Journal of Neurochemistry Raven Press, Ltd., New York 0 1992 International Society for Neurochemistry

K-252b Selectively Potentiates Cellular Actions and trk Tyrosine Phosphorylation Mediated by Neurotrophin-3 Beat Kniisel, *David R. Kaplan, ?John W. Winslow, “frnon Rosenthal, ?Louis E. Burton, Klaus D. Beck, *Stuart Rabin, tKaroly Nikolics, and Franz Hefti Department of Biological Sciences and Andrus Gerontology Center, University of Southern California, Los Angeles, California; *Eukaryotic Signal Transduction Group, ABL-Basic Research Program, NCI-Frederick Cancer Research and Development Center, FrederickxMaryland; TGenentech, Inc., South Sun Francisco, California, U.S.A.

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Abstract: K-252b, a protein kinase inhibitor, has been shown earlier to inhibit nerve growth factor actions on cholinergic neurons of the basal forebrain. In the present study, K-252b was found to prevent trophic actions of two other neurotrophins, brain-derived neurotrophic factor, and neurotrophin-3, on central cholinergic and dopaminergic neurons, peripheral sensory neurons, and PC 12 pheochromocytoma cells, when used at >2 pM concentration. Comparable actions of nonneurotrophin growth factors were not affected. Surprisingly, at 0.1-100 nM, K-252b selectively enhanced the trophic action of neurotrophin-3 on central cholinergic neurons, peripheral sensory neurons, and PC 12 cells. In PC12 cells, K-252b potentiated the neurotrophin3-induced tyrosine phosphorylation of trk, a protein

kinase responsible for transmitting neurotrophin signals. Of the three structurally related nerve growth factor inhibitors, K-252a, K-252b, and staurosporine, only the first two also mediated neurotrophin-3 potentiation. These findings indicate that K-252b generally and selectively potentiates the neurotrophic action of neurotrophin-3 and suggest that this action involves trk-type neurotrophin receptors. Key Words: Alzheimer’s disease-Brain-derived neurotrophic factor-Nerve growth factor receptor-Neurotrophin-3Protein kinase inhibitors-Parkinson’s disease. Kniisel B. et al. K-252b selectively potentiates cellular actions and trk tyrosine phosphorylation mediated by neurotrophin-3. J. Neurochem. 59, 7 15-722 ( 1 992).

The protein growth factors ofthe neurotrophin family, which includes nerve growth factor (NGF), brainderived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4, and neurotrophin-5, regulate nervous system development (Barde, 1989; Leibrock et al., 1989; Ernfors et al., 1990; Hohn et al., 1990;Jones and Reichardt, 1990; Maisonpierre et al., 1990; Rosenthal et al., 1990; Hallbook et al., 1991; Thoenen, 1991; Berkemeier et al., 1991)and are also believed to play an important role in structural maintenance, plasticity, and repair of the adult nervous system (Thoenen et al., 1987;Whittemore and Seiger, 1987; Hefti et al., 1989). At least two types of proteins are involved in the formation of functional receptors for neurotrophin growth factors. These are the low-affinity NGF receptor protein (p75-NGFR) (Chao et al., 1986; Radeke et al., 1987) and products of trk-related protooncogenes (Kaplan et al., 1990, 199la,b;

Squint0 et al., 1990, 1991;Cordon-Cardoet al., 1991; Hempstead et al., 1991; Lamballe et al., 1991; Soppet et al., 1991). The trk gene products, but not the p75NGFR, exhibit protein kinase activity (Martin-Zanca et al., 1989). Individual neurotrophins bind to and stimulate tyrosine phosphorylation of different subsets of trk receptors. trk binds to NGF, but not BDNF, and trkB binds to BDNF, but not NGF (CordonCardo et al., 1991). NT-3, in vitro, is capable of interacting with trk and trkB receptors and with trkC (Cordon-Cardo et al., 1991; Lamballe et al., 1991). Cholinergk neurons of embryonic rat basal forebrain in culture are known to respond to NGF, BDNF, basic fibroblast growth factor (bFGF), insulin, and the insulin-like growth factors I and I1 (Ferrari et al., 1989; Alderson et al., 1990; Kniisel et al., 1990a,b, 1991). Recently, we also found that treatment with NT-3, albeit only at very high concen-

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Received December 19, 199 1; revised manuscript received January 30, 1992; accepted February 13, 1992. Address correspondence and reprint requests to Dr. B. Kniisel at University of Southern California, Andrus Gerontology Center, University Park, MC-0191, Los Angeles, CA 90089, U.S.A. Abbreviations used: BDNF, brain-derived neurotrophic factor;

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bFGF, basic fibroblast growth factor; ChAT, choline acetyltransferase; des-IGF-I , des-( I-3)-insulin-like growth factor-I; DMSO, dimethyl sulfoxide; DRG, dorsal root ganglion; EGF, epidermal growth factor; NGF, nerve growth factor; NT-3, neurotrophin-3; SDS-PAGE, sodium dodecyl sulfate-pol yacrylamidegel electrophoresis.

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trations of this neurotrophin, increases the activity of the cholinergic marker enzyme choline acetyltransferase (ChAT)in these cultures (unpublishedobservations). Although BDNF, bFGF, and the insulin family of growth factors also stimulate central dopaminergic and, possibly, y-aminobutyric acid (GABA)ergic neurons, stimulation by NGF and NT-3 seems to be selective to the basal forebrain cholinergic neurons (Kniisel et al., 1990a, 1991, and unpublished data). K-252a and K-252b, two related alkaloid-likecompounds from microbial origin and known to interfere with protein kinase activities in cell-free systems, inhibit several biological actions of NGF (Kase et al., 1987; Koizumi et al., 1988; Matsuda and Fukuda, 1988).K-252a prevents the NGF-induced morphological transformation of proliferating PC 12 pheochromocytoma cells into neuron-like cells and inhibits the NGF-stimulated, but not the bFGF- or epidermal growth factor (EGF)-stimulated, phosphorylation of selected proteins (Smith et al., 1989). K-252a, in the absence of exogenouslyprovided trophic proteins, has also been shown to exhibit neurotrophic-likeactivity in chick dorsal root ganglion (DRG) cultures (Borasio, 1990) and on dopaminergic neurons of the ventral mesencephalon in culture (Kniisel and Hefti, 1991). We recently demonstrated that K-252a and K-252b inhibit NGF-mediated actions on cholinergic neurons in cell culture (Knusel and Hefti, 1991). Both compounds completely and selectively prevented the trophic action of NGF on these cells. K-252b was effective over a wide range of concentrations without being cytotoxic or neurotrophic for the cultures. The discovery of the protein family of neurotrophins prompted us to investigate whether K-252b inhibits biological actions of neurotrophins other than NGF. We found that K-252b selectively inhibits growth factors of the neurotrophin family, but surprisingly, at lower concentrations, potentiates NT-3 actions. K-252b enhanced the trophic activity of NT-3 on primary neurons and PC 12 cells and stimulated the NT3-mediated tyrosine phosphorylation of trk in PC12 cells, while inhibiting similar effects of NGF. The structurally closely related NGF inhibitor K-252a, but not the similarly related staurosporine, also potentiated NT-3. Our results suggestthat K-252b stimulates and inhibits selective cellular effects of the neurotrophins and that these actions of K-252b are due to direct or indirect interaction with the trk signal transduction pathway.

MATERIALS AND METHODS Growth factors and other materials The preparations of human recombinant neurotrophins that were used in the present study were produced in a Chinese hamster ovary cell line as described in detail previously (Knusel et al., 1990b; Rosenthal et al., 1990, 1991). The neurotrophins were purified to >95% purity using chromatographic procedures. The neurotrophins were initially J. Neurochem., Val. 59, No. 2, I992

contained at a concentration of 0.2-1 mg/ml at pH 3 and were further diluted in culture medium immediately before use. Biological activity for all tested neurotrophin preparations was determined with chick DRG and nodose ganglion assays. Recombinant human des-( 1-3)-insulin-like growth factor-1 (des-IGF-1) was obtained from Dr. S. E. Builder at Genentech and initially contained in 100 mM sodium acetate. K-252b was a gift from Dr. Y. Matsuda, Kyowa Hakko Kogyo Co., Tokyo, Japan. It was dissolved in dimethyl sulfoxide (DMSO) at 2 mM concentrations. Aliquots of this solution were kept at -70°C. Maximal concentration of DMSO in the medium was 0.0 1%, which was found not to affect the cultures (data not shown).

Primary cultures of fetal rat brain neurons Primary cultures of fetal rat brain septal and mesencephalic cells were prepared as described in detail elsewhere (Kniisel et al., 1990a,b, 1991). In brief, defined areas were dissected from fetal rat brains (Wistar, E 15-16; Charles River). The septal area contained the cholinergic neurons from septum, diagonal band of Broca, and nucleus basalis. The ventral mesencephalon contained the dopaminergic neurons of the substantia nigra and the ventral tegmental area, and was dissected and dissociated mechanically. The cells were plated in 16-mm multiwell plates, precoated with polyethylenimine ( 1 mg/ml, 37"C, overnight), containing 0.5 ml of modified L-15 medium supplemented with 5% heat-inactivated horse serum and 0.5% heat-inactivated fetal calf serum. Modified L-15 was prepared as described earlier (Kniisel et al., 1 9 9 0 ~by ) adding various amino acids, vitamins, antibiotics, glucose, and NaHCO, to Leibovitz's L-15 medium (GIBCO). Plating densities were 4 X 1O5 cells/ cm2 for basal forebrain cultures and 3 X lo5 cells/cm2 for mesencephalic cultures. Of these cells, 0.5- 1% were cholinergic or dopaminergic, respectively. Growth factors were typically added on day 2 of culture and the cells were grown for 2 5 days. For ChAT assays, tissue was homogenized in 250 pl of 50 mMTris-HC1 buffer, pH 6.0, with 0.3% Triton X-100. [ l-'4C]Acetyl-CoA(NEN) concentration was 20 pM and specific activity, 4.09 Ci/mol. To measure dopamine uptake, cultures were preincubated for 5 min at 37°C with 250 p1 of incubation solution (5 m M glucose, 1 mM ascorbic acid in phosphate-bufferedsaline) containing I M p a r gyline. [3H]Dopamine(37 Ci/mmol) was then added to give a final concentration of 50 nMand the cultures were incubated for another 15 min. Blanks were obtained by incubating cells at 0°C.

Primary cultures of chick DRG neurons DRGs of embryonic day 9 chicks were dissected and dissociated using enzymatic and mechanical procedures as described in detail elsewhere (Rosenthal et al., 1990, 1991). Neurons were plated, 1,800 per well, in 96-well tissue-culture plates pretreated with polyornithine (500 pg/ml) and laminin (10 pg/ml). Cells were incubated for 48 h with or without growth factors and the indicated concentrations of K-252b. Phase-bright cells with elaborated neurites fivefold the diameter of the cell bodies were then counted.

Neurite outgrowth and trk phosphorylation in PC12 cells PC12 cells were grown as described earlier (Kaplan et al., 1990, 1991ah). To assess effects on neurite outgrowth, cells were incubated for 48 h with K-252b (50 nM) and NT-3 or NGF (50 ng/ml). Neurites were scored if they were a length

NT-3 POTENTIATION BY K-252b of one cell body or more. To measure the status of trk tyrosine phosphorylation, PC12 cells were lysed and immunoprecipitated with anti-trk serum. trk proteins were subjected to 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed by immunoblotting with antiphosphotyrosine antibodies as described in detail elsewhere (Kaplan et al., 1990, 1991a,b). Tyrosine phosphorylation of cellular proteins was assayed by probing western blots of lysates of PC12 cells incubated with K-252b and NT-3 or NGF with antiphosphotyrosine antibodies. Phospholipase C 7-1 was identified in these blots as described elsewhere (Vetter et al., 1991).

RESULTS Inhibition of neurotrophin actions by K-252b in primary cultures of brain neurons Recombinant human NGF, BDNF, and NT-3, in the presence or absence of K-252b, were added to primary cultures of fetal rat basal forebrain or ventral mesencephalon, containing cholinergic and dopaminergic neurons, respectively. A broad range of concentrations of K-252b were tested with 50 ng/ml of NGF, 200 ng/ml of BDNF, and 200 ng/ml of NT-3, growth factor concentrations producing trophic actions on the basal forebrain cholinergic neurons (Kniisel et al., 1990b, 1991, and unpublished observations). The trophic action was measured by determining the activity of ChAT, a parameter reflecting both survival and transmitter-specific differentiation of cholinergic cells (Hartikka and Hefti, 1988). K-252b, at concentrations higher than 200 nM, inhibited the ChAT activity increase mediated by all three neurotrophins (Fig. 1). The concentration requirements for the inhibitions of NGF, BDNF, and NT-3 actions ap-

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FIG. 1. ChAT activity in cultures of rat basal forebrain treated with NGF, BDNF, or NT-3, and K-252b. Growth factors were added on day 2 of culture and the cells were grown for 5 days. Growth factor concentrations producing maximal elevations of ChAT activity were used. 0,control; 0,50 ng/ml of NGF; A, 200 ng/ml of BDNF; V, 200 ng/rnl of NT-3. The ChAT stimulation mediated by all three neurotrophinswas inhibited by K-252b at concentrations >200 nM. At concentrationsbetween 100 pM and 30 nM, K-252b potentiated the actions of NT-3 without affecting those of NGF or BDNF. n = 4 per data point. Error bars represent SEMs and were omitted where they would have appeared smaller than the symbol.

717

peared identical. The minimal concentration of K-252b that was required to completely abolish the trophic responses was - 5 pM (Fig. 1). In agreement with our earlier findings (Knusel and Hefti, 199I), the increases in ChAT activity mediated by bFGF, insulin, and insulin-like growth factor- 1 were not affected by K-252b (data not shown). BDNF, but not NGF or NT-3, trophically acts on dopaminergic neurons as reflected by an increase in the uptake rate for dopamine by these cells (Hyman et al., 1991;Kniisel et al., 1991). Similar to its actions on cholinergic neurons, K-252b prevented the increase in dopamine uptake rate mediated by BDNF (Table 1). Dopamine uptake is also stimulated by other growth factors including bFGF, EGF, insulin, insulin-like growth factors-1 and 2 (Ferrari et al., 1989; Kniisel et al., 1990a). We earlier showed that K-252b does not inhibit the stimulatory action of bFGF and insulin (Knusel and Hefti, 1991). For the present study, des-IGF- 1 was used as an additional control, and K-252b was found not to inhibit its stimulatory action on dopamine uptake (Table 1). The findings obtained on central cholinergic and dopaminergic neurons showed that K-252b, at concentrations >200 nM, selectivelyblocks the actions of all neurotrophins stimulating these cells in primary cultures, whereas comparable effects of nonneurotrophin growth factors are not inhibited. Potentiation of NT-3 actions by K-252b on brain neurons Surprisingly, the detailed dose-response analysis of K-252b actions on cholinergic neurons revealed that the compound strongly enhanced the trophic effect of NT-3 on these cells at concentrations lower than those producing inhibitory effects. In the presence of 3-30 nM of K-252b, NT-3, which by itself elevated ChAT activity by only -20% of the NGF-induced elevation, produced the same stimulatory effect as NGF (Fig. 1). The stimulatory effects of NGF or BDNF on cholinergic neurons were not potentiated by these low concentrations of K-252b (Fig. 1). Similarly, the low concentrations did not potentiate the action of BDNF on dopaminergic neurons. Dose-response analysis for NT-3 revealed that K-252b, at 50 nM, increased both potency and maximal efficacy of the trophic action of NT-3. Potentiation is observed at as low as 10 ng/ml of NT-3 and maximal effect is at 30-300 ng/ml of the neurotrophin (Fig. 2). Corresponding experiments with NGF and BDNF did not show any potentiation of the effects of these neurotrophins, even at subsaturating concentration (data not shown). K-252b potentiates NT-3-mediated survival of chick sensory neurons and neurite outgrowth of PCI 2 cells To generally establish that K-252b, at concentrations lower than required to inhibit neurotrophin actions, selectively enhances cellular effects of NT-3, cultures of different neurotrophin responsive cell popJ. Neurochem., Vol. 59, No. 2, 1992

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TABLE 1. K-2526 inhibits the stirnulatory action of BDNF, but not des-IGF-I, on doparnine uptake in cultures of ventral mesencephalon With K-252b (5 PW

NO K-252b

Growth factor treatment

Mean f SEM

% of control

Control BDNF des-IGF- 1

15.3 k 3.6 28.0 + 0.3" 26.3 1.1"

183 172

Mean

k

% of control

SEM

11.4 + 0.2 13.6 f 0.7 22.9 k 1.0"

119 20 1

Dopamine uptake is given as femtomoles per minute per culture dish. Cultures were grown in 24-well plates for 7 days in L-15 medium with 5% horse and 0.5% fetal bovine serum and treated with growth factors and K-252b from day 2 of culture. BDNF, 200 ng/ml; des-IGF- I , 1 pg/ml; n = 4. The experiment shown is a representative case of a total of five independent experiments. See legend of Fig. 1 for experimental details. " Different from respective control group, p < 0.01 (Student's t test). The apparent difference between control cultures grown in the presence and absence of K-252b was statistically not significant.

ulations were tested. Survival and neurite outgrowth of chick DRG neurons is supported by NGF, BDNF, and NT-3, and the extent of the effect and the subpopulation supported by each factor is a function of the embryonic age of the animals used to prepare the cultures (Davies et al., 1986). In our DRG cultures of embryonic day 9, NGF is most effective in promoting neuronal survival, whereas NT-3 only produces a moderate effect. Similar to the findings obtained in primary cultures of brain cholinergic neurons, low concentration of K-252b (50 nM)potentiated the survival promoting action of a maximally effective concentration of NT-3 (Fig. 3). The number of neurons supported by the combination of NT-3 and 50 nM K-252b was identical to that supported by NGF. The same concentration of K-252b failed to influence survival mediated by NGF (Fig. 3) or BDNF (data not

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FIG. 2. K-252b (50 nM) increases potency and efficacy of NT-3 stimulation of ChAT activity in cultures of rat basal forebrain. Halfmaximal concentrations were calculated by nonlinear fit of a sigmoid curve. Broken line: NT-3 alone, ED,, = 175 k 72.6 ng/ml (mean k SE); solid line: NT-3 K-252b, ED,, = 14.3 f 1.6 ng/ml; n = 8 per symbol. Culture conditions were as described in legend of Fig. 1.

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shown). High concentrations of K-252b (10 p M ) inhibited the action of NGF on sensory neurons as found for cholinergic neurons (Fig. 3). K-252b alone did not enhance the survival of the DRG neurons. Similar results were also seen in cultures of dissociated chick sympathetic neurons (data not shown). Experiments on PC12 cells confirmed observations made on primary neuron cultures. PC12 cells incubated for 48 h in the presence of NT-3 (50 ng/ml) and K-252b (50 nM) showed significantly more neurite outgrowth activity than cells incubated with NT-3 alone (Fig. 4A). At the concentration of K-252b used in this experiment, NGF-induced neurite outgrowth was reduced approximately threefold, whereas in the primary neuron cultures no significant inhibition was detected at this concentration (Fig. 1). This result is similar to a previous observation with K-252a. Higher concentration of this inhibitor is also required to block the NGF action in cholinergic neurons in pn-

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FIG. 3. Survival of dissociated chick DRG neurons in presence of NGF (100 ng/ml) or NT-3 (100 ng/ml) and K-252b. Concentrations on horizontal axis are for K-252b. Columns represent the mean f SEM.

NT-3 POTENTIATION BY K-252b 2

1001

NT-3 NI-3

NGF N$F

K-252b

8

1

tfk

K-252b

2

3

4

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2

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FIG. 4. Treatment with K-252b stimulates NT-3-induced neurite outgrowth and trk tyrosine phosphorylation in PC12 cells while inhibiting NGF-induced effects. PC12 cells were incubated for 48 h with K-252b (50 nM) and NT-3 (50 ng/ml) or NGF (50 ng/ml). A Percentage of cells with neurites after 48 h of incubation. Neurites were scored if they were a length one cell body or more. B: PC12 cells were subsequently lysed and immunoprecipitated with anti-frk serum. trk proteins were subjected to SDS-PAGE and analyzed by immunoblotting with antiphosphotyrosine antibodies. Lane 1, NT-3 + K-252b; lane 2, NT-3; lane 3, NGF K-252b; lane 4, NGF. C PC12 cells were incubated for 1 h in K-252b (50 nM or 10 pM) and with NT-3 or NGF for 5 min before lysis. Other methods were the same as in B. Lane 1, NT-3 + 10 f l K-252b; lane 2, NT-3 50 nM K-252b; lane 3, NT-3; lane 4, NGF + 10 f l K-252b; lane 5, NGF + 50 nM K-2526; lane 6, NGF; lane 7, untreated control.

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719

NT-3 induces only low levels of trk tyrosine phosphorylation and neurite outgrowth in these cells (Fig. 4A and Berkemeier et al., 1991). PC 12 cells that were exposed to NT-3 or NGF for 48 h in the presence or absence of K-252b (Fig. 4A) were also analyzed for pl40trk tyrosine phosphorylation (Fig. 4B). NGF (50 ng/ml) strongly stimulated trk tyrosine phosphorylation. No phosphorylation was detectable if the cells were treated with NT-3 alone (Fig. 4B). Similarly, cells treated with K-252b alone were identical to untreated controls (data not shown). However, a clear increase in trk tyrosine phosphorylation was seen in cells grown in the simultaneouspresence of 50 ng/ml of NT-3 and 50 nMK-252b (Fig. 4B, lane 1). Similar enhancement of trk tyrosine phosphorylation was observed when cells were acutely treated for 1 h with K-252b followed, during the last 5 min of K-252b treatment, with NT-3 (Fig. 4C). Again, stimulation of tyrosine phosphorylation by NT-3 alone was minimal, but was greatly enhanced by the simultaneous presence of K-252b. In this assay, NGF-mediated tyrosine phosphorylation was inhibited twofold or fivefold by 50 nM or 10 pM K-252b, respectively. Tyrosine phosphorylation of other cellular proteins was examined in PC12 cells treated with NT-3 or NGF in the presence of K-252b. The tyrosine phosphorylation of phospholipase C y-1, a direct target of the trk tyrosine kinase (Vetter et al., 1991), and several other cellular proteins, was inhibited by 10 pM K-252b in NGF-treated cells. In contrast, the NT-3mediated tyrosine phosphorylation of these proteins in PC12 cells was enhanced by 50 nM K-252b (data not shown). The tyrosine phosphorylation of EGF receptors in PC12 cells treated with 100 ng/ml of EGF for 5 min was not inhibited by 10 pM K-252b (data not shown). Structural requirements for NT-3potentiation To determine the structural features of the K-252b molecule that are responsible for selective potentia-

- 45 3 '= 0

mary cultures than is required to suppress NGF effects in PC12 cells (Koizumi et al., 1988; Smith et al., 1989; Kniisel and Hefti, 1991). Effects of K-252b on tyrosine phosphorylation of tvk The fact that K-252b inhibits various protein kinases in cell-free systems (Kase et al., 1987) and the recent discovery that trk protooncogenesare involved in the formation of high-affinity neurotrophin receptors (Kaplan et al., 1990, 1991a,b; Squint0 et al., 1990, 1991;Hempstead et al., 1991;Cordon-Cardoet al., 1991; Soppet et al., 1991) suggested that K-252b exerts its neurotrophin inhibitory and stimulatory actions by directly interfering with protein kinases of the trk protein family. NGF has been shown to stimulate the tyrosine phosphorylation of p 140trk within minutes of addition to PC I2 cells (Kaplan et al., 1991b).

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FIG. 5. K-252aand K-252b, but not the closely relatedNGF inhibitor staurosporine, potentiate the NT-3 action on cholinergic neurons. Cultures were untreated (control) or treated with 200 ng/ml of NT-3 and increasing concentrationsof the inhibitors.0, control (untreated cultures); V, NT-3 only; 0 , K-252a NT-3; u, K-252b NT-3; A, staurosporine NT-3; n = 8 per symbol. Culture conditions were as described in legend of Fig. 1.

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tion of NT-3 and, in particular, whether these features are different from the ones mediating neurotrophin inhibition, two close structural relatives of K-252b were studied. We showed earlier that besides K-252b, K-252a and staurosporine inhibit the NGF action on central cholinergic neurons (Kniisel and Hefti, 1991). We now tested these two compounds for possible NT3 potentiation at lower concentrations than necessary for NGF inhibition. Of the two K-252b analogues, only K-252a also induced potentiation, whereas the other NGF inhibitor, staurosporine, was ineffective (Fig. 5). DISCUSSION The findings of the present study demonstrate that K-252b selectively modifies actions of the neurotrophin growth factor family. At nanomolar concentrations, K-252b selectively potentiates the actions of NT-3. At micromolar concentrations, the compound inhibits the actions of all three neurotrophins tested, without interfering with the effects of nonneurotrophin growth factors. Our finding of parallel effects of K-252b on neurite outgrowth and trk tyrosine autophosphorylation in PC 12 cells in the presence of NT3 or NGF, respectively, suggests that the selective inhibitory and stimulatory actions of K-252b on neurotrophin effects involve interaction with trk-type receptor mechanisms. It remains to be determined, however, whether such interaction is by direct effect on protein kinase activity of the trk protein as has been demonstrated recently for inhibition by K-252a (Berg et al., 1992). Cordon-Cardo et al. (1991) have shown that NT-3 can act as a ligand for the trk receptor and induce biological function. A possible explanation for the observation of increased NT-3 effects is that K-252b modifies trk in a manner so that this receptor will interact with NT-3, but not NGF, more efficiently. Although K-252a does not interfere with binding of '251-NGFto PC12 cells (Koizumi et al., 1988),it seems possible that the related K-252b might affect binding of selected neurotrophins to specific active sites. Such an effect could also involve other proteins that are believed to be part of neurotrophin receptors, like the p75-NGFR low-affinity NGF receptor protein (Rodriguez-Tebar et al., 1990; Bothwell, 1991). K-252b inhibits protein kinase C, as well as cyclic AMP- and cyclic GMP-dependent protein kinases with Ki values in the 10- 100 nM range as shown by in vitro assay systems (Kase et al., 1987). Given this rather broad spectrum of inhibitory actions, the selective inhibition of neurotrophin effects is surprising. The two related molecules, K-252b and K-252a, inhibit similar protein kinases with only slightly different substrate specificities in cell-free assay systems (Kase et al., 1987),whereas there are pronounced differences between their actions on intact cells (Knusel and Hefti, 1991). In contrast to K-252a, K-252b is a J. Neurochem.. Vol. 59, No. 2, 1992

hydrophilic molecule with a free carboxylic acid residue and, therefore, does not freely pass the cell membrane (Nagashima et al., 1991). Because of this property, the intracellular concentration and distribution of K-252b in different compartments of living cells may be very different from that of K-252a. The selectivity of the effects on neurotrophin actions could be explained if only very limited amounts of K-252b enter the cells and if K-252b is more potent in modifying trk protein kinase activity than other protein kinases, including other growth factor receptor kinases. Alternatively, K-252b, in intact cells, might interact with an ecto-protein kinase (Ehrlich et al., 1986) or, specifically, with extracellularor transmembranal domains of trk proteins, without access to intracellular protein kinases. This possibility is supported by recent findings showing that K-252b inhibits the protein kinase activity of the platelet-derived growth factor receptor in cell-free preparations, but not in intact cells (D. R. Kaplan, unpublished observations). Different cellular distribution might also explain the absence of neurotrophic-like effects of K-252b as they are observed with K-252a in chick DRG cells (Borasio, 1990) or in rat dopaminergic neurons (Knusel and Hefti, 199I), or with staurosporine in PC 12 cells (Hashimoto and Hagino, 1989; Tischler et al., 1990).Our discovery of NT-3 potentiation by K-252b invites the speculation that neurotrophic-like effects of K-252a and staurosporine could involve enhancement of the action of an endogenous neurotrophin that might be present in the respective cultures at low concentration. If true, this neurotrophin would be different from NT-3 because no similar trophic effects have been found with K-252b in the absence of exogenously provided trophic protein (Knusel and Hefti, 199 1). Potentiation of biochemical responses to different trophic factors by K-252a has indeed been shown. Koizumi et al. (1988) found K-252a to enhance the actions of EGF and bFGF on the activity of ornithine decarboxylase and of EGF on phosphatidylinositol breakdown in PC 12 cells. Interestingly, receptors for EGF and bFGF are also of the tyrosine kinase type. The dose requirements for NT-3 potentiation on basal forebrain cholinergic neurons by K-252a and K-252b seem to be identical, whereas the latter compound is approximately threefold less potent in inhibiting NGF effects on the same neurons (Knusel and Hefti, 1991). This observation suggests that the structural requirements for NT-3 stimulation and for NGF inhibition are different, evidence being added by the fact that staurosporine, which inhibits NGF actions on the same neurons with high potency (Kniisel and Hefti, 199l), does not enhance the NT-3 action. Our findings suggest K-252b as a unique tool to study the mechanisms of action of neurotrophins and to demonstrate biological actions of these proteins in vivo and in vitro. Compared with the related com-

NT-3 POTENTIATION BY K-252b

pounds, K-252a and staurosporine, which show complex patterns of activity and are cytotoxic (Kniiseland Hefti, 199l), K-252b is a nontoxic and highly selective modifier of neurotrophin actions in vitro. Most importantly, comparison of the results with three structurallyrelated inhibitors suggeststhat the requirements for NGF inhibition and for NT-3 potentiation are different from each other. Our findings raise the possibility of development of compounds of even higher selectivity, able to inhibit or potentiate actions of individual neurotrophins. They identify K-252b as a lead compound for further structural modifications and the development of other selective molecules. Such molecules would be valuable tools to investigate biological functions of neurotrophins. Furthermore, it is possible that, in the future, highly selective drugs with agonistic or antagonistic actions on neurotrophin mechanisms could become therapeutically useful in the treatment of neurodegenerative diseases and nervous system injury and, possibly, pathological cell proliferation. Acknowledgment: This study was supported by USPHS Grants NS22933 and AG09793, NSF Grant BNS 9021255, State of California Alzheimer’s Disease Program contract 90- 11297, and grants from the National Parkinson Foundation (Miami, FL,U.S.A.), and by the National Cancer Institute, DHHS, under contract no. NO 1-CO-7410 1 with ABL (S.R. and D.R.K.).

REFERENCES Alderson R. F., Alterman A. L., Barde Y. A., and Lindsay R. M. ( 1990) Brain-derived neurotrophic factor increases survival and differentiated functions of rat septal cholinergic neurons in culture. Neuron 5,297-306. Barde Y. A. (1989) Trophic factors and neuronal survival. Neuron 2, 1525-1534. Berg M. M., Sternberg D. W., Parada L. F., and Chao M. V. (1992) K-252a inhibits nerve growth factor-induced trk proto-oncogene tyrosine phosphorylation and kinase activity. J. Biof. Chem. 267,13-16. Berkemeier L. R., Winslow J. W., Kaplan D. R., Nikolics K., Goeddel D. V., and Rosenthal A. (1991) Neurotrophin-5: a novel neurotrophic factor that activates trk and trkB. Neuron 7,857866. Borasio G. D. (1990) Differential effects ofthe protein kinase inhibitor K-252a on the in vitro survival of chick embryonic neurons. Neurosci. Lett. 108, 207-2 12. Bothwell M. (199 1) Keeping track of neurotrophin receptors. Cell 65,9 15-9 18. Chao M. V., Bothwell M. S., Ross H., Koprowski A. A,, Lanahan C . R., Buck C . R., and Sehgal A. (1986) Gene transfer and molecular cloning of the human NGF receptor. Science 232, 5 18-521. Cordon-Cardo C., Tapley P., Jing S., Nandure V., O’Rourke E., Lamballe F., Kovary K., Klein R., Jones K. R., Reichardt L. F., and Barbacid M. (1991) The trk tyrosine protein kinase mediates the mitogenic properties of nerve growth factor and neurotrophin-3. Cell 66, 173-1 83. Davies A. M., Thoenen H., and Barde Y. A. (1986) The response of chick sensory neurons to brain-derived neurotrophic factor. J. Neurosci. 6, 1897-1904. Ehrlich Y. H., Davis T. B., Bock E., Kornecki E., and Lenox R. H.

721

(1986) Ecto-protein kinase activity on the external surface of neural cells. Nature 320, 67-70. Ernfors P., Wetmore C., Olson L., and Persson H. (1990) Identification of cells in rat brain and peripheral tissues expressing mRNA for members ofthe nervegrowth factor family. Neuron 5,5 1 1-526. Ferrari G., Minozzi M. C., Toffano G., Leon A., and Skaper S. D. (1989) Basic fibroblast growth factor promotes the survival and development of mesencephalic neurons in culture. Dev. Biol. 133, 140-147. Hallbook F., Ibanez C. F., and Persson H. (1991) Evolutionary studies on the nerve growth factor family reveal a novel member abundantly expressed in Xenopus ovary. Neuron 6, 845-858. Hartikka J. and Hefti F. (1988) Development of septal cholinergic neurons in culture, plating density and glial cells modulate effects of NGF on survival, fiber growth and expression of transmitter-specific enzymes. J. Neurosci. 8, 2967-2985. Hashimoto S. and Hagino A. (1989) Blockage of nerve growth factor action in PC12h cells by staurosporine, a potent protein kinase inhibitor. J. Neurochem. 53, 1675-1685. Hefti F., Hartikka J., and Kniisel B. (1989) Function of neurotrophic factors in the adult and aging brain and their possible use in the treatment of neurodegenerativediseases. Neurobiol.Aging 10,515-533. Hempstead B. L., Martin-Zanca D., Kaplan D. R., Parada L. F., and Chao M. V. (199 I ) High-affinity NGF binding requires coexpression of the trk proto-oncogene and the low-affinity NGF receptor. Nature 344, 339-34 1. Hohn A., Leibrock J., Bailey K., and Barde Y. A. ( I 990) Identification and characterization of a novel member of the nerve growth factor/brain-derived neurotrophic factor family. Nature 344, 339-341. Hyman C., Hofer M., Barde Y. A., Juhasz M., Yancopoulos G. D., Squint0 S. P., and Lindsay R. M. (1991) BDNF is a neurotrophic factor for dopaminergic neurons of the substantia nigra. Nature 350,230-233. Jones K. R. and Reichardt L. F. (1 990) Molecular cloning of a human gene that is a member of the nerve growth factor family. Proc. Natl. Acad. Sci. USA 87, 8060-8064. Kaplan D. R., Momson D. K., Wong G., McCormick F., and Williams L. T. (1990) PDGF beta receptor stimulates tyrosine phosphorylation of GAP and association of GAP with a signaling complex. Cell 61, 125-133. Kaplan D. R., Hempstead B. L., Martin-Zanca D., Chao M. V., and Parada L. F. (199 l a ) The trk proto-oncogene product: a signal transducing receptor for nerve growth factor. Science 252, 554-558. Kaplan D. R., Martin-Zanca D., and Parada L. F. ( 19916) Tyrosine phosphorylation and tyrosine kinase activity of the trk protooncogene product induced by NGF. Nature 350, 158-160. Kase H., Iwahashi K., Nakanishi S., Matsuda Y., Yamada K., Takahishi M., Murakata C., Sat0 A., and Kaneko M. (1987) K-252 compounds, novel and potent inhibitors of protein kinase C and cyclic nucleotide-dependent protein kinases. Biochern. Biophys. Res. Commun. 142,436-440. Kniisel B. and Hefti F. (1 99 1) K-252b is a selective and nontoxic inhibitor of nerve growth factor action on cultured brain neurons. J. Neurochem. 57,955-962. Kniisel B., Burton L. E., Longo F. M., Mobley W. C., Koliatsos V. E., Price D. L., and Hefti F. (1990~)Trophic actions of recombinant human nerve growth factor on cultured rat embryonic CNS cells. Exp Neurol. 110, 274-383. Kniisel B., Michel P. P., SchwaberJ., and Hefti F. (19906) Selective and nonselective stimulation of central cholinergic and dopaminergic development in vitro by nerve growth factor, basic fibroblast growth factor, epidermal growth factor, insulin and the insulin-like growth factors 1 and 2. J. Neurosci. 10, 558570. Kniisel B., Winslow J. W., Rosenthal A., Burton L. E., Seid D. P., Nikolics K., and Hefti F. (199 I ) Promotion ofcentral cholinergic and dopaminergic neuron differentiation by brain-derived

J. Neurochem., Vol. 59, No. 2. 1992

722

B. KNUSEL. ET AL.

neurotrophic factor but not neurotrophin-3. Proc. Natl. Acad. Sci. USA 88,961-965. Koizumi S., Contreras M. L., Matsuda Y., Hama T., Lazarovici P., and Guroff G. (1988) K-252a: a specific inhibitor of the action of nerve growth factor on X I 2 cells. J. Neurosci. 8,7 15-72 1. Lamballe F., Klein R., and Barbacid M. (1991) TrkC, a new member of the trk family of tyrosine protein kinases, is a recep tor for neurotrophin-3. Cell 66,967-979. Leibrock J., Lottspeich F., Hohn A., Hofer M., Hengerer B., Masiakowski P., Thoenen H., and Barde Y. A. (1989) Molecular cloning and expression of brain-derived neurotrophic factor. Nature 341, 149-152. Maisonpierre P. C., Belluscio L., Squinto S., Ip N. Y., Furth M. E., Lindsay R. M., and Yancopoulos G. D. (1990) Neurotrophin3: a neurotrophic factor related to NGF and BDNF. Science 247, 1446-1451. Martin-Zanca D., Oskam R., Mitra G., Copeland T., and Barbacid M. (1989) Molecular and biochemical characterization of the human trk proto-oncogene. Mol. Cell. Biol. 9, 24-33. Matsuda Y. and Fukuda J. (1988) Inhibition by K-252a, a new inhibitor of protein kinase, of nerve growth factor-induced neurite outgrowth of chick embryo dorsal root ganglion cells. Neurosci. Lett. 87, 295-301. Nagashima K., Nakanishi S., and Matsuda Y. (1991) Inhibition of nerve growth factor-induced neurite outgrowth of PC12 cells by a protein kinase inhibitor which does not permeate the cell membrane. FEBS Lett. 293, 1 19-123. Radeke M. J., Misko T. P., Hsu C., Henenberg L. A., and Shooter E. M. (1987) Gene transfer and molecular cloning of the rat nerve growth factor receptor: a new class of receptors. Nature 325,593-597. Rodriguez-Tebar A., Dechant G., and Barde Y. A. (1990) Binding of brain-derived neurotrophic factor to the nerve growth factor receptor. Neuron 4,487-492. Rosenthal A., Goeddel D. V., Nguyen T., Lewis M., Shih A., Laramee G. R., Nikolics K., and Winslow J. W. (1990) Primary structure and biological activity of a novel human neurotrophic factor. Neuron 4,767-773. Rosenthal A., Goeddel D. V., Nguyen T., Martin E., Burton L. E., Shih A., and Winslow J. W. (199 1) Primary structure and biological activity of human brain-derived neurotrophic factor. Endocrinology 129, 1289-1294.

J. Neurochem., Vol, 59, No. 2, 1992

Sano M., Nishiyama K., and Kitajima S. (1990) A nerve growth factor-dependent protein kinase that phosphorylates microtubule-associated proteins in vitro: possible involvement of its activity in the outgrowth of neurites from PC12 cells. J. Neurochem. 55,427-435. Smith D. S., King C. S., Pearson E., Gittinger C. K., and Landreth G. (1989) Selective inhibition of nerve growth factor-stimulated protein kinases by K-252a and 5'-9methyladenosine in PC12 cells. J. Neurochem. 53, 800-806. Soppet D., Escandon E., Maragos J., Kaplan D. R., Hunter T., Nikolics K., and Parada L. F. (1991) The neurotrophic factors BDNF and NT-3 are ligands for the trkB tyrosine kinase recep tor. Cell 65, 895-903. Squinto S. P., Aldrich T. H., Lindsay R. M., Momssey D. M., Panayotatos N., Bianco S. M., Furth M. E., and Yancopoulos G. D. ( 1990) Identification of functional receptors for ciliary neurotrophic factor on neuronal cell lines and primary neurons. Neuron 5,757-766. Squinto S. P., Stitt T. N., Aldrich T. H., Davis S. M., Bianco S. M., Rdziejewski C., Glass D. J., Masiakowski P., Furth M. E., Valenzuela D. M., DiStefano P. S., and Yancopoulos G. D. ( 1991) trkB encodes a functional receptor for brain-derived neurotrophic factor and neurotrophin-3 but not NGF. Ce1165, 885-893. Thoenen H. (1991) The changing scene of neurotrophic factors. Trends Neurosci. 14, 165- 170. Thoenen H., Bandtlow C., and Heumann R. (1987) Thephysiological function of nerve growth factor in the central nervous system: comparison with the periphery. Rev.Physiol. Biochem. Pharrnacol. 109, 145-178. Tischler A. S., Ruzicka L. A., and Perlman R. L. (1990) Mimicry and inhibition of nerve growth factor effects: interactions of staurosporine,forskolin, and K-252a in PC12 cells and normal rat chromaffin cells in vitro. J. Neurochem. 55, 1159-1 165. Vetter M. L., Martin-Zanca D., Parada L. F., Bishop J. M., and Kaplan D. R. (1991) Nerve growth factor rapidly stimulates tyrosine phosphorylation of phospholipase C-gamma1 by a kinase activity associated with the product of trk protooncogene. Proc. Natl. Acad. Sci. USA 88, 5650-5654. Whittemore S. R. and Seiger A. (1987) The expression,localization and functional significance of beta-nerve growth factor in the central nervous system. Brain Res. Rev. 12,439-464.