Neurite Outgrowth Stimulated by the Tyrosine Kinase Inhibitor Herbimycin A Requires Activation of Tyrosine Kinases and Protein Kinase C

Joiirnal of Neurochemistry Raven Press, Ltd., New York 0 1994 International Society for Neurochernistry

Neurite Outgrowth Stimulated by the Tyrosine Kinase Inhibitor Herbimycin A Requires Activation of Tyrosine Kinases and Protein Kinase C

Patrick Doherty, Josie Furness, Emma J. Williams, and Frank S. Walsh

Department of Experimental Pathology, UMDS, Guy’s Hospital, London, England

Abstract: Activation of tyrosine kinases is established as an important mechanism for controlling growth cone motility and neurite outgrowth. We have tested the effects of a range of tyrosine kinase inhibitors on neurite outgrowth from postnatal day 4 cerebellar granule cells cultured over confluent monolayers of 3T3 fibroblasts. The only agent that had any effect was herbimycin A, which stimulated neurite outgrowth. The response is shown to be attribut- able to a direct effect of this tyrosine kinase inhibitor on neurones. The neurite outgrowth response to herbimycin A was inhibited by two other tyrosine kinase inhibitors, which on their own did not affect neurite outgrowth. The data suggest that the response to herbimycin A reflects either a direct or indirect activation of one or more protein tyrosine kinases. Independent signalling events downstream from tyrosine kinase activation underlying the neurite outgrowth response to herbimycin A include increased activity of protein kinase C and calcium influx into neurones through both N- and L-type calcium channels. Key Words: Tyrosine kinases-Neurite outgrowth-Herbimy- cin A-Protein kinase C-Calcium channels. J. Neurochem. 62,2124-21 31 (1 994).

Neuronal growth cones are capable of recognising a wide range of environmental signals, and this is cru- cial for pathway finding and target innervation. Correct growth and guidance most likely require the integration of information arising from up to three classes of molecule: soluble factors, extracellular matrix molecules, and cell surface glycoproteins. Several of these molecules have been reported to influence growth cones by promoting motility and extension, causing collapse and retraction, or influencing direc- tionality of growth in an attractive or repulsive man- ner (reviewed by Schwab, 1990; Bixby and Hams, 199 1 ; Lumsden and Cohen, 199 1 ; Reichardt and To- maselli, 199 1 ; Doherty and Walsh, 1992).

Molecules that promote and inhibit axonal growth have been shown to operate by binding to receptors and activating second messenger pathways in neurones. For example, cell-contact-dependent axonal growth stimulated by cell adhesion molecules

(CAMs) depends on the activation of a second messenger pathway rather than on adhesion per se (Saffell et al., 1992; Williams et al., 1992). The first step in the cascade appears to be the activation of a tyrosine kinase (Williams et al., 1993). The response can also be inhibited by pertussis toxin and agents that inhibit or negate the effects of calcium influx into neurones through N- and L-type calcium channels (Doherty et al., 199 1; Doherty and Walsh, 1992). Indeed, calcium is widely recognised as a key second messenger controlling growth cone motility (Kater and Mills, 199 1). Agents other than CAMs, such as neurotransmitters, can also modulate neurite outgrowth by modulating calcium levels or fluxes in growth cones. Several agents that cause growth cone collapse and neurite retraction also operate by activating a pertussis toxin- sensitive G protein (Igarishi et al., 1993). At least one membrane-derived factor, which inhibits neurite outgrowth over oligodendrocytes, induces a relatively large increase in calcium levels in growth cones, and this is responsible for its growth cone-collapsing activity (Bandtlow et al., 1993). Thus, pertussis toxin-sensitive G proteins and intracellular calcium are key second messengers involved in both the promotion and inhibition of axonal growth.

Several observations suggest that a wide range of receptor and nonreceptor tyrosine kinases can stimulate neuritic growth. Soluble factors such as nerve growth factor (NGF) and fibroblast growth factor (FGF) can promote neurite outgrowth following bind-

Received September 2, 1993; revised manuscript received Oc- tober 13, 1993; accepted October 13, 1993.

Address correspondence and reprint requests to Dr. P. Doherty at Department of Experimental Pathology, UMDS, Guy’s Hospital, London Bridge, London SE 1 9RT, U.K.

Abbreviations used: BAPTA-AM, (bis-0-aminophen0xy)ethane- N,N,N’,N‘-tetraacetic acid/acetoxymethyl ester; CAM, cell adhesion molecule; DAG, diacylglycerol; EGF, epidermal growth factor; FGF, fibroblast growth factor; GAP-43, 43-kDa growth-associated protein; NGF, nerve growth factor; PKA, protein kinase A; PKC, protein kinase C; PLA,, phospholipase A,; PLC, phospholipase C; PMA, phorbol 12-mynstate 13-acetate.

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ing to and activating receptors that have intrinsic tyrosine kinase domains (Chao, 1992; Schlessinger and Ullrich, 1992). Also, nonreceptor tyrosine kinases such as c-src can be localised to the growth cone and are expressed at high levels during periods of growth and regeneration (Maness et al., 1988; LeBeau et al., 199 1). The transforming oncogene v-src can directly induce neurite outgrowth from PC12 cells (Cox and Maness, 1991), and experiments with blocking anti- bodies suggest that src activation is downstream from NGF and FGF receptor activation in the pathway leading to induction of the neuronal phenotype in PC 12 cells (Kremer et al., 199 1 ). NGF-stimulated neurite outgrowth does not, however, depend on activation of heterotrimeric G proteins or calcium influx into PC12 cells (Doherty et al., 1991).

Several tyrosine kinase inhibitors have been reported to stimulate neurite outgrowth, suggesting that some tyrosine kmases tonically suppress neurite outgrowth (Bixby and Jhabvala, 1992; Miller et al., 1993). One possibility is that they do so by suppress- ing the activity of tyrosine kinases that promote neurite outgrowth. For example, the c-src tyrosine kinase can be negatively regulated by a tyrosine kinase (Nada et al., 1991). In the present study we show that the tyrosine kinase inhibitor herbimycin A (Uehara et al., 1989; June et al., 1990; Weiss and Nuccitelli, 1992) can stimulate neurite outgrowth from cerebellar granule cells. This response was inhibited by two other tyrosine kinase inhibitors, which on their own did not directly affect neurite outgrowth. These results suggest that herbimycin A stimulates neurite outgrowth by directly or indirectly activating one or more tyrosine kinases. A major part of the neurite outgrowth response to herbimycin A was similar to the response stimulated by CAMs in that it could be inhibited by pertussis toxin and agents that block or negate the effects of calcium influx into neurons via N- and L- type calcium channels. However, in contrast to CAMs, a component of the herbimycin A response can be attributed to activation of protein kinase C (PKC), and this is not inhibited by calcium channel antagonists or pertussis toxin.

MATERIALS AND METHODS Cell culture and neurite outgrowth

Rat cerebellar neurons isolated at postnatal day 4 were cultured for 16 h on confluent monolayers of parental 3T3 cells (Williams et al., 1992). Cocultures were established by seeding 3,000 cerebellar neurons onto confluent monolayers of 3T3 cells established in individual wells of eight- chamber Lab-Tek slides. The coculture medium was SAT0 supplemented with 2% fetal calf serum with Ca2+ and Mg2+ at 4 and 0.25 mM, respectively. Cerebellar neurones were allowed to attach to 3T3 monolayers for 4 h before drugs were added to discount effects on initial adhesion. Cocul- tures were fixed with paraformaldehyde and methanol-per- meabilised, and the cerebellar neurones were stained for 43-kDa growth-associated protein (GAP-43). The average length of the longest neurite on each neurone was deter-

mined using a Sight System Image Manager (Sight Systems, Newbury, U.K.) as previously described (Doherty et al., 1991).

Tyrosine kinase inhibitors Several commercially available tyrosine kinase inhibitors

were tested in this study. Lavendustin A (Onoda et al., 1989), the stable methyl 2,5-dihydroxycinnamate analogue of erbstatin (hereafter referred to as the erbstatin analogue), which inhibits epidermal growth factor (EGF) receptor au- tophosphorylation in cultured cells (Umezawa et al., 1990), and herbimycin A (Uehara et al., 1989) were obtained from Calbiochem Novabiochem, Ltd. (Nottingham, U.K.). All these agents were diluted in medium from dimethyl sulphoxide stock solutions and added to the cultures 4 h after plating the neurones. Controls for these experiments in- cluded dimethyl sulphoxide diluted in medium to the same extent as the drug stocks.

Protein extraction and western blotting for phosphotyrosine-modified proteins

Cultures of cerebellar neurones were established by seeding 5 X lo6 cells into polylysine-coated 35-mm-diameter tissue culture plates. After 20 h the cultures were washed twice with ice-cold phosphate-buffered saline and harvested on ice into 20 mM Tris-HC1 (pH 7.4) containing 1 mM EDTA, 1% Nonidet P-40, 1% aprotinin, 1 Mphenyimeth- ylsulphonyl fluoride, and 1 mM vanadate. Proteins were resolved by electrophoresis on 7.5% polyacrylamide mini- gels and transferred to nitrocellulose membranes as previously described (Williams et al., 1992). For immunoblotting, the nitrocellulose membranes were incubated for 4 h with phosphate-buffered saline containing soya milk (2%) and Tween 20 (0.2%). The antiphosphotyrosine primary antibody was PY72 (obtained from Dr. Bart Sefton and used at a 1:300 dilution ofascitic fluid). The secondary antibody was horseradish peroxidase-conjugated goat anti- mouse (from Amersham at a 1:3,000 dilution). Both anti- bodies were diluted into phosphate-buffered saline/Tween and incubated for 1 h at room temperature. Immunoreac- tive bands were visualised using an ECL detection kit (Amersham, U.K.).

PKC assay Cultures of cerebellar neurones were established as for

protein extraction and western blotting (see above). After 16 h control and treated (see text) cultures were assayed ac- cording to the supplier's instructions for PKC using a commercially available kit (Amersham, U.K.).

RESULTS

Herbimycin A stimulates neurite outgrowth from cerebellar neurones

At low-micromolar concentrations herbimycin A has been reported to be a highly specific inhibitor of several tyrosine kinases (see, e.g., Uehara et al., 1989). We tested the effects of herbimycin A on neurite outgrowth from postnatal day 4 cerebellar granule cells cultured at low density on confluent monolayers of 3T3 fibroblasts. This represents a good model for studying neurite outgrowth as several tyrosine kinase inhibitors, including herbimycin A, are extremely toxic when added to low-density cultures of cerebellar

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2126 P. DOHERTY ET AL.

FIG. 1. Cerebellar neurones extend longer neurites in herbimycin A-treated cultures. Cerebellar neurones were cultured for 16 h over confluent monolayers of 3T3 cells in control media (a and b) or, alternatively, media supplemented with 0.5 pM herbimycin A (c and d). Cerebellar neurones were immunostained using a rabbit anti-GAP-43 polyclonal serum. Bar = 50 pm.

neurones cultured on collagen or laminin (authors’ unpublished data). After 16 h cultures of cerebellar neurones maintained in control media or media supplemented with herbimycin A (0.08-2.5 p M ) were fixed and immunostained for GAP-43. Examples of neurones from control cultures and cultures treated with 0.625 pM herbimycin A are shown in Fig. 1. There was a clear increase in neurite outgrowth in the herbimycin A-treated cultures. We quantified this by measuring the length of the longest neurite on each cerebellar granule cell, and these data are shown for a representative experiment in Fig. 2. A small but significant increase in mean length of the longest neurite was found in cultures treated with herbimycin A at 0.08 pA4. The response was dose dependent and started to plateau at 0.625-2.5 pM. Similar dose-response curves were obtained in two other experiments. In a total of five independent experiments the mean k SEM length of the longest neurite increased from a basal value of 3 1.9 k 2.8 pm in control media to 77.3 & 5.2 pm in media containing 0.5 pA4 herbimycin A (n = 5) . We can conclude that herbimycin A stimulates neurite outgrowth, and this contrasts with results obtained with several other tyrosine kinase inhibitors (genistein, lavendustin A, the erbstatin analogue, and tyrphostins 23,25,34, and 47), which have

no effect on the mean length of neurites extended by postnatal day 4 cerebellar granule cells over monolayers of 3T3 fibroblasts (Williams et al., 1993).

To test if herbimycin was increasing neurite outgrowth by directly acting on the neurones, suspen- sions of cerebellar neurones were pretreated with herbimycin A (0.5 pM for 4 h) before being washed once and cultured in control media over 3T3 monolayers. The mean f SEM length of the longest neurite was, on average, 51.8 k 5.4% greater for the herbimycin A-pretreated neurons as compared with the corre- sponding control neurones; this contrasts with an increase of 137.9 -t 9.2% for neurones cultured in the continuous presence of herbimycin A (both values determined from three independent experiments). Both responses were statistically significant ( p < 0.005). In contrast, pretreatment of monolayers with 0.5 pA4 herbimycin A for up to 8 h (again with one wash) had no effect on their ability to support neurite outgrowth (data not shown). These data suggest that herbimycin A stimulates neurite outgrowth by directly acting on the cerebellar neurones. Herbimycin A effects on the levels of phosphotyrosine-modified proteins

The effects of herbimycin A (0.5 pM for 16 h) on the level of phosphotyrosine-modified proteins was

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FIG. 2. Herbimycin A induces a dose-dependent increase in neurite outgrowth from cerebellar neurones. Cerebellar neurones were cultured on monolayers of 3T3 fibroblasts in control media containing twofold dilutions of herbimycin A over the range 2.5- 0.08 pM. After -16 h the cultures were fixed and stained for GAP-43 immunoreactivity. The results show the mean 1 SEM (bars) length of the longest neurite per cell, and each value was determined by sampling 120-1 80 neurones from replicate cultures.

determined in high-density cultures of cerebellar neurones by immunoblotting using the PY72 antiphosphotyrosine antibody. The results of a representative experiment are shown in Fig. 3. Major phosphotyrosine bands were seen at 21 1, 119 (a doublet), 98, 85, 80, and 60 kDa. The major difference between herbimycin A-treated and control cultures was in the level of the band at 21 1 kDa and in other bands in the 150-200-kDa range, which were greatly increased in cultures treated with herbimycin A. In contrast, there was no difference in the level of the doublet at 119 kDa or the major bands at 85 and 80 kDa. There was an apparent reduction in the band at 60 kDa; however, this band was particularly labile and was not always detected by immunoblotting.

In contrast to herbimycin A, the erbstatin analogue, which inhibits the EGF receptor tyrosine kinase (Umezawa et al., 1990), reduced the level of several phosphotyrosine-modified proteins (Fig. 3, lane

FIG. 3. Herbimycin A modulates the level of phosphotyrosine-modified proteins in neurones. Cultures of cerebellar neurones were maintained for 16 h in media supplemented with 0.5 pM herbimycin A (lane A), control media (lane b), or media supplemented with 10 pg/ml of the erbstatin analogue (lane C). Phosphotyrosine-modified proteins were detected by immunoblotting using the PY72 antibody (see Mate- rials and Methods). Molecular masses were determined against premarked standards obtained from Sigma.

A B C

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FIG. 4. Neurite outgrowth stimulated by herbimycin A is reduced by tyrosine kinase inhibitors. The effects of herbimycin A (0.5 pM) on neurite outgrowth were determined as in Fig. 2 for cultures further supplemented with the erbstatin analogue at 10 pg/ml (columns B) or lavendustin A at 20 pLM (columns C). Control, columns A. The results show the length of the longest neurite per cell in the absence (open columns) or presence (hatched columns) of herbimycin A. The results are pooled to show the mean 1 SEM (bars) values from three independent experiments.

C). The fact that herbimycin A can increase the level of tyrosine phosphorylation on some proteins suggests that it can inhibit a phosphatase, activate a kinase, or inhibit a kinase that tonically suppresses a second tyrosine kinase. The latter possibility is the only one in keeping with the known pharmacological properties of herbimycin A.

The erbstatin analogue and lavendustin A inhibit the neurite outgrowth response induced by herbimycin A

If herbimycin A stimulates neurite outgrowth by indirectly activating a tyrosine kinase, it follows that more general tyrosine kinase inhibitors should block the response. We have previously shown that the erbstatin analogue and lavendustin A (see Onoda et al., 1989) do not inhibit or stimulate neurite outgrowth from cerebellar granule cells (Williams et al., 1993). The effects of the erbstatin analogue (10 pg/ml) and lavendustin A (20 pM) on neurite outgrowth stimulated by herbimycin A were determined in three independent experiments, and the pooled results are summarised in Fig. 4. Whereas the erbstatin analogue and lavendustin A had no effect on basal neurite outgrowth, they both inhibited the herbimycin A response by -75%. The erbstatin analogue does not inhibit neurite outgrowth over 3T3 monolayers induced by activation of protein kinase A (PKA) or PKC (Williams et al., 1993). This confirms its relative specificity as a tyrosine kinase inhibitor and also shows that it has no direct effect on the ability of 3T3 cells to support neurite outgrowth. Thus, the above results suggest that herbimycin A stimulates neurite outgrowth by activating one or more tyrosine kinases.

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FIG. 5. Neurite outgrowth stimulated by herbimycin A is inhibited by agents that block or negate the effects of calcium influx into neurones and also pertussis toxin. The ability of herbimycin A (6 pld) to stimulate neurite outgrowth from cerebellar neurones cultured on 3T3 monolayers was determined as in Fig. 2 in media containing extracellular calcium at 0.25 mM (column A), media supplemented with an L-type calcium channel antagonist at 10 pV (nifedipine, diltiazem, or verapamil) together with o-conotoxin at 0.25 pM (column B), media containing BAPTA-AM (column C). or media containing 850 ng/ml of pertussis toxin (column D). None of these had any significant effect on basal neurite outgrowth in control media (data not shown, but see Williams et al., 1992), but all substantially inhibited the herbimycin A response. The results show the percent inhibition determined from the given number of independent experiments, and bars show the SEM values. In this particular set of experiments herbimycin A increased the mean f SEM length of the longest neurite from 33.8 -C 2.1 to 68.9 k 7.2 pm (both values from seven experiments). The same results were obtained when herbimycin was used at lower concentrations.

Neurite outgrowth stimulated by herbimycin A is partly dependent on calcium influx into neurones

Tyrosine kinases can directly phosphorylate cyto- skeletal proteins, and this might account for their ability to modulate neurite outgrowth. Alternatively, and particularly in the case of receptor tyrosine kinases, phosphorylation of effector molecules leads to the generation of other second messengers that can modulate neurite outgrowth. Calcium is established as a key modulator of growth cone motility (see introductory section). In the present study reducing the extracellular calcium concentration to 0.25 mM inhibited herbimycin A-induced neurite outgrowth by 76.3 f 5% (Fig. 5, column A). In control media the herbimycin A response could also be substantially inhibited by three agents that specifically block L-type calcium channels (verapamil, diltiazem, or nifedipine, all at 10 pM) or by w-conotoxin (0.25 pW), which blocks N- type calcium channels. There was little difference between results obtained with these three agents added alone or together (data not shown), and the pooled results from five independent experiments showed an -60% inhibition of the herbimycin response with a combination of an L-type calcium channel blocker

and w-conotoxin (Fig. 5, column B). Additional supporting evidence for calcium influx into neurones being a trigger for the herbimycin A response was obtained by preloading neurones with (bis-0-amino- phenoxy)ethane-N,N,N',N'-tetraacetic acid/acetoxymethyl ester (BAPTA-AM) (Koike et al., 1989; Wil- liams et al., 1992). This calcium chelator is membrane permeant and can be sequestered and trapped exclusively in the neurones following hydrolysis of its acetoxymethyl ester group. Results pooled from four experiments showed treatment with BAPTA-AM (20 pA4) to again inhibit herbimycin A-induced neurite outgrowth by -60% (Fig. 5 , column C).

Pertussis toxin inactivates both Go and Gi members of the heterotrimeric G protein family by ribosylation of the a subunit, which prevents dissociation of the complex. Such a G protein has been implicated as an important step upstream of calcium influx in the CAM pathway by virtue of the fact that pertussis toxin can completely inhibit neurite outgrowth stimulated by NCAM, N-cadherin, and L1 but not that stimulated by K+ depolarisation, integrin receptor stimulation, or treatment with NGF or agents that activate PKA or PKC (reviewed by Doherty and Walsh, 1992). In the present study results pooled from four independent experiments showed that pertussis toxin can also substantially inhibit neurite outgrowth stimulated by herbimycin A (Fig. 5, column D). These data clearly show that neurite outgrowth stimulated by herbimycin A depends to a large extent on calcium influx into neurones and that herbimycin A acts at a site upstream of a pertussis toxin-sensitive G protein.

Herbimycin A increases the activity of PKC Stimulation of receptor tyrosine kinase can lead to

the activation of PKC following tyrosine phosphorylation of phospholipase C (PLC) and the subsequent generation of diacylglycerol (DAG) (see, e.g., Schles- singer and Ullrich, 1992). Activation of PKC by low concentrations of phorbol esters stimulates neurite outgrowth from various neurones (see, e.g., Burstein et al., 1982; Bixby, 1989). However, higher concentrations of phorbol esters do not stimulate neurite outgrowth or activate PKC (see below). PKC activity was measured in cultures of cerebellar neurones treated with herbimycin A, high concentrations of the phorbol ester phorbol 12-myristate 13-acetate (PMA), or herbimycin A plus high PMA, and these results are summarised in Table 1.

The results show a significant increase in PKC activity in cultures treated with herbimycin A alone. In contrast, PMA reduced PKC activity by -20%. In cultures treated simultaneously with herbimycin A and PMA, the level of PKC remained below the value found in untreated control cultures. In contrast to herbimycin A, the erbstatin analogue had no effect on PKC activity; however, this agent completely inhibited the increase in PKC activity induced by herbimy-

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TABLE 1. Herbimvcin A increases the activitv of PKC

Drug PKC activity (% of control)

Herbimycin A 163 k 4.3 (4) PMA 78 k 3.5 (3) Herbimycin A + PMA 96 * 5.4 (3) Erbstatin analogue 99 * 1.5 (4) Herbimycin A + erbstatin analogue 98 * 9.4 (3)

Postnatal day 4 cerebellar neurons were cultured for 16 h in control media or media supplemented with 0.5 pM herbimycin A, I pM PMA, the erbstatin analogue at 10 pglml, or herbimycin A together with either PMA or the erbstatin analogue. The activity of PKC in control and treated cultures was then determined. The results show PKC activity in treated cultures expressed as a percent- age of that measured in untreated cultures. Data are mean ? SEM values (no. of indpendent experiments). PKC activity in control cultures was 14,756 f 1,244 pmol ofphosphate transferred per 6.25 X los cells plated (mean 1?I SEM from four experiments).

cin A (Table 1). Thus, herbimycin A increases the activity of PKC, and this is clearly downstream from activation of an erbstatin-sensitive tyrosine kinase.

Neurite outgrowth stimulated by herbimycin A is partly dependent on activation of PKC

High concentrations of PMA can prevent herbimycin A from increasing PKC activity to above the value found in control cultures (Table 1). We therefore tested the effects of high concentrations of PMA on the neurite outgrowth response to herbimycin A, with the results from three independent experiments summarised in Fig. 6. On its own, PMA at high concentrations (1 p M ) did not stimulate neurite outgrowth; however, PMA substantially inhibited the herbimycin A response. Neurite outgrowth over 3T3 monolayers can also be stimulated by activation of PKA (by cholera toxin) or by directly stimulating calcium influx into neurons by K+ depolarisation (Williams et al., 1992, 1993). These responses are not inhibited by high concentrations ofPMA (E. W. and P. D., unpublished data). We can therefore conclude that PMA does not in any way impair the ability of the monolayer cells to support neurite outgrowth.

DISCUSSION

The results of the present study show that herbimycin A can stimulate neurite outgrowth from cerebellar granule cells. In this respect herbimycin A is relatively unique in that a wide variety of other tyrosine kinase inhibitors neither stimulates nor inhibits basal neurite outgrowth from these neurones cultured over confluent monolayers of 3T3 cells (Williams et al., 1993). Pretreatment of neurones, but not the monolayers, with herbimycin A was sufficient to promote a neurite outgrowth response, suggesting a direct effect on neurones rather than an indirect effect via changes in the monolayer cells. Herbimycin A can also increase the rate of neurite extension from primed PC 12 cells cul-

tured on a collagen-coated substratum (Miller et al., 1993), further supporting the notion that it directly affects neurite outgrowth.

Herbimycin A-induced neurite outgrowth was inhibited by two independent tyrosine kinase inhibitors. The erbstatin analogue binds to the substrate binding site of tyrosine kinases and inhibits tyrosine phosphorylation in cerebellar granule cells. In the same model the erbstatin analogue has no inhibitory effect on PKA or PKC, as judged by its failure to inhibit neurite outgrowth stimulated by cholera toxin or low concentrations of PMA (Williams et al., 1993). Lavendustin A is also a specific tyrosine kinase inhibitor that binds to the ATP site on protein tyrosine kinases (Onoda et al., 1989). Both of these agents substantially inhibited neurite outgrowth induced by herbimycin A at concentrations that had no effects on the monolayer's ability to support basal neurite outgrowth. To our knowledge there is no evidence that herbimycin A can directly activate a tyrosine kinase. There is, however, clear evidence that tyrosine kinases can be inhibited by phosphorylation on negative regu- latory sites by other tyrosine kinases. For example, a tyrosine kinase that inhibits .src activity has been cloned (Nada et al., 1991). Thus, a simple model in keeping with the known properties of herbimycin A is that it stimulates neurite outgrowth by inhibiting a tyrosine kinase that tonically suppresses the activity of a second tyrosine kinase. The latter kinase is, however, unlikely to be src, as herbimycin causes an irre- versible loss of the catalytic activity of v-src in trans- formed cells (Uehara et al., 1989).

The nature of the tyrosine kinase that is indirectly

0 Control

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FIG. 6. Neurite outgrowth stimulated by herbimycin A is inhibited by high concentrations of PMA. Cerebellar neurones were cultured on monolayers of 3T3 fibroblasts in control media (column A) or media supplemented with 1 pM PMA (column B), 0.5 pM herbimycin A (column C), or PMA together with herbimycin A (column D). After 16 h the cultures were fixed, and the mean length of the longest neurite per cell was determined. The results are pooled to show mean k SEM (bars) neurite length determined in three independent experiments.

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activated by herbimycin A remains to be determined. Clues as to its identity can clearly come from under- standing the downstream signalling events that lead to neurite outgrowth. For example, differentiation of naive PC12 cells to a neuronal phenotype can be induced by both NGF and FGF, and this involves a linear pathway of growth factor + tyrosine kinase receptor -P src -P ras * raf- + -P neurite outgrowth (reviewed by Chao, 1992). This pathway is not blocked by the erbstatin analogue, pertussis toxin, or calcium channel antagonists (Doherty et al., 1991; authors’ unpublished data) and does not require the activation of PKC (Burstein et al., 1982). Thus, the evidence suggests that herbimycin A does not induce neurite outgrowth by indirectly activating the src --* ras --f raf pathway.

Cell-contact-dependent neurite outgrowth induced by NCAM, N-cadherin, and L1 involves the following sequence: CAM binding + activation of a tyrosine kinase (erbstatin sensitive) - calcium influx through N- and L-type channels (sensitive to calcium channel antagonists) + + neurite outgrowth (Doherty and Walsh, 1992; Williams et al., 1993; authors’ unpublished data). A pertussis toxin-sensitive G protein is also involved in the pathway upstream of calcium influx. At least part of the response to herbimycin A can be attributed to the activation of this or a very similar pathway. For example, like the CAM response, the herbimycin A response can be inhibited by the erbstatin analogue, by pertussis toxin, and by agents that block or negate the effects of calcium influx into the neurons. The main trigger for neurite outgrowth in this pathway is calcium influx through N- and L-type calcium channels, as the response can be fully mim- icked by K+ depolarisation (Saffell et al., 1992; Wil- liams et al., 1992). The rate of cycling rather than absolute level of calcium may be important (Doherty et al., 1993; S. Harper, S. Bolsover, F. S. Walsh, and P. Doherty, unpublished data). There is no evidence for direct activation of calcium channels by G proteins and no evidence for PKC or PKA acting as second messengers in the CAM pathway. However, arachidonic acid and one of its metabolites (leukotriene B4) can directly activate voltage-gated calcium channels (Huang et al., 1992). Arachidonic acid can be gener- ated by phospholipase A, (PLA,) or the sequential activities of PLC to generate DAG and then DAG lipase. We have recently found that mastoporan can stimulate neurite outgrowth via the sequential activation of a pertussis toxin-sensitive G protein, PLA,, and N- and L-type calcium channels (E. Williams, F. S. Walsh, and P. Doherty, manuscript in prepara- tion). However, the response to CAMS and herbimycin A is not blocked by a PLA, inhibitor, suggesting that the activation of PLC may lead to the activation of calcium channels. In this context it is clear that several receptor tyrosine kinases can bind and activate PLC-7 (Schlessinger and Ullrich, 1992), and in some instances activation of PLC-7 by tyrosine phos-

phorylation can be inhibited by pertussis toxin (Yang et al., 199 1). Whereas the CAM response can be completely inhibited by calcium channel antagonists, the response to herbimycin A is blocked by only 60%. This suggests an additional site of action for herbimycin A.

The activation of PLC would generate DAG, and this is the physiological activator of PKC. Evidence that herbimycin A might stimulate this pathway was obtained by showing that PKC activity is increased in cultures treated with herbimycin A. This response was fully inhibited by the erbstatin analogue, clearly establishing that it is downstream from activation of a tyrosine kinase. High concentrations of the phorbol ester PMA prevented the induction of PKC to above control values and also substantially inhibited the neurite outgrowth response induced by herbimycin A. Furthermore, neurite outgrowth stimulated by direct activation of PKC is not inhibited by the erbstatin analogue, confirming that the erbstatin-sensitive tyrosine kinase resides upstream of PKC activation. Neu- rite outgrowth induced by PKC does not require calcium influx into neurons, and conversely calcium influx does not cause neurite outgrowth by activating PKC, as high concentrations of PMA do not inhibit this response. Thus, neurite outgrowth induced by herbimycin A most likely depends on the activation of a bifurcating pathway, one arm of which involves increased PKC activity and the other calcium influx through N- and L-type channels. In this respect DAG is a good candidate for being an important messenger in the pathway because it can directly activate PKC and, by acting as a substrate for DAG lipase, lead to the production of arachidonic acid and its metabolites. As discussed above, the latter can stimulate neurite outgrowth by directly activating N- and L-type calcium channels.

In summary, the results of the present study show that the tyrosine kinase inhibitor herbimycin A can stimulate neurite outgrowth. The response most likely requires the indirect activation of a tyrosine kinase, which in turn can stimulate neurite outgrowth via a bifurcating pathway. One branch of this pathway involves activation of PKC, whereas the other involves calcium influx via N- and L-type calcium channels. Future experiments will be directed at iden- tifying the tyrosine kinase and determining if it stimulates neurite outgrowth by activating PLC.

Acknowledgment: We would like to thank Bart Sefton for the PY72 antibody and Hendrika Rickard for typing the manuscript. The work was supported by grants from the Medical Research Council, the Wellcome Trust, and the Dunhill Medical Trust.

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Neurite Outgrowth Stimulated by the Tyrosine Kinase Inhibitor Herbimycin A Requires Activation of Tyrosine Kinases and Protein Kinase C