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Rap1 GTPase drives assembly of the Mig-10/RIAM/Lamellipodin (MRL protein)-integrin-talin (MIT) complex that enables integrin-dependent lymphocyte functions. Here we used tandem affinity tag-based proteomics to isolate and analyze the MIT complex and reveal that Phostensin (Ptsn), a regulatory subunit of protein phosphatase 1, is a component of the complex. Ptsn mediates dephosphorylation of Rap1, thereby preserving the activity and membrane localization of Rap1 to stabilize the MIT complex. CRISPR/Cas9-induced deletion of PPP1R18, which encodes Ptsn, markedly suppresses integrin activation in Jurkat human T cells. We generated apparently healthy Ppp1r18-/- mice that manifest lymphocytosis and reduced population of peripheral lymphoid tissues ascribable, in part, to defective activation of integrins αLβ2 and α4β7. Ppp1r18-/- T cells exhibit reduced capacity to induce colitis in a murine adoptive transfer model. Thus, Ptsn enables lymphocyte integrin-mediated functions by dephosphorylating Rap1 to stabilize the MIT complex. As a consequence, loss of Ptsn ameliorates T cell-mediated colitis., (© 2022 Lee et al.)

Autoimmune disorders are the third most common diseases in the United States, and affect the daily lives of millions of people. In this study, we analyzed patient samples, utilized a transgenic mouse model and human B cells to reveal Natural Killer Cell Transcript 4 (NK4) as a novel regulator that promotes the development of autoimmune disorders. NK4 was significantly elevated in samples from patients with Sjӧgren's Syndrome (SS). SS patients show elevated NK4 levels. There is a strong and positive correlation between the increased levels of NK4 and the duration of SS. Interestingly, transgenic expression of NK4 in a mouse model led to the development of autoantibodies and lymphocytic infiltration in salivary glands similar to those in SS patients. Those phenotypes were associated with increased B1a cells in the peritoneum, plasma cells in the spleen, and increased IgM, IgA, and IgG2a in serum of the NK4 transgenic mice. The autoimmune phenotypes became more severe in older mice. Moreover, after NK4 transfection, human naïve B cells were activated and memory B cells differentiation into IgG and IgA-plasmablasts, resulting in an increased production of autoantibodies.NK4 regulated the differentiation and activation of B cells through activating Rap1 activity. NK4 also promoted B cell migration in a paracrine fashion through an induction of CXCL13 in endothelial cells. Collectively, these findings identify NK4 as a promoter of the development of autoimmune disorders through its roles on B cells. Therefore, NK4 may be a novel therapeutic target for the treatment of autoimmune diseases., (Published by Elsevier Ltd.)

Background: MicroRNAs play a crucial role in diabetic peripheral neuropathic pain (DPNP). miR-590-3p is a novel miRNA and involved in multiple diseases. However, the pathological mechanism of miR-590-3p in DPNP needs to be elucidated., Materials and Methods: The db/db mice and db/m mice were selected to mimic diabetes and control, respectively, for in vivo studies. The miR-590-3p agomir was injected into db/db mice and pain-related behavioral tests were performed. The interaction of miR-590-3p with target gene was confirmed by dual-luciferase reporter assay. The expression of target gene was determined by qRT-PCR and western blot assay. The levels of inflammatory cytokines were measured by enzyme-linked immunosorbent assay (ELISA)., Results: miR-590-3p was down-regulated in diabetic peripheral neuropathy mice. More importantly, miR-590-3p agomir alleviated pain-related behavior, reduced TNF-α, IL-1β and IL-6 concentrations, and inhibited neural infiltration by immune cells in db/db mice. Interestingly, RAP1A was predicted to be the target of miR-590-3p by Targetscan, and was actually regulated by miR-590-3p. Finally, the rescue experiments proved that overexpression of RAP1A partially abrogated the suppressive impact of miR-590-3p on T cells proliferation and migration., Conclusion: miR-590-3p ameliorates DPNP via targeting RAP1A and inhibiting T cells infiltration, indicating that exogenous miR-590-3p may be a potential candidate for clinical treatment of DPNP.

Neutrophil extravasation is a critical step of the innate immune system's response to inflammation. This multistep process is tightly regulated by adhesion and signaling molecules in the endothelium and neutrophils. Activation of the β 2 integrin LFA-1 is critical for adhesion of leukocytes to postcapillary venules. This step requires coordinated activation of signaling pathways in chemokine-stimulated neutrophils, including GTPase activation and cytoskeletal remodeling, leading to conformational changes in LFA-1. Hematopoietic cell-specific lyn substrate 1 (HS1) is a cortactin-related and leukocyte-specific actin-binding protein (ABP) that regulates several processes in various immune cells. It has been shown in vitro that HS1 is important for neutrophil chemotaxis and transendothelial migration of NK cells, but its role in neutrophil extravasation in vivo has not been investigated yet. Intravital microscopy of CXCL1-stimulated cremaster venules revealed an increased rolling velocity and reduced neutrophil adhesion and transmigration in HS1 knockout (KO) mice. CXCL1-induced rapid neutrophil arrest in vivo and adhesion under flow conditions in vitro were also reduced significantly. Whereas random motility of neutrophils was unaffected, chemotaxis toward a CXCL1 gradient was reduced in the absence of HS1. Further analysis of the underlying mechanisms demonstrated that HS1 controls CXCL1-induced activation of the small GTPases Ras-related C3 botulinum toxin substrate 1 (Rac1) and Ras-related protein 1 (Rap1), thus supporting LFA-1-mediated neutrophil adhesion. Importantly, with the use of Rac1 KO neutrophils, we could show that Rac1 acts upstream of Rap1. Our results establish HS1 as an important regulator of proper Rac1 and Rap1 activation and neutrophil extravasation., (© Society for Leukocyte Biology.)

Regulation of integrins is critical for lymphocyte adhesion to endothelium and trafficking through secondary lymphoid organs. Inside-out signaling to integrins is mediated by the small GTPase Rap1. Two effectors of Rap1 regulate integrins, RapL and Rap1 interacting adaptor molecule (RIAM). Using mice conditionally deficient in both Rap1a and Rap1b and mice null for RIAM, we show that the Rap1/RIAM module is not required for T- or B-cell development but is essential for efficient adhesion to intercellular adhesion molecule (ICAM) 1 and vascular cell adhesion molecule (VCAM) 1 and for proper trafficking of lymphocytes to secondary lymphoid organs. Interestingly, in RIAM-deficient mice, whereas peripheral lymph nodes (pLNs) were depleted of both B and T cells and recirculating B cells were diminished in the bone barrow (BM), the spleen was hypercellular, albeit with a relative deficiency of marginal zone B cells. The abnormality in lymphocyte trafficking was accompanied by defective humoral immunity to T-cell-dependent antigens. Platelet function was intact in RIAM-deficient animals. These in vivo results confirm a role for RIAM in the regulation of some, but not all, leukocyte integrins and suggest that RIAM-regulated integrin activation is required for trafficking of lymphocytes from blood into pLNs and BM, where relatively high shear forces exist in high endothelial venules and sinusoids, respectively., (© 2015 by The American Society of Hematology.)

Rap1-GTP activates leukocyte function-associated antigen-1 (LFA-1) to induce arrest on the high endothelial venule (HEV). Here we show that Rap1-GDP restrains rolling behaviours of T cells on the peripheral lymph node addressin (PNAd), P-selectin and mucosal addressin cell adhesion molecule-1 (MadCAM-1) by inhibiting tether formation. Consequently, Rap1 deficiency impairs homing of naive T cells to peripheral lymph nodes, but accelerates homing of TH17 and TH1 cells to the colon, resulting in spontaneous colitis with tumours. Rap1-GDP associates with and activates lymphocyte-oriented kinase, which phosphorylates ERM (ezrin, radixin and moesin) in resting T cells. Phosphomimetic ezrin reduces the rolling of Rap1-deficient cells, and thereby decreases their homing into the colon. On the other hand, chemokines activate Rap1 at the plasma membrane within seconds, and Rap1-GTP binds to filamins, which diminishes its association with the β2 chain of LFA-1 and results in LFA-1 activation. This Rap1-dependent regulation of T-cell circulation prevents the onset of colitis.

Dendritic cells (DC) are one target for immune suppression by regulatory T cells (Treg), because their interaction results in reduced T cell stimulatory capacity and secretion of inhibitory cytokines in DC. We show that DC in the presence of Treg are more mobile as compared with cocultures with conventional CD4(+) T cells and form DC-Treg aggregates within 2 h of culture. The migration of DC was specifically directed toward Treg, as Treg, but not CD4(+) T cells, attracted DC in Boyden chambers. Treg deficient for the ectonucleotidase CD39 were unable to attract DC. Likewise, addition of antagonists for A2A adenosine receptors abolished the formation of DC-Treg clusters, indicating a role for adenosine in guiding DC-Treg interactions. Analysis of the signal transduction events in DC after contact to Treg revealed increased levels of cAMP, followed by activation of Epac1 and the GTPase Rap1. Subsequently activated Rap1 localized to the subcortical actin cytoskeleton in DC, providing a means by which directed locomotion of DC toward Treg is facilitated. In aggregate, these data show that Treg degrade ATP to adenosine via CD39, attracting DC by activating Epac1-Rap1-dependent pathways. As a consequence, DC-Treg clusters are formed and DC are rendered less stimulatory. This adenosine-mediated attraction of DC may therefore act as one mechanism by which Treg regulate the induction of immune responses by DC., (Copyright © 2015 by The American Association of Immunologists, Inc.)

CD28 is a coreceptor expressed on T lymphocytes. Signaling downstream of CD28 promotes multiple T cell functions such as proliferation, survival, and cytokine secretion. Adhesion to APCs is another function of T cells; however, little is known with regard to the role of CD28 in this process. Our previous studies have shown that CD28 inhibits T cell adhesion, but the underlying mechanism that mediates this process remains unknown. In the present study we discovered that signaling downstream of CD28 resulted in inhibition of Rap1 activity and decreased LFA-1-mediated adhesion. We showed that this was regulated by the recruitment of calcium-promoted Ras inactivator (CAPRI), a GTPase-activating protein, to the plasma membrane downstream of CD28 signaling. CAPRI trafficking to the plasma membrane was secondary to calcium influx and was mediated by its C2A and C2B domains. We conclude that CD28 inhibits Rap1-mediated adhesion by recruiting the protein CAPRI to the plasma membrane., (Copyright © 2015 by The American Association of Immunologists, Inc.)

Adaptive immunity depends on lymphocyte adhesion that is mediated by the integrin lymphocyte functional antigen 1 (LFA-1). The small guanosine triphosphatase Rap1 regulates LFA-1 adhesiveness through one of its effectors, Rap1-interacting adapter molecule (RIAM). We show that RIAM was recruited to the lymphocyte plasma membrane (PM) through its Ras association (RA) and pleckstrin homology (PH) domains, both of which were required for lymphocyte adhesion. The N terminus of RIAM inhibited membrane translocation. In vitro, the RA domain bound both Rap1 and H-Ras with equal but relatively low affinity, whereas in vivo only Rap1 was required for PM association. The PH domain bound phosphoinositol 4,5-bisphosphate (PI(4,5)P(2)) and was responsible for the spatial distribution of RIAM only at the PM of activated T cells. We determined the crystal structure of the RA and PH domains and found that, despite an intervening linker of 50 aa, the two domains were integrated into a single structural unit, which was critical for proper localization to the PM. Thus, the RA-PH domains of RIAM function as a proximity detector for activated Rap1 and PI(4,5)P(2).

Altered Ras superfamily guanine nucleotide triphosphatase signaling may contribute to the activation of autoreactive T cells in diseases such as rheumatoid arthritis and systemic lupus erythematosus. Here, we show that transgenic expression of activated Rap1, a Ras-related protein which is protective in murine arthritis, in both wildtype (WT) and 2D2 mice, enhances autoreactive T cell activation by myelin oligodendrocyte glycoprotein peptide in vitro and in vivo. However, RapV12 reduces the number of autoreactive T cells in both WT and 2D2 mice, and increases murine survival in experimental autoimmune encephalitis, suggesting Rap1 activation restricts autoimmune T cell-mediated pathology through enhancing tolerance., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Chemokines regulate T cell trafficking into secondary lymphoid organs and migration across endothelial cells in response to inflammatory signals. The small guanosine triphosphatase Rap1 is a critical regulator of chemokine signaling in T cells, but how chemokines activate Rap1 has been unclear. A study showed that Abl family tyrosine kinases were essential for chemokine-induced Rap1 activation, T cell polarization, and migration. Abl family kinases promoted Rap1 activation by phosphorylating the adaptor protein human enhancer of filamentation 1 (HEF1), thus establishing a critical Abl-HEF1-Rap1 signaling axis for chemokine-induced T cell migration.

Rap1, which is closely related to ras, plays a key role in T-cell receptor (TCR)-signaling. TCR-stimulation without costimulation leads to constitutively activated rap1, which may mediate T-cell anergy via inhibition of ras-dependent induction of extracellular signal-regulated kinases (ERK). This activation is mediated by a second protein kinase b-Raf. Rap1-GTP is thought to activate ERK in a ras-independent manner by binding b-raf. Generally, T cells do not express b-raf while they express the adaptor protein raf-1, which is usually sequestered by rap1 leading to inhibition of ras-mediated ERK activation. In this study, we demonstrate that in rap1-deficient T cells, signaling by the ERK and p38 kinases is increased following activation by different stimuli leading to increased intracellular accumulation and secretion of cytokines. In addition, in a hypersensitivity model rap1-deficient mice demonstrated reduced contact dermatitis compared to wildtype mice, demonstrating the impact of rap1-deficiency on the inflammatory response in vivo., (Copyright © 2012 Elsevier Inc. All rights reserved.)

RAPL (an alternative spliced form of Rassf5) is a critical Ras-related protein1 (Rap1) effector that regulates lymphocyte adhesion. Here, we have shown that in addition to this previously described function, RAPL also negatively controls lymphocyte proliferation and prevents autoimmunity and lymphoma. RAPL-deficient mice experienced age-related lupus-like glomerulonephritis and developed B cell lymphomas. RAPL-deficient lymphocytes showed hyperproliferation by enhanced S phase entry after antigen receptor ligation. Compared to wild-type cells, RAPL-deficient naive lymphocytes had a 2- to 3-fold increase in Cdk2 kinase activity with a cytoplasmic mislocalization of the cyclin-dependent kinase inhibitor p27(kip1). RAPL was found to suppress the phosphorylation of p27(kip1) on serine 10 (S10) and promoted p27(kip1) nuclear translocation. An S10A mutation in p27(kip1) corrected its cytoplasmic accumulation, reduced hyperproliferation in RAPL-deficient lymphocytes, and suppressed glomerulonephritis and development of B cell lymphoma. Thus, RAPL serves as a checkpoint for S phase entry to prevent lymphoproliferative disorders through the spatial regulation of p27(kip1)., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Objective: 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)., Methods: 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., Results: 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., Conclusion: 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., (Copyright © 2010 by the American College of Rheumatology.)

The hematopoietic progenitor kinase 1 (HPK1) signals into MAPK and NFκB pathways downstream of immunoreceptors, but enigmatically is a negative regulator of leukocytes. Here, we report a novel role for HPK1 in regulating the activation of the adhesion molecule leukocyte function-associated antigen-1 (LFA-1). Upon TCR stimulation, mediated by binding of adhesion and degranulation promoting adaptor protein (ADAP) to SLP-76, a ternary complex composed of ADAP/55-kDa src kinase associated phosphoprotein (SKAP-55) and RIAM translocates to the membrane and causes membrane recruitment of the active small GTPase Ras-related protein 1 (Rap1). Active Rap1, via its binding to RapL (regulator for cell adhesion and polarization enriched in lymphoid tissues), mediates LFA-1 integrin activation. We show here that HPK1, which also binds SLP-76, compete with ADAP for SLP-76 binding. In addition, HPK1 dampens Rap1 activation, resulting in decreased LFA-1 activity. Analysis of HPK1-deficient T cells revealed increased ADAP recruitment to SLP-76 and elevated Rap1 activation in those cells, leading to increased adhesion to ICAM-1 and cell spreading. Altogether, these results describe a novel function for HPK1 in linking TCR signaling to cell adhesion regulation and provide a mechanistic explanation for the negative regulatory role of HPK1 in T-cell biology.

The monomeric GTPase Rap1 controls functional activation of beta2 integrins in leukocytes. In this article, we describe a novel mechanism by which the chemoattractant fMLP activates Rap1 and inside-out signaling of beta2 integrins. We found that fMLP-induced activation of Rap1 in human polymorphonuclear leukocytes or neutrophils and differentiated PLB-985 cells was blocked by inhibitors of the NO/guanosine-3',5'-cyclic monophosphate-dependent protein kinase (cGKI) pathway [N-(3-(aminomethyl)benzyl)acetamidine, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, DT-3 peptide, 8-(4-chlorophenylthio)guanosine 3',5'-cyclic monophosphothioate, Rp-isomer triethylammonium salt-guanosine-3',5'-cyclic monophosphate], indicating that the downstream signaling events in Rap1 activation involve the production of NO and guanosine-3',5'-cyclic monophosphate, as well as the activation of cGKI. Silencing the expression of vasodilator-stimulated phosphoprotein (VASP), a substrate of cGKI, in resting PLB-985 cells or mice neutrophils led to constitutive activation of Rap1. In parallel, silencing VASP in differentiated PLB-985 cells led to recruitment of C3G, a guanine nucleotide exchange factor for Rap1, to the plasma membrane. Expression of murine GFP-tagged phosphodeficient VASP Ser235Ala mutant (murine serine 235 of VASP corresponds to human serine 239) in PLB-985 cells blunted fMLP-induced translocation of C3G to the membrane and activation of Rap1. Thus, bacterial fMLP triggers cGKI-dependent phosphorylation of human VASP on serine 239 and, thereby, controls membrane recruitment of C3G, which is required for activation of Rap1 and beta2 integrin-dependent antibacterial functions of neutrophils.

Although essential for T cell function, the identity of the T cell receptor "inside-out" pathway for lymphocyte function-associated antigen 1 (LFA-1) adhesion has proved elusive. Here, we define the "inside-out" pathway mediated by N-terminal SKAP1 (SKAP-55) domain binding to the C-terminal SARAH domain of RapL. TcR induced Rap1-RapL complex formation and LFA-1 binding failed to occur in Skap1(-/-) primary T cells. SKAP1 generated a SKAP1-RapL-Rap1 complex that bound to LFA-1, whereas a RapL mutation (L224A) that abrogated SKAP1 binding without affecting MST1 disrupted component colocalization in vesicles as well as T cell-dendritic cell (DC) conjugation. RapL expression also "slowed" T cell motility in D011.10 transgenic T cells in lymph nodes (LNs), an effect reversed by the L224A mutation with reduced dwell times between T cells and DCs. Overall, our findings define a TCR "inside-out" pathway via N-SKAP1-C-RapL that regulates T cell adhesion, motility, and arrest times with DCs in LNs., (Copyright 2010 Elsevier Inc. All rights reserved.)

Cytotoxic T lymphocyte antigen 4 (CTLA-4) is a T cell co-stimulation receptor that delivers inhibitory signals upon activation. This inhibitory effect by CTLA-4 requires activation of small GTPase Rap-1. However, the precise mechanism underlying these negative signals remains unclear. Here, we show that CTLA-4-induced suppression of IL-2 production correlates with rapid destabilization of immunological synapse (IS) formation in murine normal T cell clones. Overexpression of Spa-1, a Rap-1-specific GTPase activating protein (GAP), abolished both Rap-1 activation and IL-2 suppression induced by CTLA-4. Although we failed to find any specific inhibition of activation of early signals upon CTLA-4 engagement, we found that CTLA-4 specifically up-regulates cell motility and suppresses prolonged accumulation of Talin at the contact area with antigen presenting cells upon antigen stimulation. These results suggest that Rap-1 is activated upon CTLA-4 ligation and mediates inhibitory signals through prevention of IS formation.

Zoledronic acid (ZOL), an effective nitrogen-containing bisphosphonate against excessive bone loss, has been shown affecting the function of cells of both innate and acquired immunity. In this study, we tested the effect of ZOL on differentiation and maturation of human myeloid dendritic cells (DC). When ZOL (1.1 to 10 microM) was added to the culture of starting monocytes, but not to immature DC, the recovery rate of DC was markedly reduced in a concentration-dependent manner. The mature DC differentiated in the presence of ZOL had fewer and shorter cell projections. ZOL treatment affected DC differentiation and maturation in terms of lower expression of CD1a, CD11c, CD83, CD86, DC-SIGN, HLA-DR, and, in contrast, higher expression of CD80. IL-10 production by DC was inhibited by ZOL treatment whereas IL-12p70 secretion remained unchanged. Interestingly, ZOL augmented the allostimulatory activity of DC on naive CD4(++)CD45(+)RA(++) T cells in terms of their proliferation and interferon-gamma production. Addition of geranylgeraniol abrogated the effect of ZOL on DC differentiation and prenylation of Rap1A. It suggests that ZOL redirects DC differentiation toward a state of atypical maturation with allostimulatory function and this effect may go through prevention of Rap1A prenylation.

Phagocytosis of IgG-opsonized microbes via the Fc gamma receptor (Fc gammaR) requires the precise coordination of a number of signaling molecules, including the low-molecular mass GTPases. Little is known about the Ras-family GTPase Rap1 in this process. We therefore investigated its importance in mediating Fc gammaR-dependent phagocytosis in NR8383 rat alveolar macrophages. Pulldown of active Rap1 and fluorescence microscopic analysis of GFP-RalGDS (Ral guanine dissociation stimulator)-transfected macrophages revealed that Rap1 is indeed activated by Fc gammaR crosslinking. Inhibition of Rap1 activity, both by Rap1GAP (GTPase-activating protein) expression and liposome-delivered blocking Ab, severely impaired the ability of cells to ingest IgG-opsonized targets. Fc gammaR-induced Rap1 activation was found to be independent of both cAMP and Ca(2+), suggesting a role for the second messenger-independent guanosine exchange factor, C3G. This was supported by the facts that 1) liposome-delivered blocking Ab against C3G inhibited both Fc gammaR-dependent phagocytosis and Rap1 activation, and 2) both active Rap1GTP and C3G were found to translocate to the phagosome. Taken together, our data demonstrate a novel role for Rap1 and its exchange factor C3G in mediating Fc gammaR-dependent phagocytosis.

Integrins are critical for the migration of T cells to lymphoid organs and to sites of inflammation and are also necessary for productive interactions between T cells and antigen-presenting cells. Integrin activation is enhanced following T-cell receptor (TCR) engagement, as signals initiated by the TCR increase affinity and avidity of integrins for their ligands. This process, known as inside-out signaling, has been shown to require several molecular components including the cytosolic adapter proteins adhesion and degranulation-promoting adapter protein and Src homology 2 domain-containing adapter protein of 55 kDa, the low molecular weight guanosine triphosphatase Rap1, and the Rap1 effector proteins Rap1 guanosine triphosphate-interacting adapter molecule, regulator of adhesion and cell polarization enriched in lymphoid tissues, and protein kinase D1. Herein, we review recent findings about how the TCR is linked to integrin activation through inside-out signaling.

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.

Extravasation of leukocytes is a crucial process in the immunological defense. In response to a local concentration of chemokines, circulating leukocytes adhere to and migrate across the vascular endothelium toward the inflamed tissue. The small guanosinetriphosphatase Rap1 plays an important role in the regulation of leukocyte adhesion, polarization, and chemotaxis. We investigated the role of a guanine nucleotide exchange protein for Rap1 directly activated by cAMP (Epac1) in adhesion and chemotaxis in a promonocytic cell line and in primary monocytes. We found that Epac1 is expressed in primary leukocytes, platelets, CD34-positive hematopoietic cells, and the leukemic cell lines U937 and HL60. Epac activation with an Epac-specific cAMP analog induced Rap1 activation, beta1-integrin-dependent cell adhesion, and cell polarization. In addition, activated Epac1 enhanced chemotaxis of U937 cells and primary monocytes. Similar to activation of Epac1, stimulation of cells with serotonin to induce cAMP production resulted in Rap1 activation, increased cell adhesion and polarization, and enhanced chemotaxis. The effects of serotonin on U937 cell adhesion were dependent on cAMP production but could not be blocked by a protein kinase A inhibitor, implicating Epac in the regulation of serotonin-induced adhesion. In summary, our work reveals the existence of previously unrecognized cAMP-dependent signaling in leukocytes regulating cell adhesion and chemotaxis through the activation of Epac1.

An important aspect of tumor progression is the ability of cancer cells to escape detection and clearance by the immune system. Recent studies suggest that several tumors express soluble factors interfering with the immune response. Here, we show that semaphorin-3A (Sema-3A), a secreted member of the semaphorin family involved in axonal guidance, organogenesis, and angiogenesis, is highly expressed in several tumor cells. Conditioned media of Sema-3A-transfected COS-7 cells or human recombinant Sema-3A inhibited primary human T-cell proliferation and cytokines production under anti-CD3 plus anti-CD28 stimulating conditions. Sema-3A also inhibited the activation of nonspecific cytotoxic activity in mixed lymphocyte culture (MLC), as measured against K-562 cells. In contrast, suppression of Sema-3A in tumor cells with a small interfering RNA (siRNA) augmented T-cell activation. The inhibitory effect of Sema-3A in T cells is mediated by blockade of Ras/mitogen-activated protein kinase (MAPK) signaling pathway. The presence of Sema-3A increased the activation of the Ras family small GTPase Rap1 and introduction of the dominant-negative mutant of Rap1 (Rap1N17) blunted the immunoinhibitory effects of Sema-3A. These results suggest that Sema-3A inhibits primary T-cell activation and imply that it can contribute to the T-cell dysfunction in the tumor microenvironment.

SPA-1 is involved in the regulation of T cell activation in response to antigens through the control of Rap1 GTPase signaling. In this study, the subcellular localization of SPA-1 in the T cells was examined by using anti-SPA-1 antibody and GFP-SPA-1. While SPA-1 was detected diffusely at the surface cortical region in the floating unpolarized T cells, it was concentrated at the matrix-adhesion region with dense actin-cytoskeleton. Upon interaction with specific antigen-presenting cells, SPA-1 was highly concentrated at the immunological synapse closely co-localizing with actin. By yeast two-hybrid system, SPA-1 was shown to interact with an actin-bundling protein alpha-actinin, and it was indicated that SPA-1 co-localized with alpha-actinin at the immunological synapse. The results have suggested that SPA-1 in the T cells is selectively recruited to the immunological synapse with dense actin-cytoskeletal reorganization and keeps restraining the levels of Rap1GTP at the local TCR-signaling complex for the T cell activation.

Immunoblotting with a monoclonal Rap1 antibody, we found that elevation of cyclic AMP, with forskolin and IBMX or CPT-cAMP, led to a rapid reduction in the levels of Rap1 protein associated with particulate, nuclear/perinuclear fractions from PC12 and COS1 cells. In contrast, cytoplasmic levels of Rap1 remained constant following cyclic AMP stimulation. To gain independent confirmation that cyclic AMP promoted loss of Rap1 in nuclear/perinuclear fractions we used a polyclonal Rap1 antibody, which gave similar results to the monoclonal antibody. This demonstrated that the loss in Rap1 immunoreactivity was not due to phosphorylation-dependent changes that alter immunorecognition. The reduction in Rap1 levels was blocked by PKA inhibitors and by a Rap1 serine to alanine PKA-phosphorylation site mutant (S180A). Peptide inhibitors of the proteasome, cathespin, and calpain II also inhibited the decrease in Rap1 levels, indicating that proteolytic degradation may contribute to maintaining Rap1 levels in the nuclear/perinuclear fraction of cells.

Mammalian telomeres consist of TTAGGG repeats, telomeric repeat binding factor (TRF), and other proteins, resulting in a protective structure at chromosome ends. Although structure and function of the somatic telomeric complex has been elucidated in some detail, the protein composition of mammalian meiotic telomeres is undetermined. Here we show, by indirect immunofluorescence (IF), that the meiotic telomere complex is similar to its somatic counterpart and contains significant amounts of TRF1, TRF2, and hRap1, while tankyrase, a poly-(ADP-ribose)polymerase at somatic telomeres and nuclear pores, forms small signals at ends of human meiotic chromosome cores. Analysis of rodent spermatocytes reveals Trf1 at mouse, TRF2 at rat, and mammalian Rap1 at meiotic telomeres of both rodents. Moreover, we demonstrate that telomere repositioning during meiotic prophase occurs in sectors of the nuclear envelope that are distinct from nuclear pore-dense areas. The latter form during preleptotene/leptotene and are present during entire prophase I.