InteractiveFly: GeneBrief

Ras GTPase activating protein 1 : Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

BIOLOGICAL OVERVIEW

During normal eye development cone cell precursors are competent to become R7 photoreceptors and will adopt this cell fate if their Ras1 activity level is sufficiently high. Gap1, a guanine nucleotide exchange factor, functions in cone cell precursors to down-regulate Ras1 (Buckles, 1992; Gaul, 1992, and Rogge, 1992). Ras activity in these cells is likely elevated to a moderate degree by EGF receptor activity. Loss of Gap1 function causes the cone cell precursors to aberrantly adopt the fate of R7 cells. The number of supernumerary R7 cells produced within each ommatidium depends on the severity of the Gap1 allele (Buckles, 1992) indicating a sensitivity to different levels of Gap1 activity that can be exploited for the purposes of a structure-function analysis (Powe, 1999).

Ras signaling can be thought of in simple terms as occuring downstream of receptor tyrosine kinases, triggered by association of adaptor proteins with the phosphorylated cytoplasmic tails of these receptors. The analysis of Gap1 belies this simple picture, pointing to the involvement of Ca2+ and lipid signaling in moderating the strength and duration of the Ras signal. The Gap1 catalytic domain alone is insufficient for in vivo activity of Gap1, indicating a requirement for the additional domains. An inositol-1,3,4,5-tetrakisphosphate (IP4)-sensitive extended PH domain is essential for Gap1 activity, while Ca2+-sensitive C2 domains and a glutamine-rich region contribute equally to full activity in vivo. Furthermore, a strong positive genetic interaction occurs between Gap1 and phospholipase Cgamma (PLCgamma), an enzyme that generates inositol-1,4,5-trisphosphate, a precursor for IP4 and a second messenger for intracellular Ca2+ release. These results suggest that Gap1 activity in vivo is stimulated under conditions of elevated intracellular Ca2+and IP4 . Since receptor tyrosine kinases (RTKs) trigger an increase in intracellular Ca2 + and IP4 concentration through stimulation of PLCgamma, RTKs may stimulate not only activation of Ras but also its deactivation by Gap1 (Powe, 1999),

Several GAPs have been identified in both mammals and Drosophila, including neurofibromin, p120Gap and Gap1, which all share a catalytic domain required for stimulating Ras GTPase activity. However, GAPs possess additional non-catalytic domains that distinguish them from one another and that could be involved in their differential regulation or activity. Mammalian neurofibromin appears to constitutively down-regulate Ras, but the Drosophila homolog of this Gap protein might function upstream of Protein kinase A or in a parallel pathway, failing to interact with the Ras pathway (The, 1997). Nf1 and PKA appear to interact in a pathway that controls the overall growth of Drosophila (The, 1997). Mammalian p120Gap is required for moderating the extent and the duration of Ras activation after growth factor stimulation. Drosophila p120Gap has been implicated in the function of Breathless and Heartless, two Drosophila receptor tyrosine kinases of the fibroblast growth factor receptor family that are involved in wing morphogenesis, but no effect of RasGAP overexpression on photoreceptor development in the eye can be detected (Feldmann, 1999). Both Drosophila FGF receptors have exact matches to the optimal binding site (phospho YxxPxD, where x is any amino acid) for the SH2 domains of mammalian p120 Ras-GAP. The in vivo effects of RasGAP overexpression require intact SH2 domains, indicating that intracellular localization of RasGAP through SH2-phosphotyrosine interactions is important for RasGAP activity (Feldmann, 1999). Activation of mammalian p120Gap in response to growth factor stimulation may be achieved by both membrane translocation and stimulation of its catalytic activity: p120Gap contains SH2 domains that can be induced to bind to the activated RTK, thereby leading to the translocation of the cytoplasmic protein to the membrane. In addition, the N-terminal region of p120Gap appears to be required for full in vitro activity of the C-terminal catalytic domain (Powe, 1999 and references).

Gap1, which was first isolated in Drosophila as a negative regulator of RTK signaling, has two homologs in mammals (Gaul, 1992; Maekawa, 1994; Cullen, 1995). Apart from the catalytic domain in the middle of the molecule, all Gap1 family members share two N-terminal C2 domains and a C-terminal extended PH domain. Biochemical studies with the two mammalian homologs, Gap1^m and Gap1^IP4BP, have shown that the PH domain, which is required for membrane localization of an N-terminally truncated Gap1^IP4BP, binds IP4 or phosphatidylserine (PS) in a mutually exclusive manner (Fukuda, 1996; Cullen, 1997), suggesting that an increase in intracellular IP4 levels leads to the release of the PH domain from the membrane. IP4 binding to the PH domain also appears to stimulate GAP activity in vitro (Cullen, 1995; Fukuda, 1996), implying that release of the PH domain from the membrane is accompanied by activation of the catalytic domain. The N-terminal C2a domain has been shown to bind phospholipids in a Ca2+-dependent manner (Fukuda, 1996 and Fukuda, 1997), while the role of the C2b domain remains unclear. However, the physiological relevance of any of these domains for Gap1 in vivo function has not been established. Resolution of these problems has been approached in Drosophila by carrying out an in vivo functional analysis of Drosophila Gap1 in the context of cone cell determination in the developing Drosophila eye. Under basal conditions Drosophila Gap1 is localized to the membrane through its extended PH domain in a less active form; upon elevation of Ca2+ and IP4 concentrations, the PH domain binds to IP4 thereby stimulating catalytic activity, while at the same time the C2a domain takes over the role of membrane tether. This model is supported by the observation that Gap1 appears to be constitutively associated with the plasma membrane (Powe, 1999).

To introduce altered Drosophila Gap1 transgenes into flies, the sevenless (sev) enhancer/promoter cassette, an eye-specific promoter, was used to drive expression in cone cell precursors at appropriate levels. Expression of one copy of wild type Gap1 under control of the sev enhancer/ promoter cassette (sevGap1) rescues the complete loss-of-function eye phenotype, while creating no phenotypic defects when placed in a wild type background containing two copies of the endogenous Gap1 gene. By contrast, high levels of Gap1 expression under sev enhancer/promoter control using amplification by the UAS/GAL4 system in an otherwise wild type background results in a rough eye phenotype due to loss of photoreceptors (Rørth, 1996). Therefore, fusing altered Gap1 transgenes directly to the sev enhancer/promoter cassette provides sufficient levels of expression without interfering with development. To assess their rescue ability, transgenes were placed into two different Gap1 mutant backgrounds: the partial loss-of-function allele Gap1^S1-11 and the complete loss-of-function allele Gap1^B2 (Gaul, 1992). Gap1 transgenes containing only the catalytic domain (sevGapM) fail to rescue either the partial or complete loss-of-function phenotype of Gap1. These findings clearly indicate, in contrast to other studies, that the catalytic domain of Drosophila Gap1 requires additional parts of the protein for in vivo activity (Powe, 1999).

Since the catalytic domain alone could not rescue the loss-of-function phenotype, a test was performed to see whether the C- or N-terminal portions are required along with the catalytic domain for in vivo function. In the C-terminus of the molecule two regions can be distinguished. Immediately adjacent to the catalytic domain lies a large stretch of sequence similarity that is shared by all Gap1 family members. This conserved region consists of a PH domain, followed by a Cys3His1 zinc finger, referred to as the BTK motif. The PH and BTK sequences are followed by a stretch of 123 amino acids with no known homology to other proteins and poor homology within the Gap1 family; however, since a sequence of eight amino acids (KYGSxxxPIGD) at the C-terminal part of this region is conserved among Gap1 family members, this 123 aa stretch is designated the KYG region. In mammalian Gap1, the BTK/KYG region is required for specific high affinity binding of IP4 by the PH domain (Fukuda, 1996). C-terminal to the KYG domain is a glutamine-rich stretch found only in the Drosophila homolog (Powe, 1999).

Transgenes containing the catalytic domain and the entire C-terminus (sevGapdeltaN) show a 50% rescue of the Gap1 partial loss-of-function phenotype. The expression level of the sevGapdeltaN transgene providing the most complete rescue is higher than that of the best transgene expressing full length Gap1, indicating that the inability to rescue completely is due to an alteration of intrinsic activity. Thus, in conjunction with the C-terminus, the catalytic domain is sufficient for partial rescue activity. However, the N-terminus is necessary for full in vivo activity, since transgenes lacking the N-terminus do not completely rescue loss-of-function mutants. The N-terminal region of Drosophila Gap1 possesses two domains with known function, namely C2a and C2b domains. Various transgenes containing just the N-terminus and the catalytic domain all essentially fail to rescue the mutant phenotype. These results demonstrate that, unlike the C-terminus, the N-terminus does not confer the ability to rescue onto the minimal catalytic domain, but is necessary for full rescue activity (Powe, 1999).

Attempts were made to determine which part of the C-terminus is responsible for endowing the catalytic domain with the ability to rescue. The importance of the glutamine-rich region was tested. Transgenes without the glutamine-rich region provide 50% rescue. Hence, the glutamine-rich region is required for full rescue, but not essential for in vivo activity. Next, the importance of the PH/BTK/KYG region was tested, beginning with BTK and KYG motifs. The sevGa-pdeltaBTK-Q transgenes lack the entire KYG domain and the last cysteine of the BTK zinc finger as well as the glutamine-rich region, and show virtually no rescue. This finding clearly demonstrates that the BTK and KYG regions are critical for rescue activity. To examine the role of the PH domain, a point mutation (sevGap1^R786C), that severely reduces IP4 binding in mammalian Gap1 (Fukuda, 1996), was introduced and rescue ability was evaluated. Like the full length sevGap1 transgenes, sevGap1^R786C transgenes are able to fully rescue the partial loss-of-function situation. However, unlike sevGap1, when placed in the complete loss-of-function background, sevGap1^R786C only partially rescues (~70%) the phenotype. In the complete loss-of-function background, the average number of R7s per ommatidium for sevGap1^R786C is 1.6, compared to 1.0 for sevGap1 and 2.9 in the absence of a Gap1 transgene. Thus, the R786C mutation does compromise Gap1 in vivo activity, even if only moderately. The effect of this point mutation is masked by residual activity in the partial loss-of-function situation and is only uncovered in the complete loss-of-function situation. In general, the rescuing ability of Gap1 transgenes is lower in the complete loss-of-function background than in the partial loss-of-function background (with the exception of the sevGapdeltaC2a transgene), suggesting that the residual activity in Gap1^S1-11 partially compensates for the loss of activity in mutant Gap1 constructs (Powe, 1999).

These results demonstrate that the N-terminus of Gap1 is necessary for full in vivo activity, but only in the presence of critical C-terminal regions, suggesting a modulatory role for the N-terminus. It was therefore necessary to determine which of the two domains in the N-terminal region, C2a or C2b, is involved in the modulation of activity. This was done by separately deleting the entire C2a domain or half of the C2b domain. The C2a domain is required in the N-terminal region for full in vivo activity of Gap1. The presence of the C2b domain in the absence of the C2a domain confers a dominant negative activity on the protein. This finding in turn suggests that Gap1 molecules can physically interact either with one another or with other molecules. Deletion of the C2b domain leads to a complete loss of rescue activity in both the partial and the complete loss-of-function background, suggesting that the presence of the C2b domain is crucial for Gap1 in vivo activity. While it is possible that the presence of the C2a domain without the C2b domain inhibits the activity of the rest of the molecule, it seems more likely that the disruption of the C2b domain has a deleterious effect on the remainder of the protein, perhaps through misfolding (Powe, 1999).

Since the C2 and PH domains, which constitute potential regions of interaction with Ca2 + and IP4, are required for Gap1 in vivo activity, it became of interest to see whether genes involved in Ca2+ and phosphoinositide signaling regulate Gap1 function in vivo. One such candidate is the Drosophila gene small wing (sl), which encodes a phospholipaseCgamma (PLCgamma) homolog. PLCgamma comprises a catalytic core, two PH, two SH2, and one SH3 domains and a conserved tyrosine phosphorylation site, all of which act as signal-dependent regulators of enzymatic activity. Subsequent to RTK stimulation, PLCgamma is recruited to the receptor through its SH2 domains and then itself activated by tyrosine phosphorylation. Upon activation, PLCgamma converts phosphatidylinositol-4,5-bisphosphate into diacylglycerol and IP3; IP3, in turn, serves as a precursor for IP4, both of which act as signals for mobilization of intracellular Ca2+. Loss-of-function mutations in the sl locus display a phenotype that is consistent with PLCgamma playing a role as a negative regulator of Egfr signaling, similar to Gap1 (Thackeray, 1998). In the eye, sl complete loss-of-function mutations cause a supernumerary R7 phenotype in approximately 30% of ommatidia; this is qualitatively similar to the mutation caused by Gap1, but much less severe. In hemizygous sl¹ mutants, the average number of R7s is 1.3. Thus, the sl complete loss-of-function phenotype is extremely mild compared to that of Gap1 partial or complete loss-of-function mutations. To probe the relationship between PLCgamma and Gap1, a test was performed to determine whether they genetically interact. In a wildtype background, Gap1/+ flies are phenotypically wildtype, i.e. all ommatidia have only one R7 cell (Gaul, 1992; Lai, 1992), while flies hemizygous for sl¹ and heterozygous for Gap1 have a supernumerary R7 phenotype greatly enhanced over that seen in hemizygous sl¹ flies; greater than 80% of ommatidia have multiple R7 cells. Thus, the phenotype of mutants hemizygous for sl and heterozygous for Gap1 closely resembles that of the partial loss-of-function Gap1^S1-11 allele, demonstrating a synergistic positive genetic interaction between sl and Gap1. Given PLCgamma’s role in Ca2+ and phosphoinositide signaling and Gap1’s requirement for its Ca 2+ and IP4-sensitive domains, this result suggests that Gap1 activity is positively regulated by PLCgamma (Powe, 1999).

In order to examine the distribution of Gap1 protein in vivo, full length Gap1 was expressed under the control of the GMR promoter, which drives high levels of expression in all cells posterior to the morphogenetic furrow. Confocal microscopy of immunohistochemically stained eye discs reveals that full length Gap1 protein is concentrated at the plasma membrane in all cells, both inside and outside of developing ommatidial clusters. Examination of the surface of the eye disc shows Gap1 immunoreactivity marking the apical profiles of all cells posterior to the morphogenetic furrow. Using antibodies against HRP to mark the membrane of neuronal cells, Gap1 is found to colocalize with the HRP antigen in the apical microvillar protrusions of photoreceptor cells. These protrusions have been shown to be enriched for membrane-bound signaling molecules, such as Sevenless. Gap1 also appears to colocalize with the HRP antigen throughout the rest of the cell membrane, including axons. These findings suggest that Gap1 is constitutively localized to the plasma membrane, apparently independent of RTK signaling. In addition to its requirement for in vivo activity, the C-terminus, which includes the extended PH domain, is shown to be essential for membrane localization (Powe, 1999).

These findings argue for a link between RTK/Ras signaling and the second messengers Ca2+ and inositol phosphates. The in vivo activity of Gap1 is positively regulated by Ca2+-sensitive and IP4-sensitive domains that are adjacent to the Gap1 catalytic domain, thereby increasing the intrinsic GTPase activity of Ras and hence negatively regulating Ras activity. These results strongly suggest a negative regulation of RTK/Ras signaling by Ca2+ and IP4. Intriguingly, one of the signaling events responsible for increases in cytosolic Ca2+ and IP4 in non-excitable cells is the activation of RTKs themselves. Studies in vertebrates show that growth factor-stimulated RTKs, including Egfr, activate PLCgamma through association with the activated receptor and subsequent phosphorylation. Given the structural conservation of Egfr and PLCgamma between vertebrates and Drosophila, as well as the functional requirement for both in cone cell precursor development, this regulatory relationship is likely preserved. The observed interaction between Gap1 and PLCgamma, in conjunction with these findings, suggests that Egfr through PLCgamma activation could positively influence Gap1 activity. The RTK Torso, whose activity, together with Gap1 and Ras, determines terminal cell fates in the Drosophila embryo, may provide another example for a positive regulatory relationship between RTK, PLCgamma and Gap1. Torso possesses a conserved PLCgamma binding site that binds mammalian PLCgamma and whose removal leads to overactivity of in vivo signaling similar to removal of Gap1, suggesting that activation of the Torso RTK stimulates PLCgamma, which, by stimulation of Gap1, could result in the suppression of Ras1 activity. Thus, if RTKs trigger both activation and deactivation of Ras, then the duration and strength of the Ras signal in response to growth factor stimulation will depend on the kinetics of the pathways involved. Activation of Ras is thought to be achieved largely by membrane localization (and activation) of Sos, which is likely to be fast, whereas the deactivation of Ras by Gap1 may require a more complex series of events, including recruitment and stimulation of PLCgamma and the IP3-mediated increase in intracellular Ca2+, which has been shown to be slow and sustained in response to RTK stimulation (Powe, 1999 and references).

REGULATION

Protein Interactions

Both full-length Sprouty and a truncated Sprouty containing residues 1-369 (i.e., without the cys-rich domain and C-terminal residues) were assayed for their ability to bind in vitro translated members of the Ras pathway. Strong interactions are detected between Sprouty and Drk (an SH2-SH3 containing adaptor protein homologous to mammalian Grb2), and between Sprouty and Gap1 (a Ras GTPase-activating protein). No interactions were seen between Sprouty and several other proteins involved in the Ras pathway: Sos, Dos, Csw, Ras1, Raf, and Leo (14-3-3). The interactions with Drk and Gap1 do not require the presence of the C-terminal cysteine-rich domain, the region of Sprouty most conserved between flies and humans. Since the well-conserved cysteine-rich domain of Sprouty is not required for binding to Drk or Gap1, it might instead target the protein to the plasma membrane. To test this, two truncated forms of Sprouty were expressed in cultured cells. One form lacks the conserved cysteine-rich domain, whereas a second exclusively comprises the cysteine-rich domain. The form with the cysteine-rich domain is membrane associated and is indistinguishable from the wild-type protein. In sharp contrast, the form lacking the cysteine-rich domain is distributed uniformly throughout the cell, with no specific localization to membranes. Cell fractionation confirms these results. It is concluded that the 147-residue cysteine-rich domain in Sprouty, which corresponds to the most conserved region in the published human ESTs, is responsible for the specific localization of Sprouty to the plasma membrane (Casci, 1999).

DEVELOPMENTAL BIOLOGY / EFFECTS OF MUTATION

A Drosophila gene with similarity to the mammalian Ras GTPase activating protein has been isolated in screens for mutations that affect eye development. Inactivation of the locus, Gap1, mimics constitutive activation of the Sevenless receptor tyrosine kinase and eliminates the need for a functional Sevenless protein in the R7 cell. These results suggest that Gap1 acts as a negative regulator of signaling by Sevenless by down-regulating the activity of the Ras1 protein, which has been shown to be a key element in Sevenless signaling (Gaul, 1992). Germline clonal analysis shows that Gap1 is required in the somatic follicle cells and not the germ line for embryonic dorsoventral polarity determination (Chou, 1993).

The two central photoreceptor neurons of the Drosophila eye, R7 and R8, form a retinotopic map in the optic lobe of the fly brain. A technique has been developed that allows the visualization of the projections of these neurons with high resolution. Using this technique, a new mutant, mip (more inner photoreceptors) has been identified in which this map shows a striking hyperinnervation. The extra terminals in the brain derive from an excessive recruitment of sevenless-independent R7 photoreceptor cells during eye development. The original R7, however, remains Sevenless responsive. The behavior of this gene suggests that recruitment to the R7 pathway, and possibly to multiple programs in ommatidial assembly, is partially regulated by inhibition (Buckles, 1992).

M Loss of one copy of the recently isolated gene sextra (sxt) promotes R7 photoreceptor cell development in a genetically sensitized background, while loss of both copies results in precursors of non-neuronal cone cells transforming into R7 cells. The requirement for sxt function is cell-autonomous. The transformation of cone-cell precursors into R7 cells occurs independent of the Sevenless signal. However, the R7 precursor becomes neuronal in an sxt/sxt mutant only in a wild-type sevenless background. The genetic analysis of sxt suggests that it plays an inhibitory role, preventing cone cells from becoming neuronal. Additionally, sxt functions in R7 precursors, but the Sevenless signal is essential for specification of this fate, since loss of sextra alone is unable to impart a neural fate to this cell (Rogge, 1992).

EVOLUTIONARY HOMOLOGS

A novel GTPase-activating protein (GAP) for Ras has been purified: it is immunologically distinct from the known Ras GAPs, p120GAP and neurofibromin. On the basis of the partial amino acid sequence, a cDNA has been obtained that encodes the novel Ras GAP. The predicted protein consists of 847 amino acids whose calculated molecular mass (96,369 Da) is close to the apparent molecular mass of the novel Ras GAP (100 kDa). The amino acid sequence shows a high degree of similarity to the entire sequence of the Drosophila melanogaster Gap1 gene. When the catalytic domain of the novel GAP is compared with those of Drosophila Gap1, p120GAP, and neurofibromin, the highest degree of similarity is again observed with Gap1. Thus, this gene has been designated Gap1m, a mammalian counterpart of the Drosophila Gap1 gene. Expression of Gap1m is relatively high in brain, placenta, and kidney tissues, and it is expressed at low levels in other tissues. A recombinant protein consisting of glutathione-S-transferase and the GAP-related domain of Gap1m stimulates GTPase of normal Ras but not that of Ras having valine at the 12th residue. Expression of the same region in Saccharomyces cerevisiae suppresses the ira2- phenotype. In addition to the GAP catalytic domain, Gap1m has two domains with sequences closely related to those of the phospholipid-binding domain of synaptotagmin and a region with similarity to the unique domain of Btk tyrosine kinase. These results clearly show that Gap1m is a novel Ras GAP molecule of mammalian cells (Maekawa, 1994).

Inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4] is produced rapidly from inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] in stimulated cells. Despite extensive experimentation, no clearly defined cellular function has yet been described for this inositol phosphate. Binding sites specific for Ins(1,3,4,5)P4 have been identified in several tissues, and one such protein has been purified to homogeneity. Its high affinity for Ins(1,3,4,5)P4, and its exquisite specificity for this isomeric configuration, suggest it may be an Ins(1,3,4,5)P4 receptor. The cloning and characterization of this protein reveals it to be a GTPase-activating protein, specifically a member of the GAP1 family. In vitro it shows GAP activity against both Rap and Ras, but only the Ras GAP activity is inhibited by phospholipids and is specifically stimulated by Ins(1,3,4,5)P4 (Cullen, 1995).

Gap1(IP4BP), one member of the family of Ras GTPase-activating proteins, has been identified as a specific inositol 1,3,4,5-tetrakisphosphate (IP4)-binding protein. Gap1(m), which is closely related to Gap1(IP4BP), is also an IP4-binding protein; the pleckstrin homology domain (PH) is the central IP4-binding domain. In addition to the PH domain, an adjacent GAP-related domain and carboxyl terminus are required for high affinity specific IP4 binding. The PH domain is highly conserved in the Gap1 family and also has striking homology to the amino-terminal region of Bruton's tyrosine kinase. Substitution of Cys for Arg at position 628 in the PH domain corresponding to the mutation of Bruton's tyrosine kinase observed in X-linked immunodeficiency mice results in a dramatic reduction of IP4 binding activity as well as the phospholipid binding capacity of Gap1(m). This mutant also shows the GAP activity against Ha-Ras is similar to that of the wild type Gap1(m). These results suggest that the PH domain of Gap1(m) functions as a modulatory domain of GAP activity by binding IP4 and phospholipids (Fukuda, 1996).

A high affinity isomerically specific inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4]-binding protein has been purified and cloned that, because it is clearly a member of the GAP1 family of Ras GTPase-activating proteins (GAP), has been termed GAP1(IP4BP). Expressed full-length GAP1(IP4BP) binds Ins(1,3,4, 5)P4 with an affinity and specificity similar to that of the originally purified protein, a binding activity that is dependent on a functional PH/Btk domain. Furthermore, a fundamental distinction between GAP1(IP4BP) and its homolog GAP1(m) has been highlighted: both proteins function as Ras GAPs but only GAP1(IP4BP) displays Rap GAP activity (Bottomley, 1998).

Addition of Ins(1,3,4,5)P4 to permeabilized L1210 cells increases the amount of Ca2+ mobilized by a submaximal concentration of Ins(2,4,5)P3; it is suggested that in doing this, Ins(1,3,4,5)P4 is not working via an InsP3 receptor but indirectly via an InsP4 receptor. An investigation was carried out to see whether this effect might be mediated by GAP1(IP4BP), recently identified as a putative receptor for Ins(1,3, 4,5)P4. GAP1(IP4BP) is a protein that interacts with one or more monomeric G-proteins, so evidence was sought for involvement of monomeric G-proteins in the effects of Ins(1,3,4,5)P4 in permeabilized L1210 cells. Guanosine 5'-[gamma-thio]triphosphate (GTP[S]) enhances the effect of Ins(1,3,4,5)P4 on Ins(2,4, 5)P3-stimulated Ca2+ mobilization, but has no effect on the action of Ins(2,4,5)P3 alone. A specific enhancement of only the action of Ins(1,3,4,5)P4 is also seen with GTP[S]-loaded R-Ras or Rap1a [two G-proteins known to interact with GAP1(IP4BP)], whereas H-Ras is inactive at similar concentrations. Guanosine 5'-[beta-thio]diphosphate (GDP[S]) does not alter the action of either Ins(2,4,5)P3 or Ins(1,3,4,5)P4. Finally, the addition of exogenous GAP1(IP4BP), purified from platelets, markedly enhances the effect of Ins(1,3,4,5)P4, and again, the amount of Ca2+ mobilized by Ins(2,4,5)P3 alone is unaltered. It is concluded that the increase in Ins(2,4,5)P3-stimulated Ca2+ mobilization by Ins(1,3,4, 5)P4 may be mediated by GAP1(IP4BP) or a closely related protein [such as GAP1(m)], and if so, the action of the GAP1 is not solely to regulate GTP loading of a G-protein, but rather it acts with a G-protein to influence the G-protein's effect (Loomis-Husselbee, 1998).

GAP1(IP4BP) and GAP1(m) belong to the GAP1 family of Ras GTPase-activating proteins that are candidate InsP4 receptors. They are ubiquitously expressed in human tissues and are likely to have tissue-specific splice variants. Analysis by subcellular fractionation of RBL-2H3 rat basophilic leukemia cells confirms that endogenous GAP1(IP4BP) is primarily localized to the plasma membrane, whereas GAP1(m) appears localized to the cytoplasm (cytosol and internal membranes) but not the plasma membrane. Subcellular fractionation does not indicate a specific co-localization between membrane-bound GAP1(m) and several Ca2+ store markers, consistent with the lack of co-localization between GAP1(m) and SERCA1 upon co-expression in COS-7 cells. This difference suggests that GAP1(m) does not reside at a site where it could regulate the ability of InsP4 to release intracellular Ca2+. Since GAP1(m) is primarily localized to the cytosol of unstimulated cells, it may be spatially regulated in order to interact with Ras at the plasma membrane (Lockyer, 1999).

The activation status of the guanosine triphosphate (GTP)-binding protein Ras is dictated by the relative intensities of two opposing reactions: the formation of active Ras-GTP complexes, promoted by guanine-nucleotide exchange factors (GEFs), and their conversion to inactive Ras-GDP as a result of the deactivating action of GTPase-activating proteins (GAPs). The relevance of phosphoinositide 3-kinase (PI 3-kinase) to these processes is still unclear. The regulation of Ras activation by PI 3-kinase has been investigated in the myelomonocytic U937 cell line. These cells exhibit basal levels of Ras-GTP, which are suppressed by two PI 3-kinase inhibitors and a dominant-negative PI 3-kinase. In addition, PI 3-kinase inhibition aborts Ras activation by all stimuli tested, including fetal calf serum (FCS) and phorbol 12-myristate 13-acetate (TPA). Significantly, TPA does not activate PI 3-kinase in U937 cells, indicating that PI 3-kinase has a permissive rather than an intermediary role in Ras activation. Investigation of the mechanism of PI 3-kinase action reveals that inhibition of PI 3-kinase does not affect nucleotide exchange on Ras but abrogates Ras-GTP accumulation through an increase in GAP activity. These findings establish blockage of GAP action as the mechanism underlying a permissive function of PI 3-kinase in Ras activation (Rubio, 2000)

The increase in GAP activity induced by PI 3-kinase inhibitors indicates that resting levels of the lipids produced in the plasma membrane by PI 3-kinase inhibit the action of GAP proteins on Ras. Wortmannin pretreatment of U937 cells does not alter GAP activity as assayed from cell lysates. This suggests that membrane integrity is important for PI3-kinase-mediated inhibition of GAPs. Considering that Ras can activate PI 3-kinase through a direct interaction with the p110 catalytic subunit, these data suggest the following scenario. Active Ras could activate PI 3-kinase to induce spatially restricted generation of 3-phosphoinositides. This would promote local downregulation of relevant GAP species and thus allow basal Ras activation (Rubio, 2000).

REFERENCES

Search PubMed for articles about Drosophila Ras GTPase activating protein 1

Bottomley, J. R., et al. (1998). Structural and functional analysis of the putative inositol 1,3,4, 5-tetrakisphosphate receptors GAP1(IP4BP) and GAP1(m). Biochem. Biophys. Res. Commun. 250(1): 143-9.

Buckles, G. R., Smith, Z. D. and Katz, F. N. (1992). mip causes hyperinnervation of a retinotopic map in Drosophila by excessive recruitment of R7 photoreceptor cells. Neuron 8(6): 1015-29.

Casci, T., Vinos, J. and Freeman, M. (1999). Sprouty, an intracellular inhibitor of Ras signaling. Cell 96(5): 655-65.

Chou, T. B., Noll, E. and Perrimon, N. (1993). Autosomal P[ovoD1] dominant female-sterile insertions in Drosophila and their use in generating germ-line chimeras. Development 119(4): 1359-1369.

Cullen, P. J., et al. (1995). Identification of a specific Ins(1,3,4,5)P4-binding protein as a member of the GAP1 family. Nature 376(6540): 527-30.

Cullen, P.J., et al. (1997). Inositol 1,3,4,5-tetrakisphosphate and Ca 2 + homoeostasis: the role of GAP1IP4BP. Biochem. Soc. Trans. 25, 991-996.

Feldmann P., et al. (1999). Control of growth and differentiation by Drosophila RasGAP, a homolog of p120 ras-GTPase-activating protein. Mol. Cell. Biol. 19(3): 1928-37.

Fukuda, M. and Mikoshiba, K. (1996). Structure-function relationships of the mouse Gap1m. Determination of the inositol 1,3,4,5-tetrakisphosphate-binding domain. J. Biol. Chem. 271(31): 18838-42.

Fukuda, M., Kojima, T., Mikoshiba, K. (1997). Regulation by bivalent cations of phospholipid binding to the C2A domain of synaptotagmin III. Biochem. J. 323, 421-425.

Gaul, U., Mardon, G. and Rubin, G. M. (1992). A putative Ras GTPase activating protein acts as a negative regulator of signaling by the Sevenless receptor tyrosine kinase. Cell 68(6): 1007-19.

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