BIOLOGICAL OVERVIEW

The human neurofibromatosis type 1 (NF1) tumor suppressor protein functions as a Ras-specific guanosine triphosphatase-activating protein (Ras-GAP). Neurofibromin is the product of the NF1 gene, whose alteration is responsible for the pathogenesis of NF1, one of the most frequent genetic disorders in man. Although loss of Nf1 expression correlates with increased Ras activity in several mammalian tumor cell types, it is not known which pathways are altered to produce the diverse symptoms observed in NF1 patients, which in addition to frequent benign and infrequent malignant tumors also include short stature and learning disabilities. Drosophila homozygous for null mutations of an Nf1 homolog shows no obvious signs of perturbed Ras1-mediated signaling. However, loss of Nf1 results in a reduction in size of larvae, pupae, and adults. This size defect is not modified by manipulation of Ras1 signaling, suggesting that Drosophila Nf1 is not involved in Ras signaling (The, 1997).

Flies carrying a viable heteroallelic combination of mutant alleles of the gene encoding the PKA catalytic subunit, DC0, are reduced in size (Skoulakis, 1993). To test whether the adenosine 3'-5'-monophosphate (cAMP)-PKA pathway might represent a target for Nf1, pupae of a DC0 heteroallelic combination were tested to see whether increasing PKA activity in Nf1 mutant animals would rescue the size defect. A constitutive active murine PKA* transgene was expressed in Nf1 mutant flies. Heat shock-induced expression of this transgene results in lethality. However lower transgene expression can be achieved by growing the fly cultures at 28 degrees C. Under these conditions, statistically significant rescue of the pupal size defect is observed. Because expression of activated PKA suppresses the phenotype of NF1 null alleles, PKA appears not to function upstream of Nf1 in a simple linear pathway. Therefore, PKA must either function downstream of Nf1 or in a parallel pathway (The, 1997).

A hybrid system was used to further explore the relationship between Nf1 and the PKA pathway. PACAP38 is mammalian protein that belongs to the vasoactive intestinal polypeptide-secretin-glucagon peptide family. Mutations in Drosophila genes rutabaga, Ras1 and Raf1 eliminate the response of flies to PACAP38. PACAP38 functions as a ligand for G protein-coupled receptors in vertebrates and in flies is known to stimulate cAMP synthesis inducing a 100-fold enhancement in K+ currents by coactivating both Rutabaga-adenylyl cyclase-cAMP and Ras-Raf kinase pathways (Zhong, 1995a). Mutations in rutabaga, Ras1 and Raf1 eliminate the response to PACAP38. Activation of both cAMP and Ras-Raf pathways together, but not alone, mimics the PACAP38 response (Zhong, 1995a). The involvement of Ras in the PACAP38 response has led to an investigation into the effect of Nf1 mutations. The purpose of this study was to further test whether Nf1 acts in the Ras or PKA pathways (Guo, 1997).

The PACAP38 enhancement of potassium currents is eliminated in Nf1 mutants. Perfusion of PACAP38 to the neuromuscular junction induces an inward current followed by a 100-fold enhancement of K+ currents in wild-type larvae. In Nf1 mutants, the inward current remains mostly intact, but the enhancement of K+ currents is abolished. Because the inward current is not affected in Nf1 mutants, it appears that PACAP38 receptors are normally activated by the peptide in these mutants. To control for the involvement of potential developmental effects of Nf1 mutants, wild type heat shock inducable Nf1 was induced in Nf1 mutants and the response to PACAP38 was studied. The PACAP mediated enhancement requires heat shock induction of Nf1. Because PACAP38 is a vertebrate peptide, the response induced by endogenous PACAP38-like neuropeptide (Zhong, 1995b) was tested. High-frequency stimulation (40Hz) applied to motor axons through a suction pipette increases K+ currents, presumably by causing the release of PACAP38-like peptides (Zhong, 1995b). The evoked PACAP38-like response is also eliminated in NF1 mutants. Induced expression of constitutively active Ras or active Raf neither blocks nor mimicks the PACAP38 response, suggesting that failure to negatively regulate Ras-Raf signaling does not explain the defective PACAP38 response in Nf1 mutants (Guo, 1997).

What is the role of the cyclic AMP pathway in PACAP38 responses? Application of cAMP analogs to Nf1 mutants restores the normal response to PACAP38. The cAMP analogs are effective if applied any time before or within 2 min after application of PACAP38. Application of cAMP analogs also restores the response to PACAP38 in rutabaga mutants, but not in Ras mutants. Thus, Nf1 appears to regulate the rutabaga-encoded adenylyl cyclase rather than the Ras-Raf pathway. Moreover, the Nf1 defect is rescued by the exposure of cells to pharmacological treatment that increased concentrations of cAMP, such as forskolin, which stimulates G-protein coupled adenylyl cyclase activity. Exploration of the mechanism by which NF1 influences G protein-mediated activation of adenylyl cyclase may lead to new insights into the mechanisms of G protein-mediated signal transduction and the pathogenesis, and possibly the treatment, of human type 1 neurofibromatosis (Guo, 1997).

The mammalian neurofibromatosis type 1 (NF1) tumor suppressor protein is thought to restrict cell proliferation by functioning as a Ras-specific guanosine triphosphatase-activating protein. but is restored by expression of activated adenosine 3', 5'-monophosphate-dependent protein kinase (PKA). Thus, Nf1 and PKA appear to interact in a pathway that controls the overall growth of Drosophila (The, 1997).

The tumor-suppressor gene Neurofibromatosis 1 (Nf1) encodes a Ras-specific GTPase activating protein (Ras-GAP). In addition to being involved in tumor formation, NF1 has been reported to cause learning defects in humans and Nf1 knockout mice. However, it remains to be determined whether the observed learning defect is secondary to abnormal development. The Drosophila NF1 protein is highly conserved, showing 60% identity of its 2,803 amino acids with human NF1. Previous studies have suggested that Drosophila NF1 acts not only as a Ras-GAP but also as a possible regulator of the cAMP pathway that involves the rutabaga (rut)-encoded adenylyl cyclase. Because rut was isolated as a learning and short-term memory mutant, the hypothesis has been pursued that NF1 may affect learning through its control of the Rut-adenylyl cyclase/cAMP pathway. NF1 has been shown to affect learning and short-term memory independent of its developmental effects. G-protein-activated adenylyl cyclase activity consists of NF1-independent and NF1-dependent components, and the mechanism of the NF1-dependent activation of the Rut-adenylyl cyclase pathway is essential for mediating Drosophila learning and memory (Guo, 2000).

Olfactory associative learning of adult fruit flies was examined by using a well-defined Pavlovian procedure. Significant decrements in olfactory learning performance were shown for two independently isolated NF1 null alleles, NF1^P1 and NF1^P2, and compared with K33, the parental line for NF1 mutants with a P-element inserted nearby the NF1 locus. Olfactory avoidance and electric-shock reactivity, two sensorimotor activities necessary for performing the learning task, were similar in the mutant and control K33 flies. To consider the potential effects of genetic background on behavior, NF1 mutants and K33 were outcrossed with an isogenic line w¹¹¹⁸ (isoCJ1). Again, learning scores of NF1 mutants were significantly reduced, whereas the parameters of sensorimotor activities were not statistically different from the control with a similar genetic background. Even though learning scores and some scores for shock reactivity and odor avoidance are significantly different for K33 in different genetic backgrounds, these behavioral parameters also vary accordingly in NF1 mutants. These results indicate that NF1 is a learning mutant. This conclusion is further supported by the observation that the learning defect is rescued by induced expression of the NF1 transgene without a significant change in sensorimotor activity (Guo, 2000).

The effect of heat-shock-induced expression of the NF1 transgene was examined to determine whether the learning defect is caused by an adult requirement for NF1, or whether it is a secondary consequence of developmental abnormalities, such as the small body size of NF1 mutants. Heat-shock treatment of hsNF1 transgenic flies leads to expression of the NF1 protein. Such treatment does not affect learning scores in NF1 mutants, control flies or hsNF1; K33; however, learning scores are improved when mutant flies carrying the NF1 transgene are heat shocked (Guo, 2000).

Heterozygous transgenic NF1 (hsNF1/+; NF1 ^P2) flies were raised at room temperature (20-24°C). These flies show a learning score PI of 63 +/- 3, indicating a partial rescue because of leaky expression. To minimize the leaky expression of the heat-shock promoter-controlled NF1 transgene, flies were shifted to 18°C overnight before the test. This reduced the learning score significantly to 52 +/- 4. Learning scores of transgenic flies (hsNF1/+; NF1^P2) were improved to a better extent when flies were treated at 30°C as compared with 25°C for two hours, or when the transgenic flies were subjected to 25°C for successively longer times. Presumably, more NF1 is expressed with higher temperatures or for longer times of treatment. This is supported by data from polymerase chain reaction with reverse transcriptase (RT-PCR). Higher temperature treatment led to accumulation of more messenger RNA transcribed from the hsNF1 transgene. Thus, the level of performance improvement may be proportional to the amount of NF1 expressed (Guo, 2000).

A single heat-shock treatment during larval stages does not change the smaller body size of NF1 mutants, as quantified by measuring the length of pupal cases. hsNF1/+; NF1 ^P2pupal cases are indistinguishable after treatment from NF1 ^P2, but were smaller than those of control K33. However, repetitive or continuous heat-shock treatments rescue the developmental phenotype. Thus, the learning defect can be rescued by acute expression of the NF1 transgene during adulthood, but the developmental defect requires repetitive or continuous heat-shock treatment during development. This suggests that NF1 is essential for the learning process (Guo, 2000).

To test whether the NF1-dependent learning defect involves the cAMP pathway, learning scores of NF1 ^P2 and rut ¹ single-mutant, and rut ¹; NF1 ^P2 double-mutant flies were compared. The learning scores of all three mutant genotypes are very similar. The learning score of another double mutant, dunce (dnc) rut ¹, is reduced when compared with either single mutants, which indicates that the two mutations exert additive effects on learning even though both gene products are involved in the cAMP cascade (Rut-adenylyl cyclase (AC) for synthesizing cAMP, and Dnc-phosphodiesterase for degrading cAMP. Therefore, the absence of any further reduction of learning in the double mutant rut ¹;NF1 ^P2 suggests that both gene products function closely in the cAMP pathway (Guo, 2000).

This idea is supported by studies of NF1 mutant flies carrying a transgene encoding a mutant catalytic subunit of cAMP-dependent protein kinase (PKA*), which is constitutively active. Sustained expression of this PKA subunit rescues the small body size phenotype of NF1 mutants. Heat-shock induction of the constitutively active PKA should, in principle, bypass the requirement for the Rut-AC and all other molecules upstream of normal PKA activation. The hsp70-PKA* transgene completely rescues the learning defect of NF1^P1 when the flies are raised at room temperature. NF1^P2 mutants are partially rescued by the transgene at room temperature, but show complete rescue with heat shock (37°C, 30 min), or with a shift to 25°C overnight before being tested. In addition, NF1 mutations also cause a short-term memory defect (3- and 8-h retention) that is also fully rescued by heat-shock induction of PKA*. To determine whether expression of hsp70-PKA* induces a nonspecific enhancement of learning, leaky or induced expression of hsp-PKA* in the wild-type background does not increase the learning score even if flies are undertrained. For undertraining, flies were subjected to 3 repeats of electric shock in a single training trial instead of 12 trials. It is concluded that the PKA* effect is not nonspecific and that the learning defect observed in NF1 mutants can be rescued by induction of PKA activity. Therefore, the biochemical deficiency in the NF1 mutants must reside upstream of PKA induction in the cAMP pathway (Guo, 2000).

These behavioral analyses corroborate electrophysiological data that indicate that NF1 might exert its effect through regulation of the activation of Rut-AC. Biochemical assays provide direct evidence to support the idea. Previous experiments have shown that Rut-AC expressed in a cell line can be stimulated not only by Ca²⁺/calmodulin, but also by reagents that stimulate G-proteins, including GTPgammaS and AIF^-₄. AC activity has been examined in membrane fractions of adult brain tissues. The basal level of AC activity is very similar in the control (K33) and NF1 mutant membranes, but the GTPgammaS-stimulated AC activity is markedly reduced in NF1^P1 and NF1^P2 mutant membranes. However, significant GTPgammaS-stimulated activity occurs above the basal level in the mutants. Overexpression of NF1 in control flies does not increase AC activity, whereas the reduction in stimulated AC activity seen in NF1 mutants is mostly rescued by acutely induced expression of the NF1 transgene, indicating that NF1 is indeed able to regulate cAMP synthesis. Thus, GTPgammaS-stimulated AC activity consists of two components: one that is NF1 dependent and one that is NF1 independent. To determine whether the NF1-dependent AC activity is due to Rutabaga, rut ¹ and rut ¹;NF1 ^P2 mutant flies were examined. The basal and GTPgammaS-stimulated levels of AC activity are very similar in the single mutant, rut ¹ , and in the double mutant, rut ¹;NF1 ^P2. Thus, the NF1 mutation has no impact on AC activity in the absence of Rut-AC. In other words, the NF1-dependent cAMP activity is mediated through Rut-AC (Guo, 2000).

The Ca²⁺ dependence of the NF1 effect was examined to determine how Rut-AC is involved. Membrane fractions extracted from abdominal tissues were used because the Ca²⁺-dependent Rut-AC activity is easier to detect. The data are consistent with a report that the Ca ²⁺-dependent peak of AC activity is missing in rut mutants. Again, the NF1 mutation has no effects on AC activity across Ca ²⁺ concentrations without Rut-AC. Moreover, the Ca²⁺-dependent peak of Rut-AC activity is dependent on G-protein stimulation but not on the presence of NF1 (Guo, 2000).

Together, these results reveal a new mechanism that describes how G-proteins activate the cAMP pathway for normal learning and memory. The G-protein-activated AC activity is both NF1 dependent and NF1 independent. The NF1-dependent component involves Rut-AC. One possibility for how NF1 regulates AC activity is that NF1 acts as a GAP not only for the small G-protein Ras but also for heterotrimeric G-proteins. Thus, NF1 is required for a functional interaction between AC and heterotrimeric G-proteins, similar to the involvement of IRA, a Ras-GAP that is distantly related to NF1, in the Ras-activated cAMP pathway in yeast (Guo, 2000).

Another possibility is that NF1 regulates AC activity independent of its role as a Ras-GAP. Therefore, NF1 may be important for coordinating activities of multiple signal-transduction pathways. Nevertheless, the expression of the tumor-suppressor gene NF1 and its regulation of the Rut-AC signal-transduction pathway are critical to the biochemical processes underlying olfactory learning in Drosophila. Similar mechanisms are to be expected in vertebrates (Guo, 2000).

The receptor tyrosine kinase Alk controls neurofibromin functions in Drosophila growth and learning

Anaplastic Lymphoma Kinase (Alk) is a Receptor Tyrosine Kinase (RTK) activated in several cancers, but with largely unknown physiological functions. This study reports two unexpected roles for the Drosophila ortholog dAlk, in body size determination and associative learning. Remarkably, reducing neuronal dAlk activity increased body size and enhanced associative learning, suggesting that its activation is inhibitory in both processes. Consistently, dAlk activation reduced body size and caused learning deficits resembling phenotypes of null mutations in dNf1, the Ras GTPase Activating Protein-encoding conserved ortholog of the Neurofibromatosis type 1 (NF1) disease gene. dAlk and dNf1 co-localize extensively and interact functionally in the nervous system. Importantly, genetic or pharmacological inhibition of dAlk rescued the reduced body size, adult learning deficits, and Extracellular-Regulated-Kinase (ERK) overactivation dNf1 mutant phenotypes. These results identify dAlk as an upstream activator of dNf1-regulated Ras signaling responsible for several dNf1 defects, and they implicate human Alk as a potential therapeutic target in NF1 (Gouzi, 2011).

The prominent expression of Alk in the mammalian and Drosophila CNS and presence of the dAlk ligand Jeb in the embryonic fly CNS, provided the first indication that Alk and Jeb likely participate in the development of the nervous system. Subsequent in vitro studies demonstrated that Alk promoted neuronal differentiation of PC12 or neuroblastoma cell lines, and work in C. elegans implicated its Alk ortholog, scd-2, in the inhibition of presynaptic neuronal differentiation in vivo. Several functions have also been attributed to Drosophila dAlk, including roles in the specification of the intestinal musculature, in retinal axon targeting, and in signaling at the larval neuromuscular junction. The current results establish two novel in vivo functions for the Drosophila dAlk/Jeb receptor/ligand pair, in the regulation of organismal growth and associative learning (Gouzi, 2011).

The results presented in this study lead to a hypothesis that dAlk and dNf1 have opposite roles in controlling neuronal ERK activity during larval development, and therefore determine overall organismal size in a non-cell autonomous manner. In support of this hypothesis, dAlk and dNf1 co-localize extensively in larval neurons, both proteins control ERK activity, and both modulate growth by regulating cell size. In agreement with this conclusion, transgenic neuronal expression of the constitutively active ERK, Rl^SEM, is sufficient to reduce Drosophila size (Gouzi, 2011).

dAlk is the second active RTK implicated in Drosophila growth control. Previous work demonstrated that the fly homolog of the insulin/IGF1 receptor dInr, regulates both body and organ size. In peripheral tissues, dInr is activated by a family of Insulin-like proteins (dILPs), leading to the activation of the IRS (Chico), PI(3)K (Dp110), PTEN (dPTEN), and Akt/PKB, (dAkt1/dPKB), signaling pathway. Ablating the Insulin Producing Cells (IPCs), or silencing the function of dInr pathway components in the larval CNS resulted in severe growth defects. Notwithstanding the similar growth phenotypes, several lines of evidence argue that dAlk and dInr control growth in fundamentally different ways. Most importantly, organismal growth is impaired when dInr activity or signaling is reduced, whereas a similar phenotype is observed upon dAlk activation. Secondly, dAlk affects organism growth by specifically regulating cell size in a non cell-autonomous manner. In contrast, dInr signaling affects both cell size and number cell-autonomously and non-autonomously. Finally, expression of Jeb or dAlk transgenes in neuroendocrine IPCs using the dILP2-Gal4 driver did not modify pupal size. Thus, although both dAlk and dInr RTKs are involved in body size determination, their mechanisms and sites of action are distinct. This interpretation is consistent with results with the C. elegans Alk homolog Scd-2 shown to function in parallel with or converge with TGF-β signaling, but act independently of the Insulin cascade in dauer determination (Reiner, 2008). However, given that dInr and dAlk are members of the same subfamily of RTKs, a potential explanation for the lack of rescue of dNf1 mutant homozygous larvae with systemic administration of 100 µM of the selective inhibitor TAE684, may be off-target inhibition of dInr at the higher drug concentration (Gouzi, 2011).

Interestingly, S6K (dS6K) resides on a downstream branch of the dInr/PI(3)K signaling pathway and regulates cell size without impinging on cell number. Although the dS6K loss-of-function phenotype resembles the dAlk gain-of-function and dNf1 loss-of-function phenotypes, its mode of action is cell-autonomous. However, it is still tempting to speculate that dAlk and dNf1 ultimately affect neuroendocrine signals that affect dS6K activity in peripheral tissues (Gouzi, 2011).

Increasing signaling through the cyclic AMP (cAMP)-dependent protein kinase A (PKA) pathway has been reported to suppress the size defect of dNf1 mutants. This among other findings, have led some to propose that dNf1 regulates Ras activity and cAMP levels independently. In contrast, an investigation of the cAMP/PKA sensitive dNf1 mutant growth defect argued that aberrantly upregulated Ras/ERK signaling in Ras2-expressing larval neurons was its proximal cause. The current results further support the latter explanation implicating a Ras/ERK signaling defect downstream of dAlk as the cause of size defects in dNf1 mutants. Then, how could elevated cAMP/PKA signaling rescue decreased body size? Because neuroendocrine signals can activate the cAMP pathway, it is possible that defective dAlk/Ras/ERK signaling in dNf1 mutants may lead to a neuroendocrine deficiency, which is restored by increasing cAMP/PKA signals (Gouzi, 2011).

In Drosophila, the dAlk/Jeb receptor-ligand pair has been shown to act in an antrerograde signaling pathway essential for assembly of the neuronal circuitry of the fly visual system. However, loss of either Alk or Jeb did not appear to impair assembly of functional synapses with normal postsynaptic responses at the larval neuromuscular junction, indicating that they do not participate in CNS development. In agreement, pan-neuronal, spatially restricted attenuation or unregulated activation of Alk throughout development did not appear to yield gross structural defects in the adult brain or alter naïve behavioral responses to the stimuli used for conditioning. Hence, it is unlikely that the learning phenotypes described in this study are the result of developmental alterations in the CNS. In fact, dAlk seems to be acutely required for normal learning as limiting modulation of its activity to the adult CNS results in phenotypes on its own and also modifies the learning deficits of dNf1 mutants. Moreover, the function of dAlk and dNf1 in associative learning is independent of body size as the learning reverted to normal by dAlk abrogation in the small-sized dNf1 mutant homozygotes. Interestingly, the C. elegans Jeb homolog Hen-1 is required non-cell autonomously in the mature nervous system for sensory integration and associative learning (Ishihara, 2002). Collectively then, these studies in C. elegans, mice and the current data strongly support an evolutionary conserved role for Alk signaling in adult associative learning and memory (Gouzi, 2011).

Elevated dAlk/Jeb signaling outside the MBs impaired olfactory learning, while its abrogation increased learning efficiency. These are results are consistent with the enhanced performance in a hippocampus-dependent task described for Alk knockout mice (Bilsland, 2008). It is proposed then, that Alk signaling normally functions to limit the strength of the CS/US associations, in effect providing a putative threshold required to be overcome for specific and efficient association of the stimuli. A GABAergic neuron outside the MBs, the Anterior Paired Lateral (APL), was recently reported to similarly suppress olfactory learning and its silencing yielded enhanced performance. Interestingly, a decrease in presynaptic GABA release or abrogation of the GABA_A receptor, RDL in the post-synaptic mushroom body neurons resulted in enhanced learning. Whether Ras2-Gal4 is expressed in the APL neuron and dAlk also functions in this neuron to suppress learning are questions currently under investigation (Gouzi, 2011).

Interestingly, a recent study suggested that the learning defects of Nf1^+/− mice are attributed to increased ERK-mediated phosphorylation of synapsin I in hippocampal inhibitory neurons and concomitant increase in GABA release. In accord, a GABA_A receptor antagonist enhanced learning in Nf1^+/− mice and controls, and reversed LTP defects in the mutants. Similarly, elimination of the dAlk-mediating inhibition in Drosophila Ras2-expressing neurons enhanced learning, potentially via attenuation of ERK phosphorylation. In support of this notion, this study shows that constitutive activation of ERK in adult Ras2-expressing neurons precipitates learning deficits. Collectively, these results together with the reported learning deficits of Drosophila synapsin mutants, suggest that a mechanism similar to that proposed for vertebrates may also regulate Nf1-dependent learning in flies (Gouzi, 2011).

In mice, a decrease in Nf1 levels in heterozygous mutants increased Ras/ERK signaling and precipitated Long-Term Potentiation (LTP) and spatial learning deficits. These deficits were reversed upon genetic or pharmacological inhibition of Ras signaling. The current results demonstrate that dAlk inhibition reversed the impaired learning of dNf1 mutants and since this is the first 'kinase-active' RTK shown to be involved in this process in flies, it provides independent support for Ras/ERK hyperactivation as causal of these learning defects. Then, how can the reported phenotypic reversal of Nf1 learning deficits by expressing the PKA catalytic subunit throughout the fly be explained? It is hypothesized that the MBs are functionally downstream of the dAlk/dNf1 neurons and elevated PKA activity within the former could result in normal learning. Future work will focus on addressing the merits of these hypotheses regarding the mechanisms underlying the size and learning defects of dNf1 mutants (Gouzi, 2011).

A recent report Buchanan (2010) suggested that dNf1 mRNA is found in the mushroom bodies and in agreement, immunohistochemical results of the current study demonstrate that dNf1 is present within the mushroom body calyces. Protein synthesis-dependent memory defects in Nf1 mutants were rescued upon MB-limited expression of the same full-length transgene as was used in this study. Since memory deficits were not examined in the current work, this complements the current data and suggests a function for dNf1 within the MBs. In contrast, the current data indicate that dNf1 expression in the MBs is not sufficient for learning/3 min memory. Three common MB drivers including the most specific MB247 and the most broadly expressed OK107-Gal4, did not rescue learning in Nf1 mutants by expressing dNf1. It is suggested therefore that rescue described by Buchanan and Davis was mediated largely by c739-Gal4 transgene expression in neurons extrinsic to MBs where Elav, Ras2 and Alk(38)-Gal4 are expressed, perhaps in combination with expression within MB-intrinsic neurons. The neuronal circuits where dNf1 and dAlk are required for normal learning are the subject of ongoing investigations (Gouzi, 2011).

This study identifies dAlk as the first RTK to functionally interact with Nf1 in Drosophila, raising the important question whether a similar functional relationship exists in mammals. Suggestive evidence argues that this may indeed be the case. Thus, Alk and NF1 extensively colocalize in the mammalian CNS during the same developmental periods. Additionally, excess Alk expression or activation has been reported in astrocytomas, gliomas, neuroblastomas and pheochromocytomas, in which loss of NF1 expression has also been found. Based on identification of Alk as a bona-fide RTK that initiates a Ras/ERK cascade regulated by Nf1, this suggests that Alk inhibition may rescue not only the phenotypes reported in this study, but also other symptoms that have been previously associated with Nf1 loss and ERK over-activation. It was recently reported that knockdown of NF1 expression renders a neuroblastoma cell line resistant to retinoic acid-induced differentiation, and that NF1 deficient neuroblastoma tumors have a poor outcome. The current results suggest that Alk inhibition may provide an intervention strategy in such cases. Finally, the findings reported in this study, combined with the lack of overt abnormalities in Alk knock-out mice, provide a rationale for further explorations of Alk as a potential therapeutic target in NF1 (Gouzi, 2011).

GENE STRUCTURE

The Nf1 gene is localized to a 30 kb DNA segment between the bride of sevenless gene and the Enhancer of split complex (The, 1997).

Exons - 17 constitutive and 2 alternatively spliced

PROTEIN STRUCTURE

Amino Acids - 2802

Structural Domains

The sequence similarity between Drosophila Nf1 and mammalian NF1 is observed over the entire length of the proteins, including regions outside of the catalytic GAP-related domain (GRD) or the more extensive segment related to yeast inhibitor of RAS activity (IRA) protein. Sequence conservation within the GRD is 69%, while conservation of both the N- and C-terminal to the GRD is about 50% (The, 1997).

date revised: 23 June 2023

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