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).