Specific Proteolysis of Neuronal Protein GAP-43 by Calpain: Characterization,Regulation, and Physiological Role

The mechanism of specific proteolysis of the neuronal protein GAP-43 in axonal terminals has been investigated. In synaptic terminals in vivo and in synaptosomes in vitro GAP-43 is cleaved only at the single peptide bond formed by Ser41; this is within the main effector domain of GAP-43. Proteolysis at this site involves the cysteine calcium-dependent neutral protease calpain. The following experimental evidences support this conclusion: 1) calcium-dependent proteolysis of GAP-43 in synaptosomes is insensitive to selective inhibitor of micro-calpain (PD151746), but it is completely blocked by micro- and m-calpain inhibitor PD150606; 2) GAP-43 proteolysis in the calcium ionophore A23187-treated synaptosomes is activated by millimolar concentration of calcium ions; 3) the pattern of fragmentation of purified GAP-43 by m-calpain (but not by micro-calpain) is identical to that observed in synaptic terminals in vivo. GAP-43 phosphorylated at Ser41 by protein kinase C (PKC) is resistant to the cleavage by calpain. In addition, calmodulin binding to GAP-43 decreases the rate of calpain-mediated GAP-43 proteolysis. Our results indicate that m-calpain-mediated GAP-43 proteolysis regulated by PKC and calmodulin is of physiological relevance, particularly in axonal growth cone guidance. We suggest that the function of the N-terminal fragment of GAP-43 (residues 1-40) formed during cleavage by m-calpain consists in activation of neuronal heterotrimeric GTP-binding protein G(o); this results in growth cone turning in response to repulsive signals.     

Enhanced level of site-specific proteolysis of GAP-43 protein during early stages of brain development

GAP-43 protein of nerve terminals (B-50, F1, F57, pp46, neuromodulin) is thought to be one of key proteins involved in the control of outgrowth of neurites, release of neuromediators, synapse plasticity, etc. GAP-43 is usually considered as a whole protein. Along with the intact protein, nerve cells also contain two large native fragments of GAP-43 deprived of four or of about forty N-terminal amino acid residues (GAP-43-2 and GAP-43-3, respectively). The full-length GAP-43 is predominant in the mature brain. However, the ratio of the full-length protein and its fragments can vary under different physiological conditions. Changes in the GAP-43 proteins (the full-length protein and its fragments) were studied during embryonal and postnatal development of rat brain. The GAP-43 proteins were found to be expressed not later than on the 12-13th day of embryogenesis. Then their contents increased, and, until the 10th day after birth, GAP-43-3 dominated rather than the full-length protein. It is suggested that during this period the activity of a specific protease, which cleaves the N-terminal peptide of about 40 residues from the full-length GAP-43 molecule, is increased. The cleavage occurs in the region responsible for the interaction of GAP-43 with calmodulin. In the full-length molecule, this region is responsible also for the recognition of Ser41 residue by protein kinase C during phosphorylation. Another functionally important region that determines, in particular, the attachment of GAP-43 to the plasma membrane is cleaved from the main part of the molecule together with the N-terminal peptide. Thus, the specific fragmentation of GAP-43 that depends on developmental stage should be considered as a controlled structural rearrangement fundamentally affecting the functions of this protein.     

Identification of brain proteins BASP1 and GAP-43 in mouse oocytes and zygotes

The similarity between the calcium-activated signaling systems of oocytes and neuronal axon terminals has prompted us to test whether BASP1 and GAP-43 proteins, highly expressed in brain neurons, are present in oocytes. Using immunocytochemical techniques combined with confocal microscopy, we have for the first time demonstrated that both BASP1 and GAP-43 are present in mouse metaphase II (MII) oocytes and zygotes. BASP1 is localized to the plasma membrane and actin cortex of MII oocytes, which is similar to BASP1 distribution in neurons and other cell types. GAP-43 is generally regarded as a postmitotic membrane marker of nerve cells; however, GAP-43 in MII oocytes is associated with microtubules of the meiotic spindle. GAP-43 is also colocalized with γ-tubulin at the spindle poles (centrosomes) and at the discrete microtubule- organizing centers in the cytoplasm. The antibodies to Ser41-phosphorylated form of GAP-43 allowed for demonstration that GAP-43 in oocytes is subject to phosphorylation by protein kinase C. The presence of BASP1 and GAP-43 in oocytes is also confirmed by electrophoresis and western blotting. Microinjection of BASP1 (but not GAP-43) into the cytoplasm of mouse MII oocytes induces their exit from metaphase II arrest followed by parthenogenetic embryo development. This suggests putative BASP1 involvement in fertilization-induced oocyte activation, presumably, through regulation of local concentration of polyphosphoinositides in the plasma membrane. Recently it was found that GAP-43 is associated with centrosomes in asymmetrically dividing neuronal progenitors, which is similar to the localization of GAP-43 at the meiotic spindle and centrosomes in oocytes. Therefore we suggest that GAP-43 may be involved in regulation of spindle orientation and oocyte polarity.     

Phosphorylation-site mutagenesis of the growth-associated protein GAP- 43 modulates its effects on cell spreading and morphology

The 43-kD growth-associated protein (GAP-43) is a major protein kinase C (PKC) substrate of axonal growth cones, developing nerve terminals, regenerating axons, and adult central nervous system areas associated with plasticity. It is a cytosolic protein associated with the cortical cytoskeleton and the plasmalemma. Membrane association of GAP-43 is mediated by palmitoylation at Cys3Cys4. In vitro and in vivo, phosphorylation by PKC exclusively involves Ser41 of mammalian GAP-43 (corresponding to Ser42 in the chick protein). To identify aspects of GAP-43 function, we analyzed the actions of wild-type, membrane- association, and phosphorylation-site mutants of GAP-43 in nonneuronal cell lines. The GAP-43 constructs were introduced in L6 and COS-7 cells by transient transfection. Like the endogenous protein in neurons and their growth cones, GAP-43 in nonneuronal cells associated with the cell periphery. GAP-43 accumulated in the pseudopods of spreading cells and appeared to interact with cortical actin-containing filaments. Spreading L6 cells expressing high levels of recombinant protein displayed a characteristic F-actin labeling pattern consisting of prominent radial arrays of peripheral actin filaments. GAP-43 had dramatic effects on local surface morphology. Characteristic features of GAP-43-expressing cells were irregular cell outlines with prominent and numerous filopodia. The effects of GAP-43 on cell morphology required association with the cell membrane, since GAP-43(Ala3Ala4), a mutant that failed to associate with the cell cortex, had no morphogenetic activity. Two GAP-43 phosphorylation mutants (Ser42 to Ala42 preventing and Ser42 to Asp42 mimicking phosphorylation by PKC) modulated the effects of GAP-43 in opposite ways. Cells expressing GAP- 43(Asp42) spread extensively and displayed large and irregular membranous extensions with little filopodia, whereas GAP-43(Ala42) produced small, poorly spreading cells with numerous short filopodia. Therefore, GAP-43 influences cell surface behavior and phosphorylation modulates its activity. The presence of GAP-43 in growing axons and developing nerve termini may affect the behavior of their actin- containing cortical cytoskeleton in a regulatable manner.     

GAP-43, a protein associated with axon growth, is phosphorylated at three sites in cultured neurons and rat brain.

GAP-43 is a neuronal calmodulin-binding phosphoprotein that is concentrated in growth cones and presynaptic terminals. By sequencing tryptic and endoproteinase Asp-N phosphopeptides and directly determining the release of radioactive phosphate, we have identified three sites (serines 41 and 96 and threonine 172) that are phosphorylated, both in cultured neurons and in neonatal rat brain. These three sites account for most of the 32PO4 that was incorporated into GAP-43 in cultured neurons; 8-15% of each site was occupied with phosphate in GAP-43 isolated from neonatal rat brain. Phosphorylation of serine 41 in cultured neurons was stimulated by phorbol ester, indicating that it is the only site phosphorylated by protein kinase C. The resemblance of the sequence surrounding the other two sites suggests that they may be substrates for the same protein kinase. None of the sites phosphorylated by casein kinase II in vitro was phosphorylated in living cells or in neonatal rat brain. These results show that GAP-43 is a substrate for at least one protein kinase in addition to protein kinase C in living cells and brain.     

Mutation of Serine 41 in the Neuron-Specific Protein B-50 (GAP-43) Prohibits Phosphorylation by Protein Kinase C

The neuron-specific, calmodulin-binding protein B-50 (also known as GAP-43, F1, or neuromodulin) is an endogenous substrate of protein kinase C (PKC). PKC exclusively phosphorylates Ser residues in B-50. As potential phosphorylation sites for PKC, Ser41, Ser110, and Ser122 were indicated, of which Ser41 is contained in the sequence ASF, which matches with the sequence of a synthetic PKC substrate. N-terminally 35S-labeled B-50, produced from cDNA, was subjected to digestion with Staphylococcus aureus V8 protease (SAP). Consecutively, 35S-labeled 28- and 15-kDa fragments were formed, similar to those after digestion of 32P-labeled B-50. In a previous study, we showed that the 32P-labeled 15-kDa SAP fragment contains all 32P radioactivity. The present data indicate that it contains the N-terminus of B-50 as well. The 15-kDa fragment, with a calculated length ranging from amino acid residue 1 to 65, contains only one potential PKC phosphorylation site, at Ser41. Mutagenesis of Ser41 into Thr or Ala resulted in recombinant B-50 products with mobilities on two-dimensional electrophoresis similar to those of the nonmutated recombinant B-50 and the rat brain B-50. Only [Ser41]B-50 was phosphorylated by PKC, whereas [Thr41]- or [Ala41]B-50 did not show any phosphorylation at the positions indicated on the immunoblots. This leads us to the conclusion that Ser41 is the sole phosphorylation site for PKC in vitro.     

GAP-43 promoter elements in transgenic zebrafish reveal a difference in signals for axon growth during CNS development and regeneration

A pivotal event in neural development is the point at which differentiating neurons become competent to extend long axons. Initiation of axon growth is equally critical for regeneration. Yet we have a limited understanding of the signaling pathways that regulate the capacity for axon growth during either development or regeneration. Expression of a number of genes encoding growth associated proteins (GAPs) accompanies both developmental and regenerative axon growth and has led to the suggestion that the same signaling pathways regulate both modes of axon growth. We have tested this possibility by asking whether a promoter fragment from a well characterized GAP gene, GAP-43, is sufficient to activate expression in both developing and regenerating neurons. We generated stable lines of transgenic zebrafish that express green fluorescent protein (GFP) under regulation of a 1 kb fragment of the rat GAP-43 gene, a fragment that contains a number of evolutionarily conserved elements. Analysis of GFP expression in these lines confirms that the rat 1 kb region can direct growth-associated expression of the transgene in differentiating neurons that extend long axons. Furthermore, this region supports developmental down-regulation of transgene expression which, like the endogenous gene, coincides with neuronal maturation. Strikingly, these same sequences are insufficient for directing expression in regenerating neurons. This finding suggests that signaling pathways regulating axon growth during development and regeneration are not the same. While these results do not exclude the possibility that pathways involved in developmental axon growth are also active in regenerative growth, they do indicate that signaling pathway(s) controlling activation of the GAP-43 gene after CNS injury differ in at least one key component from the signals controlling essential features of developmental axon growth.     

GAP-43 is key to mitotic spindle control and centrosome-based polarization in neurons

In neurons, the position of the centrosome during final mitosis marks the point of emergence of the future axon. However, the molecular underpinnings linking centrosome position to axon emergence are unknown. GAP-43 is a calmodulin-binding IQ motif protein that regulates neuronal cytoskeletal architecture by interacting with F-actin in a phosphorylation dependent manner. Here we show that GAP-43 is associated with the centrosome and plays a critical role in mitosis and acquisition of neuronal polarity in cerebellar granule neurons. In the absence of GAP-43, the centrosome position is delinked from process outgrowth and is only capable of mediating morphological polarization, however molecular specification of the axonal compartment does not take place. These results show that GAP-43 is required to link centrosome position to process outgrowth in order to generate neuronal polarity in cerebellar granule cells.     

GAP-43 mediates retinal axon interaction with lateral diencephalon cells during optic tract formation

GAP-43 is an abundant intracellular growth cone protein that can serve as a PKC substrate and regulate calmodulin availability. In mice with targeted disruption of the GAP-43 gene, retinal ganglion cell (RGC) axons fail to progress normally from the optic chiasm into the optic tracts. The underlying cause is unknown but, in principle, can result from either the disruption of guidance mechanisms that mediate axon exit from the midline chiasm region or defects in growth cone signaling required for entry into the lateral diencephalic wall to form the optic tracts. Results here show that, compared to wild-type RGC axons, GAP-43-deficient axons exhibit reduced growth in the presence of lateral diencephalon cell membranes. Reduced growth is not observed when GAP-43-deficient axons are cultured with optic chiasm, cortical, or dorsal midbrain cells. Lateral diencephalon cell conditioned medium inhibits growth of both wild-type and GAP-43-deficient axons to a similar extent and does not affect GAP-43-deficient axons more so. Removal or transplant replacement of the lateral diencephalon optic tract entry zone in GAP-43-deficient embryo preparations results in robust RGC axon exit from the chiasm. Together these data show that RGC axon exit from the midline region does not require GAP-43 function. Instead, GAP-43 appears to mediate RGC axon interaction with guidance cues in the lateral diencephalic wall, suggesting possible involvement of PKC and calmodulin signaling during optic tract formation.     

Rapid changes in the distribution of GAP-43 correlate with the expression of neuronal polarity during normal development and under experimental conditions

Hippocampal neurons growing in culture initially extend several, short minor processes that have the potential to become either axons or dendrites. The first expression of polarity occurs when one of these minor processes begins to elongate rapidly, becoming the axon. Before axonal outgrowth, the growth-associated protein GAP-43 is distributed equally among the growth cones of the minor processes; it is preferentially concentrated in the axonal growth cone once polarity has been established (Goslin, K., D. Schreyer, J. Skene, and G. Banker. 1990. J. Neurosci. 10:588-602). To determine when the selective segregation of GAP-43 begins, we followed individual cells by video microscopy, fixed them as soon as the axon could be distinguished, and localized GAP-43 by immunofluorescence microscopy. Individual minor processes acquired axonal growth characteristics within a period of 30-60 min, and GAP-43 became selectively concentrated to the growth cones of these processes with an equally rapid time course. We also examined changes in the distribution of GAP-43 after transection of the axon. After an axonal transection that is distant from the soma, neuronal polarity is maintained, and the original axon begins to regrow almost immediately. In such cases, GAP-43 became selectively concentrated in the new axonal growth cone within 12-30 min. In contrast, when the axon is transected close to the soma, polarity is lost; the original axon rarely regrows, and there is a significant delay before a new axon emerges. Under these circumstances, GAP-43 accumulated in the new growth cone much more slowly, suggesting that its ongoing selective routing to the axon had been disrupted by the transection. These results demonstrate that the selective segregation of GAP-43 to the growth cone of a single process is closely correlated with the acquisition of axonal growth characteristics and, hence, with the expression of polarity.     

GAP-43 Augmentation of G Protein-Mediated Signal Transduction Is Regulated by Both Phosphorylation and Palmitoylation

Abstract: The neuronal protein GAP-43 is concentrated at the growth cone membrane, where it is thought to amplify the signal transduction process. As a model for its neuronal effects, GAP-43 protein injection into Xenopus laevis oocytes strongly augments the calcium-sensitive chloride current evoked by the G protein-coupled receptor stimulation. We have now examined a series of GAP-43 mutants in this system and determined those regions of GAP-43 required for this increase in current flux. As expected, palmitoylation inhibits signal amplification in oocytes by blocking G protein activation. Unexpectedly, a second domain of GAP-43 (residues 35–50) containing a protein kinase C phosphorylation site at residue 41 is also necessary for augmentation of G protein-coupled signals in oocytes. This region is not required for activation of isolated G_o but is necessary for GAP-43 binding to isolated calmodulin and to isolated protein kinase C. Substitution of Asp for Ser⁴¹ inactivates GAP-43 as a signal facilitator in oocytes. This mutation blocks GAP-43 binding to both protein kinase C and calmodulin. Thus, GAP-43 regulates an oocyte signaling cascade via coordinated, simultaneous G protein activation and interaction with either calmodulin or protein kinase C.     

GAP-43 as a plasticity protein in neuronal form and repair.

Neurons exhibit a remarkable plasticity of form, both during neural development and during the subsequent remodelling of synaptic connectivity. Here we review work on GAP-43 and G0, and focus upon the thesis that their interaction may endow neurons with such plasticity. We also present new data on the role of G proteins in neurite growth, and on the interaction of GAP-43 and actin. GAP-43 is a protein induced during periods of axonal extension and highly enriched on the inner surface of the growth cone membrane. Its membrane localization is primarily due to a short amino terminal sequence which is subject to palmitoylation. Binding to actin filaments may also assist in restricting the protein to specific cellular domains. Consistent with its role as a "plasticity protein," there is evidence that GAP-43 can directly alter cell shape and neurite extension, and several theses have been advanced for how it might do so. Two other prominent components of the growth cone membrane are the alpha and beta subunits of G0. GAP-43 regulates their guanine nucleotide exchange, which is an unusual role for an intracellular protein. We speculate that GAP-43 may adjust the "set point" of responsiveness for G0 stimulation by receptors, thereby altering the neuronal propensity to growth, without actually causing growth. To begin to address how G protein activity affects axon growth, we have developed a means to introduce guanine nucleotide analogs into sympathetic neurons. Stimulation of G proteins with GTP-gamma-S retards axon growth, whereas GDP-beta-S enhances it. This is compatible with G protein registration of inhibitory signals.     

Mutagenesis of ser41 to ala inhibits the association of GAP-43 with the membrane skeleton of GAP-43-deficient PC12B cells: Effects on cell adhesion and the composition of neurite cytoskeleton and membrane

To investigate the molecular basis for GAP-43 function in axon outgrowth, we produced a mutant, GAP-43 (Ala₄₁), whose interaction with calmodulin in vitro was unaffected by increasing Ca²⁺ concentrations, and stably transfected it into GAP-43-deficient PC12B cells. Several lines that expressed wild-type or mutant protein at levels that resembled endogenous GAP-43 expression in PC12 controls were subcloned and characterized. GAP-43 (Ala₄₁) was significantly more extractable with Nonidet P-40 and less tightly associated with the membrane skeleton than the wild-type protein. Furthermore, GAP-43 (Ala₄₁) expression by PC12B cells profoundly affected their phenotype: First, observation of living cells using video-enhanced microscopy revealed irregular plasma membranes with numerous blebs and protrusions and neurites that appeared thin and varicose. Second, both the cells' ability to remain attached to laminin substrates and the amount of α1β1 integrin expressed on the cell surface was significantly decreased. Finally, peripherin transport, which is abnormal in PC12B cells, could be rescued by transfection of wild-type GAP-43 but not the GAP-43 (Ala₄₁) mutant. The phenotypic abnormalities resemble other cell types in which membrane skeleton/plasma membrane interactions have been functionally decoupled, and our results are consistent with the notion that these interactions may be abnormal in GAP-43 (Ala₄₁)-expressing PC12B cells, either as a direct consequence of the mutation or arising secondarily to the altered availability of calmodulin in the growing neurite. © 1996 John Wiley & Sons, Inc.     

GAP-43 as a plasticity protein in neuronal form and repair

Neurons exhibit a remarkable plasticity of form, both during neural development and during the subsequent remodelling of synaptic connectivity. Here we review work on GAP-43 and G₀, and focus upon the thesis that their interaction may endow neurons with such plasticity. We also present new data on the role of G proteins in neurite growth, and on the interaction of GAP-43 and actin. GAP-43 is a protein induced during periods of axonal extension and highly enriched on the inner surface of the growth cone membrane. Its membrane localization is primarily due to a short amino terminal sequence which is subject to palmitoylation. Binding to actin filaments may also assist in restricting the protein to specific cellular domains. Consistent with its role as a ?plasticity protein,”? there is evidence that GAP-43 can directly alter cell shape and neurite extension, and several theses have been advanced for how it might do so. Two other prominent components of the growth cone membrane are the α and β subunits of G₀. GAP-43 regulates their guanine nucleotide exchange, which is an unusual role for an intracellular protein. We speculate that GAP-43 may adjust the ?set point”? of responsiveness for G₀ stimulation by receptors, thereby altering the neuronal propensity to growth, without actually causing growth. To begin to address how G protein activity affects axon growth, we have developed a means to introduce guanine nucleotide analogs into sympathetic neurons. Stimulation of G proteins with GTP-γ-S retards axon growth, whereas GDP-β-S enhances it. This is compatible with G protein registration of inhibitory signals. © 1992 John Wiley & Sons, Inc.     

Growth-associated protein of 43 kDa (GAP-43) is cleaved nonprocessively by the 20S proteasome.

Purified, nonubiquitinated growth-associated protein of 43 kDa (GAP-43) was attacked by purified reticulocyte 20S proteasome but not by the 26S proteasome. Cleavage yielded 12 N-terminally labelled GAP-43 fragments that could be resolved by SDS/PAGE. Inhibitor experiments suggested that proteasome beta1 activity yielded the resolved bands and that proteasomebeta5 activity generated nonresolvable fragments. Processive degradation, yielding only nonresolvable fragments, therefore did not occur. Most of the resolved fragments co-migrated with fragments formed in the reticulocyte lysate translation mixture used for GAP-43 synthesis, which suggested that the fragments were also produced in the translation mixture by the endogenous reticulocyte lysate proteasome. Consistent with this idea, the addition of proteasome inhibitors to translation mixtures blocked fragment production. Ubiquitinated GAP-43 appeared to be the source of the fragments in the presence of ATP, and nonubiquitinated GAP-43 the source in the absence of ATP. The results therefore suggest that the lack of processing seen with the 20S proteasome is not an artefact arising from the way in which the 20S proteasome was purified. In one purification protocol, the GAP-43 fragments formed in translation mixtures co-purified with full-length GAP-43. These fragments were digested to nonresolvable products upon addition of purified 20S proteasome. Addition of calmodulin or G-actin blocked the consumption of both full-length GAP-43 and the co-purified GAP-43 fragments. This showed that the resolved fragments can re-enter the proteasome and be cleaved to nonresolvable products, indicating that the lack of processivity is not a result of their resistance to further proteasome attack. The difficult step therefore appears to be the transfer of the large fragments within the proteasome from the beta1 to the beta5 activity for further attack.     

Mass spectrometric analysis of GAP-43/neuromodulin reveals the presence of a variety of fatty acylated species

GAP-43 (neuromodulin) is a protein kinase C substrate that is abundant in developing and regenerating neurons. Thioester-linked palmitoylation at two cysteines near the GAP-43 N terminus has been implicated in directing membrane binding. Here, we use mass spectrometry to examine the stoichiometry of palmitoylation and the molecular identity of the fatty acid(s) attached to GAP-43 in vivo. GAP-43 expressed in either PC12 or COS-1 cells was acetylated at the N-terminal methionine. Approximately 35% of the N-terminal GAP-43 peptides were also modified by palmitate and/or stearate on Cys residues. Interestingly, a variety of acylated species was detected, in which one of the Cys residues was acylated by either palmitate or stearate, or both Cys residues were acylated by palmitates or stearates or a combination of palmitate and stearate. Depalmitoylation of membrane-bound GAP-43 did not release the protein from the membrane, implying that additional forces function to maintain membrane binding. Indeed, mutation of four basic residues within the N-terminal domain of GAP-43 dramatically reduced membrane localization of GAP-43 without affecting palmitoylation. These data reveal the heterogeneous nature of S-acylation in vivo and illustrate the power of mass spectrometry for identification of key regulatory protein modifications.     

Effects of Gangliosides GM1 and GD1 a on GAP-43 Phosphorylation and Dephosphorylation in Isolated Growth Cones

Abstract: 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.     

Palmitoylation alters protein activity: blockade of G(o) stimulation by GAP-43.

The addition of palmitate to cysteine residues enhances the hydrophobicity of proteins, and consequently their membrane association. Here we have investigated whether this type of fatty acylation also regulates protein-protein interactions. GAP-43 is a neuronal protein that increases guanine nucleotide exchange by heterotrimeric G proteins. Two cysteine residues near the N-terminus of GAP-43 are subject to palmitoylation, and are necessary for membrane binding as well as for G(o) activation. N-terminal peptides, which include these cysteines, stimulate G(o). Monopalmitoylation reduces, and dipalmitoylation abolishes the activity of the peptides. The activity of GAP-43 protein purified from brain also is reversibly blocked by palmitoylation. This suggests that palmitoylation controls a cycle of GAP-43 between an acylated, membrane-bound reservoir of inactive GAP-43, and a depalmitoylated, active pool of protein.