Negative regulation of Rap1 activation by the Cbl E3 ubiquitin ligase

Cbl functions as an adaptor protein by interacting with other signalling molecules to form multimolecular complexes. Previous studies have proposed that Cbl is also a positive regulator of CrkL–C3G signalling, which leads to Rap1 activation. However, there is a lack of genetic evidence for a physiological function of Cbl in regulating this pathway. Here, we show that Cbl deficiency results in enhanced activation of Rap1. Cbl was shown to promote the ubiquitylation of CrkL without any apparent effect on its stability. Remarkably, the membrane translocation of C3G, its association with CrkL, and the guanine-nucleotide exchange activity of C3G were all increased in Cbl^−/− thymocytes. Consistent with a function of Rap1 in integrin activation, enhanced integrin-mediated cell adhesion was also seen in Cbl^−/− thymocytes. Thus, Cbl negatively regulates Rap1 activation, probably through a proteolysis-independent E3-ubiquitin-ligase activity of Cbl that modulates protein–protein interactions.     

Cell-cell junction formation: The role of Rap1 and Rap1 guanine nucleotide exchange factors

Rap proteins are Ras-like small GTP-binding proteins that amongst others are involved in the control of cell-cell and cell-matrix adhesion. Several Rap guanine nucleotide exchange factors (RapGEFs) function to activate Rap. These multi-domain proteins, which include C3G, Epacs, PDZ-GEFs, RapGRPs and DOCK4, are regulated by various different stimuli and may function at different levels in junction formation. Downstream of Rap, a number of effector proteins have been implicated in junctional control, most notably the adaptor proteins AF6 and KRIT/CCM1. In this review, we will highlight the latest findings on the Rap signaling network in the control of epithelial and endothelial cell-cell junctions.     

Ras and Rap1: two highly related small GTPases with distinct function

The Ras-like family of small GTPases includes, among others, Ras, Rap1, R-ras, and Ral. The family is characterized by similarities in the effector domain. While the function of Ras is, at least in part, elucidated, little is known about other members of the family. Currently, much attention is focused on the small GTPase Rap1. Initially, this member was identified as a transformation suppressor protein able to revert the morphological phenotype of Ras-transformed fibroblasts. This has led to the hypothesis that Rap1 antagonizes Ras by interfering in Ras effector function. Recent analysis revealed that Rap1 is activated rapidly in response to activation of a variety of receptors. Rap1 activation is mediated by several second messengers, including calcium, diacylglycerol, and cAMP. Guanine nucleotide exchange factors (GEFs) have been identified that mediate these effects. The most interesting GEF is Epac, an exchange protein directly activated by cAMP, thus representing a novel cAMP-induced, protein kinase A-independent pathway. Furthermore, Rap1 is inactivated by specific GTPase-activating proteins (GAPs), one of which is regulated through an interaction with Galphai. While Ras and Rap1 may share some effector pathways, evidence is accumulating that Ras and Rap1 each regulate unique cellular processes in response to various extracellular ligands. For Rap1 these functions may include the control of cell morphology.     

The M-Ras-RA-GEF-2-Rap1 pathway mediates tumor necrosis factor-alpha dependent regulation of integrin activation in splenocytes

The Rap1 small GTPase has been implicated in regulation of integrin-mediated leukocyte adhesion downstream of various chemokines and cytokines in many aspects of inflammatory and immune responses. However, the mechanism for Rap1 regulation in the adhesion signaling remains unclear. RA-GEF-2 is a member of the multiple-member family of guanine nucleotide exchange factors (GEFs) for Rap1 and characterized by the possession of a Ras/Rap1-associating domain, interacting with M-Ras-GTP as an effector, in addition to the GEF catalytic domain. Here, we show that RA-GEF-2 is specifically responsible for the activation of Rap1 that mediates tumor necrosis factor-alpha (TNF-alpha)-triggered integrin activation. In BAF3 hematopoietic cells, activated M-Ras potently induced lymphocyte function-associated antigen 1 (LFA-1)-mediated cell aggregation. This activation was totally abrogated by knockdown of RA-GEF-2 or Rap1. TNF-alpha treatment activated LFA-1 in a manner dependent on M-Ras, RA-GEF-2, and Rap1 and induced activation of M-Ras and Rap1 in the plasma membrane, which was accompanied by recruitment of RA-GEF-2. Finally, we demonstrated that M-Ras and RA-GEF-2 were indeed involved in TNF-alpha-stimulated and Rap1-mediated LFA-1 activation in splenocytes by using mice deficient in RA-GEF-2. These findings proved a crucial role of the cross-talk between two Ras-family GTPases M-Ras and Rap1, mediated by RA-GEF-2, in adhesion signaling.     

Specificity in Ras and Rap Signaling

Ras and Rap proteins are closely related small GTPases. Whereas Ras is known for its role in cell proliferation and survival, Rap1 is predominantly involved in cell adhesion and cell junction formation. Ras and Rap are regulated by different sets of guanine nucleotide exchange factors and GTPase-activating proteins, determining one level of specificity. In addition, although the effector domains are highly similar, Rap and Ras interact with largely different sets of effectors, providing a second level of specificity. In this review, we discuss the regulatory proteins and effectors of Ras and Rap, with a focus on those of Rap.Ras-like small G-proteins are ubiquitously expressed, conserved molecular switches that couple extracellular signals to various cellular responses. Different signals can activate GEFs² that induce the small G-protein to switch from the inactive, GDP-bound state to the active, GTP-bound state. This induces a conformational change that allows downstream effector proteins to bind specifically to and be activated by the GTP-bound protein to mediate diverse biological responses. Small G-proteins are returned to the GDP-bound state by hydrolyzing GTP with the help of GAPs. Ras (Ha-Ras, Ki-Ras, and N-Ras) and Rap proteins (Rap1A, Rap1B, Rap2A, Rap2B, and Rap2C) have similar effector-binding regions that interact predominantly with RA domains or the structurally similar RBDs present in a variety of different proteins. Both protein families operate in different signaling networks. For instance, Ras is central in a network controlling cell proliferation and cell survival, whereas Rap1 predominantly controls cell adhesion, cell junction formation, cell secretion, and cell polarity. These different functions are reflected in a largely different set of GEFs and GAPs. Also the downstream effector proteins operate in a selective manner in either one of the networks.     

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.     

AF6 negatively regulates Rap1-induced cell adhesion

AF6 is involved in the connection of membrane-associated proteins to the actin cytoskeleton. It binds to Ras-like small GTPases and is suggested to be an effector of both Ras and Rap. Here we show that knockdown of AF6 in T cells by RNA interference enhanced Rap1-induced integrin-mediated cell adhesion, whereas overexpression of AF6 had the opposite effect. Interestingly, AF6-induced inhibition of cell adhesion correlated with an increase in RapGTP levels. Like AF6, protein KIAA1849 contains a Ras association domain and interacted with Rap1. However, KIAA1849 did not inhibit Rap1-induced cell adhesion. We concluded that AF6 is a negative regulator of Rap-induced cell adhesion. We proposed that AF6 inhibits Rap-mediated cell adhesion by sequestering RapGTP in an unproductive complex and thus prevents the interaction of Rap1 not only with effectors that mediate adhesion but also with Rap GTPase-activating proteins. Thus, AF6 may buffer RapGTP in resting T cells and maintain them in a non-adherent state.     

Ras is required for the cyclic AMP-dependent activation of Rap1 via Epac2

Exchange proteins activated by cAMP (cyclic AMP) 2 (Epac2) is a guanine nucleotide exchange factor for Rap1, a small G protein involved in many cellular functions, including cell adhesion, differentiation, and exocytosis. Epac2 interacts with Ras-GTP via a Ras association (RA) domain. Previous studies have suggested that the RA domain was dispensable for Epac2 function. Here we show for the first time that Ras and cAMP regulate Epac2 function in a parallel fashion and the Ras-Epac2 interaction is required for the cAMP-dependent activation of endogenous Rap1 by Epac2. The mechanism for this requirement is not allosteric activation of Epac2 by Ras but the compartmentalization of Epac2 on the Ras-containing membranes. A computational modeling is consistent with this compartmentalization being a function of both the level of Ras activation and the affinity between Ras and Epac2. In PC12 cells, a well-established model for sympathetic neurons, the Epac2 signaling is coupled to activation of mitogen-activated protein kinases and contributes to neurite outgrowth. Taken together, the evidence shows that Epac2 is not only a cAMP sensor but also a bona fide Ras effector. Coincident detection of both cAMP and Ras signals is essential for Epac2 to activate Rap1 in a temporally and spatially controlled manner.     

A Highlights from MBoC Selection: Radil controls neutrophil adhesion and motility through β2-integrin activation

Integrin activation is required to facilitate multiple adhesion-dependent functions of neutrophils, such as chemotaxis, which is critical for inflammatory responses to injury and pathogens. However, little is known about the mechanisms that mediate integrin activation in neutrophils. We show that Radil, a novel Rap1 effector, regulates β1- and β2-integrin activation and controls neutrophil chemotaxis. On activation and chemotactic migration of neutrophils, Radil quickly translocates from the cytoplasm to the plasma membrane in a Rap1a-GTP–dependent manner. Cells overexpressing Radil show a substantial increase in cell adhesion, as well as in integrin/focal adhesion kinase (FAK) activation, and exhibit an elongated morphology, with severe tail retraction defects. This phenotype is effectively rescued by treatment with either β2-integrin inhibitory antibodies or FAK inhibitors. Conversely, knockdown of Radil causes severe inhibition of cell adhesion, β2-integrin activation, and chemotaxis. Furthermore, we found that inhibition of Rap activity by RapGAP coexpression inhibits Radil-mediated integrin and FAK activation, decreases cell adhesion, and abrogates the long-tail phenotype of Radil cells. Overall, these studies establish that Radil regulates neutrophil adhesion and motility by linking Rap1 to β2-integrin activation.     

KIF14 negatively regulates Rap1a–Radil signaling during breast cancer progression

The small GTPase Rap1 regulates inside-out integrin activation and thereby influences cell adhesion, migration, and polarity. Several Rap1 effectors have been described to mediate the cellular effects of Rap1 in a context-dependent manner. Radil is emerging as an important Rap effector implicated in cell spreading and migration, but the molecular mechanisms underlying its functions are unclear. We report here that the kinesin KIF14 associates with the PDZ domain of Radil and negatively regulates Rap1-mediated inside-out integrin activation by tethering Radil on microtubules. The depletion of KIF14 led to increased cell spreading, altered focal adhesion dynamics, and inhibition of cell migration and invasion. We also show that Radil is important for breast cancer cell proliferation and for metastasis in mice. Our findings provide evidence that the concurrent up-regulation of Rap1 activity and increased KIF14 levels in several cancers is needed to reach optimal levels of Rap1–Radil signaling, integrin activation, and cell–matrix adhesiveness required for tumor progression.     

RIAM, an Ena/VASP and Profilin ligand, interacts with Rap1-GTP and mediates Rap1-induced adhesion

The small GTPase Rap1 induces integrin-mediated adhesion and changes in the actin cytoskeleton. The mechanisms that mediate these effects of Rap1 are poorly understood. We have identified RIAM as a Rap1-GTP-interacting adaptor molecule. RIAM defines a family of adaptor molecules that contain a RA-like (Ras association) domain, a PH (pleckstrin homology) domain, and various proline-rich motifs. RIAM also interacts with Profilin and Ena/VASP proteins, molecules that regulate actin dynamics. Overexpression of RIAM induced cell spreading and lamellipodia formation, changes that require actin polymerization. In contrast, RIAM knockdown cells had reduced content of polymerized actin. RIAM overexpression also induced integrin activation and cell adhesion. RIAM knockdown displaced Rap1-GTP from the plasma membrane and abrogated Rap1-induced adhesion. Thus, RIAM links Rap1 to integrin activation and plays a role in regulating actin dynamics.     

Linking Rap to cell adhesion

The small GTPase Rap1 is involved in several aspects of cell adhesion, including integrin-mediated cell adhesion and cadherin-mediated cell junction formation. Recently, several effector proteins for Rap1 have been identified providing a clear link between Rap1 and actin dynamics. Furthermore, evidence is accumulating that Rap1 functions in the spatial and temporal control of cell polarity.     

Apical Accumulation of the Sevenless Receptor Tyrosine Kinase During Drosophila Eye Development Is Promoted by the Small GTPase Rap1

The Ras/MAPK-signaling pathway plays pivotal roles during development of metazoans by controlling cell proliferation and cell differentiation elicited, in several instances, by receptor tyrosine kinases (RTKs). While the internal mechanism of RTK-driven Ras/MAPK signaling is well understood, far less is known regarding its interplay with other corequired signaling events involved in developmental decisions. In a genetic screen designed to identify new regulators of RTK/Ras/MAPK signaling during Drosophila eye development, we identified the small GTPase Rap1, PDZ-GEF, and Canoe as components contributing to Ras/MAPK-mediated R7 cell differentiation. Rap1 signaling has recently been found to participate in assembling cadherin-based adherens junctions in various fly epithelial tissues. Here, we show that Rap1 activity is required for the integrity of the apical domains of developing photoreceptor cells and that reduced Rap1 signaling hampers the apical accumulation of the Sevenless RTK in presumptive R7 cells. It thus appears that, in addition to its role in cell–cell adhesion, Rap1 signaling controls the partitioning of the epithelial cell membrane, which in turn influences signaling events that rely on apico-basal cell polarity.     

Impaired fertility and spermiogenetic disorders with loss of cell adhesion in male mice expressing an interfering Rap1 mutant

The guanosine trisphosphatase Rap1 serves as a critical player in signal transduction, somatic cell proliferation and differentiation, and cell-cell adhesion by acting through distinct mechanisms. During mouse spermiogenesis, Rap1 is activated and forms a signaling complex with its effector, the serine-threonine kinase B-Raf. To investigate the functional role of Rap1 in male germ cell differentiation, we generated transgenic mice expressing an inactive Rap1 mutant selectively in differentiating spermatids. This expression resulted in a derailment of spermiogenesis due to an anomalous release of immature round spermatids from the seminiferous epithelium within the tubule lumen and in low sperm counts. These spermiogenetic disorders correlated with impaired fertility, with the transgenic males being severely subfertile. Because mutant testis exhibited perturbations in ectoplasmic specializations (ESs), a Sertoli-germ cell-specific adherens junction, we searched for expression of vascular endothelial cadherin (VE-cadherin), an adhesion molecule regulated by Rap1, in spermatogenic cells of wild-type and mutant mice. We found that germ cells express VE-cadherin with a timing strictly related to apical ES formation and function; immature, VE-cadherin-positive spermatids were, however, prematurely released in the transgenic testis. In conclusion, interfering with Rap1 function during spermiogenesis leads to reduced fertility by impairment of germ-Sertoli cell contacts; our transgenic mouse provides an in vivo model to study the regulation of ES dynamics.     

The Rap GTPase activator Drosophila PDZ-GEF regulates cell shape in epithelial migration and morphogenesis

Epithelial morphogenesis is characterized by an exquisite control of cell shape and position. Progression through dorsal closure in Drosophila gastrulation depends on the ability of Rap1 GTPase to signal through the adherens junctional multidomain protein Canoe. Here, we provide genetic evidence that epithelial Rap activation and Canoe effector usage are conferred by the Drosophila PDZ-GEF (dPDZ-GEF) exchange factor. We demonstrate that dPDZ-GEF/Rap/Canoe signaling modulates cell shape and apicolateral cell constriction in embryonic and wing disc epithelia. In dPDZ-GEF mutant embryos with strong dorsal closure defects, cells in the lateral ectoderm fail to properly elongate. Postembryonic dPDZ-GEF mutant cells generated in mosaic tissue display a striking extension of lateral cell perimeters in the proximity of junctional complexes, suggesting a loss of normal cell contractility. Furthermore, our data indicate that dPDZ-GEF signaling is linked to myosin II function. Both dPDZ-GEF and cno show strong genetic interactions with the myosin II-encoding gene, and myosin II distribution is severely perturbed in epithelia of both mutants. These findings provide the first insight into the molecular machinery targeted by Rap signaling to modulate epithelial plasticity. We propose that dPDZ-GEF-dependent signaling functions as a rheostat linking Rap activity to the regulation of cell shape in epithelial morphogenesis at different developmental stages.     

Rap1 GTPases: an emerging role in the cardiovasculature

The Ras related GTPase Rap has been implicated in multiple cellular functions. A vital role for Rap GTPase in the cardiovasculature is emerging from recent studies. These small monomeric G proteins act as molecular switches, coupling extracellular stimulation to intracellular signaling through second messengers. This member of the Ras superfamily was once described as the transformation suppressor with the ability to ameliorate the Ras transformed phenotype; however, further studies uncovered a unique set of guanine nucleotide exchange factors (GEFs), GTPase activating proteins (GAPs) and effector proteins for Rap suggesting a more sophisticated role for this small GTPase. At least three different second messengers can activate Rap, namely cyclic AMP (cAMP), calcium and diacylglycerol. More recently, an investigation of Rap in the cardiovasculature has revealed multiple pathways of regulation involving Rap in this system. Two closely related isoforms of Rap1 exist, 1a and 1b. Murine genetic models exist for both and have been described. Although thought at first to be functionally redundant, these isoforms have differing roles in the cardiovasculature. The activation of Rap1a and 1b in various cell types of the cardiovasculature leads to alterations in cell attachment, migration and cell junction formation. This review will focus on the role of these Rap1 GTPases in hematopoietic, endothelial, smooth muscle, and cardiac myocyte function, and conclude with their potential role in human disease.     

Characterization of the GbpD-activated Rap1 pathway regulating adhesion and cell polarity in Dictyostelium discoideum

The regulation of cell polarity plays an important role in chemotaxis. GbpD, a putative nucleotide exchange factor for small G-proteins of the Ras family, has been implicated in adhesion, cell polarity, and chemotaxis in Dictyostelium. Cells overexpressing GbpD are flat, exhibit strongly increased cell-substrate attachment, and extend many bifurcated and lateral pseudopodia. These cells overexpressing GbpD are severely impaired in chemotaxis, most likely due to the induction of many protrusions rather than an enhanced adhesion. The GbpD-overexpression phenotype is similar to that of cells overexpressing Rap1. Here we demonstrate that GbpD activates Rap1 both in vivo and in vitro but not any of the five other characterized Ras proteins. In a screen for Rap1 effectors, we overexpressed GbpD in several mutants defective in adhesion or cell polarity and identified Phg2 as Rap1 effector necessary for adhesion, but not cell polarity. Phg2, a serine/threonine-specific kinase, directly interacts with Rap1 via its Ras association domain.     

Rap1GAP impairs cell-matrix adhesion in the absence of effects on cell-cell adhesion

The significance of the widespread downregulation of Rap1GAP in human tumors is unknown. In previous studies we demonstrated that silencing Rap1GAP expression in human colon cancer cells resulted in sustained increases in Rap activity, enhanced spreading on collagen and the weakening of cell-cell contacts. The latter finding was unexpected based on the role of Rap1 in strengthening cell-cell adhesion and reports that Rap1GAP impairs cell-cell adhesion. We now show that Rap1GAP is a more effective inhibitor of cell-matrix compared to cell-cell adhesion. Overexpression of Rap1GAP in human colon cancer cells impaired Rap2 activity and the ability of cells to spread and migrate on collagen IV. Under the same conditions, Rap1GAP had no effect on cell-cell adhesion. Overexpression of Rap1GAP did not enhance the dissociation of cell aggregates nor did it impair the accumulation of β-catenin and E-cadherin at cell-cell contacts. To further explore the role of Rap1GAP in the regulation of cell-cell adhesion, Rap1GAP was overexpressed in non-transformed thyroid epithelial cells. Although the formation of cell-cell contacts required Rap1, overexpression of Rap1GAP did not impair cell-cell adhesion. These data indicate that transient, modest expression of Rap1GAP is compatible with cell-cell adhesion and that the role of Rap1GAP in the regulation of cell-cell adhesion may be more complex than is currently appreciated.Key words: Rap1GAP, cell adhesion, matrix adhesion, Rap, E-cadherin, β-catenin     

Phosphorylation-induced Conformational Changes in Rap1b: ALLOSTERIC EFFECTS ON SWITCH DOMAINS AND EFFECTOR LOOP*

Rap1b has been implicated in the transduction of the cAMP mitogenic response. Agonists that increase intracellular cAMP rapidly activate (i.e. GTP binding) and phosphorylate Rap1b on Ser¹⁷⁹ at its C terminus. cAMP-dependent protein kinase (PKA)-mediated phosphorylation of Rap1b is required for cAMP-dependent mitogenesis, tumorigenesis, and inhibition of AKT activity. However, the role of phosphorylation still remains unknown. In this study, we utilized amide hydrogen/deuterium exchange mass spectroscopy (DXMS) to assess potential conformational changes and/or mobility induced by phosphorylation. We report here DXMS data comparing exchange rates for PKA-phosphorylated (Rap1-P) and S179D phosphomimetic (Rap1-D) Rap1b proteins. Rap1-P and Rap1-D behaved exactly the same, revealing an increased exchange rate in discrete regions along the protein; these regions include a domain around the phosphorylation site and unexpectedly the two switch loops. Thus, local effects induced by Ser¹⁷⁹ phosphorylation communicate allosterically with distal domains involved in effector interaction. These results provide a mechanistic explanation for the differential effects of Rap1 phosphorylation by PKA on effector protein interaction.Rap1b, a member of the Ras superfamily of small G proteins, is a GTPase that acts as a molecular on/off switch for the transduction of several external stimuli by alternating from an inactive GDP-bound to an active GTP-bound state (1, 2). Rap1 activation is mediated by several second messengers, growth factors, cytokines, and cell adhesion molecules. The steady-state level of Rap1-GTP is tightly regulated by a family of guanine nucleotide exchange factors that catalyze the otherwise slow dissociation of GDP (i.e. activation) and GTPase-activating proteins, which stimulate the rather slow intrinsic GTPase catalytic activity (i.e. inactivation) (3). GTP binding is coupled to conformational changes in two well defined regions, the switch I (residues 30–40) and switch II (residues 60–76) domains, responsible for high affinity interaction with effector molecules (4, 5) and thus downstream signal transduction.cAMP is one among several pathways leading to Rap1 activation (6). cAMP exerts both mitogenic and anti-mitogenic responses in different cell types, and Rap1 activation is required downstream of cAMP in both scenarios (7, 8). Elevation of intracellular cAMP levels activates cAMP-dependent protein kinase (PKA)⁴ and Epac (exchange protein activated by cAMP), a Rap guanine nucleotide exchange factor (9). Expression of Rap1b in cells where cAMP is mitogenic is associated with an increase in cAMP-mediated G₁/S phase entry (7, 10), and both biochemical events, Rap activation and phosphorylation at Ser¹⁷⁹, are synergistically required for this action (11).PKA substrates able to modulate Rap1 activity (i.e. Src/C3G recruitment and GTPase-activating protein) were recently reported (12, 13). However, the role of PKA-dependent Rap1 phosphorylation at Ser¹⁷⁹ is still unknown. Rap1 phosphorylation does not affect its overall intracellular localization, its basal GTP/GDP exchange reaction, its intrinsic rate of GTP hydrolysis, or its ability to be stimulated by a cytosolic Rap GTPase-activating protein (10); however, several reports suggest that Rap1 phosphorylation is able to modulate its association with some binding partners, namely cytochrome b₅₅₈ (14) and Raf1 (15). The mechanism by which a modification of Ser¹⁷⁹ at the C-terminal end of the molecule affects the regions involved with effector interaction at its N terminus is for the moment unclear.In this study, we report a global assessment of the effects of Ser¹⁷⁹ phosphorylation on conformational change/mobility analyzed by hydrogen/deuterium exchange mass spectrometry (DXMS). The results are consistent with an allosteric effect of the C terminus (containing Ser¹⁷⁹) to the switch loops/effector domain.     

小分子量G蛋白Rap2信号途径的生物学功能

Rap2与Rap1同属于Ras超家族小分子量GTP结合蛋白的Rap亚家族,Rap2的氨基酸序列与Rap1具有60%的同源性,推测二者可能具有相似的信号途径和相近的生物学功能,包括细胞的增殖、分化、粘附和细胞骨架重排。然而,Rap2位于效应因子结构域的第39位的苯丙氨酸不同于Rap1及Ras的丝氨酸,这个关键差异表明其可能通过特异的下游信号分子调控独特的生物学功能。最近,随着Rap2特异效应因子的不断发现,Rap2特异的信号通路及功能受到了更多的关注,Rap2具有多样的生物学功能,除调控细胞粘附及细胞骨架动态组装外、Rap2调节中枢神经突触的可塑性以及非洲爪蟾发育中背腹轴特化。此外,也有报道显示Rap2的表达增强与多种肿瘤的形成具有相关性。本文主要针对Rap2的信号途径和生物学功能研究的最新进展进行介绍。