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.     

Bromodomain protein Brd4 binds to GTPase-activating SPA-1, modulating its activity and subcellular localization

Brd4 is a mammalian protein that contains a double bromodomain. It binds to chromatin and regulates cell cycle progression at multiple stages. By immunopurification and mass spectrometry, we identified a Rap GTPase-activating protein (GAP), signal-induced proliferation-associated protein 1 (SPA-1), as a factor that interacts with Brd4. SPA-1 localizes to the cytoplasm and to a lesser degree in the nucleus, while Brd4 resides in the nucleus. Bifluorescence complementation revealed that Brd4 and SPA-1 interact with each other in the nucleus of living cells. Supporting the functional importance of the interaction, Brd4 enhanced Rap GAP activity of SPA-1. Furthermore ectopic expression of SPA-1 and Brd4 redirected subcellular localization of the partner and disrupted normal cell cycle progression. These effects were, however, reversed by coexpression of the two proteins, indicating that a proper balance between Brd4 and SPA-1 in G2 is required for cell division. This work reveals a novel link between Brd4 and a GTPase-dependent mitogenic signaling pathway.     

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.     

Downregulation of Rap1GAP in Human Tumor Cells Alters Cell/Matrix and Cell/Cell Adhesion

Rap1GAP expression is decreased in human tumors. The significance of its downregulation is unknown. We show that Rap1GAP expression is decreased in primary colorectal carcinomas. To elucidate the advantages conferred on tumor cells by loss of Rap1GAP, Rap1GAP expression was silenced in human colon carcinoma cells. Suppressing Rap1GAP induced profound alterations in cell adhesion. Rap1GAP-depleted cells exhibited defects in cell/cell adhesion that included an aberrant distribution of adherens junction proteins. Depletion of Rap1GAP enhanced adhesion and spreading on collagen. Silencing of Rap expression normalized spreading and restored E-cadherin, β-catenin, and p120-catenin to cell/cell contacts, indicating that unrestrained Rap activity underlies the alterations in cell adhesion. The defects in adherens junction protein distribution required integrin signaling as E-cadherin and p120-catenin were restored at cell/cell contacts when cells were plated on poly-l-lysine. Unexpectedly, Src activity was increased in Rap1GAP-depleted cells. Inhibition of Src impaired spreading and restored E-cadherin at cell/cell contacts. These findings provide the first evidence that Rap1GAP contributes to cell/cell adhesion and highlight a role for Rap1GAP in regulating cell/matrix and cell/cell adhesion. The frequent downregulation of Rap1GAP in epithelial tumors where alterations in cell/cell and cell/matrix adhesion are early steps in tumor dissemination supports a role for Rap1GAP depletion in tumor progression.Mammalian Rap proteins Rap1a/b and Rap2a/b/c are members of the Ras superfamily of small GTPases. Rap proteins are active when bound to GTP and inactive when bound to GDP. Cellular Rap activity is regulated by the concerted action of guanine nucleotide exchange factors that activate Rap (RapGEFs) and Rap-specific GTPase-activating proteins (RapGAPs) that inactivate Rap (reviewed in reference 10). The Rap1GAP family is composed of several members, including Rap1GAP, Rap1GAPII, Spa-1/SIPA1, and E6TP1/SIPA1L1. Several lines of evidence suggest that RapGAPs function as tumor and/or invasion suppressors. Downregulation of E6TP1 by human papillomavirus protein E6 contributes to cervical cancer (20, 21), and Spa-1 deficiency in mice induces a spectrum of myelodysplastic disorders similar to chronic myelogenous leukemia (26). The SPA1 gene was identified as a candidate for the metastasis efficiency modifier locus in mice (38). Although the relevance of this observation to humans is not yet clear, single-nucleotide polymorphisms in the SPA1 gene in human breast tumors have been associated with lymph node involvement and poor survival (15). Intriguingly, Spa-1 interacts with Brd4 (18) and Rrp-1b (13), the protein products of genes associated with patterns of extracellular matrix protein gene expression characteristic of metastatic tumors (14).The RAP1GAP gene maps to 1p35-36, a chromosomal region subject to copy number alterations in human tumors (36, 49). Rap1GAP protein levels are decreased in pancreatic adenocarcinomas (53), papillary thyroid carcinomas (37, 47, 57), and melanomas (56). Rap1GAP downregulation has been shown to arise as a consequence of proteasomal degradation (46), loss of heterozygosity (37, 53), and promoter methylation (56, 57). Mutations of unknown significance in RAP1GAP have been identified in breast cancer (42). Although downregulation of Rap1GAP is frequent in human tumors, the functional significance of decreased Rap1GAP expression is unknown. Up to now, studies assessing the role of Rap1GAP in tumor cells have relied exclusively on overexpression experiments. Overexpression of Rap1GAP in oropharyngeal squamous cell (54) and pancreatic (53) carcinoma lines impaired tumor formation in mouse xenograft models. In vitro, overexpression of Rap1GAP impaired tumor cell proliferation (34, 47, 53, 54, 56) and enhanced apoptosis (34, 53, 56). In some instances, overexpression of Rap1GAP inhibited tumor cell migration and invasion (3, 47, 53, 56), while in others, it enhanced invasion (34). While these studies provide insight into cellular processes that can be deregulated by overexpression, they do not assess the significance of depletion of endogenous Rap1GAP in human tumors.Colorectal cancer (CRC) is one of the leading causes of cancer deaths worldwide. The majority of CRC deaths arise as a consequence of distant metastases, most frequently to the liver. While the genetic basis of CRC is well understood (19, 48), less is known about the events that trigger the transition to metastatic disease. We report that Rap1GAP is highly expressed in normal colonic epithelium and that its expression is profoundly decreased in primary colorectal carcinomas. As one strategy to assess the significance of Rap1GAP depletion, the expression of Rap1GAP was silenced in human colon carcinoma cells. Silencing of Rap1GAP induced marked increases in Rap1 and Rap2 activity, the first evidence that Rap1GAP is an essential negative regulator of Rap GTPases in colon cancer. Rap1 regulates inside-out signaling through integrins (reviewed in references 8, 9, and 11) and is a target of outside-in signaling via cadherins (reviewed in reference 30). Downregulation of Rap1GAP induced profound alterations in cell/matrix and cell/cell adhesion. Suppressing Rap1GAP expression enhanced adhesion and spreading on collagen. Unexpectedly, based on the role of Rap1 in promoting cell/cell adhesion, silencing of Rap1GAP impaired cell/cell adhesion. These findings demonstrate a requirement for regulated Rap activity in the maintenance of epithelial cell structure and demonstrate a heretofore unappreciated role for Rap1GAP in the regulation of cell/cell adhesion. As the dissemination of tumor cells requires the weakening of cell/cell adhesion and an enhanced ability to adhere to collagen-rich interstitial matrices, our studies identify a potential mechanism through which loss of Rap1GAP contributes to tumor progression.     

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.     

Spa-1 regulates the maintenance and differentiation of human embryonic stem cells

Human embryonic stem cells (hESCs) are pluripotent, whereby they can proliferate endlessly and differentiate into many different cell types. At the molecular level, little is known of the mechanisms underlying their capability for self-renewal and differentiation. In the present study, we established two new hESC lines (AMC-hES1 and AMC-hES2) and demonstrated the existence of a regulator that may be a key molecule in hESC dynamics. Spa-1 is a principal Ras-proximate 1 (Rap1) GTPase-activating protein in hematopoietic progenitor cells that regulates Rap1-related signal transduction and is expressed restrictively in human adult tissues (bone marrow, thymus, and spleen). To investigate its functions in hESCs, we examined spa-1 expression profiles during hESC differentiation and used RNA interference (RNAi) to downregulate spa-1 in these cells. Our results show that Spa-1 is expressed in undifferentiated hESCs and is downregulated during hESC differentiation. In addition, the process of passing from the mode of self-renewal to that of differentiation in hESCs was regulated by spa-1 via Rap1/Raf/mitogen-activated protein kinase kinase/extracellular signal-related kinase signaling. An RNAi expression vector against spa-1 (pSUPER.retro.puro) was transfected into hESCs, which were seen to differentiate into three germ layers in spite of being in the undifferentiated condition. Based on our findings, therefore, it appears that spa-1 may be involved in hESC dynamics, and our results provide fundamental information regarding the self-renewal and differentiation of hESCs.     

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.     

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.     

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 COOH-terminal domain of the Rap1A (Krev-1) protein is isoprenylated and supports transformation by an H-Ras:Rap1A chimeric protein.

Although the Rap1A protein resembles the oncogenic Ras proteins both structurally and biochemically, Rap1A exhibits no oncogenic properties. Rather, overexpression of Rap1A can reverse Ras-induced transformation of NIH 3T3 cells. Because the greatest divergence in amino acid sequence between Ras and Rap1A occurs at the COOH terminus, the role of this domain in the opposing biological activities of these proteins was examined. COOH-terminal processing and membrane association of Rap1A were studied by constructing and expressing a chimeric protein (composed of residues 1 to 110 of an H-Ras activated by a Leu-61 mutation attached to residues 111 to 184 of Rap1A) in NIH 3T3 cells and a full-length human Rap1A protein in a baculovirus-Sf9 insect cell system. Both the chimeric protein and the full-length protein were synthesized as a 23-kDa cytosolic precursor that rapidly bound to membranes and was converted into a 22-kDa form that incorporated label derived from [3H]mevalonate. The mature 22-kDa form also contained a COOH-terminal methyl group. Full-length Rap1A, expressed in insect cells, was modified by a C20 (geranylgeranyl) isoprenoid. In contrast, H-Ras, expressed in either Sf9 insect or NIH 3T3 mouse cells contained a C15 (farnesyl) group. This suggests that the Rap1A COOH terminus is modified by a prenyl transferase that is distinct from the farnesyl transferase that modifies Ras proteins. Nevertheless, in NIH 3T3 cells the chimeric Ras:Rap1A protein retained the transforming activity conferred by the NH2-terminal Ras61L domain. This demonstrates that the modifications and localization signals of the COOH terminus of Rap1A can support the interactions between H-Ras and membranes that are required for transformation.     

Identification of Rgl3 as a potential binding partner for Rap-family small G-proteins and profilin II

A cDNA encoding a RalGDS-related protein, Rgl3, was isolated by yeast two-hybrid screening using a small G-protein, Rap1, as a bait. Rgl3 mRNA is commonly detectable in several visceral organs (e.g. kidney, heart, liver, and lung) in the mouse and human. The Rgl3 protein mainly localizes in the cytoplasm when expressed in fibroblasts. Yeast two-hybrid assay indicated that Rgl3 could interact with Rap1, Rap2, H-Ras, N-Ras, and R-Ras but failed to interact efficiently with Ral and Rho. Interestingly, Rgl3 was found to affect cell morphology in two assay systems in culture. First, Rgl3 suppressed cell-spreading induced by Rap1, R-Ras, or C3G-CAAX (a membrane-targeted Rap/R-Ras activator) in HEK-293 cells. Second, Rgl3 enhanced the focus-formation induced by oncogenic H-Ras and N-Ras mutants in NIH3T3 cells. Moreover, we identified profilin II as a potential binding partner for Rgl3 by yeast two-hybrid screening. This interaction requires the characteristic proline cluster in the Rgl3 amino-terminal domain. Profilin II and Rgl3 co-operated in enhancing the N-Ras-induced focus-formation. These findings raise the possibility that Rgl3 mediates interaction between Ras/Rap-family proteins and profilin II, an important activator of actin polymerization.     

Cyclic AMP-mediated inhibition of cell growth requires the small G protein Rap1

In many normal and transformed cell types, the intracellular second messenger cyclic AMP (cAMP) blocks the effects of growth factors and serum on mitogenesis, proliferation, and cell cycle progression. cAMP exerts these growth-inhibitory effects via inhibition of the mitogen-activated protein (MAP) kinase cascade. Here, using Hek293 and NIH 3T3 cells, we show that cAMP's inhibition of the MAP kinase cascade is mediated by the small G protein Rap1. Activation of Rap1 by cAMP induces the association of Rap1 with Raf-1 and limits Ras-dependent activation of ERK. In NIH 3T3 cells, Rap1 is required not only for cAMP's inhibition of ERK activation but for inhibition of cell proliferation and mitogenesis as well.     

Adipocytes contain a novel complex similar to the tuberous sclerosis complex

Recently we identified a novel 250 kDa protein in adipocytes that is a substrate for the insulin-activated protein kinase Akt. We refer to this protein as AS250 for Akt substrate of 250 kDa. AS250 has a predicted GTPase activating protein (GAP) domain at its carboxy terminus. This domain shows some homology to the GAP domains for Rheb at the carboxy terminus of the protein tuberin and for Rap1 in the protein Rap1 GAP. The present study further characterizes AS250. The cDNA sequence for human AS250 is reported, and the sites that undergo phosphorylation upon insulin treatment of adipocytes have been identified by tandem mass spectrometry. We have found that in adipocytes AS250 exists as a complex with a novel protein of 1484 amino acids known as KIAA1219. The complex of AS250 with KIAA1219 is notably similar to the important regulatory complex of the protein tuberin with hamartin (the tuberous sclerosis complex), in the size of its subunits, the location of the GAP domain, and its phosphorylation by Akt. In an effort to detect the cellular role of the AS250/KIAA1219 complex, we generated 3T3-L1 adipocytes that largely lack AS250 by shRNA knockdown and examined several insulin-dependent effects. The knockdown of AS250 had no effect on insulin activation of the kinases, Akt, 70 kDa S6 kinase, or ERK1/2, or on insulin-stimulated actin bundling, and it had only a slight effect on insulin-stimulated GLUT4 translocation.     

GAP1 family members constitute bifunctional Ras and Rap GTPase-activating proteins

GAP1(IP4BP) is a member of the GAP1 family of Ras GTPase-activating proteins (Ras GAPs) that includes GAP1(m), CAPRI, and RASAL. Composed of a central Ras GAP domain, surrounded by amino-terminal C(2) domains and a carboxyl-terminal pleckstrin homology/Bruton's tyrosine kinase domain, GAP1(IP4BP) has previously been shown to possess an unexpected GAP activity on the Ras-related protein Rap, besides the predicted Ras GAP activity (Cullen, P. J., Hsuan, J. J., Truong, O., Letcher, A. J., Jackson, T. R., Dawson, A. P., and Irvine, R. F. (1995) Nature 376, 527-530). Here we have shown that GAP1(IP4BP) is indeed an efficient Ras/Rap GAP, having K(m)s of 213 and 42 microm and estimated k(cat)s of 48 and 16 s(-1) for Ras and Rap, respectively. For this dual activity, regions outside the Ras GAP domain are required, as the isolated domain (residues 291-569) retains a pronounced Ras GAP activity yet has very low activity toward Rap. Interestingly, mutagenesis of the Ras GAP arginine finger, and surrounding residues important in Ras binding, inhibit both Ras and Rap GAP activity of GAP1(IP4BP). Although the precise details by which GAP1(IP4BP) can function as a Rap GAP remain to be determined, these data are consistent with Rap associating with GAP1(IP4BP) through the Ras-binding site within the Ras GAP domain. Finally, we have established that such dual Ras/Rap GAP activity is not restricted to GAP1(IP4BP). Although GAP1(m) appears to constitute a specific Ras GAP, CAPRI and RASAL display dual activity. For CAPRI, its Rap GAP activity is modulated upon its Ca(2+)-induced association with the plasma membrane.     

Integrin beta 1 cytoplasmic domain dominant negative effects revealed by lysophosphatidic acid treatment.

Integrin receptors localize to focal contact sites and interact with the cytoskeleton via the beta 1 cytoplasmic domain. To study the role of this domain in adhesion, we have expressed in NIH 3T3 cells a cDNA consisting of the interleukin 2 receptor alpha subunit extracellular and transmembrane domains, connected to the integrin beta 1 cytoplasmic domain (IL2R-beta 1). Since the extracellular domain of the chimeric protein has no role in adhesion, this protein could uncouple adhesion from intracellular events. As expected, in a cell line expressing IL2R-beta 1, this chimera was directed to focal contact sites. Unexpectedly, the cells exhibited normal adhesion to fibronectin (FN). However, when a rapid reorganization of the cytoskeleton was induced using lysophosphatidic acid (LPA), IL2R-beta 1 cells detached from FN in contrast to wild-type cells. The detachment in response to LPA could be prevented with cytochalasin D, an inhibitor of actin polymerization. These results imply that a beta 1 cytoplasmic domain, which is uncoupled from adhesion, can compete with the cytoplasmic domain of native integrin beta 1 for cytoskeletal proteins. As a consequence, the IL2R-beta 1 protein acts as a dominant negative effector of adhesion by disrupting the integrin-cytoskeleton connection.     

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.     

Characterization of proteins that interact with the GTP-bound form of the regulatory GTPase Ran in Arabidopsis

Ran, a small soluble GTP-binding protein, has been shown to be essential for the nuclear translocation of proteins and it is also thought to be involved in regulating cell cycle progression in mammalian and yeast cells. Genes encoding Ran-like proteins have been isolated from different higher plant species. Overexpression of plant Ran cDNAs, similarly to their mammalian/yeast homologues, suppresses the phenotype of the pim46-1 cell cycle mutant in yeast cells. The mammalian/yeast Ran proteins have been shown to interact with a battery of Ran-binding proteins, including the guanidine nucleotide exchange factor RCC1, the GTPase-activating Ran-GAP, nucleoporins and other Ran-binding proteins (RanBPs) specific for Ran-GTP. Here, the characterization of the first Ran-binding proteins from higher plants is reported. The yeast two-hybrid system was used to isolate cDNA clones encoding proteins of approximately 28 kDa (At-RanBP1a, At-RanBP1b) that interact with the GTP-bound forms of the Ran1, Ran2 and Ran3 proteins of Arabidopsis thaliana . The deduced amino acid sequences of the At-RanBP1s display high similarity (60%) to mammalian/yeast RanBP1 proteins and contain the characteristic Ran-binding domains. Furthermore, interaction of the plant Ran and RanBP1 proteins, is shown to require the acidic C-terminal domain (-DEDDDL) of Ran proteins in addition to the presence of an intact Ran-binding domain. In whole cell extracts, the GST-RanBP1a fusion protein binds specifically to GTP-Ran and will not interact with Rab/Ypt-type small GTP-binding proteins. Finally, in good agreement with their proposed biological function, the At-Ran and the At-RanBP genes are expressed coordinately and show the highest level of expression in meristematic tissues.     

Cloning and characterization of a cDNA encoding Ran binding protein from wheat.

Ran, which functions in nucleocytoplasmic transport and mitosis, binds to and is regulated in part by Ran binding protein (RanBP). A RanBP cDNA (TaRanBP1) was isolated from a wheat cDNA library using RT-PCR product as a probe. The predicted amino acid sequence of TaRanBP1 is over 60% identity to AtRanBP1 from Arabidopsis and also with considerable similarity to human and fungi RanBPs. TaRanBP1 gene was expressed ubiquitously in roots, leaves and stems, with a similar abundance in these tissues. Phylogenetic reconstruction of TaRanBP1 with 32 other RanBPs from 26 species of organisms revealed that RanBPs from plants, animals and fungi clustered as the distinct groups, intraspecies isoforms were not developed for RanBPs, contrast with most other ancestral genes. Structural analysis revealed that all RanBPs were highly conserved in the middle region of their amino acid sequence, which included Ran binding domain and the three conserved motifs that have the essential roles in binding with Ran protein and promotion of GTP hydrolysis by the Ran/RanGAP/RanBP complex. However, N-terminus and C-terminus exhibited very low similarity between the different RanBPs. The different structures in N-terminus and C-terminus of RanBPs are likely to direct the Ran into the specific physiological processes and subsequently exhibit the different roles in different organisms.     

Overexpression of Nd1-s, a variant form of new kelch family protein, perturbs the cell cycle progression of fibroblasts

The murine Nd1 gene encodes two forms of protein, Nd1-L and Nd1-S, both of which share the BTB/POZ domain, but Nd1-S lacks the kelch repeats. Although Nd1-L ubiquitously expresses, localizes in the cytoplasm and functions as a stabilizer of actin filaments, expression and function of Nd1-S were unknown. Here we show that Nd1-S were expressed in all tissues examined and localized in the nucleus as a speckled-like pattern. Furthermore, overexpression of Nd1-S perturbed cell growth of NIH3T3 cells at the G1/S phase of the cell cycle. These results suggest that Nd1-S may play a role in cell cycle progression in the nucleus.