Monoclonal antibodies show that kinase C phosphorylation of GAP-43 during axonogenesis is both spatially and temporally restricted in vivo.

To study the role of kinase C phosphorylation in the distribution and function of GAP-43 we have generated a panel of mAbs that distinguish between GAP-43 that has been phosphorylated by kinase C and forms that have not. One class of antibodies, typified by 2G12/C7, reacts with only the phosphorylated form of GAP-43; it recognizes the peptide IQAS(PO4)FR equivalent to residues 38-43 that includes the single kinase C phosphorylation site at serine. Another, exemplified by 10E8/E7, reacts with both phosphorylated and nonphosphorylated forms. We have used the antibodies to study the distribution of kinase C-phosphorylated GAP-43 during axonogenesis and in the adult nervous system. Two major findings emerge. First, there is a lag between the initiation of axon outgrowth and the phosphorylation of GAP-43 by kinase C. The extent of this lag period varies between the different structures studied. In some cases, e.g., the trigeminal nerve, our result suggest that kinase C phosphorylation may be correlated with proximity of the growing axon to its target. Second, kinase C-phosphorylated GAP-43 is always spatially restricted to the distal axon. It is never seen either proximally or in cell bodies, even those with high levels of GAP-43 protein. This result also implies that GAP-43 is axonally transported in the non-kinase C phosphorylated form. Thus, kinase C phosphorylation of GAP-43 is not required for axon outgrowth or growth cone function per se and may be more related to interactions of the growth cone with its environment.

Altered GAP-43 immunoreactivity in regenerating sciatic nerve of diabetic rats.

Experimental diabetes in the rat is associated with impaired axon regeneration. Successful regeneration depends on the construction of axonal growth cones and establishment of appropriate target connections. The growth-associated protein (GAP)-43 is a major component of the axonal growth cone, and its synthesis and axonal transport are markedly increased during regeneration. The purpose of this study was to determine the effect of experimental diabetes on the synthesis and axonal transport of GAP-43 in regenerating sciatic nerves. Rats were rendered diabetic with 50 mg/kg streptozotocin i.p. Four weeks later, the rats were anesthetized, and one sciatic nerve was crushed to induce regeneration. After 2 weeks, nerves were ligated, and 6 h later, nerve pieces proximal to the ligature and dorsal root ganglia were removed, and proteins were separated by PAGE. Western blots of gels were probed with antibody 10E8/E7 against GAP-43. The presence of GAP-43 was confirmed by immunohistochemistry of nerve sections. Densitometric analysis of the blots showed a 45% reduction in native GAP-43 immunoreactivity in nerve pieces proximal to the ligature (P < 0.05; n = 7). Northern blots of total RNA extracted from pooled dorsal root ganglia were probed with a 32P-radiolabeled cDNA probe for GAP-43. There was no significant difference in the amount of GAP-43 mRNA between diabetic and nondiabetic rats. Immunohistochemistry of sciatic nerve confirmed the reduction in GAP-43 immunoreactivity. We conclude that a defect in turnover or axonal transport of GAP-43 may contribute to the impaired peripheral nerve regeneration in diabetes.

On the formation of neuromata in the primary olfactory projection.

Olfactory axons have been shown to grow aberrantly and form dense collections of axons, termed neuromas, in the olfactory epithelium of rats in which the olfactory bulb was ablated. Likewise, in human olfactory mucosa, collections of neurites have been noted in a variety of disease states, including Alzheimer's disease. We report here an immunohistochemical and electron microscopic analysis of aberrant axonal growth in the rat olfactory mucosa induced by experimental lesion. In particular, we have used the monoclonal antibody 2G12, which binds to the phosphorylated form of GAP-43, as an extremely sensitive marker for neuromatous axons, because it does not label neuronal cell bodies. In unilaterally bulbectomized rats, neuromas form in posterior olfactory epithelium on the operated side. Several lines of evidence, including serial section reconstruction, indicate that olfactory axons are induced to grow back into the epithelium at a distance from their point of origin as a consequence of bulbectomy, and are accompanied by glial cells from the olfactory nerve. Avulsion of a part of the olfactory nerve has similar effects as destruction of the olfactory bulb. Intraepithelial neuromas also develop in the olfactory mucosa of rats simultaneously exposed to methyl bromide gas and injected with 3-methyl indole; this treatment severely damages the olfactory epithelium directly. Exposure to methyl bromide alone causes milder damage, and the neuromas that form are transient. The evidence indicates that neuromas form after the epithelium is directly damaged because axons are trapped in the epithelium. Both of the mechanisms identified here should be taken into account when considering the findings in the human olfactory mucosa.

Demonstration of presynaptic protein kinase C activation following long-term potentiation in rat hippocampal slices.

Pharmacological and biochemical evidence implicate the Ca2+ and phospholipid-dependent protein kinase C in long-term potentiation. The in vitro hippocampal slice preparation was used to demonstrate redistribution of protein kinase C from cytosol to membrane and protein kinase C-dependent phosphorylation of the presynaptic growth-associated protein-43 substrate following long-term potentiation induction in area CA1. Protein kinase C translocation was assessed using both quantitative immunoblotting with a monoclonal antibody recognizing a common epitope in the alpha and beta isoforms of protein kinase C and Ca2+ and phospholipid-dependent phosphorylation of exogenous histone substrate. Slices examined 5 min after tetanus-induced spike potentiation showed no change in protein kinase C redistribution, whereas slices examined at 15-, 30- and 60-min intervals all showed a similar degree of protein kinase C translocation to membrane, although only at 15 min was the effect statistically significant. Additionally, an increase in protein kinase C-dependent growth-associated protein 43 phosphorylation was observed 10 min after high-frequency stimulation. The translocation of protein kinase C and phosphorylation of growth-associated protein 43 were dependent upon high-frequency (repetitive 400 Hz) afferent stimulation, as no effects were observed in slices receiving low-frequency (1 Hz) or no stimulation. The N-methyl-D-aspartate receptor antagonist, DL-2-amino-5-phosphonovaleric acid (50 microM), inhibited induction of long-term potentiation, redistribution of protein kinase C and phosphorylation of growth-associated protein 43. A significant redistribution of the predominantly presynaptic protein kinase C isoform, protein kinase C-alpha, was also detected 15 min after induction of long-term potentiation using an alpha-isoform-specific monoclonal antibody. These observations support a presynaptic role for protein kinase C and growth-associated protein 43 in the early maintenance phase of LTP, and further suggest that a retrograde messenger produced postsynaptically following N-methyl-D-aspartate receptor activation mediates these effects.

Increase of GAP-43 expression following kainic acid injection into whisker barrel cortex.

Plasticity after microinjection of kainic acid (KA) into the adult rat whisker barrel cortex was investigated with immunohistochemical staining of phosphorylated growth-associated protein (GAP)-43. After mapping the barrel cortex with the technique of intrinsic signal optical imaging, a small volume of KA was injected into one barrel. Rats were sacrificed at 2 days, 3 days, 1 week, and 6 weeks after lesioning. GAP-43 staining demonstrated intense immunoreactivity (IR) at the injected barrel which spread to the inter-barrel septa and the surrounding barrels. Elevated IR of GAP-43 was visible 2 days after KA injection, and increased gradually at least 6 weeks following the lesion. This model has the possibility of offering a simple and reliable tool for studying cortical plasticity.

Axonal transport of protein in motor fibres of experimentally diabetic rats--fast anterograde transport.

Fast axonal transport of radiolabelled proteins in motor fibres of rat sciatic nerves was studied after 14 days of streptozotocin-induced diabetes. The rate of fast transport as measured at two time intervals after application of [3H]leucine to the motor neurone cell bodies in the spinal cord was reduced by 21% in diabetic rats. There was no significant change in the time between injection of isotope and the start of fast transport. The amount of axonal transport of radiolabelled proteins as measured by accumulation of proteins proximal to a ligation on the sciatic nerve was also unchanged. The reduction in fast transport rate in the diabetic rats was eliminated by maintenance of normal blood glucose levels in twice daily insulin administration. The results are discussed with regard to the known effects of experimental diabetes on axonal transport in sensory fibres and to the role of fast axonal transport in peripheral neuropathies in general.

Targeted disruption of GAP-43 in P19 embryonal carcinoma cells inhibits neuronal differentiation. As well as acquisition of the morphological phenotype.

GAP-43 is expressed in proliferating neuroblasts in vivo and in vitro, but its role during early neurogenesis has not been investigated. Here we show that neuroectodermal differentiation stimulated by retinoic acid (RA) in the embryonal carcinoma (EC) line P19 is accompanied by upregulation of GAP-43 expression in neuroepithelial precursor cells. In contrast, when upregulation of GAP-43 expression was prevented in 3 independent P19 lines because of a targeted insertion into the gene, generation of neuroepithelial precursors was inhibited. Consequently, neuronal number was significantly decreased, neuronal morphology was abnormal and fewer than 20% of all neurons were able to initiate neuritogenesis. Extracellular matrix (ECM) was unable to rescue initiation of neuritogenesis in the mutant cells, however those neurites that were extended responded normally to ECM-stimulated neurite outgrowth-promoting signals. These data suggest that GAP-43 function is required for commitment to a neuronal phenotype as well as initiation of neurite extension. However, stimulation of neurite outgrowth by ECM in P19s occurs independently of GAP-43.

Nerve growth factor induced modification of presynaptic elements in adult visual cortex in vivo.

Nerve growth factor (NGF) has been shown to play important roles in neuronal survival, growth and differentiation. Recently, we have found that intracortical infusion of NGF into adult cat visual cortex can recreate ocular dominance plasticity, suggesting that NGF is also involved in activity-dependent modification of synaptic connectivity in the adult brain. To further explore the mechanisms of NGF-induced plasticity in adult visual cortex, we studied two presynaptic markers: GAP-43 and synaptophysin. Immunocytochemical staining showed that NGF-treatment of adult visual cortex selectively increased the level of the phosphorylated form of GAP-43, while the total level of GAP-43 was not changed. These results demonstrate that NGF-treatment stimulates phosphorylation processes of GAP-43 in vivo. In addition, NGF-treatment of adult visual cortex increased the level of synaptophysin immunoreactivity. Since the phosphorylated form of GAP-43 is known to be enriched in the membrane skeleton of growth cones and of developing synapses, and the phosphorylation of GAP-43 has been linked with events that underlie synaptic plasticity, and since synaptophysin is a major component of presynaptic vesicles, our results suggest that NGF-treatment of adult visual cortex modulates presynaptic terminals, possibly by inducing axonal sprouting and formation of new synapses, and that these changes may play a role in the NGF-induced functional plasticity.

Alterations in hippocampal GAP-43 phosphorylation and protein level following contextual fear conditioning.

C57BL/6 (B6) mice display better contextual learning than the DBA/2 (D2) mice. The possibility that GAP-43, is differentially affected as a function of strain and learning was investigated in the present study. No basal difference between C57BL/6J (B6) and DBA/2J (D2) mice in the amount of hippocampal GAP-43 was observed, but naive D2 mice have slightly lower basal levels of GAP-43 phosphorylation than do B6 mice. Interestingly, alterations in hippocampal GAP-43 protein levels and phosphorylation state in response to training for contextual learning were observed only in B6 mice. Immediate-shocked mice, serving as nonlearning controls, showed no GAP-43 alterations, nor did D2 mice subjected to either training condition. These results suggest that modulation of hippocampal GAP-43 may be important for contextual learning and that strain-specific alterations in GAP-43 may be part of a disrupted pathway in D2 mice that is essential for learning.

Limitations to the usefulness of N-succinimidyl propionate for the study of retrograde axonal transport.

Retrograde axonal transport of radiolabelled proteins was studied in rat sciatic nerve, after direct application of [3H]N-succinimidyl propionate. Waves of radiolabelled proteins were observed but only two proteins were predominantly labelled, one of molecular weight 68 kilodaltons (K) and the other of 19K. There was no evidence to confirm the waves as representing retrograde axonal transport of identifiable proteins, and the tendency of the covalent label to bind selectively in vivo to a small number of prominent proteins limits its usefulness for the detection of retrogradely transported proteins in general.

GAP-43 is essential for the neurotrophic effects of BDNF and positive AMPA receptor modulator S18986.

Positive alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor modulators include benzamide compounds that allosterically modulate AMPA glutamate receptors. These small molecules that cross the blood-brain barrier have been shown to act as a neuroprotectant by increasing the levels of endogenous brain-derived neurotrophic factor (BDNF). Positive AMPA receptor modulators have also been shown to increase the levels of growth-associated protein-43 (GAP-43). GAP-43 plays a major role in many aspects of neuronal function in vertebrates. The goal of this study was to determine whether GAP-43 was important in mediating the actions of positive AMPA receptor modulator (S18986) and BDNF. Using cortical cultures from GAP-43 knockout and control mice, we show that (1) GAP-43 is upregulated in response to S18986 and BDNF in control cultures; (2) this upregulation of GAP-43 is essential for mediating the neuroprotective effects of S18986 and BDNF; (3) administration of S18986 and BDNF leads to an increase in the expression of the glutamate transporters GLT-1 and GLAST that are key to limiting excitotoxic cell death and this increase in GLT-1 and GLAST expression is completely blocked in the absence of GAP-43. Taken together this study concludes that GAP-43 is an important mediator of the neurotrophic effects of S18986 and BDNF on neuronal survival and plasticity, and is essential for the success of positive AMPA receptor modulator-BDNF-based neurotrophin therapy.

Nerve growth factor stimulation of GAP-43 phosphorylation in intact isolated growth cones.

Phosphorylation of the nervous system-specific growth cone protein GAP-43 by kinase C in vivo occurs exclusively in growth cones and distal axons, and the onset of this phosphorylation is delayed relative to the onset of axonogenesis, with the delay predicted on the time needed for axons to reach the vicinity of their targets (Meiri et al., 1991). We have used a subcellular fraction of intact growth cones (IGCs) to investigate whether this induction of GAP-43 phosphorylation can be influenced by target-derived substances, and show here that increased phosphorylation of GAP-43 can be both stimulated and maintained by NGF at concentrations of 2 x 10(-10) M. This low concentration of NGF and the subsequent phosphorylation of GAP-43 are both consistent with the interpretation that phosphorylation is due to the binding of NGF to a biologically active high-affinity receptor. Second, we used the monoclonal antibody 2G12 to show that the NGF-stimulated phosphorylation of GAP-43 occurs on serine, the kinase C phosphorylation site, consistent with the results seen in vivo. Levels of phosphorylated GAP-43 in the intact IGCs are also modulated by calcium-stimulated dephosphorylation that could be inhibited by EGTA but not okadaic acid and that therefore resembled the calcineurin-stimulated dephosphorylation reported in vitro. The results suggest that the spatial and temporal regulation of GAP-43 phosphorylation that occurs during axonogenesis in vivo can be regulated by target-derived neurotropic molecules, specifically NGF.

Effects of gangliosides GM1 and GD1a on GAP-43 phosphorylation and dephosphorylation in isolated growth cones.

Phosphorylation of the nervous system-specific protein GAP-43 in growth cones in vivo increases as the growth cones near their targets, at a time when the gangliosides GM1 and GD1a are being accumulated in the growth cone membrane, thus raising the possibility that the gangliosides could modulate GAP-43 behavior. We used a subcellular fraction of intact isolated growth cones to show that both GM1 and GD1a affected the calcium-dependent posttranslational regulation of GAP-43 in several similar ways. Both gangliosides induced rapid incorporation of phosphate into GAP-43; however, the induction was undetectable with our antibody 2G12 that is specific for kinase C-phosphorylated GAP-43. Furthermore, neither ganglioside stimulated kinase C activity in isolated growth cones, suggesting that the rapid phosphorylation may not be on Ser41, the kinase C site. However, both gangliosides did induce a slower accumulation of GAP-43 phosphorylated on Ser41, apparently by inhibiting a phosphatase. Finally, calcium-dependent proteolysis of GAP-43 was also stimulated by both GM1 and GD1a. In contrast, GD1a, but not GM1, caused the redistribution of GAP-43 into the isolated growth cone cytoskeleton. The results demonstrate that both gangliosides can modulate the calcium-dependent regulation of GAP-43.

GAP-43 phosphorylation is dynamically regulated in individual growth cones.

In vivo, kinase C phosphorylation of the growth-associated protein GAP-43 is spatially and temporally associated with the proximity of growing axons to their targets. Here we have used dissociated dorsal root ganglia (DRG)s and an antibody specific for the phosphorylated form of GAP-43 to demonstrate that neurite regeneration in culture also begins in the absence of detectable levels of phosphorylated GAP-43. Since the beta isoform of kinase C was found to be enriched in growth cones before stably phosphorylated GAP-43 was detected, it may normally be inactive during initial neurite outgrowth; however, premature phosphorylation of GAP-43 could be stimulated in newly dissociated DRGs by plating them on cultures in which phosphorylation had already been initiated media conditioned by such cultures caused no response suggesting an effect of either cell-cell or cell-substrate contact. Increased GAP-43 phosphorylation correlated with a reduced extent of neurite outgrowth but not with the rate at which individual growth cones translocated so that motile growth cones contained very low levels of phosphorylated GAP-43, whereas stationary growth cones showed much more immunoreactivity. Downregulation of kinase C by phorbol ester prevented increased GAP-43 phosphorylation and led to growth cone collapse. Finally, phosphorylated GAP-43 was found to be differently distributed within growth cones. Increased immunoreactivity was frequently observed in the neck of the growth cone and was heterogeneously distributed in lamellae and filopodia. These results, which demonstrate the dynamic regulation of GAP-43 phosphorylation in individual growth cones, are discussed with reference to the association between changes in growth cone shape and the ability to translocate and change direction.

Distribution of phosphorylated GAP-43 (neuromodulin) in growth cones directly reflects growth cone behavior.

Phosphorylation of GAP-43 (neuromodulin) by protein kinase C (PKC) occurs at a single site, serine41. In vivo, phosphorylation is induced after initiation of axonogenesis and is confined to distal axons and growth cones. Within individual growth cones, phosphorylation is nonuniformly distributed. Here, we have used high-resolution video-enhanced microscopy of cultured dorsal root ganglia neurons together with immunocytochemistry with a monoclonal antibody that recognizes PKC-phosphorylated GAP-43 to correlate the distribution of phosphorylated GAP-43 with growth cone behavior. In "quiescent," nontranslocating growth cones, phosphorylated GAP-43 was confined to the proximal neurite and the central organelle-rich region, and was low in organelle-poor lamellae. However, levels in lamellae were elevated when they became motile. Conversely, levels of phosphorylated GAP-43 were low in either lamellae that were actively retracting or in the central organelle-rich region and proximal neurite of growth cones that had totally collapsed. The results suggest a mechanism whereby phosphorylation of GAP-43 by PKC, potentially in response to extracellular signals, could direct the functional behavior of the growth cone.

Growth-associated protein, GAP-43, a polypeptide that is induced when neurons extend axons, is a component of growth cones and corresponds to pp46, a major polypeptide of a subcellular fraction enriched in growth cones.

Growth-associated protein, GAP-43, is a polypeptide that is induced in neurons when they grow axons. We show by means of subcellular fractionation and immunohistochemical localization that GAP-43 is a component of neuronal growth cones as well as growing neurites; it is similar to a major phosphoprotein, pp46, of a growth cone-enriched subcellular fraction. These conclusions are consistent with the possibility that the induction of GAP-43/pp46 is an important event in the establishment of a productive growth state in which a neuron is competent to extend an axon.

GAP-43 in growth cones is associated with areas of membrane that are tightly bound to substrate and is a component of a membrane skeleton subcellular fraction.

To ascertain the subcellular localization of the growth-associated protein GAP-43 in growth cones, we isolated growth cones from the forebrains of neonatal rats. Anti-GAP-43 immunoreactivity in these isolated growth cones (IGCs) resembled that seen in cultured growth cones in 2 respects: First, in substrate-attached IGCs, as in cultured growth cones, immunoreactivity was intracellular, punctate, and extended throughout the IGCs and their processes. Second, when IGCs were dislodged from this substrate, patches of membrane that were highly immunoreactive for GAP-43 were left behind. Extracting IGCs in nonionic detergents revealed that almost all the particulate GAP-43 colocalized with a membrane skeleton fraction: It was not present in the cytoskeleton. The association of GAP-43 with the membrane skeleton was not due to nonspecific aggregation, nor was it calcium dependent. Examination of this fraction under the electron microscope showed it to consist of membrane fragments associated with amorphous material that could be visualized with tannic acid. Immunoelectron microscopy showed that anti-GAP-43 immunoreactivity was localized to this amorphous material. Fodrin, talin, and a-actinin immunoreactivity could be detected in the membrane skeleton fraction, and actin and tubulin were also identifiable from SDS gels. The association of GAP-43 with the membrane skeleton, which by analogy with other cell types is involved in the dynamic regulation of cell shape, implies that GAP-43 in growth cones may be involved in this function.

Modulation of actin filament behavior by GAP-43 (neuromodulin) is dependent on the phosphorylation status of serine 41, the protein kinase C site.

Synthesis of GAP-43 (also known as neuromodulin) in neurons is induced during axon growth, and high concentrations (estimated between 50 and 100 microM) accumulate in the growth cone. GAP-43 is tightly associated with the growth cone membrane skeleton, the structure that transduces extracellular guidance cues into alterations in morphology by spatially regulating polymerization of actin filaments, thereby causing directional changes in axon growth. GAP-43 cosediments with actin filaments, and its phosphorylation on serine 41 by PKC, too, is spatially regulated so that phosphorylated GAP-43 is found in areas where growth cones make productive, stable contacts with other cells. In contrast, unphosphorylated GAP-43, which binds calmodulin, is always found in parts of the growth cone that are retracting. Here we have used a cell-free assay to investigate how the phosphorylation status of GAP-43 affects its interactions with actin and show that both phosphorylated and unphosphorylated GAP-43 have different, independent effects on actin filament structure. Phosphorylated GAP-43 stabilizes long actin filaments (Kd = 161 nM), and antibodies to phosphorylated GAP-43 inhibit binding of actin to phalloidin, implying a lateral interaction with filaments. In contrast, unphosphorylated GAP-43 reduces filament length distribution (Kd = 1.2 microM) and increases the critical concentration for polymerization. Prebinding calmodulin potentiates this effect. The results show that spatially regulated post-translational modifications of GAP-43 within the growth cone, which can be regulated in response to extracellular signals, have the ability to directly influence the structure of the actin cytoskeleton.

Failure to express GAP-43 during neurogenesis affects cell cycle regulation and differentiation of neural precursors and stimulates apoptosis of neurons.

GAP-43 is first expressed in proliferating neuroblasts and is required for maturation of neurons. When GAP-43 is not expressed in differentiating embryonal carcinoma P19 cells, reduced numbers of neurons were generated. Here we show that neuronal differentiation is initially disrupted at the onset of cell-cycle arrest in aggregated, proliferating neuronal precursors. The ratio of nestin:beta-tubulin-labeled progeny generated at this stage suggests that the differentiation is asymmetric. Apoptosis of immature neurons subsequently produced was also significantly induced. In vivo, too, proliferation of neuroblasts was significantly reduced in cortex of GAP-43(-/-) mice at E14.5. These data demonstrate that when GAP-43 is not expressed in proliferating neuroblasts, neural differentiation is not initiated appropriately, inducing apoptosis. Moreover, the concurrent inhibition of Ca2+-dependent adhesion between differentiating P19 cells in aggregates implicates GAP-43 in CAM-mediated signaling during neurogenesis, as has been previously shown in growth cones.

Distribution and phosphorylation of the growth-associated protein GAP-43 in regenerating sympathetic neurons in culture.

Sympathetic neurons regenerating in culture were studied in order to gain further insight into the intracellular distribution and phosphorylation of GAP-43, a protein that has been suggested to have a role in axonal outgrowth and neuronal plasticity (Willard et al., 1987). Superior cervical ganglion neurons from embryonic rats were highly reactive with a polyclonal antibody against the growth-associated protein GAP-43 soon after they were placed in culture on a laminin substrate. As these neurons extended neurites, the distribution of GAP-43 reactivity changed. The cell body became progressively less reactive, whereas the growth cone at the tip of the growing neurite reacted strongly. The pattern of immunofluorescence was punctate both in the growth cone and the adjacent neurite, but appeared more diffusely distributed in the cell body. The antibody reacted only with cells that had been subjected to treatment that permeabilized the plasma membrane. When antibody was supplied in the medium of growing neurons, it neither bound to the cells nor altered normal neurite initiation or elongation. Of the different types of cells in these cultures, the antibody reacted only with neurons; it did not react with Schwann cells or fibroblasts. The stimulation of protein kinase C in these cultures resulted in a 7-fold stimulation of the phosphorylation of a protein of similar electrophoretic mobility to GAP-43. These observations demonstrate that GAP-43 is neuron-specific, is present throughout the neuron but at higher levels in the growth cone, and is a major substrate of protein kinase C. The high concentration of GAP-43 in the growth cones may necessitate its increased synthesis in neurons with elongating axons. Its location and phosphorylation by kinase C suggest that it could perform a function in the growth cone that is modulated by extracellular signals, such as those used in pathfinding or in the control of axonal elongation.