Downregulation of Rap1GAP contributes to Ras transformation

Although abundant in well-differentiated rat thyroid cells, Rap1GAP expression was extinguished in a subset of human thyroid tumor-derived cell lines. Intriguingly, Rap1GAP was downregulated selectively in tumor cell lines that had acquired a mesenchymal morphology. Restoring Rap1GAP expression to these cells inhibited cell migration and invasion, effects that were correlated with the inhibition of Rap1 and Rac1 activity. The reexpression of Rap1GAP also inhibited DNA synthesis and anchorage-independent proliferation. Conversely, eliminating Rap1GAP expression in rat thyroid cells induced a transient increase in cell number. Strikingly, Rap1GAP expression was abolished by Ras transformation. The downregulation of Rap1GAP by Ras required the activation of the Raf/MEK/extracellular signal-regulated kinase cascade and was correlated with the induction of mesenchymal morphology and migratory behavior. Remarkably, the acute expression of oncogenic Ras was sufficient to downregulate Rap1GAP expression in rat thyroid cells, identifying Rap1GAP as a novel target of oncogenic Ras. Collectively, these data implicate Rap1GAP as a putative tumor/invasion suppressor in the thyroid. In support of that notion, Rap1GAP was highly expressed in normal human thyroid cells and downregulated in primary thyroid tumors.     

The residues of Ras and Rap proteins that determine their GAP specificities.

The oncogenic transformation of a normal fibroblast by mutated Ras genes can be reversed by overexpression of a Ras-related gene called Rap1A (or Krev1). Both Ras and Rap1A proteins are G proteins and appear to serve as signal transducers only in the GTP-bound form. Therefore, GAP1 and GAP3, which stimulate the intrinsic GTPase activities of normal Ras and Rap1A proteins, respectively, serve as attenuators of their signal transducing activities. In this paper, we describe the enzymatic properties of several mutated Rap1A and chimeric Ras/Rap1A (or -1B) proteins which lead to the following conclusions: (i) the GAP3-dependent activation of both Rap1A and -1B GTPases requires Gly12, but neither Thr61 nor Gln63; (ii) residues 64 to 70 of the Rap1 GTPases are sufficient to determine their specificities for GAP3; and (iii) residues 61 to 65 of the Ras GTPases are sufficient for determining their specificities for GAP1. Thus, the domains of the Ras or Rap1 proteins that determine whether their signals are attenuated by GAP1 or GAP3 are distinct from the N-terminal domain (residues 21 to 54) that determines whether their signals are oncogenic or antioncogenic. The Arg12 mutant of chimeric HaRas(1-54)/Rap1A(55-184) protein has been previously reported to be oncogenic (Zhang, K., Noda, M., Vass, W. C., Papageorge, A.G., and Lowy, D.R. (1990) Science 249, 162-165). In this paper, we show that the Val12 mutant of chimeric HaRas(1-54)/Rap1B(55-184) protein is also oncogenic, suggesting that the C-terminal geranylgeranylation of the Rap 1B protein can replace functionally the C-terminal farnesylation of the Ras protein to allow the G protein to be oncogenic.     

Tumor Cell Migration and Invasion Are Enhanced by Depletion of Rap1 GTPase-activating Protein (Rap1GAP)

The functional significance of the widespread down-regulation of Rap1 GTPase-activating protein (Rap1GAP), a negative regulator of Rap activity, in human tumors is unknown. Here we show that human colon cancer cells depleted of Rap1GAP are endowed with more aggressive migratory and invasive properties. Silencing Rap1GAP enhanced the migration of confluent and single cells. In the latter, migration distance, velocity, and directionality were increased. Enhanced migration was a consequence of increased endogenous Rap activity as silencing Rap expression selectively abolished the migration of Rap1GAP-depleted cells. ROCK-mediated cell contractility was suppressed in Rap1GAP-depleted cells, which exhibited a spindle-shaped morphology and abundant membrane protrusions. Tumor cells can switch between Rho/ROCK-mediated contractility-based migration and Rac1-mediated mesenchymal motility. Strikingly, the migration of Rap1GAP-depleted, but not control cells required Rac1 activity, suggesting that loss of Rap1GAP alters migratory mechanisms. Inhibition of Rac1 activity restored membrane blebbing and increased ROCK activity in Rap1GAP-depleted cells, suggesting that Rac1 contributes to the suppression of contractility. Collectively, these findings identify Rap1GAP as a critical regulator of aggressive tumor cell behavior and suggest that the level of Rap1GAP expression influences the migratory mechanisms that are operative in tumor cells.     

Ras interaction with the GTPase-activating protein (GAP)

Biologically active forms of Ras complexed to GTP can bind to the GTPase-activating protein (GAP), which has been implicated as possible target of Ras in mammalian cells. In order to study the structural features of Ras required for this interaction, we have evaluated a series of mutant ras proteins for the ability to bind GAP and a series of Ras peptides for the ability to interfere with this interaction. Point mutations in the putative effector region of Ras (residues 32-40) that inhibit biological activity also impair Ras binding to GAP. An apparent exception is the Thr to Ser substitution at residue 35; [Ser-35]Ras binds to GAP as effectively as wild-type Ras even though this mutant is biologically weak in both mammalian and S. cerevisiae cells. In vitro, [Ser-35]Ras can also efficiently stimulate the S. cerevisiae target of Ras, adenylyl cyclase, indicating that other factors may influence Ras/protein interactions in vivo. Peptides having Ras residues 17-44 and 17-32 competed with the binding of Ras to E. coli-expressed GAP with IC50 values of 2.4 and 0.9 microM, respectively, whereas Ras peptide 17-26 was without effect up to 400 microM. A related peptide from the yeast GTP-binding protein YPT1 analogous to Ras peptide 17-32 competed with an IC50 value of 19 microM even though the YPT1 protein itself is unable to bind to GAP. These results suggest that determinants within Ras peptide 17-32 may be important for Ras binding to GAP.     

The GTPase-activating protein Rap1GAP: a new player to modulate Ret signaling

Glial cell line-derived neurotrophic factor (GDNF) plays a critical role in orchestrating the development and maintenance of different populations of central a...     

Rap1 GTPase-activating protein SPA-1 negatively regulates cell adhesion.

Rap1 GTPase is activated by a variety of stimulations in many types of cells, but its exact functions remain unknown. In this study we have shown that SPA-1 interferes with Rap1 activation by membrane-targeted C3G, C3G-F, in 293T cells through the GTPase activating protein (GAP) activity. SPA-1 transiently expressed in HeLa cells was mostly localized at the cortical cytoskeleton and induced rounding up of the cells, whereas C3G-F conversely induced extensive cell spreading. Conditional SPA-1 overexpression in HeLa cells by tetracycline-regulative system suppressed Rap1 activation upon plating on dishes coated with fibronectin and resulted in the reduced adhesion. When SPA-1 was conditionally induced after the established cell adhesion, the cells gradually rounded up and detached from the dish. Both effects were counteracted by exogenous fibronectin in a dose-dependent manner. Retroviral overexpression of SPA-1 in promyelocytic 32D cells also inhibited both activation of Rap1 and induction of cell adhesion by granulocyte colony stimulating factor without affecting differentiation. These results have indicated that Rap1 GTP is required for the cell adhesion induced by both extracellular matrix and soluble factors, which is negatively regulated by SPA-1.     

The Ras/p120 GTPase-activating protein (GAP) interaction is regulated by the p120 GAP pleckstrin homology domain

Pleckstrin homology domains are structurally conserved functional domains that can undergo both protein/protein and protein/lipid interactions. Pleckstrin homology domains can mediate inter- and intra-molecular binding events to regulate enzyme activity. They occur in numerous proteins including many that interact with Ras superfamily members, such as p120 GAP. The pleckstrin homology domain of p120 GAP is located in the NH(2)-terminal, noncatalytic region of p120 GAP. Overexpression of the noncatalytic domains of p120 GAP may modulate Ras signal transduction pathways. Here, we demonstrate that expression of the isolated pleckstrin homology domain of p120 GAP specifically inhibits Ras-mediated signaling and transformation but not normal cellular growth. Furthermore, we show that the pleckstrin homology domain binds the catalytic domain of p120 GAP and interferes with the Ras/GAP interaction. Thus, we suggest that the pleckstrin homology domain of p120 GAP may specifically regulate the interaction of Ras with p120 GAP via competitive intra-molecular binding.     

Cyclic nucleotide-dependent protein kinases inhibit binding of 14-3-3 to the GTPase-activating protein Rap1GAP2 in platelets

GTPase-activating proteins are required to terminate signaling by Rap1, a small guanine nucleotide-binding protein that controls integrin activity and cell adhesion. Recently, we identified Rap1GAP2, a GTPase-activating protein of Rap1 in platelets. Here we show that 14-3-3 proteins interact with phosphorylated serine 9 at the N terminus of Rap1GAP2. Platelet activation by ADP and thrombin enhances serine 9 phosphorylation and increases 14-3-3 binding to endogenous Rap1GAP2. Conversely, inhibition of platelets by endothelium-derived factors nitric oxide and prostacyclin disrupts 14-3-3 binding. These effects are mediated by cGMP- and cAMP-dependent protein kinases that phosphorylate Rap1GAP2 at serine 7, adjacent to the 14-3-3 binding site. 14-3-3 binding does not change the GTPase-activating function of Rap1GAP2 in vitro. However, 14-3-3 binding attenuates Rap1GAP2 mediated inhibition of cell adhesion. Our findings define a novel crossover point of activatory and inhibitory signaling pathways in platelets.     

Activation of Rap1B by G(i) family members in platelets

It has become increasingly appreciated that receptors coupled to G(alpha)(i) family members can stimulate platelet aggregation, but the mechanism for this has remained unclear. One possible mediator is the small GTPase, Rap1, which has been shown to contribute to integrin activation in several cell lines and to be activated by a calcium-dependent mechanism in platelets. Here, we demonstrate that Rap1 is also activated by G(alpha)(i) family members in platelets. First, we show that platelets from mice lacking the G(alpha)(i) family member G(alpha)(z) (which couples to the alpha(2A) adrenergic receptor) are deficient in epinephrine-stimulated Rap1 activation. We also show that platelets from mice lacking G(alpha)(i2), which couples to the ADP receptor, P2Y12, exhibit reduced Rap1 activation in response to ADP. In contrast, platelets from mice that lack G(alpha)(q) show no decrease in the ability to activate Rap1 in response to epinephrine but show a partial reduction in ADP-stimulated Rap1 activation. This result, combined with studies of human platelets treated with ADP receptor-selective inhibitors, indicates that ADP-stimulated Rap1 activation in human platelets is dependent on both the G(alpha)(i)-coupled P2Y12 receptor and the G(alpha)(q)-coupled P2Y1 receptor. G(alpha)(i)-dependent activation of Rap1 in platelets does not appear to be mediated by enhanced intracellular calcium release because no increase in intracellular calcium concentration was detected in response to epinephrine and because the calcium response to ADP was not diminished in platelets from the G(alpha)(i2)-/- mouse. Finally, using human platelets treated with selective inhibitors of phosphatidylinositol 3-kinase (PI3K) and mouse platelets selectively lacking the G(beta)(gamma)-activated form of his enzyme (PI3Kgamma), we show that G(i)-mediated Rap1 activation is PI3K-dependent. In summary, activation of Rap1 can be stimulated by G(alpha)(i)- and PI3K-dependent mechanisms in platelets and by G(q)- and Ca(2+)-dependent mechanisms, both of which may play a role in promoting platelet activation.     

Localized Diacylglycerol-dependent Stimulation of Ras and Rap1 during Phagocytosis

We describe a role for diacylglycerol in the activation of Ras and Rap1 at the phagosomal membrane. During phagocytosis, Ras density was similar on the surface and invaginating areas of the membrane, but activation was detectable only in the latter and in sealed phagosomes. Ras activation was associated with the recruitment of RasGRP3, a diacylglycerol-dependent Ras/Rap1 exchange factor. Recruitment to phagosomes of RasGRP3, which contains a C1 domain, parallels and appears to be due to the formation of diacylglycerol. Accordingly, Ras and Rap1 activation was precluded by antagonists of phospholipase C and of diacylglycerol binding. Ras is dispensable for phagocytosis but controls activation of extracellular signal-regulated kinase, which is partially impeded by diacylglycerol inhibitors. By contrast, cross-activation of complement receptors by stimulation of Fcγ receptors requires Rap1 and involves diacylglycerol. We suggest a role for diacylglycerol-dependent exchange factors in the activation of Ras and Rap1, which govern distinct processes induced by Fcγ receptor-mediated phagocytosis to enhance the innate immune response.Receptors that interact with the constant region of IgG (FcγR)⁴ mediate the recognition and elimination of soluble immune complexes and particles coated (opsonized) with immunoglobulins. Clustering of FcγR on the surface of leukocytes upon attachment to multivalent ligands induces their activation and subsequent internalization. Soluble immune complexes are internalized by endocytosis, a clathrin- and ubiquitylation-dependent process (1). In contrast, large, particulate complexes like IgG-coated pathogens are ingested by phagocytosis, a process that is contingent on extensive actin polymerization that drives the extension of pseudopods (2). In parallel with the internalization of the opsonized targets, cross-linking of phagocytic receptors triggers a variety of other responses that are essential components of the innate immune response. These include degranulation, activation of the respiratory burst, and the synthesis and release of multiple inflammatory agents (3, 4).Like T and B cell receptors, FcγR possesses an immunoreceptor tyrosine-based activation motif that is critical for signal transduction (3, 4). Upon receptor clustering, tyrosyl residues of the immunoreceptor tyrosine-based activation motif are phosphorylated by Src family kinases, thereby generating a docking site for Syk, a tyrosine kinase of the ZAP70 family (3, 4). The recruitment and activation of Syk in turn initiates a cascade of events that include activation of Tec family kinases, Rho- and ARF-family GTPases, phosphatidylinositol 3-kinase, phospholipase Cγ (PLCγ), and a multitude of additional effectors that together remodel the underlying cytoskeleton, culminating in internalization of the bound particle (5, 6).Phosphoinositide metabolism is thought to be critical for FcγR-induced phagocytosis (7, 8). Highly localized and very dynamic phosphoinositide changes have been observed at sites of phagocytosis: phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P₂) undergoes a transient accumulation at the phagocytic cup, which is rapidly superseded by its complete elimination from the nascent phagosome (7). The secondary disappearance of PtdIns(4,5)P₂ is attributable in part to the localized generation of phosphatidylinositol 3,4,5-trisphosphate, which has been reported to accumulate at sites of phagocytosis (9). Activation of PLCγ is also believed to contribute to the acute disappearance of PtdIns(4,5)P₂ in nascent phagosomes. Indeed, the generation of diacylglycerol (DAG) and inositol 1,4,5-trisphosphate has been detected by chemical means during FcγR-evoked particle ingestion (10, 11). Moreover, imaging experiments revealed that DAG appears at the time and at the precise site where PtdIns(4,5)P₂ is consumed (7).Two lines of evidence suggest that the DAG generated upon engagement of phagocytic receptors modulates particle engulfment. First, antagonists of PLC severely impair phagocytosis by macrophages (7, 12). This inhibition is not mimicked by preventing the associated [Ca²⁺] transient, suggesting that DAG, and not inositol 1,4,5-trisphosphate, is the crucial product of the PLC (13). Second, the addition of exogenous DAG or phorbol esters, which mimic the actions of endogenous DAG, augment phagocytosis (14, 15).Selective recognition of DAG by cellular ligands is generally mediated by specific regions of its target proteins, called C1 domains (16). Proteins bearing C1 domains include, most notably, members of the classical and novel families of protein kinase C (PKC), making them suitable candidates to account for the DAG dependence of phagocytosis. Indeed, PKCα, a classical isoform, and PKCϵ and PKCδ, both novel isoforms, are recruited to phagosomes (12, 15, 17, 18). Although the role of the various PKC isoforms in particle engulfment has been equivocal over the years, Cheeseman et al. (12) convincingly demonstrated that PKCϵ contributes to particle uptake in a PLC- and DAG-dependent manner.PKCs are not the sole proteins bearing DAG-binding C1 domains. Similar domains are also found in several other proteins, including members of the RasGRP family, chimaerins, and Munc-13 (19–21). One or more of these could contribute to the complex set of responses elicited by FcγR-induced DAG production. The RasGRP proteins are a class of exchange factors for the Ras/Rap family of GTPases (22). There are four RasGRP proteins (RasGRP1 to -4), and emerging evidence has implicated RasGRP1 and RasGRP3 in T and B cell receptor signaling (23–27).The possible role of DAG-mediated signaling pathways other than PKC in phagocytosis and the subsequent inflammatory response has not been explored. Here, we provide evidence that DAG stimulates Ras and Rap1 at sites of phagocytosis, probably through RasGRPs. Last, the functional consequences of Ras and Rap1 activation were analyzed.     

Mechanism of the spatio-temporal regulation of Ras and Rap1

Epidermal growth factor (EGF) activates Ras and Rap1 at distinct intracellular regions. Here, we explored the mechanism underlying this phenomenon. We originally noticed that in cells expressing Epac, a cAMP-dependent Rap1 GEF (guanine nucleotide exchange factor), cAMP activated Rap1 at the perinuclear region, as did EGF. However, in cells expressing e-GRF, a recombinant cAMP-responsive Ras GEF, cAMP activated Ras at the peripheral plasma membrane. Based on the uniform cytoplasmic expression of Epac and e-GRF, GEF did not appear to account for the non-uniform increase in the activities of Ras and Rap1. In contrast, when we used probes with reduced sensitivity to GTPase-activating proteins (GAPs), both Ras and Rap1 appeared to be activated uniformly in the EGF-stimulated cells. Furthermore, we calculated the local rate constants of GEFs and GAPs from the video images of Ras activation and found that GAP activity was higher at the central plasma membrane than the periphery. Thus we propose that GAP primarily dictates the spatial regulation of Ras family G proteins, whereas GEF primarily determines the timing of Ras activation.     

CalDAG-GEFIII activation of Ras, R-ras, and Rap1

We characterized a novel guanine nucleotide exchange factor (GEF) for Ras family G proteins that is highly homologous to CalDAG-GEFI, a GEF for Rap1 and R-Ras, and to RasGRP/CalDAG-GEFII, a GEF for Ras and R-Ras. This novel GEF, referred to as CalDAG-GEFIII, increased the GTP/GDP ratio of Ha-Ras, R-Ras, and Rap1 in 293T cells. CalDAG-GEFIII promoted the guanine nucleotide exchange of Ha-Ras, R-Ras, and Rap1 in vitro also, indicating that CalDAG-GEFIII exhibited the widest substrate specificity among the known GEFs for Ras family G proteins. Expression of CalDAG-GEFIII was detected in the glial cells of the brain and the glomerular mesangial cells of the kidney by in situ hybridization. CalDAG-GEFIII activated ERK/MAPK most efficiently, followed by CalDAG-GEFII and CalDAG-GEFI in 293T cells. JNK activation was most prominent in cells expressing CalDAG-GEFII, followed by CalDAG-GEFIII and CalDAG-GEFI. Expression of CalDAG-GEFIII induced neuronal differentiation of PC12 cells and anchorage-independent growth of Rat1A cells less efficiently than did CalDAG-GEFII. Thus, co-activation of Rap1 by CalDAG-GEFIII apparently attenuated Ras-MAPK-dependent neuronal differentiation and cellular transformation. Altogether, CalDAG-GEFIII activated a broad range of Ras family G proteins and exhibited a biological activity different from that of either CalDAG-GEFI or CalDAG-GEFII.     

Rap1GAP interacts with RET and suppresses GDNF-induced neurite outgrowth

Glial cell line-derived neurotrophic factor (GDNF) was originally recognized for its ability to promote survival of midbrain dopaminergic neurons, but it has since been demonstrated to be crucial for the survival and differentiation of many neuronal subpopulations, including motor neurons, sympathetic neurons, sensory neurons and enteric neurons. To identify possible effectors or regulators of GDNF signaling, we performed a yeast two-hybrid screen using the intracellular domain of RET, the common signaling receptor of the GDNF family, as bait. Using this approach, we identified Rap1GAP, a GTPase-activating protein (GAP) for Rap1, as a novel RET-binding protein. Endogenous Rap1GAP co-immunoprecipitated with RET in neural tissues, and RET and Rap1GAP were co-expressed in dopaminergic neurons of the mesencephalon. In addition, overexpression of Rap1GAP attenuated GDNF-induced neurite outgrowth, whereas suppressing the expression of endogenous Rap1GAP by RNAi enhanced neurite outgrowth. Furthermore, using co-immunoprecipitation analyses, we found that the interaction between RET and Rap1GAP was enhanced following GDNF treatment. Mutagenesis analysis revealed that Tyr981 in the intracellular domain of RET was crucial for the interaction with Rap1GAP. Moreover, we found that Rap1GAP negatively regulated GNDF-induced ERK activation and neurite outgrowth. Taken together, our results suggest the involvement of a novel interaction of RET with Rap1GAP in the regulation of GDNF-mediated neurite outgrowth.     

Chaperone-mediated specificity in Ras and Rap signaling

Abstract

Ras and Rap proteins are closely related small guanosine triphosphatase (GTPases) that share similar effector-binding domains but operate in a very different signaling networks; Ras has a dominant role in cell proliferation, while Rap mediates cell adhesion. Ras and Rap proteins are regulated by several shared processes such as post-translational modification, phosphorylation, activation by guanine exchange factors and inhibition by GTPase-activating proteins. Sub-cellular localization and trafficking of these proteins to and from the plasma membrane are additional important regulatory features that impact small GTPases function. Despite its importance, the trafficking mechanisms of Ras and Rap proteins are not completely understood. Chaperone proteins play a critical role in trafficking of GTPases and will be the focus of the discussion in this work. We will review several aspects of chaperone biology focusing on specificity toward particular members of the small GTPase family. Understanding this specificity should provide key insights into drug development targeting individual small GTPases.     

Interactions of phosphatidylinositol kinase, GTPase-activating protein (GAP), and GAP-associated proteins with the colony-stimulating factor 1 receptor.

The interactions of the macrophage colony-stimulating factor 1 (CSF-1) receptor with potential targets were investigated after ligand stimulation either of mouse macrophages or of fibroblasts that ectopically express mouse CSF-1 receptors. In Rat-2 cells expressing the mouse CSF-1 receptor, full activation of the receptor and cellular transformation require exogenous CSF-1, whereas NIH 3T3 cells expressing mouse c-fms are transformed by autocrine stimulation. Activated CSF-1 receptors physically associate with a phosphatidylinositol (PI) 3'-kinase. A mutant CSF-1 receptor with a deletion of the kinase insert region was deficient in its ability to bind functional PI 3'-kinase and to induce PI 3'-kinase activity precipitable with antiphosphotyrosine antibodies. In fibroblasts, CSF-1 stimulation also induced the phosphorylation of the GTPase-activating protein (GAP)-associated protein p62 on tyrosine, although GAP itself was a relatively poor substrate. In contrast to PI 3'-kinase association, phosphorylation of p62 and GAP was not markedly affected by deletion of the kinase insert region. These results indicate that the kinase insert region selectively enhances the CSF-1-dependent association of the CSF-1 receptor with active PI 3'-kinase. The insert deletion mutant retains considerable transforming activity in NIH 3T3 cells (G. Taylor, M. Reedijk, V. Rothwell, L. Rohrschneider, and T. Pawson, EMBO J. 8:2029-2037, 1989). This mutant was more seriously impaired in Rat-2 cell transformation, although mutant-expressing Rat-2 cells still formed small colonies in soft agar in the presence of CSF-1. Therefore, phosphorylation of GAP and p62 through activation of the CSF-1 receptor does not result in full fibroblast transformation. The interaction between the CSF-1 receptor and PI 3'-kinase may contribute to c-fms fibroblast transformation and play a role in CSF-1-stimulated macrophages.     

The purification of a Rap1 GTPase-activating protein from bovine brain cytosol.

Two GTPase-activating proteins (GAPs) have been detected in extracts from bovine brain: GAP-1, which is specific for the activation of ras GTPases, and GAP-3, which is specific for the activation of the rap1 GTPases. We present a strategy for the purification to homogeneity of a cytosolic form of GAP-3 from bovine brain. The 100,000 x g supernatant from homogenized brains was chromatographed sequentially on DEAE Fast Flow, green H-E4BD Sepharose, Bio-Gel A1.5, hydroxyapatite, and phenyl-Sepharose prior to high resolution separation on Mono Q HR 5/5, phenyl-Superose HR 5/5, Mono Q PC 1.6/5, and Superose 12 PC 3.2/30. This procedure resulted in an approximately 18,000-fold purification, yielding 50 micrograms of GAP-3 from 1.6 kg of tissue. Purified cytosolic GAP-3 migrated as a single band of apparent Mr 55,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. However, on gel filtration cytosolic GAP-3 chromatographed as a dimer with an apparent Mr 92,000. Purified GAP-3 does not activate ras or rho GTPases and possesses no intrinsic GTPase activity. Amino acid sequence data indicated a proline-rich N terminus. The amino acid sequences of peptides generated by Staphylococcus aureus V8 digestion of reduced and pyridine-ethylated GAP-3 showed no similarity to the predicted primary structure of GAP-1 or any other proteins in the nucleic acid or protein data bases. By comparison with the data of Rubinfeld et al. (Rubinfeld, B., Munemitsu, S., Clark, R., Conroy, L., Watt, K., Crosier, W.J., McCormick, F., and Polakis, P. (1991) Cell 65, 1033-1042), it appears that the membrane-associated (Mr 85,000-95,000) and cytosolic forms of GAP-3 are derived from equivalent, or closely related, genes.     

Specific changes of Ras GTPase-activating protein (GAP) and a GAP-associated p62 protein during calcium-induced keratinocyte differentiation.

Induction of tyrosine phosphorylation occurs as an early and specific event in keratinocyte differentiation. A set of tyrosine-phosphorylated substrates which transduce mitogenic signals by tyrosine kinases has previously been identified. We show here that of these substrates, the Ras GTPase-activating protein, GAP, is specifically affected during calcium-induced keratinocyte differentiation. As early as 10 min after calcium addition to cultured primary mouse keratinocytes, GAP associates with tyrosine-phosphorylated proteins and translocates to the membrane. In addition, a GAP-associated protein of approximately 62 kDa (p62) becomes rapidly and heavily tyrosine phosphorylated in both membrane and cytosolic fractions. This protein corresponds to the major tyrosine-phosphorylated protein that is induced in differentiating keratinocytes as early as 5 min after calcium addition. p62 phosphorylation was not observed after exposure of these cells to epidermal growth factor, phorbol ester, or transforming growth factor beta. In contrast, PLC gamma and P13K were tyrosine phosphorylated after epidermal growth factor, but not calcium, stimulation. Thus, changes of Ras GAP and an associated p62 protein occur as early and specific events in keratinocyte differentiation and appear to involve a calcium-induced tyrosine kinase.     

The Ability of GAP1IP4BP To Function as a Rap1 GTPase-Activating Protein (GAP) Requires Its Ras GAP-Related Domain and an Arginine Finger Rather than an Asparagine Thumb

GAP1^IP4BP is a member of the GAP1 family of Ras GTPase-activating proteins (GAPs) that includes GAP1^m, CAPRI, and RASAL. Composed of a central Ras GAP-related domain (RasGRD), surrounded by amino-terminal C2 domains and a carboxy-terminal PH/Btk domain, these proteins, with the notable exception of GAP1^m, possess an unexpected arginine finger-dependent GAP activity on the Ras-related protein Rap1 (S. Kupzig, D. Deaconescu, D. Bouyoucef, S. A. Walker, Q. Liu, C. L. Polte, O. Daumke, T. Ishizaki, P. J. Lockyer, A. Wittinghofer, and P. J. Cullen, J. Biol. Chem. 281:9891-9900, 2006). Here, we have examined the mechanism through which GAP1^IP4BP can function as a Rap1 GAP. We show that deletion of domains on either side of the RasGRD, while not affecting Ras GAP activity, do dramatically perturb Rap1 GAP activity. By utilizing GAP1^IP4BP/GAP1^m chimeras, we establish that although the C2 and PH/Btk domains are required to stabilize the RasGRD, it is this domain which contains the catalytic machinery required for Rap1 GAP activity. Finally, a key residue in Rap1-specific GAPs is a catalytic asparagine, the so-called asparagine thumb. By generating a molecular model describing the predicted Rap1-binding site in the RasGRD of GAP1^IP4BP, we show that mutagenesis of individual asparagine or glutamine residues that lie in close proximity to the predicted binding site has no detectable effect on the in vivo Rap1 GAP activity of GAP1^IP4BP. In contrast, we present evidence consistent with a model in which the RasGRD of GAP1^IP4BP functions to stabilize the switch II region of Rap1, allowing stabilization of the transition state during GTP hydrolysis initiated by the arginine finger.The Ras-like family of small GTPases are ubiquitously expressed, evolutionarily conserved proteins that, by undergoing conformational changes in response to the alternate binding of GDP and GTP, function as binary switches (28, 31, 35). The GDP-bound “off” state and the GTP-bound “on” state recognize distinct effector proteins, thereby allowing the regulation of a variety of downstream signaling events (28, 31, 35). While Ras is the best-known and best-studied Ras-like GTPase, Rap1 has recently attracted considerable attention (reviewed in reference 20).Rap1 was originally identified through its ability, when overexpressed, to reverse the phenotype of K-Ras-transformed NIH 3T3 cells (19). As Ras and Rap1 have very similar effector regions, the ability of Rap1 to reverse the transformed phenotype appeared to arise through an ability to compete with K-Ras effectors. For example, Rap1 binds the Ras effector Raf1 but this does not lead to its activation (11). This is consistent with a simple model in which Rap1 functions as a Ras antagonist (6, 37). However, recent work has challenged this view. Increasing evidence points to Rap1 interacting with its own panel of effectors through which it controls cell-cell adhesion and cell-matrix interactions (reviewed in reference 20).Like that of other GTPases, the activation of Ras and Rap1 is regulated through guanine nucleotide exchange factors, which control activation by stimulating the exchange of GDP for GTP. Inactivation is driven by GTPase-activating proteins (GAPs). These enhance the intrinsic GTPase activity of Ras and Rap1, thereby leading to GTP hydrolysis. A wide variety of guanine nucleotide exchange factors and GAPs specific for these GTPases have been identified (14). Through the arrangement of different modular domains, these proteins are regulated following the activation of cell surface receptors. This occurs either through direct association with the activated receptor or indirectly through second messengers (4, 5, 14, 41).Mammalian proteins capable of functioning as Ras GAPs include NF1 (3, 27, 40), p120^GAP (38), the semaphorin 4D receptor plexin-B1 (29), and members of the GAP1 (reviewed in reference 41) and SynGAP (DAB2IP, nGAP, and SynGAP) families (10, 18, 39). These function as Ras GAPs by supplying a catalytic arginine residue—the arginine finger—into the active site of Ras. This stabilizes the transition state of the GTPase reaction, increasing the reaction rate by more than 1,000-fold (1, 33, 34).Rap1 GAPs include Rap GAPs I and II, the SPA-1 family (SPA-1, SPAR, SPAL, and E6TP1), and tuberin (16, 17, 26, 32). Unlike Ras, Rap1 does not possess the catalytic glutamine residue that is critical for GTP hydrolysis in Ras. This fundamental difference means that the mechanisms by which Ras and Rap1 GAPs function are distinct. Rap1 GAPs do not employ a catalytic arginine residue (8, 9); instead, they provide a catalytic asparagine—the asparagine thumb—to stimulate GTP hydrolysis (15). Here the asparagine carboxamide side chain has a function similar to that of the glutamine residue in Ras, stabilizing the position of the nucleophilic water and γ-phosphate in the transition complex (15, 36).Given such distinct catalytic mechanisms, surprisingly, some Ras GAPs, while having no detectable sequence homology with any Rap1 GAPs, are capable of stimulating the GTPase activity of Rap1. The first protein found to display such dual activities was GAP1^IP4BP (13) (also known as RASA3, GAPIII, and R-Ras GAP). This is a member of the GAP1 family, which also comprises GAP1^m, CAPRI, and RASAL (2, 23-25). These proteins are characterized by a domain architecture comprising amino-terminal tandem C2 domains, a highly conserved central Ras GAP-related domain (RasGRD), and a carboxy-terminal pleckstrin homology (PH) domain that is associated with a Bruton''s tyrosine kinase (Btk) motif (41). Consistent with the presence of the RasGRD, all proteins display Ras GAP activity, although each is differentially regulated following receptor stimulation (41). With the notable exception of GAP1^m, all GAP1 proteins also possess efficient Rap1 GAP activity (22). Such dual specificity is not restricted solely to GAP1 proteins. Recently, C2 domain-containing SynGAP—a neuronal Ras GAP—has also been shown to display Rap1 GAP activity (21), an activity that appears to require, alongside the RasGRD, the presence of a single C2 domain (30).Here we have examined the mechanism behind the dual Ras and Rap1 GAP activities of GAP1^IP4BP. Through the generation of a series of GAP1^IP4BP/GAP1^m chimeras, we have established that while the C2 domains of GAP1^IP4BP are required to stabilize the RasGRD, these domains do not supply catalytic residues required for Rap1 GAP activity. Rather, the Rap1 GAP catalytic machinery appears to reside solely within the RasGRD. By the site-directed mutagenesis of selected asparagine and glutamine residues within this domain—selected following the generation of a predicted molecular model of the GAP1^IP4BP RasGRD-Ras(Rap1) complex—we establish that the ability of GAP1^IP4BP to function as a Rap1 GAP does not occur via a mechanism that utilizes a classic asparagine thumb. Rather, we suggest that the GAP1^IP4BP RasGRD functions to stabilize the switch II region of Rap1 in a manner that allows a catalytic arginine finger from GAP1^IP4BP to drive the hydrolysis of GTP.