Neurofibromin 1: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation | References

Gene name - Neurofibromin 1

Synonyms -

Cytological map position - 96F11--96F11

Function - Neurofibromin\like

Keywords - PKA pathway

Symbol - Nf1

FlyBase ID:FBgn0015269

Genetic map position -

Classification - GAP-related domain

Cellular location -



NCBI links: Entrez Gene
BIOLOGICAL OVERVIEW

Recent literature
King, L. B., Koch, M., Murphy, K., Velazquez, Y., Ja, W. W. and Tomchik, S. M. (2016). Neurofibromin loss of function drives excessive grooming in Drosophila. G3 (Bethesda) [Epub ahead of print] PubMed ID: 26896440
Summary:
Neurofibromatosis I is a common genetic disorder that results in tumor formation and predisposes individuals to a range of cognitive/behavioral symptoms, including deficits in attention, visuospatial skills, learning, language development, sleep, and autism spectrum disorder-like traits. The nf1-encoded neurofibromin protein (Nf1) exhibits high conservation, from the common fruit fly, Drosophila melanogaster, to humans. Drosophila provide a powerful platform to investigate the signaling cascades upstream and downstream of Nf1, and the fly model exhibits similar behavioral phenotypes to mammalian models. In order to understand how loss of Nf1 affects motor behavior in flies, traditional activity monitoring was combined with video analysis of grooming behavior. In nf1 mutants, spontaneous grooming was increased up to 7x. This increase in activity was distinct from previously-described dopamine-dependent hyperactivity, as dopamine transporter mutants exhibited slightly decreased grooming. Finally, relative grooming frequencies can be compared in standard activity monitors that measure infrared beam breaks, enabling the use of activity monitors as an automated method to screen for grooming phenotypes. Overall, these data suggest that loss of nf1 produces excessive activity that is manifested as increased grooming, providing a platform to dissect the molecular genetics of neurofibromin signaling across neuronal circuits.

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, NF1P1 and NF1P2, 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/+; NF1P2) 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 NF1P1 when the flies are raised at room temperature. NF1P2 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 NF1P1 and NF1P2 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).


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).

cDNA clone length - 9750

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).


Neurofibromin 1: Evolutionary Homologs | Developmental Biology | Effects of Mutation | References

date revised: 22 July 2000

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