The catalytic domain of the neurofibromatosis type 1 gene product stimulates ras GTPase and complements ira mutants of S. cerevisiae

Sequencing of the neurofibromatosis gene (NF1) revealed a striking similarity among NF1, yeast IRA proteins, and mammalian GAP (GTPase-activating protein). Using both genetic and biochemical assays, we demonstrate that this homology domain of the NF1 protein interacts with ras proteins. First, expression of this NF1 domain suppressed the heat shock-sensitive phenotype of yeast ira1 and ira2 mutants. Second, this NF1 domain, after purification as a glutathione S-transferase (GST) fusion protein, strongly stimulated the GTPase activity of yeast RAS2 and human H-ras proteins. The GST-NF1 protein, however, did not stimulate the GTPase activity of oncogenic mutant ras proteins, H-rasVal-12 and yeast RAS2Val-19 mutants, or a yeast RAS2 effector mutant. These results establish that this NF1 domain has ras GAP activity similar to that found with IRA2 protein and mammalian GAP, and therefore may also regulate ras function in vivo.     

The NF1 locus encodes a protein functionally related to mammalian GAP and yeast IRA proteins

The von Recklinghausen neurofibromatosis locus, NF1, encodes a protein with homology restricted to the catalytic region of the RAS GTPase-activating protein, GAP, and with extensive homology to the IRA1 and IRA2 gene products of the yeast S. cerevisiae. A segment of the NF1 cDNA gene, expressed in yeast, can complement loss of IRA function and can inhibit both wild-type and mutant activated human H-ras genes that are coexpressed in yeast. Yeast expressing the NF1 segment have increased H-ras GTPase-stimulating activity. These studies indicate that the NF1 gene product can interact with RAS proteins and demonstrate structural and functional similarities and differences among the GAP, IRA1, IRA2, and NF1 proteins.     

S. cerevisiae genes IRA1 and IRA2 encode proteins that may be functionally equivalent to mammalian ras GTPase activating protein

The IRA1 and IRA2 genes of S. cerevisiae encode closely related proteins that also share homology with mammalian GAP (ras GTPase activating protein). The RAS1 and RAS2 proteins overexpressed in ira mutants accumulated in the GTP-bound form, whereas in the wild-type strain the proteins were found mostly in the GDP-bound form, indicating that IRA1 and IRA2 negatively regulate the level of RAS-GTP. In contrast, the RAS2Val-19 or RAS2Thr-66 mutant protein was bound to GTP in high amounts irrespective of the IRA genotype. Overexpression of bovine GAP suppressed the phenotypes of ira mutants by reducing the level of RAS-GTP, suggesting that IRA proteins may be functionally analogous to mammalian GAP.     

IRA2, a second gene of Saccharomyces cerevisiae that encodes a protein with a domain homologous to mammalian ras GTPase-activating protein.

The IRA1 gene is a negative regulator of the RAS-cyclic AMP pathway in Saccharomyces cerevisiae. To identify other genes involved in this pathway, we screened yeast genomic DNA libraries for genes that can suppress the heat shock sensitivity of the ira1 mutation on a multicopy vector. We identified IRA2, encoding a protein of 3,079 amino acids, that is 45% identical to the IRA1 protein. The region homologous between the IRA1 protein and ras GTPase-activating protein is also conserved in IRA2. IRA2 maps 11 centimorgans distal to the arg1 locus on the left arm of chromosome XV and was found to be allelic to glc4. Disruption of the IRA2 gene resulted in (i) increased sensitivity to heat shock and nitrogen starvation, (ii) sporulation defects, and (iii) suppression of the lethality of the cdc25 mutant. Analysis of disruption mutants of IRA1 and IRA2 indicated that IRA1 and IRA2 proteins additively regulate the RAS-cyclic AMP pathway in a negative fashion. Expression of the IRA2 domain homologous with GAP is sufficient for complementation of the heat shock sensitivity of ira2, suggesting that IRA down regulates RAS activity by stimulating the GTPase activity of RAS proteins.     

sar1, a gene from Schizosaccharomyces pombe encoding a protein that regulates ras1.

Proper ras1 function is required for normal sexual function in the yeast Schizosaccharomyces pombe. We have found a gene in S. pombe, sar1, that encodes a product capable of regulating ras1 function. sar1 is a member of an expanding family of RAS GTPase-activating proteins (GAPs) that includes mammalian GAP, the yeast Saccharomyces cerevisiae IRA proteins, and the product of the human neurofibromatosis locus, NF1 sar1, like these other proteins, can complement the loss of IRA function in S. cerevisiae. Computer analysis shows that the highest degree of sequence conservation is restricted to a very small number of diagnostic residues represented by the motif Phe-Leu-Arg-X-X-X-Pro-Ala-X-X-X-Pro. We find no evidence that sar1 is required for the effector function of ras1.     

The GAP-related domain of the neurofibromatosis type 1 gene product interacts with ras p21

The neurofibromatosis type 1 (NF1) protein contains a region of significant sequence similarity to ras p21 GTPase-activating protein (GAP) and the yeast IRA1 gene product. A fragment of NF1 cDNA encoding the GAP-related domain (NF1 GRD) was expressed, immunoaffinity purified, and assayed for effects on N-ras p21 GTPase activity. The GTPase of wild-type ras p21 was stimulated by NF1 GRD, but oncogenic mutants of ras p21 (Asp-12 and Val-12) were unaffected, and the GTPase of an effector mutant (Ala-38) was only weakly stimulated. NF1 GRD also down-regulated RAS function in S. cerevisiae. The affinity of NF1 GRD for ras p21 was estimated to be 250 nM: this is more than 20-fold higher than the affinity of GAP for ras p21. However, its specific activity was about 30 times lower. These kinetic measurements suggest that NF1 may be a significant regulator of ras p21 activity, particularly at low ras p21 concentrations.     

Complete human NF1 cDNA sequence: two alternatively spliced mRNAs and absence of expression in a neuroblastoma line.

Neurofibromatosis type 1 (NF1) is caused by mutations in a large gene on chromosome 17q11.2. Previously described partial cDNAs for this gene predicted a protein related to yeast IRA1/IRA2 and the mammalian RAS GTPase activator protein GAP. To initiate a detailed study of the role of this gene in NF1, we have characterized a set of overlapping cDNAs that represent its complete coding sequence. Our results show that two differentially expressed human NF1 mRNAs differ by a 63-bp insertion in the GAP-related domain. These mRNAs predict two 2,818- and 2,839-amino acid proteins with calculated molecular masses of approximately 317 and 319 kD. Extensive similarity to IRA proteins is evident in a 1,450-amino-acid central segment, roughly between amino acids 900 and 2,350. However, the remainder of the NF1 protein is not significantly similar to other proteins. Interestingly, the SK-N-SH human neuroblastoma line expresses no detectable NF1 mRNA, indicating that expression of NF1 is not essential for viability of this neural crest-derived tumor cell line.     

The GTPase stimulatory activities of the neurofibromatosis type 1 and the yeast IRA2 proteins are inhibited by arachidonic acid.

Three proteins, GTPase activating protein (GAP), neurofibromatosis 1 (NF1) and the yeast inhibitory regulator of the RAS-cAMP pathway (IRA2), have the ability to stimulate the GTPase activity of Ras proteins from higher animals or yeast. Previous studies indicate that certain lipids are able to inhibit this activity associated with the mammalian GAP protein. Inhibition of GAP would be expected to biologically activate Ras protein. In these studies arachidonic acid is shown also to inhibit the activity of the catalytic fragments of the other two proteins, mammalian NF1 and the yeast IRA2 proteins. In addition, phosphatidic acid (containing arachidonic and stearic acid) was inhibitory for the catalytic fragment of NF1 protein, but did not inhibit the catalytic fragments of GAP or IRA2 proteins. These observations emphasize the biochemical similarity of these proteins and provide support for the suggestion that lipids might play an important role in their biological control, and therefore also in the control of Ras activity and cellular proliferation.     

A conserved alternative splice in the von Recklinghausen neurofibromatosis (NF1) gene produces two neurofibromin isoforms, both of which have GTPase-activating protein activity.

Sequence analysis has shown significant homology between the catalytic regions of the mammalian ras GTPase-activating protein (GAP), yeast Ira1p and Ira2p (inhibitory regulators of the RAS-cyclic AMP pathway), and neurofibromin, the protein encoded by the NF1 gene. Yeast expression experiments have confirmed that a 381-amino-acid segment of neurofibromin, dubbed the GAP-related domain (GRD), can function as a GAP. Using the RNA polymerase chain reaction with primers flanking the NF1-GRD, we have identified evidence for alternative splicing in this region of the NF1 gene. In addition to the already published sequence (type I), an alternative RNA carrying a 63-nucleotide insertion (type II) is present in all tissues examined, although the relative amounts of types I and II vary. The insertion is conserved across species but is not present in GAP, IRA1, or IRA2. GenBank searches have failed to identify significant similarity between the inserted sequence and known DNA or protein sequences, although the basic amino acid composition of the insertion shares features with nuclear targeting sequences. Expression studies in yeasts show that despite the partial disruption of the neurofibromin-IRA-GAP homology by this insertion, both forms of the NF1-GRD can complement loss of IRA function. In vivo assays designed to compare the GAP activity of the two alternatively spliced forms of the NF1-GRD show that both can increase the conversion of GTP-bound ras to its GDP-bound form, although the insertion of the 21 amino acids weakens this effect. The strong conservation of this alternative splicing suggests that both type I and II isoforms mediate important biological functions of neurofibromin.     

The neurofibromatosis type 1 gene encodes a protein related to GAP

cDNA walking and sequencing have extended the open reading frame for the neurofibromatosis type 1 gene (NF1). The new sequence now predicts 2485 amino acids of the NF1 peptide. A 360 residue region of the new peptide shows significant similarity to the known catalytic domains of both human and bovine GAP (GTPase activating protein). A much broader region, centered around this same 360 amino acid sequence, is strikingly similar to the yeast IRA1 product, which has a similar amino acid sequence and functional homology to mammalian GAP. This evidence suggests that NF1 encodes a cytoplasmic GAP-like protein that may be involved in the control of cell growth by interacting with proteins such as the RAS gene product. Mapping of the cDNA clones has confirmed that NF1 spans a t(1;17) translocation mutation and that three active genes lie within an intron of NF1, but in opposite orientation.     

Identification of a GTPase-activating protein homolog in Schizosaccharomyces pombe.

Loss of function of the Schizosaccharomyces pombe gap1 gene results in the same phenotypes as those caused by an activated ras1 mutation, i.e., hypersensitivity to the mating factor and inability to perform efficient mating. Sequence analysis of gap1 indicates that it encodes a homolog of the mammalian Ras GTPase-activating protein (GAP). The predicted gap1 gene product has 766 amino acids with relatively short N- and C-terminal regions flanking the conserved core sequence of GAP. Genetic analysis suggests that S. pombe Gap1 functions primarily as a negative regulator of Ras1, like S. cerevisiae GAP homologs encoded by IRA1 and IRA2, but is unlikely to be a downstream effector of the Ras protein, a role proposed for mammalian GAP. Thus, Gap1 and Ste6, a putative GDP-GTP-exchanging protein for Ras1 previously identified, appear to play antagonistic roles in the Ras-GTPase cycle in S. pombe. Furthermore, we suggest that this Ras-GTPase cycle involves the ra12 gene product, another positive regulator of Ras1 whose homologs have not been identified in other organisms, which could function either as a second GDP-GTP-exchanging protein or as a factor that negatively regulates Gap1 activity.     

Interactions between adenylate cyclase and the yeast GTPase-activating protein IRA1.

The adenylate cyclase system of the yeast Saccharomyces cerevisiae contains many proteins, including the CYR1 polypeptide, which is responsible for catalyzing the formation of cyclic AMP from ATP, RAS1 and RAS2 polypeptides, which mediate stimulation of cyclic AMP synthesis by guanine nucleotides, and the yeast GTPase-activating protein analog IRA1. We have previously reported that adenylate cyclase is only peripherally bound to the yeast membrane. We have concluded that IRA1 is a strong candidate for a protein involved in anchoring adenylate cyclase to the membrane. We base this conclusion on the following criteria: (i) a disruption of the IRA1 gene produced a mutant with very low membrane-associated levels of adenylate cyclase activity, (ii) membranes made from these mutants were incapable of binding adenylate cyclase in vitro, (iii) IRA1 antibodies inhibit binding of adenylate cyclase to the membrane, and (iv) IRA1 and adenylate cyclase comigrate on Sepharose 4B.     

Overexpression of RPI1, a novel inhibitor of the yeast Ras-cyclic AMP pathway, down-regulates normal but not mutationally activated ras function.

A high-copy-number plasmid genomic library was screened for genes that when overexpressed down-regulate Ras protein activity in Saccharomyces cerevisiae. We report on the structure and characterization of one such gene, RPI1, which potentially encodes a novel 46-kDa negative regulator of the Ras-cyclic AMP pathway. Three lines of evidence suggest that the RPI1 gene product operates upstream to negatively regulate the activity of normal but not mutationally activated Ras proteins: (i) overexpressed RPI1 lowers cyclic AMP levels in wild-type yeast cells but not in yeast cells carrying the RAS2Val-19 mutation, (ii) overexpressed RPI1 suppresses the heat shock sensitivity phenotype induced by overexpression of normal RAS2 but does not suppress the same phenotype induced by RAS2Val-19, and (iii) disruption of RPI1 results in a heat shock sensitivity phenotype which can be suppressed by mutations that lower normal Ras activity. Thus, RPI1 appears to encode an inhibitor of Ras activity that shares a common feature with Ras GTPase-activating proteins in that it fails to down-regulate activated RAS2Val-19 function. We present evidence that the down-regulatory effect of RPI1 requires the presence of one of the two Ras GTPase activators, IRA1 and IRA2.     

Characterization of Saccharomyces cerevisiae strains expressing ira1 mutant alleles modeled after disease-causing mutations in NF1

The 2818 amino acids of neurofibromin, the product of the human NF1 gene, include a 230 amino acid Ras-GAP related domain (GRD). Functions which may be associated with the rest of the protein remain unknown. However, many NF1 mutations in neurofibromatosis 1 patients are found downstream of the GRD, suggesting that the C-terminal region of the protein is also functionally important. Since the C-terminal region of neurofibromin encompassing these mutations is homologous with the corresponding regions in the two Saccharomyces cerevisiae Ras-GAPs, Ira1p and Ira2p, we chose yeast as a model system for functional exploration of this region (Ira-C region). Three missense mutations that affect the Ira-C region of NF1 were used as a model for the mutagenesis of IRA1. The yeast phenotypes of heat shock sensitivity, iodine staining, sporulation efficiency, pseudohyphae formation, and GAP activity were scored. Even though none of the mutations directly affected the Ira1p-GRD, mutations at two of the three sites resulted in a decrease in the GAP activity present in ira1 cells. The third mutation appeared to disassociate the phenotypes of sporulation ability and GAP activity. This and other evidence suggest an effector function for Ira1p.     

Functional significance of lysine 1423 of neurofibromin and characterization of a second site suppressor which rescues mutations at this residue and suppresses RAS2Val-19-activated phenotypes.

Lysine 1423 of neurofibromin (neurofibromatosis type I gene product [NF1]) plays a crucial role in the function of NF1. Mutations of this lysine were detected in samples from a neurofibromatosis patient as well as from cancer patients. To further understand the significance of this residue, we have mutated it to all possible amino acids. Functional assays using yeast ira complementation have revealed that lysine is the only amino acid that produced functional NF1. Quantitative analyses of different mutant proteins have suggested that their GTPase-activating protein (GAP) activity is drastically reduced as a result of a decrease in their Ras affinity. Such a requirement for a specific residue is not observed in the case of other conserved residues within the GAP-related domain. We also report that another residue, phenylalanine 1434, plays an important role in NF1 function. This was first indicated by the finding that defective NF1s due to an alteration of lysine 1423 to other amino acids can be rescued by a second site intragenic mutation at residue 1434. The mutation partially restored GAP activity in the lysine mutant. When the mutation phenylalanine 1434 to serine was introduced into a wild-type NF1 protein, the resulting protein acquired the ability to suppress activated phenotypes of RAS2Val-19 cells. This suppression, however, does not involve Ras interaction, since the phenylalanine mutant does not stimulate the intrinsic GTPase activity of RAS2Val-19 protein and does not have an increased affinity for Ras proteins.     

Differential activation of yeast adenylate cyclase by wild-type and mutant RAS proteins

In these experiments we demonstrate that purified RAS proteins, whether derived from the yeast RAS1 or RAS2 or the human H-ras genes, activate yeast adenylate cyclase in the presence of guanine nucleotides. These results confirm the prediction of earlier genetic and biochemical data and for the first time provide a complete biochemical assay for RAS protein function. Furthermore, we observe a biochemical difference between the RAS2 and RAS2val19 proteins in their ability to activate adenylate cyclase after preincubation with GTP.     

The Ras/cAMP/protein kinase A pathway regulates glucose-dependent assembly of the vacuolar (H+)-ATPase in yeast

Vacuolar (H+)-ATPases (V-ATPases) are ubiquitous, ATP-driven proton pumps that acidify organelles or the extracellular space. A rapid and effective mechanism for regulating V-ATPase activity involves reversible dissociation of the two functional domains of the pump, V1 and V0. This process is best characterized in yeast, where V-ATPases are reversibly disassembled in response to glucose depletion. To identify regulators that control this process in vivo, a genetic screen was performed in yeast to search for mutants that cannot disassemble their V-ATPases when grown in the absence of glucose. This screen identified IRA1 (inhibitory regulator of the Ras/cAMP pathway 1) and IRA2 as essential genes for regulating V-ATPase dissociation in vivo. IRA1 and IRA2 encode GTPase-activating proteins that negatively regulate Ras in nutrient-poor conditions. Down-regulation of Ras lowers cAMP levels by reducing adenylate cyclase activity. Decreased cAMP levels in turn lead to reduced activity of protein kinase A (PKA). Our results show that targeted deletion of IRA2 results in defective disassembly of the V-ATPase in response to glucose depletion, and reexpression of the gene rescues this phenotype. Glucose-dependent dissociation is also blocked in strains expressing the dominant active RAS2val19 allele or in strains deficient for the regulatory subunit of PKA, both of which lead to constitutively active PKA. These results reveal a role for PKA in controlling glucose-dependent V-ATPase assembly in yeast.     

IRA1, an inhibitory regulator of the RAS-cyclic AMP pathway in Saccharomyces cerevisiae.

A mutation in the gene IRA1 (formerly called PPD1) was originally characterized as a deficiency of a phosphoprotein phosphatase. The IRA1 gene has been cloned and sequenced. A large open reading frame (8,817 base pairs) which can encode a protein of 2,938 amino acids was found. Northern (RNA) blot analysis detected a message of about 10 kilobases, and nuclease S1 protection demonstrated mRNA start points at 97 and 98 base pairs upstream from the putative initiator ATG codon. Disruption of the IRA1 gene resulted in sensitivity to nitrogen starvation and heat shock. Diploids homozygous for the disrupted IRA1 gene were deficient in sporulation. Disruption of the IRA1 gene suppressed the lethality of the cdc25 mutation but did not suppress the lethality of either the ras1 ras2 or the cyr1 mutations. Deficiency of the phosphoprotein phosphatase was not reproducible in the disruption mutant of the IRA1 gene. Moreover, the ira1 mutant showed an increased level of cyclic AMP. Our results suggest that the IRA1 protein inhibits the function of the RAS proteins in a fashion antagonistic to the function of the CDC25 protein in the RAS-cyclic AMP pathway in Saccharomyces cerevisiae.     

NF1 modulates the effects of Ras oncogenes: evidence of other NF1 function besides its GAP activity

Neurofibromin (NF1) (the product of Nf1 gene) is a large cytosolic protein known as a negative regulator of Ras. A fragment of some 400 residues located at the center of the NF1 GAP-Related Domain (NF1-GRD) has strong identity with other molecules of the GAP family, which comprises, among others, the mammalian proteins NF1 and p120GAP, and the yeast proteins IRA1 and IRA2. GAP family members are known by their ability to promote the GTPase activity of Ras proteins, facilitating the transit of those proteins to their inactive state. Recent findings (Tong et al., 2002, Nat Neurosci 5:95-96) indicate that NF1 may be involved in the regulation of adenyl cyclase activity. Our results show that NF1-GRD cooperates with Ras in the anchorage-independent growth capacity of Ras-expressing fibroblasts, without affecting: (i) their ability to grow in low serum, (ii) their cellular adhesion capability, or (iii) the expression of key proteins involved in cell-cell and cell-matrix interactions. On the other hand, NF1 overexpression induces an increase in the expression levels of the focal adhesion kinase (FAK), and specific changes in the activation status of the mitogen-activated protein kinases (MAPKs). These results suggest the existence of a Ras-independent NF1-dependent pathway able to modify the levels of expression of FAK and the levels of activation of MAPKs. Because FAK and many proteins recently found to bind NF1 have a role in the cytoskeleton, this pathway may involve rearrangement of cytoskeletal components that facilitate anchorage independence.     

Centaurin-alpha(1) associates with and is phosphorylated by isoforms of protein kinase C

Centaurin-alpha(1) is a member of the family of ADP-ribosylation factors (ARF) GTPase activating proteins (GAPs), although ARF GAP activity has not yet been demonstrated. The human homologue, centaurin-alpha(1) functionally complements the ARF GAP activity of Gcs1 in yeast. Although Gcs1 is involved in the formation of actin filaments in vivo, the function of centaurin remains elusive. We have identified a number of novel centaurin-alpha(1) binding partners; including CKIalpha and nucleolin. In this report, we have focused on the interaction of centaurin-alpha(1) with PKC. All groups of PKC associate directly through their cysteine rich domains. Centaurin-alpha(1) is also a substrate for all PKC classes and we have identified the two sites of phosphorylation. This is the first report of a kinase that phosphorylates centaurin-alpha(1).