiation. In PC-12 cells, agents such as NGF that induce neuronal differentiation lead to the persistent activation (5, 33) and subsequent nuclear translocation (26) of the enzyme. In contrast, receptors for other growth factors, such as EGF, that only transiently activate MAP kinase and do not induce its nuclear translocation do not result in neuronal differentiation (26) . Such correlative data were significantly extended in recent experiments showing that expression in PC-12 cells of a constitutively active MEK mutant can itself induce differentiation (24) , suggesting that overactivation of this pathway is sufficient to induce this morphological phenotype. Moreover, transfection of PC-12 cells with a mutant MEK in which the two phosphorylation sites required for activation (serine 218/222) were substituted with alanine produced a dominant interfering phenotype in which NGF did not induce differentiation (24) . Although such experiments strongly suggest that MEK activation is essential for this cellular action of NGF, it is important to note that overexpression of dominant interfering mutants such as these are likely to bind but not dissociate from upstream activating kinases, preventing these kinases from phosphorylating their endogenous substrates. Thus, these data indicate an essential role for MEK kinases in neuronal differentiation, leaving open the issue of the necessity for MEK itself.

In order to answer this question, we have utilized a novel, small molecular weight inhibitor of MEK activity, PD98059. This compound can block the activity of MEK assayed both in vitro with recombinant enzyme and in a variety of intact cells. This inhibition appears to be noncompetitive with respect to ATP. Moreover, the compound has not been found to block a number of other protein serine/threonine or tyrosine kinases, even at higher concentrations. We show here that PD98059 can completely inhibit the activation of MAP kinase by NGF in PC-12 cells, presumably due to the blockade of its upstream activator. In contrast, the compound has no effect on early phosphorylation events catalyzed by the pp140 NGF receptor. Interestingly, at these concentrations, this inhibition is accompanied by the complete blockade of NGF-induced neuronal differentiation, without impairing cell viability. In contrast, the compound does not block differentiation of other tissue culture cells, including 3T3-L1 adipocytes or L-6 myocytes (data not shown). These data provide strong additional support for the requirement of the MAP kinase pathway in PC-12 cell differentiation.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Sugen, Inc., 515 Galveston Dr., Redwood City, CA 94063.

To whom correspondence should be addressed: Dept. of Signal Transduction, Parke-Davis Pharmaceutical Research/Warner-Lambert Co., 2800 Plymouth Rd., Ann Arbor, MI 48105. Tel.: 313-996-3960; Fax: 313-996-5668.

The abbreviations used are: NGF, nerve growth factor; MAP kinase, mitogen-activated protein kinase; MEK, MAP kinase kinase; PAGE, polyacrylamide gel electrophoresis; PI, phosphatidylinositol; MAP-2, microtubule-associated protein-2.

Dudley, D. T., Pang, L., Decker, S. J., Bridges, A. J., and Saltiel, A. R. (1995) Proc. Natl. Acad. Sci. U. S. A., in press.


ACKNOWLEDGEMENTS

We thank Drs. David Dudley, Alex Bridges, and Kunliang Guan for helpful discussions.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.