Selective disactivation of neurofibromin GAP activity in neurofibromatosis type 1

doi:10.1093/hmg/7.8.1261

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Human Molecular Genetics Pages 1261-1268

Selective disactivation of neurofibromin GAP activity in neurofibromatosis type 1 (NF1)
Introduction
Results And Discussion
   Identification of R1276P as the disease-causing mutation in a family with typical NF1 features
   Mutation R1276P affects GAP activity but not protein structure
   Evaluation of GRD missense mutations based on the crystal structure
   Complete loss of function by mutation R1276P
   Further support for the upstream model
Materials And Methods
   DNA analysis
   mRNA analysis
   Neurofibromin quantification
   Recombinant proteins
   Fluorescence measurements
Acknowledgements
References

Selective disactivation of neurofibromin GAP activity in neurofibromatosis type 1 (NF1)

Anja Klose^1,+, M. Reza Ahmadian^2,+, Markus Schuelke^3,+, Klaus Scheffzek², Sven Hoffmeyer⁴, Andreas Gewies¹, Frank Schmitz², Dieter Kaufmann⁴, Hartmut Peters¹, Alfred Wittinghofer², Peter Nürnberg^1,*

¹Institut für Medizinische Genetik, Universitätsklinikum Charité-Medizinische Fakultät der Humboldt-Universität zu Berlin, Campus Charité Mitte, D-10098 Berlin, Germany, ²Max-Planck-Institut für Molekulare Physiologie, Rheinlanddamm 201, D-44139 Dortmund, Germany, ³Kinderklinik, Universitätsklinikum Charité-Medizinische Fakultät der Humboldt-Universität zu Berlin, Campus Virchow-Klinikum, Augustenburger Platz 1, D-13353 Berlin, Germany and ⁴Abteilung Humangenetik, Universität Ulm, Albert-Einstein-Allee 11, D-89070 Ulm, Germany

Received March 18, 1998; Revised and Accepted May 18, 1998

Neurofibromatosis type 1 (NF1) is a common familial tumour syndrome with multiple clinical features such as neurofibromas, café-au-lait spots (CLS), iris Lisch nodules, axillary freckling, optic glioma, specific bone lesions and an increased risk of malignant tumours. It is caused by a wide spectrum of mutations affecting the NF1 gene. Most mutations result in the loss of one allele at the DNA, mRNA or protein level and thus in the loss of any function of the gene product neurofibromin. The idea of the simultaneous loss of several different neurofibromin functions has been postulated to explain the pleiotropic effects of its loss. However, we have identified a novel missense mutation in a family with a classical multi-symptomatic NF1 phenotype, including a malignant schwannoma, that specifically abolishes the Ras-GTPase-activating function of neurofibromin. In this family, Arg1276 had mutated into proline. Based on complex biochemical studies as well as the analysis of the crystal structure of the GTPase-activating protein (GAP) domain of p120GAP in the presence of Ras, we unequivocally identified this amino acid as the arginine finger of the neurofibromin GAP-related domain (GRD)-the most essential catalytic element for RasGAP activity. Here, we present data demonstrating that the mutation R1276P, unlike previously reported missense mutations of the GRD region, does not impair the secondary and tertiary protein structure. It neither reduces the level of cellular neurofibromin nor influences its binding to Ras substantially, but it does completely disable GAP activity. Our findings provide direct evidence that failure of neurofibromin GAP activity is the critical element of NF1 pathogenesis. Thus, therapeutic approaches aimed at the reduction of Ras·GTP levels in neural crest-derived cells can be expected to relieve most of the NF1 symptoms.

INTRODUCTION

Neurofibromatosis type 1 (NF1) is one of the most common autosomal dominant disorders in man, with an incidence of ~1/3500. It is a multisystemic disease, with the nervous system and the skin being the tissues primarily affected. Neurofibromas constitute an important diagnostic criterion of this complex genetic disorder. However, café-au-lait spots (CLS), axillary freckling, optic glioma, Lisch nodules of the iris and specific bone lesions are also common clinical features. Furthermore, ~5% of NF1 patients develop malignant tumours (1). NF1, or von Recklinghausen disease, is caused by inherited or de novo mutations of the NF1 gene, which has been identified by positional cloning on chromosome 17q11.2 (2). Most mutations identified in the large 60 exon NF1 gene are truncating mutations with dramatically reduced levels or total absense of the mutant transcript and/or protein. However, missense mutations of unsettled pathological significance have also been reported (3,4). Alternative splicing gives rise to several different transcripts of the NF1 gene (5). The major species is an 11-13 kb ubiquitously expressed transcript that encodes a protein called neurofibromin (2). The only region of the neurofibromin molecule to which a biological function has been clearly ascribed is a central domain (residues D1248-F1477) which possesses a high degree of similarity to Ras-specific GTPase-activating proteins (GAPs) (6), and is therefore called the GAP-related domain (GRD). The GRD stimulates the slow intrinsic GTPase activity of Ras up to 10⁵-fold (2). The existence of additional functional domains is a matter of ongoing speculation (1,7-9). It has been thought that the loss of the different functions of the neurofibromin molecule might explain the many and varied clinical symptoms.

Recent progress in analysing the crystal structure of the GAP domain of p120GAP in the absence (10) and in the presence (11) of Ras as well as new biochemical data about the catalytic process of the GTPase reaction (12,13) offer new insights into the role of individual amino acids in the GRD and the consequences of their substitution. With this in mind, we screened a panel of NF1 patients for mutations in the GRD and identified an arginine to proline mutation of the most essential catalytic element for RasGAP activity, the GRD arginine finger, in a family with a classical NF1 phenotype, one of whose members had a malignant schwannoma. The biochemical and structural characterization of this functionally most interesting mutation against the background of the clinically well studied family with several affecteds allows us to infer that failure of neurofibromin GAP activity is the critical element of NF1 pathogenesis. Therefore, elevated Ras·GTP levels in neural crest-derived cells of NF1 patients might be a reasonable target for therapeutic approaches.

RESULTS AND DISCUSSION

Identification of R1276P as the disease-causing mutation in a family with typical NF1 features

A total of 170 unrelated patients diagnosed according to the NF1 diagnostic criteria (19) were screened for sequence alterations in various exons of the NF1 gene by temperature gradient gel electrophoresis (TGGE) of PCR-amplified genomic DNA fragments (14,15) using intron-based primers (20). DNA samples with abnormal TGGE band patterns were subjected to sequence analysis. Using this approach, we identified a female patient (B362) with a sequence abnormality in exon 22 which she shared with all affected family members but not with her healthy relatives (Fig. 1a). Sequencing of exon 22 revealed a G->C transversion at cDNA nucleotide 3827 which results in a substitution of proline for arginine at amino acid 1276 of neurofibromin (Fig. 1b). In addition to exon 22, ~40 further exons, including exon 5 (Fig. 1a), were analysed by TGGE, and no other mutation was found in the patient's DNA. In order to exclude at least the frequent truncating mutations in the remaining exons, we also applied the protein truncation test (PTT) to the patient's mRNA (16). In the PTT, not only nonsense and frameshift mutations are detected but also aberrant splice products. Neither a premature termination codon nor a splicing anomaly were observed. This also excludes the possibility of the missense mutation R1276P inducing skip of exon 22 as recently reported for a silent mutation in exon 51 of the fibrillin-1 gene (21). As in our previous studies (22), we also looked for the expression of the two NF1 alleles. Mutant and wild-type messengers were distinguished by the polymorphic RsaI restriction site in exon 5 of the NF1 gene (cf. Fig. 1a and ref. 23). Both alleles were found to be expressed equally (Fig. 1c). Western blot analysis of immunoprecipitated neurofibromin also revealed unreduced levels of gene product (Fig. 1d). Thus, the mutant and wild-type neurofibromin molecules are co-existing in the patients' cells at a similar level.

Figure 1. Identification of NF1 mutation R1276P and study of mutant allele expression. (a) Pedigree analysis of sequence variations in exons 22 and 5 by TGGE. The altered nucleotide (nt) positions are given below each TGGE panel. Underlined nucleotides constitute a haplotype. The grey arrowheads indicate the mutant nucleotide. The black arrowhead represents the index patient (B362). (b) Sequence analysis of genomic DNA of patient B362. The arrowheads indicate the mutant nucleotide and amino acid. (c) RT-PCR analysis for the determination of allele-specific NF1 gene expression levels. PCR products including exon 5 were derived from cDNA of the two older sons of patient B362 (lanes 2 and 3). RsaI-digested PCR products were separated on an agarose gel and stained by ethidium bromide. The densitometrically determined allele ratios A/G were 1.2 and 1.38, i.e. within normal range (22). A 100 bp ladder was used as a size marker (lane 1). (d) Determination of neurofibromin content by Western blot analysis. Normal neurofibromin levels were found in the lymphocytes of the two older sons of patient B362 (lanes 1 and 2). Lanes 3 and 4 represent the reduced neurofibromin levels of two NF1 patients with truncating mutations [patients 0550 (15) and 0545 (14), respectively]. Lymphocytes of healthy donors were used as controls (lanes 5 and 6).

The female patient B362 was born as the first child of unaffected, non-consanguineous parents. Two younger sisters do not show any NF1-related symptoms (Fig. 1a). The patient developed multiple CLS within the first year of life. Her language and motor development were mildly retarded, and she complained of incoordination throughout her life. Around puberty, multiple cutaneous neurofibromas developed which worsened at the time of each of her three pregnancies. At the age of 31 years, a routine magnetic resonance imaging (MRI) scan of the brain revealed multiple areas of increased T₂ signal intensity in the midbrain (24) and a small optic glioma. Because of recurrent paraesthesias in her left leg, an MRI scan of the spine was done 2 years later which revealed multiple schwannomas within the vertebral foramina. The largest tumour in the lumbar region, with a volume of ~8 ml, was surgically removed. Histologically there was no evidence of malignancy at that time. Eight months later, the patient suffered a relapse with rapid tumour growth. At the time of re-operation, the retroperitoneal tumour had reached a volume of 800 ml and showed numerous necrotic and anaplastic areas with a proliferation rate up to 60%. Radiotherapy was tried without success and the patient died of widespread metastatic disease at the age of 34 years. Her three male children (age 4, 8 and 12 years at the time of examination) all fulfil the NF1 diagnostic criteria (19). The two elder brothers are macrocephalic. Language and motor development of all children were retarded to a similiar extent and on the same time scale as in their mother. The childrens' IQ, measured with the Kaufmann Assessment Battery for Children (KAB/C) (25), ranged from 80 to 89. This is in good agreement with the average IQ of 85 found in 76 NF1 children evaluated with the KAB/C (M. Schuelke, unpublished results). A cranial MRI scan in the two elder brothers revealed similiar increased T₂ signal intensities as in their mother.

Mutation R1276P affects GAP activity but not protein structure

Missense mutations are rare in the NF1 gene and only a few within the catalytic domain of neurofibromin have been tested functionally so far (4,26). R1276P is a novel mutation which we are able to evaluate based on our recent data about the structure-function relationship relevant for GAP-mediated GTPase reaction of Ras. Solving by X-ray crystallography the structure of the Ras·GDP·AlF₃·RasGAP complex, which is believed to mimic the transition state of the GTPase reaction, conclusively revealed that R789 from p120GAP (corresponding to R1276 in neurofibromin) participates in the catalytic process by stabilizing the transition state via its positively charged side chain (11). Final proof that indeed R789, and not the other invariant R903 (R1391 in neurofibromin), is the long sought `arginine finger' came from biochemical analyses of wild-type and mutant NF1-333 and GAP-334, the relevant catalytic domains of neurofibromin and p120GAP (13). It could be shown that R1276 of neurofibromin is crucial for catalysis and cannot be substituted by other amino acids such as A, K, Q or N. As studied here, substitution by P slightly reduces the binding affinity for Ras (6.6-fold), but compromises GAP-stimulated GTP hydrolysis 8000-fold (Fig. 2a), more severely than any other substitution investigated (13). This supports earlier findings that the arginine finger of RasGAP is involved in the chemical cleavage of [gamma]-phosphate rather than in the binding of substrate (13,27). Taking advantage of our observations that aluminium fluoride binds to Ras·GDP only in the presence of stoichiometric amounts of GAP, thereby mimicking the transition state of the GTPase reaction (12), we show in Figure 2b that R1276P is unable to stabilize aluminium fluoride binding, as has been demonstrated earlier for other mutations of R1276 (12,13). In order to assess the influence of the R1276P mutation on the three-dimensional structure, circular dichroism (CD) (Fig. 2c) and fluorescence spectra (Fig. 2d) of wild-type and mutant NF1-333 were compared. No significant changes were observed, indicating that the substitution of proline for the arginine finger does not impair the secondary and tertiary protein structure.

Figure 2. Biochemical characterization of mutation R1276P. (a) Rate constants for the reaction of Ras·mGTP with wild-type and mutant (R1276P) NF1-333. The intrinsic GTPase activity of Ras is 0.00047/s at 37°C. K_D values have been calculated from the association rate constants of Ras·mant-GTP and the dissociation rate constants of Ras·mant-GppNHp (K_D= k_off/k_on), assuming that the dissociation rate constants are similar for Ras·mant-GTP and Ras·mant-GppNHp. (b) Analysis of aluminium fluoride binding to Ras·mant-GDP in the presence of NF1-333 proteins. Fluorescence emission spectra of Ras·mant-GDP with wild-type NF1-333 and mutant NF1-333 (R1276P) in the presence or absence of aluminium fluoride are shown. Red, 0.1 µM Ras·mant-GDP; green, after addition of 1 µM wild-type NF1-333 or 5 µM R1276P-NF1-333, respectively; blue, after addition of 30 µM AlCl₃and 2 mM NaF or 150 µM AlCl₃ and 10 mM NaF for wild-type NF1-333 or R1276P-NF1-333, respectively. (c) CD spectra of the wild-type NF1-333 (blue) and R1276P-NF1-333 (red). The spectra were measured in 10 mM potassium phosphate buffer, pH 7.0, at 25°C at a protein concentration of 200 µM for wild-type NF1-333 and 220 µM for R1276P-NF1-333. (d) Fluorescence emission spectra of wild-type NF1-333 (blue) and R1276P-NF1-333 (red). The spectra were recorded at a protein concentration of 1 µM.

Evaluation of GRD missense mutations based on the crystal structure

We have modelled the effect of the R1276P and of other missense mutations (Fig. 3a) using the structure of the homologous catalytic domain of p120GAP (GAP-334) complexed with Ras (11). Assuming slight rearrangement of the finger loop that contains the catalytic arginine, proline could be accommodated in position 789 of GAP-334 (Fig. 3b), explaining why binding is not severely impaired by the mutation. Using the structure, we have also analysed the possible effects of other missense mutations of the catalytic domain found in NF1 patients, such as R1391S (4) (R903 in p120GAP), K1419R (28) (I931), K1419Q (4) (I931), K1423E (4,26) (K935), L1425P (H. Peters, unpublished data) (V937) and S1468G (4) (L980) (Fig. 3b).

Figure 3. Ras-RasGAP structural elements. (a) RasGAP sequence alignment of human neurofibromin (GenBank accession no. 89914), yeast IRA2 (M33779) and human p120GAP (M23379). Boxes indicate the similarity blocks. Below the alignment, the secondary structure elements of GAP-334 have been assigned in red and green. Conserved residues are highlighted in blue. Selected residues that have been found mutated in neurofibromin from patients with NF1 are marked by arrows. (b) Ras-RasGAP complex. Catalytic domains of GAP-334 and Ras are shown in red and yellow, respectively. The finger loop of GAP-334 is shown in green. For GDP and AlF₃, ball-and-stick models are used. Marked amino acid positions have been subject to missense mutations of neurofibromin in NF1 patients or represent oncogenic mutation sites of Ras as found in tumours. The wild-type p120GAP sequence is shown, with the exception of R789 (grey) for which proline (yellow) has been substituted.

K1423E and R1391S affect two invariant amino acid positions of particular structural importance. R1391 is part of the most highly conserved fingerprint FLR...PA...P-motif diagnostic for RasGAPs. In the structure of GAP-334, the corresponding R903 stabilizes the orientation of the arginine finger without directly contacting the active site of Ras (10,11). Introduction of serine into position 903 (1391 in neurofibromin) would abrogate a number of interactions important for stabilization of the loop carrying the arginine finger, and thereby accounts for the significantly reduced GAP activity (4). K935 (K1423 in neurofibromin) forms an intramolecular salt bridge with E950 (E1437 in neurofibromin) of the GAP KE-motif in the variable loop, the lysine residue of which is an important element of the Ras-RasGAP interaction. Replacement of K935 by glutamate would disrupt the salt bridge and would introduce additional negative charges which may lead to unfavourable interactions.

The effect of the NF1 patient mutations K1419R, K1419Q, L1425P and S1468G are more difficult to predict. Mutation of I931 (K1419 in neurofibromin) to arginine or glutamine might lead to a polar interaction of this residue with E37 of Ras (as judged from the complex structure), a proline replacing V937 (L1425) might destabilize helix [alpha]7c in which it occurs (Fig. 3a). In the GAP-334 structure, the mutation corresponding to S1468G would change a very C-terminal residue of the central domain GAPc, L980, into glycine. From the structure, it is difficult to estimate the effect on the function of the protein.

Complete loss of function by mutation R1276P

Among all patients with missense mutations in the GRD reported to date, B362 is the first who presented with a malignant schwannoma. A reason for this might be that the inactivation of the GAP activity by the R1276P mutation is de facto complete. Previously, effects on function were only tested for K1423E and R1391S. Conflicting evidence has been reported for the effect of the K1423E mutation. Whereas Li et al. (26) found the binding to be unaffected and the k_cat to be decreased 200-fold, Poullet et al. (29) found the affinity to be decreased >100-fold. For the R1391S mutation, a 300-fold decrease of GAP activity was reported (4). In comparison with the 8000-fold reduction which we found for R1276P, both cases still show a remarkable residual activity that may be sufficient under in vivo conditions to suppress tumour formation if the wild-type allele becomes inactivated somatically. Indeed, for all patients with any of the missense mutations listed above, the occurrence of a malignant tumour as part of the NF1 phenotype was definitely excluded if clinical data were reported for these cases (n [ge] 14). Possibly due to their young age, a malignancy has also not been detected in any of the three affected sons of patient B362 up to the present moment. The same reason may account for the lack of neurofibromas in her children who were all prepubertal when examined. We are aware, however, of the high degree of variable expression even within families. Hence, the actual risk of the boys developing such a severe NF1 phenotype with malignancies as did their mother is not expected to be higher than for any other NF1 patient with a truncating NF1 gene mutation. A classical complex NF1 phenotype is present in this family. All or most affected family members showed CLS, freckling and scoliosis, while a learning disability and minor mental retardation, typical features of NF1 children, were observed in all affected children of this family.

Further support for the upstream model

Two pathogenic models involving neurofibromin and Ras have been proposed to lead to the various clinical symptoms of NF1 (1,2,30). In the upstream negative Ras regulatory model of neurofibromin function, decreased neurofibromin and hence decreased RasGAP activity are predicted to lead to higher levels of activated Ras·GTP and therefore to increased cell proliferation and tumour formation. In the downstream model, neurofibromin acts as an effector of Ras·GTP in signaling pathways controlling cell proliferation or differentiation. It is tempting to explain the complex and variable picture of NF1 pathology and the pleiotropic effects of neurofibromin action by different functions attributable to the same or different domains of the large molecule. However, our finding of selective neurofibromin GAP inactivation in NF1 patients is in line with accumulating evidence about neurofibromin acting first of all as a control element for Ras·GTP levels in certain cell types such as neural crest-derived cells. Obviously, these data define the direction of the future treatment of NF1. The GAP activity has to be restored in all cells where neurofibromin is the only or the major active GAP. We have shown that Ras is an inefficient enzyme that needs to be complemented by Ras-specific GAPs to form the actual catalytic centre for efficient GTP hydrolysis on Ras (11-13). It is conceivable that ingeniously designed small molecules are able to mimic the GAP action and, if properly applied, may serve as anti-tumour and anti-NF1 drugs.

Support for the upstream model came from studies of neurofibrosarcoma cell lines derived from patients with NF1. These cells showed elevated levels of Ras·GTP and decreased biochemical GAP activity (31,32). Recent progress in elucidating the role of neurofibromin in juvenile chronic myelogeneous leukaemia (33,34) also provides convincing evidence for a selective decrease in NF1-like RasGAP activity and elevated Ras·GTP levels in primary leukaemic cells from children with NF1. Furthermore, application of a new tissue-based assay revealed a substantial increase of Ras·GTP in neurogenic sarcomas (15 times) and benign neurofibromas (four times) from NF1 patients compared with schwannomas from non-NF1 patients (35). Other findings have been interpreted in favour of the downstream model or a Ras-independent mechanism of neurofibromin function that may apply only for specific cell types (7,8). However, alternative interpretations of these data commensurate with the upstream model are conceivable. For instance, increased Ras·GTP levels were not observed in serum-stimulated neurofibromin-deficient neuroblastomas and melanomas (7), but Ras·GTP levels were only measured 10 h after serum stimulation. By this time, any difference between wild-type and neurofibromin-deficient cells may have disappeared, as suggested by experiments with growth factor-stimulated myeloid cell lines (33). Alternatively, Ras deregulation may be provoked only by stimulation with specific cytokines as also shown for myeloid cell lines (34). Interestingly, different ligands can activate the Ras pathway in the same cell while transmitting quite different signals. This has been observed in the neural crest-derived PC12 pheochromocytoma cell line (36). In PC12 cells, both epidermal growth factor (EGF) and nerve growth factor (NGF) activate the Ras pathway, yet EGF is a mitogen while NGF induces differentiation. Biochemical characterization revealed that EGF stimulation results in transient activation of the Ras pathway whereas NGF causes sustained activation. Hence, in most tissues, neurofibromin defects might cause only moderate and transient Ras·GTP level increases in response to activation of a particular as yet unidentified receptor of neural crest cells. This selective hypersensitivity to a specific growth factor may stimulate lineage-specific growth. Such a scenario is consistent with recent data demonstrating neurotrophin-independent growth and enhanced sensitivity to NGF of fetal neurons from Nf1^-/- mice (37). It also offers some plausible explanations for putative functions that the non-GRD regions of the large neurofibromin molecule might fulfil in direct conjunction with the down-regulation of Ras·GTP by its central GAP activity.

In summary, the NF1 mutation R1276P is unique for its selective and complete inactivation of the neurofibromin GAP activity without impairing any other possible function of neurofibromin. Despite this, most of the typical NF1 features were expressed in an age-dependent manner in the present family. This suggests that all of the pathologically relevant cellular processes depend exclusively on the GAP activity of the neurofibromin molecule. There is no evidence for a significant difference in phenotype caused by a 50% reduction in the level of neurofibromin, as is found in most of the other NF1 cases, or by the missense mutation R1276P described here. The latter, however, generates a faulty protein which is disabled only for one specific function: its GAP activity. Thus, a failure in reduction of Ras·GTP levels is to be considered the main pathological problem in NF1 as proposed by the upstream model of neurofibromin function. This finding has major implications for future therapeutic approaches in NF1.

MATERIALS AND METHODS

DNA analysis

Genomic DNA was prepared from peripheral blood according to standard procedures. Individual exons were amplified with primers annealing to the flanking intronic sequences, and PCR products were subjected to TGGE as described elsewhere (14,15). Silver staining was used for visualization of bands. Prior to sequencing, PCR products were isolated from agarose gels (NEEO Roth) and extracted following the manufacturer's instructions (QIAquick Gel extraction Kit; Qiagen). The purified PCR fragments were sequenced by the dideoxy chain termination method (Thermo Sequenase cycle sequencing kit; Amersham) using 5[prime]-terminal ³³P-labelled sequencing primers.

mRNA analysis

Total cellular RNA was obtained from cultured peripheral lymphocytes using the RNeasy total RNA purification kit (Qiagen). cDNA synthesis subsequently was performed using the SuperScript pre-amplification system (Gibco BRL). RT-PCR reactions to amplify five overlapping segments covering the entire coding region and in vitro transcription/translation reactions for the protein truncation test (PTT) were carried out as described previously (16). Allele-specific expression level studies were performed by RT-PCR reactions using the following primer pair to amplify exon 5: 5[prime]-TAGTCGCATTTCTACCAGGTTA-3[prime] (sense primer) and 5[prime]-GAGAATGGCTTACTTGGATTAAA-3[prime] (antisense primer). Thirty PCR cycles of 1 min at 92°C, 1 min at 54°C and 1 min at 72°C were employed. After RsaI restriction, the fragments were analysed by agarose gel electrophoresis and ethidium bromide staining. Subsequently, the band intensities were measured densitometrically using a Phosphoimager (Molecular Dynamics).

Neurofibromin quantification

Immunoprecipitations from equal amounts of total protein (300-700 µg) obtained from cultured peripheral lymphocytes were carried out as described (17). The resulting protein bands were evaluated quantitatively by using a video-densiometric system. The suitability of the method for quantitative measurements was proven by preparing standard curves with various amounts of protein. A linear response was obtained between 50 and 800 µg of protein (correlation coefficient [ge] 0.98). A 20-30% difference between neurofibromin contents of different lysates can be ascertained by this method.

Recombinant proteins

The NF1-333 mutation was made in cDNA of the catalytic domain of neurofibromin (NF1-333) as described (13). Wild-type and the R1276P mutant of NF1-333 were isolated from Escherichia coli CK600 using the pLMM expression system in E.coli. Recombinant H-Ras was prepared from E.coli using a ptac expression system. Ras·mant-GDP, Ras·mant-GTP and Ras·mant-GppNHp were prepared as described (18).

Fluorescence measurements

Stopped-flow experiments using mant-GTP and mant-GppNHp bound to Ras were performed to measure individual rate constants for the interaction of NF1-333 with Ras on an Applied Photophysics SX16MV apparatus as described (18). All the reactions were followed in 30 mM Tris-HCl, pH 7.5, 5 mM MgCl₂, 5 mM dithioerythritol (DTE) at 25°C using an excitation wavelength of 360 nm and a cut-off-filter (408 nm) in front of the emission monochromator. Exponential and hyperbolic fits to the data were done using the program Grafit (Erithakus software), and more complex kinetic situations were analysed with the program Scientist as described (18). Fluorescence emission spectra (from 390 to 550 nm) of 0.1 µM mant-GDP-bound Ras were recorded in 30 mM Tris-HCl, pH 7.5, 5 mM MgCl₂, 5 mM DTE at 25°C on an LS50B Perkin-Elmer spectrofluorometer with an excitation wavelength of 366 nm as described (12). Fluorescence emission spectra (from 300 to 450 nm) of 1 µM wild-type NF1-333 and 1 µM R1276P-NF1-333 were recorded in 30 mM Tris-HCl, pH 7.5, 5 mM MgCl₂, 5 mM DTE at 25°C on an LS50B Perkin-Elmer spectrofluorometer with an excitation wavelength of 270 nm. CD spectra were measured in 10 mM potassium phosphate buffer, pH 7.0, at 25°C at a protein concetration of 200 µM wild-type NF1-333 and 220 µM NF1-333 (R1276P). The spectra were measured 10 times and averaged in 0.1 mm cells on a Jsco Spectropolarimeter J710.

ACKNOWLEDGEMENTS

We thank the NF1 patients and their family for participating in the study; N. Kücükceylan, I. Beilfuß, C. Mischung, B. Heyne, R. Müller and P. Stege for technical assistance; and S. Tinschert for helpful discussions. This work was supported by grants from the Deutsche Krebshilfe 70-1919-Kr2.

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*To whom correspondence should be addressed. Tel: +49 30 2802 8341; Fax: +49 30 2802 1286; Email: p.nuernberg{at}genetik.charite.hu-berlinl.de
⁺These authors contributed equally to this work

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				M. E. Lush, Y. Li, C.-H. Kwon, J. Chen, and L. F. Parada Neurofibromin Is Required for Barrel Formation in the Mouse Somatosensory Cortex J. Neurosci., February 13, 2008; 28(7): 1580 - 1587. [Abstract] [Full Text] [PDF]

				I. S. Ho, F. Hannan, H.-F. Guo, I. Hakker, and Y. Zhong Distinct Functional Domains of Neurofibromatosis Type 1 Regulate Immediate versus Long-Term Memory Formation J. Neurosci., June 20, 2007; 27(25): 6852 - 6857. [Abstract] [Full Text] [PDF]

				J. A. Walker, A. V. Tchoudakova, P. T. McKenney, S. Brill, D. Wu, G. S. Cowley, I. K. Hariharan, and A. Bernards Reduced growth of Drosophila neurofibromatosis 1 mutants reflects a non-cell-autonomous requirement for GTPase-Activating Protein activity in larval neurons Genes & Dev., December 1, 2006; 20(23): 3311 - 3323. [Abstract] [Full Text] [PDF]

				F.-C. Yang, S. Chen, T. Clegg, X. Li, T. Morgan, S. A. Estwick, J. Yuan, W. Khalaf, S. Burgin, J. Travers, et al. Nf1+/- mast cells induce neurofibroma like phenotypes through secreted TGF-{beta} signaling Hum. Mol. Genet., August 15, 2006; 15(16): 2421 - 2437. [Abstract] [Full Text] [PDF]

				F. Hannan, I. Ho, J. J. Tong, Y. Zhu, P. Nurnberg, and Y. Zhong Effect of neurofibromatosis type I mutations on a novel pathway for adenylyl cyclase activation requiring neurofibromin and Ras Hum. Mol. Genet., April 1, 2006; 15(7): 1087 - 1098. [Abstract] [Full Text] [PDF]

				R. R. Mattingly, J. M. Kraniak, J. T. Dilworth, P. Mathieu, B. Bealmear, J. E. Nowak, J. A. Benjamins, M. A. Tainsky, and J. J. Reiners Jr. The Mitogen-Activated Protein Kinase/Extracellular Signal-Regulated Kinase Kinase Inhibitor PD184352 (CI-1040) Selectively Induces Apoptosis in Malignant Schwannoma Cell Lines J. Pharmacol. Exp. Ther., January 1, 2006; 316(1): 456 - 465. [Abstract] [Full Text] [PDF]

				K. Hiatt, D. A. Ingram, H. Huddleston, D. F. Spandau, R. Kapur, and D. W. Clapp Loss of the Nf1 Tumor Suppressor Gene Decreases Fas Antigen Expression in Myeloid Cells Am. J. Pathol., April 1, 2004; 164(4): 1471 - 1479. [Abstract] [Full Text] [PDF]

				L Messiaen, V Riccardi, J Peltonen, O Maertens, T Callens, S L Karvonen, E-L Leisti, J Koivunen, I Vandenbroucke, K Stephens, et al. Independent NF1 mutations in two large families with spinal neurofibromatosis J. Med. Genet., February 1, 2003; 40(2): 122 - 126. [Full Text] [PDF]

				R. Kemkemer, S. Schrank, W. Vogel, H. Gruler, and D. Kaufmann Increased noise as an effect of haploinsufficiency of the tumor-suppressor gene neurofibromatosis type 1 in vitro PNAS, October 15, 2002; 99(21): 13783 - 13788. [Abstract] [Full Text] [PDF]

				R. M. Costa and A. J. Silva Review Article : Molecular and Cellular Mechanisms Underlying the Cognitive Deficits Associated With Neurofibromatosis 1 J Child Neurol, August 1, 2002; 17(8): 622 - 626. [Abstract] [PDF]

				P Nokelainen and J Flint Genetic effects on human cognition: lessons from the study of mental retardation syndromes J. Neurol. Neurosurg. Psychiatry, March 1, 2002; 72(3): 287 - 296. [Abstract] [Full Text] [PDF]

				K. K. Hiatt, D. A. Ingram, Y. Zhang, G. Bollag, and D. W. Clapp Neurofibromin GTPase-activating Protein-related Domains Restore Normal Growth in Nf1-/- Cells J. Biol. Chem., March 2, 2001; 276(10): 7240 - 7245. [Abstract] [Full Text] [PDF]