Molecular cloning of a novel mitogen-inducible nuclear protein with a Ran GTPase-activating domain that affects cell cycle progression.

We have cloned a novel cDNA (Spa-1) which is little expressed in the quiescent state but induced in the interleukin 2-stimulated cycling state of an interleukin 2-responsive murine lymphoid cell line by differential hybridization. Spa-1 mRNA (3.5 kb) was induced in normal lymphocytes following various types of mitogenic stimulation. In normal organs it is preferentially expressed in both fetal and adult lymphohematopoietic tissues. A Spa-1-encoded protein of 68 kDa is localized mostly in the nucleus. Its N-terminal domain is highly homologous to a human Rap1 GTPase-activating protein (GAP), and a fusion protein of this domain (SpanN) indeed exhibited GAP activity for Rap1/Rsr1 but not for Ras or Rho in vitro. Unlike the human Rap1 GAP, however, SpanN also exhibited GAP activity for Ran, so far the only known Ras-related GTPase in the nucleus. In the presence of serum, stable Spa-1 cDNA transfectants of NIH 3T3 cells (NIH/Spa-1) hardly overexpressed Spa-1 (p68), and they grew as normally as did the parental cells. When NIH/Spa-1 cells were serum starved to be arrested in the G1/G0 phase of the cell cycle, however, they, unlike the control cells, exhibited progressive Spa-1 p68 accumulation, and following the addition of serum they showed cell death resembling mitotic catastrophes of the S phase during cell cycle progression. The results indicate that the novel nuclear protein Spa-1, with a potentially active Ran GAP domain, severely hampers the mitogen-induced cell cycle progression when abnormally and/or prematurely expressed. Functions of the Spa-1 protein and its regulation are discussed in the context of its possible interaction with the Ran/RCC-1 system, which is involved in the coordinated nuclear functions, including cell division.     

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.     

AF-6 controls integrin-mediated cell adhesion by regulating Rap1 activation through the specific recruitment of Rap1GTP and SPA-1

In the present study, we showed that SPA-1, a Rap1 GTPase-activating protein (GAP), was bound to a cytoskeleton-anchoring protein AF-6. SPA-1 and AF-6 were co-immunoprecipitated in the 293T cells transfected with both cDNAs as well as in normal thymocytes. In vitro binding studies using truncated fragments and their mutants suggested that SPA-1 was bound to the PDZ domain of AF-6 via probable internal PDZ ligand motif within the GAP-related domain. The motif was conserved among Rap1 GAPs, and it was shown that rapGAP I was bound to AF-6 comparably with SPA-1. RapV12 was also bound to AF-6 via the N-terminal domain, and SPA-1 and RapV12 were co-immunoprecipitated only in the presence of AF-6, indicating that they could be brought into close proximity via AF-6 in cells. Immunostaining analysis revealed that SPA-1 and RapV12 were co-localized with AF-6 at the cell attachment sites. In HeLa cells expressing SPA-1 in a tetracycline-regulatory manner, expression of AF-6 inhibited endogenous Rap1GTP and beta(1) integrin-mediated cell adhesion to fibronectin in SPA-1-induced conditions, whereas it affected neither of them in SPA-1-repressed conditions. These results suggested that AF-6 could control integrin-mediated cell adhesion by regulating Rap1 activation through the recruitment of both SPA-1 and Rap1GTP via distinct domains.     

Aquaporin-2 trafficking is regulated by PDZ-domain containing protein SPA-1

Targeted positioning of water channel aquaporin-2 (AQP2) strictly regulates body water homeostasis. Trafficking of AQP2 to the apical membrane is critical to the reabsorption of water in renal collecting ducts. Controlled apical positioning of AQP2 suggests the existence of proteins that interact with AQP2. A biochemical search for AQP2-interacting proteins led to the identification of PDZ-domain containing protein, signal-induced proliferation-associated gene-1 (SPA-1) which is a GTPase-activating protein (GAP) for Rap1. The distribution of SPA-1 coincided with that of AQP2 in renal collecting ducts. The site of colocalization was concomitantly relocated by hydration status. AQP2 trafficking to the apical membrane was inhibited by the SPA-1 mutant lacking Rap1GAP activity and by the constitutively active mutant of Rap1. AQP2 trafficking was impaired in SPA-1-deficient mice. Our results show that SPA-1 directly binds to AQP2 and regulates at least in part AQP2 trafficking.     

Thyroid-stimulating hormone/cAMP and glycogen synthase kinase 3beta elicit opposing effects on Rap1GAP stability

Beyond regulating Rap activity, little is known regarding the regulation and function of the Rap GTPase-activating protein Rap1GAP. Tuberin and E6TP1 protein levels are tightly regulated through ubiquitin-mediated proteolysis. A role for these RapGAPs, along with SPA-1, as tumor suppressors has been demonstrated. Whether Rap1GAP performs a similar role was investigated. We now report that Rap1GAP protein levels are dynamically regulated in thyroid-stimulating hormone (TSH)-dependent thyroid cells. Upon TSH withdrawal, Rap1GAP undergoes a net increase in phosphorylation followed by proteasome-mediated degradation. Sequence analysis identified two putative destruction boxes in the Rap1GAP C-terminal domain. Glycogen synthase kinase 3beta (GSK3beta) phosphorylated Rap1GAP immunoprecipitated from thyroid cells, and GSK3beta inhibitors prevented phosphorylation and degradation of endogenous Rap1GAP. Co-expression of GSK3beta and Rap1GAP in human embryonic kidney 293 cells stimulated proteasome-dependent Rap1GAP turnover. Mutational analysis established a role for serine 525 in the regulation of Rap1GAP stability. Overexpression of Rap1GAP in thyroid cells impaired TSH/cAMP-stimulated p70S6 kinase activity and cell proliferation. These data are the first to show that Rap1GAP protein levels are tightly regulated and are the first to support a role for Rap1GAP as a tumor suppressor.     

Functional interaction between Galpha(z) and Rap1GAP suggests a novel form of cellular cross-talk

G(z) is a member of the G(i) family of trimeric G proteins whose primary role in cell physiology is still unknown. In an ongoing effort to elucidate the cellular functions of G(z), the yeast two-hybrid system was employed to identify proteins that specifically interact with a mutationally activated form of Galpha(z). One of the molecules uncovered in this screen was Rap1GAP, a previously identified protein that specifically stimulates GTP hydrolytic activity of the monomeric G protein Rap1 and thus is believed to function as a down-regulator of Rap1 signaling. Like G(z), the precise role of Rap1 in cell physiology is poorly understood. Biochemical analysis using purified recombinant proteins revealed that the physical interaction between Galpha(z) and Rap1GAP blocks the ability of RGSs (regulators of G protein signaling) to stimulate GTP hydrolysis of the alpha subunit, and also attenuates the ability of activated Galpha(z) to inhibit adenylyl cyclase. Structure-function analyses indicate that the first 74 amino-terminal residues of Rap1GAP, a region distinct from the catalytic core domain responsible for the GAP activity toward Rap1, is required for this interaction. Co-precipitation assays revealed that Galpha(z), Rap1GAP, and Rap1 can form a stable complex. These data suggest that Rap1GAP acts as a signal integrator to somehow coordinate and/or integrate G(z) signaling and Rap1 signaling in cells.     

Rap1 and SPA-1 in hematologic malignancy

Rap1 is a member of the Ras family of GTPases and, depending on the cellular context, has an important role in the regulation of proliferation or cell adhesion. In lymphohematopoietic tissues, SPA-1 is a principal Rap1 GTPase-activating protein. Mice that are deficient for the SPA-1 gene develop age-dependent progression of T-cell immunodeficiency followed by a spectrum of late onset myeloproliferative disorders, mimicking human chronic myeloid leukemia. Recent studies reveal that deregulated Rap1 activation in SPA-1-deficient mice causes enhanced expansion of the bone marrow hematopoietic progenitors, but induces progressive unresponsiveness or anergy in T cells. Rap1 and its regulator, SPA-1, could, therefore, provide unique molecular targets for the control of human hematologic malignancy.     

A Mammalian bromodomain protein,brd4, interacts with replication factor C and inhibits progression to S phase

Brd4 belongs to the BET family of nuclear proteins that carry two bromodomains implicated in the interaction with chromatin. Expression of Brd4 correlates with cell growth and is induced during early G(1) upon mitogenic stimuli. In the present study, we investigated the role of Brd4 in cell growth regulation. We found that ectopic expression of Brd4 in NIH 3T3 and HeLa cells inhibits cell cycle progression from G(1) to S. Coimmunoprecipitation experiments showed that endogenous and transfected Brd4 interacts with replication factor C (RFC), the conserved five-subunit complex essential for DNA replication. In vitro analysis showed that Brd4 binds directly to the largest subunit, RFC-140, thereby interacting with the entire RFC. In line with the inhibitory activity seen in vivo, recombinant Brd4 inhibited RFC-dependent DNA elongation reactions in vitro. Analysis of Brd4 deletion mutants indicated that both the interaction with RFC-140 and the inhibition of entry into S phase are dependent on the second bromodomain of Brd4. Lastly, supporting the functional importance of this interaction, it was found that cotransfection with RFC-140 reduced the growth-inhibitory effect of Brd4. Taken as a whole, the present study suggests that Brd4 regulates cell cycle progression in part by interacting with RFC.     

Prostaglandin E2 regulates renal cell carcinoma invasion through the EP4 receptor-Rap GTPase signal transduction pathway

Prognosis for patients with early stage kidney cancer has improved, but the treatment options for patients with locally advanced disease and metastasis remain few. Understanding the molecular mechanisms that regulate invasion and metastasis is critical for developing successful therapies to treat these patients. Proinflammatory prostaglandin E(2) plays an important role in cancer initiation and progression via activation of cognate EP receptors that belong to the superfamily of G protein-coupled receptors. Here we report that prostaglandin E(2) promotes renal cancer cell invasion through a signal transduction pathway that encompasses EP4 and small GTPase Rap. Inactivation of Rap signaling with Rap1GAP, like inhibition of EP4 signaling with ligand antagonist or knockdown with shRNA, reduces the kidney cancer cell invasion. Human kidney cells evidence increased EP4 and decreased Rap1GAP expression levels in the malignant compared with benign samples. These results support the idea that targeted inhibition of EP4 signaling and restoration of Rap1GAP expression constitute a new strategy to control kidney cancer progression.     

PLK1 and β-TrCP-Dependent Ubiquitination and Degradation of Rap1GAP Controls Cell Proliferation

Rap1GAP is a GTPase-activating protein (GAP) that specifically stimulates the GTP hydrolysis of Rap1 GTPase. Although Rap1GAP is recognized as a tumor suppressor gene and downregulated in various cancers, little is known regarding the regulation of Rap1GAP ubiquitination and degradation under physiological conditions. Here, we demonstrated that Rap1GAP is ubiquitinated and degraded through proteasome pathway in mitosis. Proteolysis of Rap1GAP requires the PLK1 kinase and β-TrCP ubiquitin ligase complex. We revealed that PLK1 interacts with Rap1GAP in vivo through recognition of an SSP motif within Rap1GAP. PLK1 phosphorylates Ser525 in conserved ₅₂₄DSGHVS₅₂₉ degron of Rap1GAP and promotes its interaction with β-TrCP. We also showed that Rap1GAP was a cell cycle regulator and that tight regulation of the Rap1GAP degradation in mitosis is required for cell proliferation.     

Regulation by Afadin of Cyclical Activation and Inactivation of Rap1, Rac1, and RhoA Small G Proteins at Leading Edges of Moving NIH3T3 Cells

Cyclical activation and inactivation of Rho family small G proteins, such as Rho, Rac, and Cdc42, are needed for moving cells to form leading edge structures in response to chemoattractants. However, the mechanisms underlying the dynamic regulation of their activities are not fully understood. We recently showed that another small G protein, Rap1, plays a crucial role in the platelet-derived growth factor (PDGF)-induced formation of leading edge structures and activation of Rac1 in NIH3T3 cells. We showed here that knockdown of afadin, an actin-binding protein, in NIH3T3 cells resulted in a failure to develop leading edge structures in association with an impairment of the activation of Rap1 and Rac1 and inactivation of RhoA in response to PDGF. Overexpression of a constitutively active mutant of Rap1 (Rap1-CA) and knockdown of SPA-1, a Rap1 GTPase-activating protein that was negatively regulated by afadin by virtue of binding to it, in afadin-knockdown NIH3T3 cells restored the formation of leading edge structures and the reduction of the PDGF-induced activation of Rac1 and inactivation of RhoA, suggesting that the inactivation of Rap1 by SPA-1 is responsible for inhibition of the formation of leading edge structures. The effect of Rap1-CA on the restoration of the formation of leading edge structures and RhoA inactivation was diminished by additional knockdown of ARAP1, a Rap-activated Rho GAP, which localized at the leading edges of moving NIH3T3 cells. These results indicate that afadin regulates the cyclical activation and inactivation of Rap1, Rac1, and RhoA through SPA-1 and ARAP1.Cell migration is a spatiotemporally regulated process involving the formation and disassembly of protrusions, such as filopodia and lamellipodia, ruffles, focal complexes, and focal adhesions. At the leading edges of moving cells, the continuous formation and disassembly of these protrusive structures are tightly regulated by the actions of the Rho family small G proteins, including RhoA, Rac1, and Cdc42. RhoA regulates the formation of stress fibers and focal adhesions, whereas Rac1 and Cdc42 regulate the formation of lamellipodia and filopodia, respectively (1, 2). In addition, both Rac1 and Cdc42 regulate the formation of focal complexes (3, 4). In order to have cells keep moving, each member of the Rho family small G proteins should cyclically be active and inactive as these leading edge structures are dynamically formed and disassembled. Rac1 and Cdc42 must be activated and RhoA must be inactivated at focal complexes, and vice versa at focal adhesions. Thus, the cyclical activation and inactivation of the Rho family small G proteins are critical for turnover of the transformation of focal complexes into focal adhesions during cell movement. The activities of these small G proteins are tightly regulated by guanine nucleotide exchange factors and GTPase-activating proteins (GAPs).² It is likely that signals from receptors and integrins cooperatively regulate the dynamics of this spatial and temporal activation and inactivation of the Rho family small G proteins. However, the molecular mechanisms of their cyclical activation and inactivation through the regulation of guanine nucleotide exchange factors and GAPs at the leading edges remain largely unknown.We recently showed that platelet-derived growth factor (PDGF) receptor (PDGFR), integrin α_vβ₃, and Necl-5 associate with each other and form a complex and that this complex is clustered at the leading edges of directionally moving NIH3T3 cells in response to PDGF (5, 6). We also demonstrated that PDGF induces the activation of Rap1, which then induces the activation of Rac1 (7). Overexpression of Rap1GAP to inactivate Rap1 inhibits the PDGF-induced formation of leading edge structures, cell movement, and activation of Rac1, suggesting that, in addition to the activation of Rap1, the subsequent activation of Rac1 and presumably the inactivation of RhoA may be critical for the PDGF-induced migration of NIH3T3 cells.Afadin is a nectin- and F-actin-binding protein that is involved in the formation of adherens junctions in cooperation with nectin and cadherin (8). Afadin has multiple domains: two Ras association (RA) domains, a forkhead-associated domain, a dilute domain, a PSD-95-Dlg-1-ZO-1 domain, three proline-rich domains, and an F-actin-binding domain at the C terminus and localizes to adherens junctions in epithelial cells (9). Afadin-knock-out mice showed impaired formation of the cell-cell junction during embryogenesis (10, 11). Although Ras small G protein was initially identified as an interacting molecule with the RA domain of afadin (12), other studies demonstrate that afadin binds GTP-bound Rap1 with a higher affinity than GTP-bound Ras or GTP-bound Rap2 (13, 14). In addition to the functional role of afadin in the organization of cell-cell adhesion, we recently found that, in NIH3T3 cells that do not form cell-cell junctions, afadin did not associate with nectin, localized at the leading edges during cell movement, and was involved in their directional, but not random, movement. The interaction of afadin with Rap1 at the leading edge was necessary for the PDGF-induced directional movement of NIH3T3 cells. Thus, in addition to that in the formation of adherens junctions, afadin plays another role in directional cell movement in NIH3T3 cells.In a series of studies using afadin-knockdown NIH3T3 cells, we found that neither lamellipodia, ruffles, nor focal complexes are formed, suggesting that Rap1 may be inactivated and, conversely, RhoA may be activated in the reduced state of afadin. Here we first examined this possibility and found that Rap1 is indeed inactivated, whereas RhoA is activated in afadin-knockdown NIH3T3 cells. To understand the mechanisms of how the activities of Rap1 and RhoA are regulated in afadin-knockdown NIH3T3 cells, we searched for afadin-interacting proteins that could potentially regulate Rap1 activity and sought Rap1 targets that might regulate RhoA activity. We focused on SPA-1 and ARAP1 and found that these proteins coordinately regulate the activities of these small G proteins. SPA-1 is a GAP for Rap1 that interacts with afadin (15), whereas ARAP1 is a Rho GAP that binds Rap1 and could be activated by virtue of this binding (16). We describe here how afadin regulates the cyclical activation and inactivation of Rap1, Rac1, and RhoA through SPA-1 and ARAP1 at the leading edges of moving NIH3T3 cells. We conclude that afadin is critical for the coordinated regulation of the activation of Rap1 and Rac1 and subsequent inactivation of RhoA necessary for cell movement.     

Activation of Gz attenuates Rap1-mediated differentiation of PC12 cells

We previously identified a specific activation-dependent interaction between the alpha subunit of the heterotrimeric G protein, G(z), and a regulator of Rap1 signaling, Rap1GAP (Meng, J., Glick, J. L., Polakis, P., and Casey, P. J. (1999) J. Biol. Chem. 274, 36663-36669). We now demonstrate that activated forms of Galpha(z) are able to recruit Rap1GAP from a cytosolic location to the membrane. Using PC12 cells as a model for neuronal differentiation, the influence of G(z) activation on Rap1-mediated cell differentiation was examined. Introduction of constitutively-activated Galpha(z) into PC12 cells markedly attenuated the differentiation process of these cells induced by a cAMP analogue. Treatment of PC12 cells expressing wild type Galpha(z) with a specific agonist to the alpha(2A)-adrenergic receptor also attenuated cAMP-induced PC12 cell differentiation, demonstrating that receptor-mediated activation of G(z) was also effective in this regard. Furthermore, activation of G(z) decreased the ability of the cAMP analogue to trigger both Rap1 and extracellular-regulated kinase (ERK) activation. Differentiation of PC12 cells induced by nerve growth factor (NGF) is also thought to be a Rap1-mediated process, and G(z) activation was found to attenuate this process as well. Rap1 activation, ERK phosphorylation, and PC12 cell differentation induced by NGF treatment were all significantly attenuated by either transfection of constitutively activated Galpha(z) or receptor-mediated G(z) activation. Based on these findings, a model is proposed in which activation of G(z) results in recruitment of Rap1GAP to the membrane where it can effectively down-regulate Rap1 signaling. The implications of these findings in regard to a possible role for G(z) in neuronal development are discussed.     

RA-RhoGAP, Rap-activated Rho GTPase-activating protein implicated in neurite outgrowth through Rho

Rap1 and Rho small G proteins have been implicated in the neurite outgrowth, but the functional relationship between Rap1 and Rho in the neurite outgrowth remains to be established. Here we identified a potent Rho GTPase-activating protein (GAP), RA-RhoGAP, as a direct downstream target of Rap1 in the neurite outgrowth. RA-RhoGAP has the RA and GAP domains and showed GAP activity specific for Rho, which was enhanced by the binding of the GTP-bound active form of Rap1 to the RA domain. Overexpression of RA-RhoGAP induced inactivation of Rho for promoting the neurite outgrowth in a Rap1-dependent manner. Knockdown of RA-RhoGAP reduced the Rap1-induced neurite outgrowth. These results indicate that RA-RhoGAP transduces a signal from Rap1 to Rho and regulates the neurite outgrowth.     

Modulation of rap activity by direct interaction of Galpha(o) with Rap1 GTPase-activating protein.

We used the yeast two-hybrid system to identify proteins that interact directly with Galpha(o). Mutant-activated Galpha(o) was used as the bait to screen a cDNA library from chick dorsal root ganglion neurons. We found that Galpha(o) interacted with several proteins including Gz-GTPase-activating protein (Gz-GAP), a new RGS protein (RGS-17), a novel protein of unknown function (IP6), and Rap1GAP. This study focuses on Rap1GAP, which selectively interacts with Galpha(o) and Galpha(i) but not with Galpha(s) or Galpha(q). Rap1GAP interacts more avidly with the unactivated Galpha(o) as compared with the mutant (Q205L)-activated Galpha(o). When expressed in HEK-293 cells, unactivated Galpha(o) co-immunoprecipitates with the Rap1GAP. Expression of chick Rap1GAP in PC-12 cells inhibited activation of Rap1 by forskolin. When unactivated Galpha(o) was expressed, the amount of activated Rap1 was greatly increased. This effect was not observed with the Q205L-Galpha(o). Expression of unactivated Galpha(o) stimulated MAP-kinase (MAPK1/2) activity in a Rap1GAP-dependent manner. These results identify a novel function of Galpha(o), which in its resting state can sequester Rap1GAP thereby regulating Rap1 activity and consequently gating signal flow from Rap1 to MAPK1/2. Thus, activation of G(o) could modulate the Rap1 effects on a variety of cellular functions.     

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.     

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.     

Trafficking mechanism of water channel aquaporin-2

Targeted positioning of the water channel AQP2 (aquaporin-2) strictly regulates body water homoeostasis. Trafficking of AQP2 to the apical membrane is critical for the reabsorption of water in renal collecting ducts. In addition to the cAMP-mediated effect of vasopressin on AQP2 trafficking to the apical membrane, other signalling cascades can also induce this sorting. Recently, AQP2-binding proteins which could regulate this trafficking have been discovered; SPA-1 (signal-induced proliferation-associated gene-1), a GAP (GTPase-activating protein) for Rap1, and the cytoskeletal protein actin. This review summarizes recent advances related to the trafficking mechanisms of AQP2.