Rap1 Expression Research Articles

Dynamic changes in Rap1 activity are required for cell retraction and spreading during mitosis

At the onset of mitosis, most adherent cells undergo cell retraction characterised by the disassembly of focal adhesions and actin stress fibres. Mitosis takes place in rounded cells, and the two daughter cells spread again after cytokinesis. Because of the well-documented ability of the small GTPase Rap1 to stimulate integrin-dependent adhesion and spreading, we assessed its role during mitosis. We show that Rap1 activity is regulated during this process. Changes in Rap1 activity play an essential role in regulating cell retraction and spreading, respectively, before and after mitosis of HeLa cells. Indeed, endogenous Rap1 is inhibited at the onset of mitosis; conversely, constitutive activation of Rap1 inhibits the disassembly of premitotic focal adhesions and of the actin cytoskeleton, leading to delayed mitosis and to cytokinesis defects. Rap1 activity slowly increases after mitosis ends; inhibition of Rap1 activation by the ectopic expression of the dominant-negative Rap1[S17A] mutant prevents the rounded cells from spreading after mitosis. For the first time, we provide evidence for the direct regulation of adhesion processes during mitosis via the activity of the Rap1 GTPase.

Protein Kinase A-mediated Gating of Neuregulin-dependent ErbB2-ErbB3 Activation Underlies the Synergistic Action of cAMP on Schwann Cell Proliferation

In Schwann cells (SCs), cyclic adenosine monophosphate (cAMP) enhances the action of neuregulin, the most potent known mitogen for SCs, by synergistically increasing the activation of two crucial signaling pathways: ERK and Akt. However, the underlying mechanism of cross-talk between neuregulin and cAMP signaling remains mostly undefined. Here, we report that the activation of protein kinase A (PKA), but not that of exchange protein activated by cAMP (EPAC), enhances S-phase entry of SCs by synergistically enhancing the ligand-dependent tyrosine phosphorylation/activation of the neuregulin co-receptor, ErbB2-ErbB3. The role of PKA in neuregulin-ErbB signaling was confirmed using PKA inhibitors, pathway-selective cAMP analogs, and natural ligands stimulating PKA activity in SCs, such as adenosine and epinephrine. Two basic observations defined the synergistic action of PKA as "gating" for neuregulin-ErbB signaling: 1) the activation of PKA was not sufficient to induce S-phase entry or the activation of either ErbB2 or ErbB3; and 2) the presence of neuregulin was strictly required to ignite ErbB activation and thereby ERK and Akt signaling. However, PKA directly phosphorylated ErbB2 on Thr-686, a highly conserved intracellular regulatory site that was required for the PKA-mediated synergistic enhancement of neuregulin-induced ErbB2-ErbB3 activation and proliferation in SCs. The gating action of PKA on neuregulin-induced ErbB2-ErbB3 activation has important biological significance, because it insures signal amplification into the ERK and Akt pathways without compromising either the neuregulin dependence or the high specificity of ErbB signaling pathways.

Rap1 Activation Plays a Regulatory Role in Pancreatic Amylase Secretion

Rap1 is a member of the Ras superfamily of small GTP-binding proteins and is localized on pancreatic zymogen granules. The current study was designed to determine whether GTP-Rap1 is involved in the regulation of amylase secretion. Rap1A/B and the two Rap1 guanine nucleotide exchange factors, Epac1 and CalDAG-GEF III, were identified in mouse pancreatic acini. A fraction of both Rap1 and Epac1 colocalized with amylase in zymogen granules, but only Rap1 was integral to the zymogen granule membranes. Stimulation with cholecystokinin (CCK), carbachol, and vasoactive intestinal peptide all induced Rap1 activation, as did calcium ionophore A23187, phorbol ester, forskolin, 8-bromo-cyclic AMP, and the Epac-specific cAMP analog 8-pCPT-2'-O-Me-cAMP. The phospholipase C inhibitor U-73122 abolished carbachol- but not forskolin-induced Rap1 activation. Co-stimulation with carbachol and 8-pCPT-2'-O-Me-cAMP led to an additive effect on Rap1 activation, whereas a synergistic effect was seen on amylase release. Although the protein kinase A inhibitor H-89 abolished forskolin-stimulated CREB phosphorylation, it did not modify forskolin-induced GTP-Rap1 levels, excluding PKA participation. Overexpression of Rap1 GTPase-activating protein, which blocked Rap1 activation, reduced the effect of 8-bromo-cyclic AMP, 8-pCPT-2'-O-Me-cAMP, and vasoactive intestinal peptide on amylase release by 60% and reduced CCK- as well as carbachol-stimulated pancreatic amylase release by 40%. These findings indicate that GTP-Rap1 is required for pancreatic amylase release. Rap1 activation not only mediates the cAMP-evoked response via Epac1 but is also involved in CCK- and carbachol-induced amylase release, with their action most likely mediated by CalDAG-GEF III.

S145 – RAP1 Expression in Squamous Cell Carcinoma of Head and Neck

Objectives Rap1 has been shown to be activated in response to various growth factors, cytokines, and chemokines that act on receptor tyrosine kinases (RTKs) or G-protein-coupled receptors, and implicated in a wide range of biochemical pathways in all eukaryotic cells through a number of distinct effectors. This study was aimed to investigate the role of RAP1 in the tumorigenesis of squamous cell carcinoma of head and neck (HNSCC). Methods Immunohistochemical analysis was performed to study RAP1, CK 10, and E-cadherin expressions on the paraffin-embedded tissue of 198 HNSCCs. The relationship between RAP1 expression and the clinical prognosis was also investigated. Results RAP1 expression was readily detected in the normal epithelium and the well-differentiated HNSCC, whereas its expression turned to be weak or undetectable in the poorly differentiated HNSCC (p < 0.001). When RAP1 expression was decreased, the expressions of CK 10 (differentiation marker) and E-cadherin protein were also down regulated. In addition, patients with high RAP1 expression had significantly longer median survival than patients with low RAP1 expression (median = 51.21 vs. 13.14 months, p <0.001) by survival analysis based on the Kaplan-Meier method. Conclusions RAP1 expression may be related to cell differentiation in HNSCC. In addition, RAP1 could be a useful biomarker for tumor progression and survival of HNSCC.

A cAMP-Dependent, Protein Kinase A-Independent Signaling Pathway Mediating Neuritogenesis through Egr1 in PC12 Cells

The neurotrophic peptide PACAP (pituitary adenylate cyclase-activating polypeptide) elevates cAMP in PC12 cells. Forskolin and dibutyryl cAMP mimic PACAP's neuritogenic and cell morphological effects, suggesting that they are driven by cAMP. Comparison of microarray expression profiles after exposure of PC12 cells to either forskolin, dibutyryl cAMP, or PACAP revealed a small group of cAMP-dependent target genes. Neuritogenesis induced by all three agents is protein kinase A (PKA)-independent [not blocked by N-[2-(4-bromocinnamylamino)ethyl]-5-isoquinoline (H89)] and extracellular signal-regulated kinase (ERK)-dependent [blocked by 1,4-diamino-2,3-dicyano-1,4-bis(methylthio) butadiene (U0126)], and therefore cAMP-dependent target genes potentially mediating neuritogenesis were selected for further analysis based on the pharmacological profile of their induction by PACAP (i.e., mimicking that of neuritogenesis). Small interfering RNA (siRNA) targeting one of these genes, Egr1, blocked PACAP-induced neuritogenesis, and siRNA targeting another, Vil2, blocked a component of the cell size increase elicited by PACAP. Neither siRNA blocked PACAP's PKA-dependent antiproliferative effects. PACAP signaling to neuritogenesis was also impaired by dominant-negative Rap1 expression but was not affected by inhibition of protein kinase C (PKC), indicating a G-protein-coupled receptor-mediated differentiation pathway distinct from the one activated by receptor tyrosine kinase ligands such as nerve growth factor (NGF), that involves both Rap1 and PKC. We have thus identified a cAMP-dependent, PKA-independent pathway proceeding through ERK that functions to up-regulate the transcription of two genes, Egr1 and Vil2, required for PACAP-dependent neuritogenesis and increased cell size, respectively. Dominant-negative Rap1 expression impairs both PACAP-induced neuritogenesis and Egr1 activation by PACAP, suggesting that cAMP elevation and ERK activation by PACAP are linked through Rap1.

Inverse Rap1 and Phospho-ERK Expression Discriminate the Maintenance Phase of Tolerance and Priming of Antigen-Specific CD4+ T Cells In Vitro and In Vivo

T cell recognition of Ag can result in priming or tolerance depending on the context in which Ag is recognized. Previously, we have reported that these distinct functional outcomes are associated with marked differences in the amplitude, kinetics, and cellular localization of activated, pERK signals at the level of individual Ag-specific T cells in vitro. Here, we show that the GTPase Rap1, which can antagonize the generation of such pERK signals and has been reported to accumulate in tolerant cells, exhibits an inverse pattern of expression to pERK in individual Ag-specific primed and tolerized T cells. Although pERK is expressed by more primed than tolerized T cells when rechallenged with Ag in vitro, Rap1 is expressed by higher percentages of tolerant compared with primed Ag-specific T cells. Moreover, whereas pERK localizes to the TCR and lipid rafts in primed cells, but exhibits a diffuse cellular distribution in tolerized cells, Rap1 colocalizes with the TCR and lipid raft structures under conditions of tolerance, but not priming, in vitro. This inverse relationship between Rap1 and pERK expression is physiologically relevant, given that we observed the same patterns in Ag-specific T cells in situ, following induction of priming and tolerance in vivo. Together, these data suggest that the maintenance of tolerance of individual Ag-specific T cells may reflect the recruitment of up-regulated Rap1 to the immune synapse, potentially resulting in sequestration of Raf-1 and uncoupling of the TCR from the Ras-ERK-MAPK cascade.

Rap1 Integrates Tissue Polarity, Lumen Formation, and Tumorigenic Potential in Human Breast Epithelial Cells

Maintenance of apico-basal polarity in normal breast epithelial acini requires a balance between cell proliferation, cell death, and proper cell-cell and cell-extracellular matrix signaling. Aberrations in any of these processes can disrupt tissue architecture and initiate tumor formation. Here, we show that the small GTPase Rap1 is a crucial element in organizing acinar structure and inducing lumen formation. Rap1 activity in malignant HMT-3522 T4-2 cells is appreciably higher than in S1 cells, their nonmalignant counterparts. Expression of dominant-negative Rap1 resulted in phenotypic reversion of T4-2 cells, led to the formation of acinar structures with correct polarity, and dramatically reduced tumor incidence despite the persistence of genomic abnormalities and baseline growth. The resulting acini contained prominent central lumina not observed when other reverting agents were used. Conversely, expression of dominant-active Rap1 in T4-2 cells inhibited phenotypic reversion and led to increased invasiveness and tumorigenicity. Thus, Rap1 acts as a central regulator of breast architecture, with normal levels of activation instructing polarity during acinar morphogenesis, and increased activation inducing tumor formation and progression to malignancy.

The Hematopoietic Isoform of Cas-Hef1-Associated Signal Transducer Regulates Chemokine-Induced Inside-Out Signaling and T Cell Trafficking

Leukocyte migration and trafficking is dynamically regulated by various chemokine and adhesion molecules and is vital to the proper function of the immune system. We describe a role for the Cas and Hef-1-associated signal transducer in hematopoietic cells (Chat-H) as a critical regulator of T lymphocyte migration, by using lentivirus-mediated RNA interference (RNAi). Impaired migration of Chat-H-depleted cells coincided with defective inside-out signaling shown by diminished chemokine-induced activation of the Rap-1 GTPase and integrin-mediated adhesion. Localization of Chat-H to the plasma membrane, association with its binding partner Crk-associated substrate in lymphocytes (CasL), and Chat-H-mediated CasL serine-threonine phosphorylation were required for T cell migration. These results identify Chat-H as a critical signaling intermediate acting upstream of Rap1 to regulate chemokine-induced adhesion and migration.

Cyclic Adenosine 5′-Monophosphate-Stimulated Neurotensin Secretion Is Mediated through Rap1 Downstream of both Epac and Protein Kinase A Signaling Pathways

Neurotensin (NT), a gut peptide, plays important roles in gastrointestinal secretion, inflammation, and growth of normal and neoplastic tissues. cAMP regulates the secretion of hormones via its effector proteins protein kinase A (PKA) or Epac (exchange protein directly activated by cAMP). The small GTPase Rap1 can be activated by both PKA and Epac; however, the role of Rap1 in hormone secretion is unknown. Here, using the BON human endocrine cell line, we found that forskolin (FSK)-stimulated NT secretion was reduced by inhibition of Rap1 expression and activity. FSK-stimulated NT secretion was enhanced by overexpression of either wild-type or constitutively active Rap1. Epac activators and wild-type Epac enhanced NT release and Rap1 activity. In contrast, overexpression of a cAMP binding mutant, EpacR279E, decreased NT release and Rap1 activity. PKA activation increased NT release and Rap1 activity. FSK-stimulated NT release was reduced by PKA inhibition and the dominant negative Rap1N17. NT secretion, stimulated by Epac activation, was reduced by PKA inhibition; NT release, stimulated by PKA activation, was enhanced by wild-type Epac but reduced by the mutant EpacR279E. Finally, prostaglandin E2 (PGE2), a physiological agent that increases cAMP, stimulated NT secretion via cAMP/PKA/Rap1. Importantly, we demonstrate that PKA and Epac mediate the cAMP-induced NT secretion synergistically by converging at the common downstream target protein Rap1. Moreover, PGE2, a potent mediator of inflammation and associated with colorectal carcinogenesis, stimulates NT release suggesting a possible link between PGE2 and NT on intestinal inflammatory disorders and colorectal cancers.

Expression of Telomere-Associated Genes as Prognostic Markers for Overall Survival in Patients with Non–Small Cell Lung Cancer

Human telomeres, which are composed of long, repetitive sequences of TTAGGG and a variety of proteins, function as a protective structure capping the ends of chromosomes. Telomere dysfunction plays important roles in cancer initiation and progression. TRF1, TRF2, POT1, and RAP1 are four major telomere proteins that regulate telomere stability and telomere length. We hypothesized that the expression of these genes would have significant predictive value for cancer development and prognosis. We compared the mRNA expression level of TRF1, TRF2, POT1, and RAP1 between tumor and adjacent normal tissues from 148 patients with non-small cell lung cancer using real-time quantitative PCR. We then estimated the prognostic value of the mRNA expression of these genes in tumors. The expression level of TRF1 was significantly lower in tumor tissues than in adjacent normal tissues (P < 0.0001); no significant difference was found for TRF2, POT1, and RAP1. The expression of RAP1 gene in tumors was highly predictive of overall survival. In the Cox proportional hazards model, patients with higher RAP1 expression were associated with a significantly better survival [hazard ratio (HR), 0.47; 95% confidence interval (95% CI), 0.24-0.91]. This improved survival was more prominent in men (HR, 0.45; 95% CI, 0.22-0.996) and in ever smokers (HR, 0.50; 95% CI, 0.24-1.02). Kaplan-Meier survival curves showed that patients with higher RAP1 expression had significantly longer median survival than patients with lower expression (median = 51.21 versus 15.34 months, P < 0.0009). The expressions of TRF2 in tumor tissues were significantly correlated with tumor grades (P = 0.0114). RAP1 expression may be a useful biomarker of tumor progression and survival.

TIMP-2 promotes cell spreading and adhesion via upregulation of Rap1 signaling

We previously demonstrated that TIMP-2 treatment of human microvascular endothelial cells (hMVECs) activates Rap1 via the pathway of paxillin-Crk-C3G. Here, we show that TIMP-2 overexpression in hMVECs by adenoviral infection enhances Rap1 expression, leading to further increase in Rap1-GTP. TIMP-2 expression, previously reported to inhibit cell migration, also leads to cell spreading accompanied with increased cell adhesion. HMVECs stably expressing Rap1 display a similar phenotype as hMVECs-TIMP-2, whereas the expression of inactive Rap1 mutant, Rap1(38N), leads to elongated appearance with greatly reduced cell adhesion. Furthermore, the phenotype of hMVECs-Rap1(38N) was not reversed by TIMP-2 overexpression. TIMP-2 greatly promotes the association of Rap1 with actin. Therefore, these findings suggest that TIMP-2 mediated alteration in cell morphology requires Rap1, TIMP-2 may recruit Rap1 to sites of actin cytoskeleton remodeling necessary for cell spreading, and enhanced cell adhesion by TIMP-2 expression may hinder cell migration.

LFA‐1 bound to ICAM‐1 homodimer regulates adhesion lifetime and outside‐in signaling

Polymorphonuclear leukocytes (PMN) arrest on inflamed endothelium following transient tethering via selectins and activation and redistribution of integrin receptors. Integrin heterodimer CD11a/CD18 (LFA-1) shifts conformation following PMN exposure to chemokines presented on the endothelial surface and becomes primed for higher affinity interaction with intercellular adhesion molecule-1 (ICAM-1). Redistribution of LFA-1 and ICAM-1 into clusters is critical to prolong high avidity adhesion. We have recently reported that allosterically converting LFA-1 from a high to an intermediate or low affinity state with small molecules results in an exponential decrease in bond lifetime of dimeric ICAM-1/LFA-1. These data support the observation that adhesion involves a dynamic regulation of both LFA-1 conformation and valence in binding to ICAM-1. We present data showing that LFA-1 binding to dimeric ICAM-1 results in local activation of cytoskeletal structures involved in signaling of PMN activation. When LFA-1 on PMNs was bound to dimeric ICAM-1, F-actin and Rap1 expression was amplified at the contact region compared to binding of LFA-1 to monomeric ICAM-1. We conclude that LFA-1 ligation of a homodimer of ICAM-1 extends bond lifetime through efficient rebinding and may result in associated cytoplasmic signaling via dimeric LFA-1. Supported by NIH AI47294.

Histone deacetylase inhibitor FK228 suppresses the Ras–MAP kinase signaling pathway by upregulating Rap1 and induces apoptosis in malignant melanoma

Histone deacetylase (HDAC) inhibitors are expected to be effective for refractory cancer because their mechanism of action differs from that of conventional antineoplastic agents. In this study, we examined the effect of the HDAC inhibitor FK228 on malignant melanoma, as well as its molecular mechanisms. FK228 was highly effective against melanoma compared with other commonly used drugs. By comparing the gene expression profiles of melanoma cells and normal melanocytes, we defined a subset of genes specifically upregulated in melanoma cells by FK228, which included Rap1, a small GTP-binding protein of the Ras family. The expression of Rap1 mRNA and protein increased in FK228-treated melanoma cells in both a dose- and a time-dependent manner. A decrease in the phosphorylation of c-Raf, MEK1/2, and ERK1/2 was accompanied by an increase in Rap1 expression in both FK228-treated and Rap1-overexpressing cells. Inhibition of Rap1 upregulation by small interfering RNA (siRNA) abrogated the induction of apoptosis and suppression of ERK1/2 phosphorylation in FK228-treated melanoma cells. These results indicate that the cytotoxic effects of FK228 are mediated via the upregulation of Rap1. Furthermore, we found that Rap1 was overexpressed and formed a complex with B-Raf in melanoma cell lines with a V599E mutation of B-Raf. The siRNA-mediated abrogation of Rap1 overexpression increased the viability of these cells, suggesting that Rap1 is also an endogenous regulator of Ras-MAP kinase signaling in melanomas.

Tissue Inhibitor of Metalloproteinase-2 Promotes Neuronal Differentiation by Acting as an Anti-Mitogenic Signal

Although traditionally recognized for maintaining extracellular matrix integrity during morphogenesis, the function of matrix metallo-proteinases (MMPs) and their inhibitors, the tissue inhibitors of metalloproteinases (TIMPs), in the mature nervous system is essentially unknown. Here, we report that TIMP-2 induces pheochromocytoma PC12 cell-cycle arrest via regulation of cell-cycle regulatory proteins, resulting in differentiation and neurite outgrowth. TIMP-2 decreases cyclins B and D expression and increases p21Cip expression. Furthermore, TIMP-2 promotes cell differentiation via activation of the cAMP/Rap1/ERK (extracellular signal-regulated kinase) pathway. Expression of dominant-negative Rap1 blocks TIMP-2-mediated neurite outgrowth. Both the cell-cycle arrest and neurite outgrowth induced by TIMP-2 was independent of MMP inhibitory activity. Consistent with the PC12 cell data, primary cultures of TIMP-2 knock-out cerebral cortical neurons exhibit significantly reduced neurite length, which is rescued by TIMP-2. These in vitro results were corroborated in vivo. TIMP-2 deletion causes a delay in neuronal differentiation, as demonstrated by the persistence of nestin-positive progenitors in the neocortical ventricular zone. The interaction of TIMP-2 with alpha3beta1 integrin in the cerebral cortex suggests that TIMP-2 promotes neuronal differentiation and maintains mitotic quiescence in an MMP-independent manner through integrin activation. The identification of molecules responsible for neuronal quiescence has significant implications for the ability of the adult brain to generate new neurons in response to injury and neurological disorders, such as Alzheimer's and Parkinson's diseases.

Modulation of specific protein expression levels by PTEN: identification of AKAP121, DHFR, G3BP, Rap1 and RCC1 as potential targets of PTEN

The tumor suppressor PTEN is mutated in a high percentage of human cancers, and is implicated in pathways regulating cell growth, proliferation, survival, and migration. Despite significant advances, our understanding of its mechanisms of action remains incomplete. We have used a high-throughput proteomic immunoblotting approach to identify proteins whose expression levels are modulated by PTEN. Out of over 800 proteins screened, 22 proteins showed significant changes in expression. Five proteins that exhibited two-fold or greater changes in expression level were further characterized. AKAP121 and G3BP expression was reduced, while dihydrofolate reductase (DHFR), Rap1 and RCC1 expression was elevated in response to PTEN expression in a PTEN-null T-cell leukemia line. The phosphatase activity of PTEN was required for these effects. However, direct inhibition of PI-3 Kinase could mimic PTEN in modulating expression of DHFR, G3BP, Rap1 and RCC1, but not AKAP121. Real-time PCR showed that the effects of PTEN were primarily post-transcriptional, and would not have been revealed by mRNA-based screens. We conclude from these data that PTEN can modulate the expression level of a number of different proteins. The identified proteins have the potential to serve as previously unrecognized effectors of PTEN, and suggest the existence of additional complexity in the modes by which PTEN can regulate cellular biology.

Cannabinoid Receptor-induced Neurite Outgrowth Is Mediated by Rap1 Activation through Gαo/i-triggered Proteasomal Degradation of Rap1GAPII

The G(alpha)o/i-coupled CB1 cannabionoid receptor induces neurite outgrowth in Neuro-2A cells. The mechanisms of signaling through G(alpha)o/i to induce neurite outgrowth were studied. The expression of G(alpha)o/i reduces the stability of its direct interactor protein, Rap1GAPII, by targeting it for ubiquitination and proteasomal degradation. This results in the activation of Rap1. G(alpha)o/i-induced activation of endogenous Rap1 in Neuro-2A cells is blocked by the proteasomal inhibitor lactacystin. G(alpha)o/i stimulates neurite outgrowth that is blocked by the expression of dominant negative Rap1. Expression of Rap1GAPII also blocks the G(alpha)o/i-induced neurite outgrowth and treatment with proteasomal inhibitors potentiates this inhibition. The endogenous G(alpha)o/i-coupled cannabinoid (CB1) receptor in Neuro-2A cells stimulates the degradation of Rap1GAPII; activation of Rap1 and treatment with pertussis toxin or lactacystin blocks these effects. The CB1 receptor-stimulated neurite outgrowth is blocked by treatment with pertussis toxin, small interfering RNA for Rap, lactacystin, and expression of Rap1GAPII. Thus, the G(alpha)o/i-coupled cannabinoid receptor, by regulating the proteasomal degradation of Rap1GAPII, activates Rap1 to induce neurite outgrowth.

Expression, activation, and subcellular localization of the Rap1 GTPase in cord blood-derived human megakaryocytes

The expression of the small GTPase Rap1 in human megakaryocytes (MKs) differentiated from cord blood (CB)-derived progenitors was investigated. High levels of Rap1 were detected in the majority of mature megakaryocytes independently of days of culture, while a very low percentage of immature megakaryocytes was found to express a small amount of the protein. Rap1 was predominantly detected on internal α-granule but not on the plasma membrane. By contrast, CD41 was clearly present on the peripheral plasma membrane, although it also displayed an intracellular localization similar to that of Rap1. Upon thrombin stimulation, both Rap1 and CD41 translocated to the periphery of the cell. At the opposite, RhoA GTPase and glycoprotein Ibα were predominantly located at the plasma membrane and did not undergo relocation upon thrombin stimulation. Thrombin induced a dose- and time-dependent activation of Rap1 in mature megakaryocytes. By using a confocal microscopy approach with a specific probe, active Rap1 was detected exclusively at the peripheral plasma membrane. These results demonstrate that expression of Rap1 occurs during maturation rather than differentiation of megakaryocytes from cord blood progenitor cells. Moreover, we demonstrate that thrombin-activated Rap1 is exclusively localized at the peripheral plasma membrane.

Somatostatin down-regulates LFA-1 activation by modulating Rap1 expression in CD4 + and CD8 + T cells

Leukocyte function-associated antigen-1 (LFA-1) is one of the integrins that are expressed on the leukocytes, and has been shown to play an important role in leukocyte trafficking. The adhesive activity of LFA-1 is governed partially by the Rap1. This study examined that the relationship between LFA-1 and Rap1 mRNA expressions by anti-CD3 and anti-CD3+SOM treatment in the CD4 + and CD8 + T cells. The LFA-1 mRNA expression levels following the anti-CD3 and anti-CD3+SOM treatment for 30 min was greater on the CD8 + T cells, and the LFA-1 expression of the CD8 + T cells with anti-CD+SOM treatment was affected more severely than that of the CD4 + T cells. The Rap1 mRNA expression patterns following anti-CD3 and anti-CD3+SOM stimulation in the CD4 + and CD8 + T cells were similar to the LFA-1 expression patterns, and the expression level following anti-CD3+SOM treatment was suppressed more significantly in the CD8 + T cells. These results suggest that the difference in the Rap1 expression level after stimulation might explain the differences in the LFA-1 expression level on the T cell subsets, and that the down-regulation of Rap1 expression following SOM treatment is closely related to the diminished LFA-1 expression.

764. Role of Actin Cytoskeleton and Small GTPase Rap1 in the Co-Receptor Activity of Activated α5β1 Integrin for Human Parvovirus B19 Entry

The extremely restricted tropism of parvovirus B19 to human erythroid progenitors cells has been suggested to be mediated by blood group P antigen, which the virus uses as a cellular receptor for binding. We reported the generation of recombinant parvovirus B19 vectors (J. Virol., 72: 5224-5230, 1998), and concluded that P antigen is necessary, but not sufficient for parvovirus B19 infection (J. Virol., 75: 4110-4116, 2001). We subsequently identified activated α5β1 integrin as a co-receptor for viral entry (Blood, 102:3927-3933, 2003). To further elucidate the mechanisms that elicit the co-receptor activity of α5β1 integrins, we investigated the mediators involved in the proposed two step-model of integrin activation (release from cytoskeletal restraints and re-engagement of the cytoskeleton and assembly of a signaling complex upon ligand binding). Low-dose cytochalasin D treatment (10 nM cytoD), which disrupts the cortical cytoskeleton and releases integrins from their cytoskeletal restraints (step 1), increased parvovirus B19 transduction in PMA-differentiated K562 cells, whereas higher concentrations of cytoD (> 2 mM) inhibited parvovirus B19 transduction, suggesting that disruption of the cytoskeleton/signaling complex (step 2) impaired viral entry. The low-dose cytoD-induced increase in viral entry was abrogated by combined cell-surface immobilization of β1 and β3, but not β2, integrins. We also investigated the role of small GTPase Rap1, which facilitates cytoskeletal re-engagement and assembly of integrin signaling complex (step 2), in parvovirus B19 entry stably transfecting K562 cells with either constitutively active (Rap63E) or dominant negative (Rap17N) Rap1 expression cassettes. Transduction of PMA-differentiated Rap63E and Rap17N transfectants was reduced by 50% and 30%, respectively, compared to PMA-differentiated control cells, and correlated with 50% and 30% reduction, respectively, in cell surface expression of β1 integrins, suggesting that GDP/GTP cycling is involved in PMA-induced integrin expression. Outside-in signaling of β1 integrins was similar in PMA-differentiated Rap63E transfectants and control cells but impaired in Rap17N transfectants, suggesting that activated Rap1 proteins are crucial for the induction of β1 integrin co-receptor activity. Interestingly, over-expression of constitutively active Rap1 protein in undifferentiated K562 cells was sufficient to render these cells permissive for parvovirus B19 infection. Transduction of undifferentiated Rap63E transfectants was sensitive to outside-in signaling through β1 integrins, as determined by antibody-mediated stabilization of the high affinity β1 integrin conformation and β1 integrin cross-linking, and was completely abrogated in the presence of function-blocking β1 integrin antibodies and cytoD. Thus, our results suggest that Rap1 protein is crucial for β1 integrin activation observed after prolonged PMA-treatment, and that active Rap1 is sufficient to convert 'default inactive' β1 integrins expressed on non-adherent erythroleukemic K562 cells into functionally active co-receptors for parvovirus B19 entry. These studies have implications in the potential use of recombinant parvovirus B19 vectors in human hematopoietic cell gene therapy.

LAD-III, a leukocyte adhesion deficiency syndrome associated with defective Rap1 activation and impaired stabilization of integrin bonds

AbstractRecently, we reported a rare leukocyte adhesion deficiency (LAD) associated with severe defects in integrin activation by chemokine signals, despite normal ligand binding of leukocyte integrins.1 We now report that the small GTPase, Rap1, a key regulator of inside-out integrin activation is abnormally regulated in LAD Epstein-Barr virus (EBV) lymphocyte cells. Both constitutive and chemokine-triggered activation of Rap1 were abolished in LAD lymphocytes despite normal chemokine signaling. Nevertheless, Rap1 expression and activation by phorbol esters were intact, ruling out an LAD defect in Rap1 guanosine triphosphate (GTP) loading. The very late antigen 4 (VLA-4) integrin abnormally tethered LAD EBV lymphocytes to its ligand vascular cell adhesion molecule 1 (VCAM-1) under shear flow due to impaired generation of high-avidity contacts despite normal ligand binding and intact avidity to surface-bound anti-VLA-4 monoclonal antibody (mAb). Thus, a defect in constitutive Rap1 activation results in an inability of ligand-occupied integrins to generate high-avidity binding to ligand under shear flow. This is a first report of an inherited Rap1 activation defect associated with a pathologic disorder in leukocyte integrin function, we herein term it “LAD-III.” (Blood. 2004;103:1033-1036)