Rap1 Mutant Research Articles

Determining the role of the Ras family GTPase Rap1 in regulating mTORC2 activity and function in cell migration

Directed cell migration is an important biological process that is necessary for the embryonic development, maintenance of multicellular organisms and immune response to external stimuli. Dysregulation of cell migration is implicated in the onset and progression of diseases including cardiovascular diseases and cancer metastasis. Many studies have identified genes and proteins that are important for directed cell migration, but the process remains incompletely understood. The mechanistic Target of Rapamycin Complex 2 (mTORC2) is a complex of proteins that have been identified as important players in cell migration. mTORC2 is known to have a role in regulating F‐actin organization and actomyosin (MyoII). Moreover, there is increasing evidence that shows a correlation between mTORC2 and cancer cell migration and invasion. Despite this knowledge, the role and regulation of mTORC2 is not fully understood. Recently, we found that Rap1, a member of the Ras superfamily of small GTPases with conserved roles in cytoskeleton remodeling and cell adhesion, is a conserved binding partner of mTORC2 component RIP3/SIN1. We previously showed that Rap1 positively regulates mTORC2 activation in response to the chemoattractant cAMP in the model organism Dictyostelium discoideum. Moreover, we recently found that expression of a constitutively active Rap1 mutant (Rap1CA) potentiates the insulin‐induced activation of mTORC2 in human HEK293 cells. We then undertook a study to investigate if Rap1 regulation of mTORC2 is part of a mechanism involved in regulating human cell migration. We observed that basal mTORC2 activity, as well as that induced by several potential promigratory signals, including insulin, epidermal growth factor (EGF), insulin‐like growth factor‐1 (IGF‐1), and lysophosphatidic acid (LPA), was increased by the overexpression of wild‐type Rap1 or Rap1CA. Therefore, these observations suggest that Rap1 plays a role in positively regulating mTORC2 activation in HEK293 cells in response to stimulation of promigratory signals, similar to our previous observations made in Dictyostelium. Ongoing studies now aim at determining how Rap1 regulates the function of mTORC2 in cell migration.

Age-Related Phenotypes Linked to Aberrant Expression and Localization of a Telomeric Protein

Telomere attrition is associated with telomere biology disorders and age-related diseases. In telomere biology disorders, telomere uncapping induces a DNA damage response that evokes cell death or senescence. However, a causal mechanism for telomere attrition in age-related diseases remains elusive. Telomere capping and integrity are maintained by shelterin, a six-protein complex. Rap1 is the only shelterin member that is not required for telomere capping and is expressed at non-telomeric genomic and cytosolic regions. The objective of this study was to determine aberrant phenotypes attributed to non-telomeric Rap1. To test this, we generated a Rap1 mutant knock-in (KI) mouse model using CRISPR/Cas9 editing, in which Rap1 at telomeres is prevented, leaving only non-telomeric Rap1. Cell fractionation/western blotting of primary fibroblasts from Rap1 KI mice demonstrated decreased Rap1 expression and Rap1 re-localization off telomeres, with an altered cellular distribution. This same difference in Rap1 is also observed in human cells with telomere erosion, indicating that aberrant Rap1 in our model may recapitulate Rap1 in aging and human telomere biology disorders. Compared to wild-type control mice, Rap1 KI mice exhibited increased body weight, altered cytokine levels, behavioral deficits, and decreased lifespan. In conclusion, our results reveal a novel mechanism by which telomere shortening may contribute to age-related pathologies by disrupting Rap1 expression and cell localization.

A DNA Damage Response-Independent Mechanism for Telomere Shortening-Elicited Age-Related Pathologies

Telomere attrition is associated with telomeropathies and age-related pathologies. In telomeropathies, telomere uncapping induces a DNA damage response (DDR) that drives apoptosis or senescence. However, a defined mechanism by which telomere attrition contributes to other age-related pathologies has not been determined. Telomere integrity is maintained by shelterin, a six-protein complex. Rap1 is the only shelterin member that is not essential for telomere capping but engages non-telomeric DNA and regulates gene transcription. We hypothesized that non-telomeric Rap1 accumulation could contribute to age-related pathologies in a DDR-independent manner. To test this, we used CRISPR/Cas9 editing to generate a Rap1 mutant mouse model in which Rap1 at telomeres is prevented, leaving only non-telomeric Rap1. Indirect immunostaining showed no differences in telomere dysfunction-induced DDR foci in Rap1 mutant compared to wild-type primary fibroblasts. Cell fractionation/western blotting of fibroblasts from Rap1 mutants demonstrated decreased Rap1 expression and Rap1 re-localization off telomeres, which mimics the same alteration of Rap1 in human cells with telomere attrition. Rap1 mutant mice exhibited increased body weight and altered metabolic and immune-response transcripts in various tissues, indicating that altered transcription could account for some of the observed phenotypes related to telomere attrition. In conclusion, telomere shortening may facilitate non-telomeric Rap1, which alters gene transcription and drives metabolic and immune dysfunction in a DDR-independent manner.

DNA binding modes influence Rap1 activity in the regulation of telomere length and MRX functions at DNA ends.

The cellular response to DNA double-strand breaks (DSBs) is initiated by the Mre11–Rad50–Xrs2 (MRX) complex that has structural and catalytic functions. MRX association at DSBs is counteracted by Rif2, which is known to interact with Rap1 that binds telomeric DNA through two tandem Myb-like domains. Whether and how Rap1 acts at DSBs is unknown. Here we show that Rif2 inhibits MRX association to DSBs in a manner dependent on Rap1, which binds to DSBs and promotes Rif2 association to them. Rap1 in turn can negatively regulate MRX function at DNA ends also independently of Rif2. In fact, a characterization of Rap1 mutant variants shows that Rap1 binding to DNA through both Myb-like domains results in formation of Rap1-DNA complexes that control MRX functions at both DSBs and telomeres primarily through Rif2. By contrast, Rap1 binding to DNA through a single Myb-like domain results in formation of high stoichiometry complexes that act at DNA ends mostly in a Rif2-independent manner. Altogether these findings indicate that the DNA binding modes of Rap1 influence its functional properties, thus highlighting the structural plasticity of this protein.

The Rap activator Gef26 regulates synaptic growth and neuronal survival via inhibition of BMP signaling

In Drosophila, precise regulation of BMP signaling is essential for normal synaptic growth at the larval neuromuscular junction (NMJ) and neuronal survival in the adult brain. However, the molecular mechanisms underlying fine-tuning of BMP signaling in neurons remain poorly understood. We show that loss of the Drosophila PDZ guanine nucleotide exchange factor Gef26 significantly increases synaptic growth at the NMJ and enhances BMP signaling in motor neurons. We further show that Gef26 functions upstream of Rap1 in motor neurons to restrain synaptic growth. Synaptic overgrowth in gef26 or rap1 mutants requires BMP signaling, indicating that Gef26 and Rap1 regulate synaptic growth via inhibition of BMP signaling. We also show that Gef26 is involved in the endocytic downregulation of surface expression of the BMP receptors thickveins (Tkv) and wishful thinking (Wit). Finally, we demonstrate that loss of Gef26 also induces progressive brain neurodegeneration through Rap1- and BMP signaling-dependent mechanisms. Taken together, these results suggest that the Gef26-Rap1 signaling pathway regulates both synaptic growth and neuronal survival by controlling BMP signaling.

Crucial Role of Rapgef2 and Rapgef6, a Family of Guanine Nucleotide Exchange Factors for Rap1 Small GTPase, in Formation of Apical Surface Adherens Junctions and Neural Progenitor Development in the Mouse Cerebral Cortex.

Cerebral neocortex development in mammals requires highly orchestrated events involving proliferation, differentiation, and migration of neural progenitors and neurons. Rapgef2 and Rapgef6 constitute a unique family of guanine nucleotide exchange factors for Rap1 small GTPase, which is known to play crucial roles in migration of postmitotic neurons. We previously reported that conditional knockout of Rapgef2 in dorsal telencephalon (Rapgef2-cKO) resulted in the formation of an ectopic cortical mass (ECM) resembling that of subcortical band heterotopia. Here we show that double knockout of Rapgef6 in Rapgef2-cKO mice (Rapgef2/6-dKO) results in marked enlargement of the ECM. While Rapgef2-cKO affects late-born neurons only, Rapgef2/6-dKO affects both early-born and late-born neurons. The Rapgef2-cKO cortex at embryonic day (E) 15.5, and the Rapgef2/6-dKO cortex at E13.5 and E15.5 show disruption of the adherens junctions (AJs) on the apical surface, detachment of radial glial cells (RGCs) from the apical surface and disorganization of the radial glial fiber system, which are accompanied by aberrant distribution of RGCs and intermediate progenitors, normally located in the ventricular zone and the subventricular zone, respectively, over the entire cerebral cortex. Moreover, intrauterine transduction of Cre recombinase into the Rapgef2flox/flox brains also results in the apical surface AJ disruption and the RGC detachment from the apical surface, both of which are effectively suppressed by cotransduction of the constitutively active Rap1 mutant Rap1G12V. These results demonstrate a cell-autonomous role of the Rapgef2/6-Rap1 pathway in maintaining the apical surface AJ structures, which is necessary for the proper development of neural progenitor cells.

Rap1-GTP Augments TGF-b-Mediated Signaling in T Lymphocytes Via a Mechanism Dependent on the b Chain of LFA-1 Integrin

Integrin-mediated adhesion of lymphocytes to antigen presenting cells (APCs) is a critical event linking innate and adaptive immunity. Integrins function as bidirectional receptors and can transmit signals from both sides of the plasma membrane, a property referred to as inside-out and outside-in signaling. Lymphocyte adhesion to APC is mainly accomplished through the principle adhesion molecule on the lymphocyte surface, the lymphocyte functional antigen 1 (LFA-1), which binds to intercellular adhesion molecule 1 (ICAM-1) on the surface of APCs. In order to mediate its function, LFA-1 must be activated via a process, which results in conformation changes of the receptor that extends the ectodomains of the α and β chains leading to a high affinity state. Among the few signaling molecules that have been implicated in integrin activation in hematopoietic cells are the small GTPase Rap1A (thereafter named Rap1) and its downstream effectors RapL and RIAM. In response to Rap1-GTP, RapL regulates LFA-1 activation by interacting with the integrin α chain, whereas RIAM mediates recruitment of talin to the cytoplasmic tail of the β chain leading to its conformational change to the high affinity state. To understand the role of Rap1 in T cell responses we generated transgenic (Tg) mice that selectively express the active, GTP-bound Rap1 mutant Rap1E63 in mature T cells. Rap1E63-Tg and littermate control mice had no statistically significant difference in absolute thymocyte numbers and differentiation profiles. In contrast, in peripheral lymphoid organs, Rap1E63-Tg mice had a significant reduction in total T cells but a 4-fold increase in the CD4+ CD103+ T cell fraction. CD103 (also known as αEβ7 integrin) defines a subset of peripherally generated Treg with potent suppressive function. TGF-β is the strongest stimulus for induction of CD103 (αEβ7). To examine whether Rap1-GTP can affect TGF-β-mediated signaling in T cells, we used stable Jurkat T cell lines expressing GTP-bound Rap1 mutant, Rap1E63, or Jurkat T cell lines in which the endogenous Rap1 was depleted by shRNA (Rap1-KD), and also primary T cells from Rap1E63-Tg mice and Rap1-KO mice. After interaction with two membrane-bound receptors, TGF-βRI and II, TGF-β propagates downstream signaling via the Smad family transcription factors. Incubation of Rap1E63 Jurkat T cells with TGF-β resulted in enhanced and sustained phosphorylation of Smad2 and Smad3, which was observed with very low concentrations of TGF-β that were incapable of inducing detectable phosphorylation of Smads in control Jurkat T cells. In contrast, diminished level and duration of Smad2 and Smad3 phosphorylation was observed in Rap1-KD Jurkat T cells. Similar patterns of responses to those observed in Rap1E63 Jurkat T cells and in Rap1-KD Jurkat T cells were observed in primary mouse T cells isolated from Rap1E63-Tg mice and Rap1-KO mice, respectively. To investigate whether the LFA-1 integrin α and/or β chain had an active role in the enhanced TGF-β-mediated signaling in the presence of Rap1-GTP, we used Rap1E63 Jurkat T cells in which RapL or RIAM were depleted by shRNA (Rap1E63/RapL-KD and Rap1E63/RIAM-KD) because these adaptors selectively regulate the LFA-1 α and the LFA-1 β chain, respectively, downstream of Rap1-GTP. Although in Rap1E63/RapL-KD cells the enhanced TGF-β-induced Smad3 phosphorylation remained unaffected, in Rap1E63/RIAM-KD cells the enhanced TGF-β-induced Smad3 phosphorylation was abrogated. To investigate the biological relevance of these observations, we used T cells from Rap1E63-Tg mice crossed with mice deficient for the LFA-1 α chain. TGF-β resulted in enhanced Smad3 phosphorylation in T cells from Rap1E63-Tg/LFA1-a KO mice similarly to T cells from Rap1E63-Tg mice, indicating that this effect was not dependent on the activation of LFA-1 α chain. In contrast, T cells from RIAMflox/flox -Lck-Cre mice, in which activation of the LFA-1 β chain is impaired, displayed abrogated activation of Smad3 in response to TGF-β. Our data reveal a novel mechanism by which Rap1 regulates T cell responses via outside-in integrin signaling and may have important implications on TGF-β-mediated T cell homeostasis, differentiation and immune quiescence. DisclosuresNo relevant conflicts of interest to declare.

Expression of Subtelomeric lncRNAs Links Telomeres Dynamics to RNA Decay in S. cerevisiae.

Long non-coding RNAs (lncRNAs) have been shown to regulate gene expression, chromatin domains and chromosome stability in eukaryotic cells. Recent observations have reported the existence of telomeric repeats containing long ncRNAs – TERRA in mammalian and yeast cells. However, their functions remain poorly characterized. Here, we report the existence in S. cerevisiae of several lncRNAs within Y′ subtelomeric regions. We have called them subTERRA. These belong to Cryptic Unstable Transcripts (CUTs) and Xrn1p-sensitive Unstable Transcripts (XUTs) family. subTERRA transcription, carried out mainly by RNAPII, is initiated within the subtelomeric Y’ element and occurs in both directions, towards telomeres as well as centromeres. We show that subTERRA are distinct from TERRA and are mainly degraded by the general cytoplasmic and nuclear 5′- and 3′- RNA decay pathways in a transcription-dependent manner. subTERRA accumulates preferentially during the G1/S transition and in C-terminal rap1 mutant but independently of Rap1p function in silencing. The accumulation of subTERRA in RNA decay mutants coincides with telomere misregulation: shortening of telomeres, loss of telomeric clustering in mitotic cells and changes in silencing of subtelomeric regions. Our data suggest that subtelomeric RNAs expression links telomere maintenance to RNA degradation pathways.

Protein Kinase A-dependent Phosphorylation of Rap1 Regulates Its Membrane Localization and Cell Migration

The small G protein Rap1 can mediate "inside-out signaling" by recruiting effectors to the plasma membrane that signal to pathways involved in cell adhesion and cell migration. This action relies on the membrane association of Rap1, which is dictated by post-translational prenylation as well as by a stretch of basic residues within its carboxyl terminus. One feature of this stretch of acidic residues is that it lies adjacent to a functional phosphorylation site for the cAMP-dependent protein kinase PKA. This phosphorylation has two effects on Rap1 action. One, it decreases the level of Rap1 activity as measured by GTP loading and the coupling of Rap1 to RapL, a Rap1 effector that couples Rap1 GTP loading to integrin activation. Two, it destabilizes the membrane localization of Rap1, promoting its translocation into the cytoplasm. These two actions, decreased GTP loading and decreased membrane localization, are related, as the translocation of Rap1-GTP into the cytoplasm is associated with its increased GTP hydrolysis and inactivation. The consequences of this phosphorylation in Rap1-dependent cell adhesion and cell migration were also examined. Active Rap1 mutants that lack this phosphorylation site had a minimal effect on cell adhesion but strongly reduced cell migration, when compared with an active Rap1 mutant that retained the phosphorylation site. This suggests that optimal cell migration is associated with cycles of Rap1 activation, membrane egress, and inactivation, and requires the regulated phosphorylation of Rap1 by PKA.

A Novel Interplay between Rap1 and PKA Regulates Induction of Angiogenesis in Prostate Cancer

Angiogenesis inhibition is an important therapeutic strategy for advanced stage prostate cancer. Previous work from our laboratory showed that sustained stimulation of Rap1 by 8-pCPT-2'-O-Me-cAMP (8CPT) via activation of Epac, a Rap1 GEF, or by expression of a constitutively active Rap1 mutant (cRap1) suppresses endothelial cell chemotaxis and subsequent angiogenesis. When we tested this model in the context of a prostate tumor xenograft, we found that 8CPT had no significant effect on prostate tumor growth alone. However, in cells harboring cRap1, 8CPT dramatically inhibited not only prostate tumor growth but also VEGF expression and angiogenesis within the tumor microenvironment. Subsequent analysis of the mechanism revealed that, in prostate tumor epithelial cells, 8CPT acted via stimulation of PKA rather than Epac/Rap1. PKA antagonizes Rap1 and hypoxic induction of 1α protein expression, VEGF production and, ultimately, angiogenesis. Together these findings provide evidence for a novel interplay between Rap1, Epac, and PKA that regulates tumor-stromal induction of angiogenesis.

SPA‐1 controls the invasion and metastasis of human prostate cancer

Recent studies suggest that SIPA1 encoding a Rap GTPase-activating protein SPA-1 is a candidate metastasis efficiency-modifying gene in human breast cancer. In this study, we investigated the expression and function of SPA-1 in human prostate cancer (CaP). Immunohistochemical studies of tumor specimens from CaP patients revealed a positive correlation of SPA-1 expression with disease progression and metastasis. The correlation was recapitulated in human CaP cell lines; LNCaP that rarely showed metastasis in SCID mice expressed an undetectable level of SPA-1, whereas highly metastatic PC3 showed abundant SPA-1 expression. Moreover, SIPA1 transduction in LNCaP caused prominent abdominal lymph node metastasis without affecting primary tumor size, whereas shRNA-mediated SIPA1 knockdown or expression of a dominant-active Rap1 mutant (Rap1V12) in PC3 suppressed metastasis. LNCaP transduced with SPA-1 (LNCaP/SPA-1) showed attenuated adhesion to the precoated extracellular matrices (ECM) including collagens and fibronectin, due to defective ECM-medicated Rap1 activation. In addition, LNCaP/SPA-1 showed a diminished level of nuclear Brd4, which is known to bind SPA-1, resulting in reduced expression of a series of ECM-related genes. These results suggest that SPA-1 plays an important role in controlling metastasis efficiency of human CaP by regulating the expression of and interaction with ECM in the primary sites.

Rap1-GTP Augments Activation of Smad and p38 Mediated Signaling Downstream of TGF-β Receptor In T Lymphocytes

AbstractAbstract 956Rap1A, a member of the Ras superfamily, was discovered as a gene product that reverted K-Ras-induced transformation. Although it was initially thought that Rap1A (thereafter referred to as Rap1) opposes Ras-induced transformation by competing for common downstream effector(s), it has now become apparent that Ras and Rap1 proteins operate in different signaling networks and mediate distinct functions. In lymphocytes Rap1 is activated by BCR and TCR mediated signals and is involved in inside-out activation of integrins. To understand the role of Rap1 in T cell responses we generated transgenic (Tg) mice that express the active Rap1 mutant Rap1E63 in T cells. Rap1E63-Tg and littermate control mice had no statistically significant difference in absolute thymocyte numbers and differentiation profiles of double positive and single positive thymocytes. In contrast to thymocyte numbers, Rap1E63-Tg mice had reduced T cell numbers in peripheral lymphoid organs. In peripheral lymph nodes and spleen, the total T cell numbers were decreased to approximately one third to one forth of those in littermate controls. Strikingly, Rap1E63-Tg mice had a 4-fold increase in the CD4+CD103+ T cell fraction in spleens and lymph nodes compared to their littermate controls. CD103 defines a subset of peripherally generated Treg with potent suppressive function. CD4+CD103+ Treg cells in RapE63-Tg mice expressed CD45RBlow, CD44high, CD54high, CD62Llow and LFA-1high, findings consistent with an effector memory phenotype. TGF-β is the strongest stimulus for induction of CD103 expression. To examine whether Rap1 affected TGF-β-mediated signaling in T cells, we used stable Jurkat T cell lines expressing Rap1E63, Jurkat T cell lines, in which endogenous Rap1 was depleted by shRNA (Rap1-KD), and primary T cells from Rap1E63-Tg mice and Rap1-KO mice. TGF-β signaling involves the interaction of two membrane-bound receptors, TGF-βRI and II. TGF-β binds the exracellular domain of TGF-βRII, which then recruits TGF-βRI resulting in transphosphorylation. TGF-βRI activation propagates downstream signaling via the Smad family proteins Smad2 and Smad3 and also via p38 MAPK, in a Smad-independent manner. Incubation of Rap1E63 Jurkat T cells with TGF-β resulted in enhanced and sustained phosphorylation of Smad2 and Smad3, which was observed with very low concentrations of TGF-β that were incapable of inducing detectable phosphorylation of Smad2 or Smad3 in control cells. In contrast, diminished level and duration of Smad2 and Smad3 phosphorylation was observed in Rap1-KD Jurkat cells. Activation of p38 downstream of TGF-βRI displayed a dramatically augmented and sustained induction, whereas the opposite effect was observed in Rap1-KD cells. Similar patterns of responses to those observed in Rap1E63 Jurkat T cells and in Rap1-KD cells were observed in primary mouse T cells isolated from Rap1E63-Tg mice and Rap1 deficient mice, respectively. Assessment of TGF-β binding on TGF-β surface receptors by using biotinylated TGF-β revealed that Rap1E63 expressing cells displayed significantly enhanced TGF-β binding compared to their relevant controls, whereas reduced TGF-β binding was observed in Rap1 deficient cells. These results indicate that active Rap1 modulates TGF-β receptor binding and enhances TGF-β mediated signaling, gene transcription, and functional outcome. Our data reveal a novel and unexpected mechanism by which Rap1 regulates T cell responses that may have important implications on TGF-β-mediated T cell homeostasis and maintenance of immune quiescence. Disclosures:No relevant conflicts of interest to declare.

Sustained T cell Rap1 signaling is protective in the collagen‐induced arthritis model of rheumatoid arthritis

Defective activation of T cell receptor-proximal signaling proteins, such as the small GTPase Rap1, is thought to contribute to the pathologic behavior of rheumatoid arthritis (RA) synovial T cells. This study was undertaken to determine whether maintaining Rap1 signaling in murine T cells modifies disease onset or severity in collagen-induced arthritis (CIA). CIA experiments were conducted using wild-type and RapV12-transgenic mice, which express an active mutant of Rap1 in the T cell compartment. Mice were assessed using macroscopic, microscopic, and radiologic measures, and serum levels of anticollagen antibodies were measured by enzyme-linked immunosorbent assay. Phenotypic and functional characterization of wild-type and RapV12-transgenic T cells under homeostatic conditions and during disease onset was performed by flow cytometry. Disease incidence and severity, synovial infiltration, joint destruction, and anticollagen antibody production were significantly reduced in RapV12-transgenic mice. Although the numbers and percentages of CD3+, CD4+, and CD8+ (naive, effector, and memory) T cells, Treg cells, and Th17 cells were equivalent in wild-type and RapV12-transgenic mice, a significant decrease in the percentage of tumor necrosis factor α-secreting CD8+ T cells was observed in RapV12-transgenic mice during CIA. RapV12-transgenic T cells also inefficiently expressed inducible costimulator and CD40L costimulatory proteins involved in B cell immunoglobulin class switching. Our findings indicate that maintenance of T cell Rap1 signaling in murine T cells reduces disease incidence and severity in CIA, which are associated with specific defects in T cell effector function. Therefore, the restoration of Rap1 function in RA synovial T cells may have therapeutic benefit in RA.

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.

Rap signaling is crucial for the competence of IL-7 response and the development of B-lineage cells

Rap family GTPases consist of multiple members with substantial functional redundancy. With the use of transgenic mice conditionally expressing a bona fide dominant-negative Rap1 mutant, Rap1A17, capable of inhibiting the activation of all Rap family members in B-lineage cells (mb.1-Rap1A17 Tg), we demonstrate that these mice show a defective generation of pre-B cells in bone marrow, resulting in a significant diminution of peripheral mainstream B cells. The effect is attributed to the impaired survival and expansion of B-lineage progenitors in response to IL-7, despite normal IL-7Ralpha expression. The pre-B cells from mb.1-Rap1A17 Tg mice showed a significantly reduced expression of c-myc and E2A, and the competence of IL-7 response was restored by the transduction of c-myc, but not by constitutively active (CA) Stat5a, CA PI3K-p100, or bcl-2. The residual follicular B cells with complete Cre-mediated recombination proliferated normally in response to B-cell receptor stimulation and showed efficient germinal center reaction in vivo. These results show that endogenous Rap signaling plays a crucial role selectively in B-lineage cell development by sustaining the competence for IL-7 response, whereas it is dispensable for mature B-cell function.

Rap1 maintains adhesion between cells to affect Egfr signaling and planar cell polarity in Drosophila

The small GTPase Rap1 affects cell adhesion and cell motility in numerous developmental contexts. Loss of Rap1 in the Drosophila wing epithelium disrupts adherens junction localization, causing mutant cells to disperse, and dramatically alters epithelial cell shape. While the adhesive consequences of Rap1 inactivation have been well described in this system, the effects on cell signaling, cell fate specification, and tissue differentiation are not known. Here we demonstrate that Egfr-dependent cell types are lost from Rap1 mutant tissue as an indirect consequence of DE-cadherin mislocalization. Cells lacking Rap1 in the developing wing and eye are capable of responding to an Egfr signal, indicating that Rap1 is not required for Egfr/Ras/MAPK signal transduction. Instead, Rap1 regulates adhesive contacts necessary for maintenance of Egfr signaling between cells, and differentiation of wing veins and photoreceptors. Rap1 is also necessary for planar cell polarity in these tissues. Wing hair alignment and ommatidial rotation, functional readouts of planar cell polarity in the wing and eye respectively, are both affected in Rap1 mutant tissue. Finally, we show that Rap1 acts through the effector Canoe to regulate these developmental processes.

Cyclic AMP acts through Rap1 and JNK signaling to increase expression of cutaneous smooth muscle α2C-adrenoceptors

Cold increases cutaneous vasoconstriction by unmasking the contractile activity of alpha(2C)-adrenoceptors (alpha(2C)-ARs) in vascular smooth muscle cells (VSMCs), which is mediated by the cold-induced mobilization of alpha(2C)-ARs from the transGolgi to the cell surface. The expression of alpha(2C)-ARs in human cutaneous VSMCs is under dual regulation by cyclic AMP: gene transcription is inhibited by cyclic AMP acting through protein kinase A but is increased by cyclic AMP acting through the exchange protein directly activated by cyclic AMP (EPAC) and the GTP-binding protein Rap1. Experiments were performed to further characterize the Rap1 signaling pathway. Forskolin (10 muM), the selective EPAC activator, 8-pCPT-2'-O-Me-cyclic AMP (CMC; 100 microM), or a constitutively active mutant of Rap1 (Rap1CA) increased the activity of c-Jun NH(2)-terminal kinase (JNK) in human cutaneous VSMCs. This was associated with the increased phosphorylation of c-Jun and activation of an activator protein (AP)-1 reporter construct, which were inhibited by the JNK inhibitor SP600125 (3 microM). Rap1CA increased the activity of an alpha(2C)-AR promoter-reporter construct, which was inhibited by SP600125 (3 microM) or by the mutation of an AP-1 binding site in the alpha(2C)-AR promoter. Furthermore, forskolin (10 microM) or CMC (100 microM) increased the expression of the alpha(2C)-AR protein, and these effects were inhibited by SP600125 (3 microM). Therefore, cyclic AMP increases the expression of alpha(2C)-ARs in cutaneous VSMCs by activating a novel Rap1 signaling pathway, mediated by the activation of JNK, AP-1, and the subsequent transcriptional activation of the alpha(2C)-AR gene. By increasing the expression of cold-responsive alpha(2C)-ARs, this pathway may contribute to enhanced cold-induced vasoconstriction in the cutaneous circulation, including Raynaud's phenomenon.

Yeast Rap1 contributes to genomic integrity by activating DNA damage repair genes

Rap1 (repressor-activator protein 1) is a multifunctional protein that controls telomere function, silencing and the activation of glycolytic and ribosomal protein genes. We have identified a novel function for Rap1, regulating the ribonucleotide reductase (RNR) genes that are required for DNA repair and telomere expansion. Both the C terminus and DNA-binding domain of Rap1 are required for the activation of the RNR genes, and the phenotypes of different Rap1 mutants suggest that it utilizes both regions to carry out distinct steps in the activation process. Recruitment of Rap1 to the RNR3 gene is dependent on activation of the DNA damage checkpoint and chromatin remodelling by SWI/SNF. The dependence on SWI/SNF for binding suggests that Rap1 acts after remodelling to prevent the repositioning of nucleosomes back to the repressed state. Furthermore, the recruitment of Rap1 requires TAF(II)s, suggesting a role for TFIID in stabilizing activator binding in vivo. We propose that Rap1 acts as a rheostat controlling nucleotide pools in response to shortened telomeres and DNA damage, providing a mechanism for fine-tuning the RNR genes during checkpoint activation.

RAP-1 and the RAL-1/exocyst pathway coordinate hypodermal cell organization in Caenorhabditis elegans

The small Ras-like GTPase Rap1 has been identified as a regulator of integrin activation and cadherin-mediated cell-cell contacts. Surprisingly, null mutants of RAP-1 in Caenorhabditis elegans are viable and fertile. In a synthetic lethal RNAi screen with C. elegans rap-1 mutants, the Ras-like GTPase ral-1 emerged as one of seven genes specifically required for viability. Depletion of exoc-8 and sec-5, encoding two putative RAL-1 effectors and members of the exocyst complex, also caused lethality of rap-1 mutants, but did not affect wild-type worms. The RAP-1 and the RAL-1/exocyst pathway appear to coordinate hypodermal cell movement and elongation during embryonic development. They mediate their effect in part through targeting the alpha-catenin homologue HMP-1 to the lateral membrane. Genetic interactions show that the RAP-1 and RAL-1/exocyst pathway also act in parallel during larval stages. Together these data provide in vivo evidence for the exocyst complex as a downstream RAL-1 effector in cell migration.

How Smurfs Control Axon Formation

In the course of development, hippocampal neurons normally produce multiple equivalent neurites, only one of which eventually becomes the axon of a polarized neuron. The small guanosine triphosphatase (GTPase) Rap1B is a necessary and sufficient regulator of this process and is initially present in each of the neurites, but is later present only in the neurite destined to become the axon. Results from Schwamborn et al . show that this critical loss of Rap1B is mediated by ubiquitin-dependent degradation of the GTPase. Treatment of cultured hippocampal neurons from rat embryos with various inhibitors of the proteasome caused formation of extra axons. In transfected cells or in vitro binding assays, a catalytically inactive mutant of the ubiquitin ligase Smurf2 could be immunoprecipitated with a dominant-negative mutant of Rap1 and associated with nucleotide-free or GDP-bound Rap1, but not with Rap1 bound to a nonhydrolyzable analog of GTP. These results indicate that the ligase recognizes only inactive Rap1. Depletion of Smurf2 also caused formation of supernumerary axons in the cultured neurons. Thus, the authors propose that the ubiquitination and proteasome-dependent degradation of Rap1B is required for selection of a single neurite that becomes the axon. In their model, through a mechanism not yet revealed, one of the neurites would have higher activity of Rap1. Because the active GTPase is resistant to degradation, the Smurf-induced degradation would occur primarily in the remaining neurites with low Rap1B activity, thus converting the quantitative differences in Rap1B activity to a switchlike mechanism that allows only a single neurite to go on to form the axon. J. C. Schwamborn, M. Müller, A. H. M. Becker, A. W. Püschel, Ubiquitination of the GTPase Rap1B by the ubiquitin ligase Smurf2 is required for the establishment of neuronal polarity. EMBO J . 26 , 1410-1422 (2007). [Online Journal]