Role of ICAM-3 in Intercellular Adhesion and Activation of T Lymphocytes

The immune system requires a fine regulation of intercellular communication for its normal function. There are several regulated molecular pathways involved in leukocyte cell interactions (Springer, 1990; Hynes, 1992). Among them, the interaction of the leukocyte integrin LFA-1 (CDlla/CD18) with its ligands provides multiple accessory adhesion signals of capital importance during different functions of the immune response, such as antigen presentation (Harding and Unanue, 1991), T-B lymphocyte interaction (Moy and Brian, 1992), cellular cytotoxicity (Makgoba et al., 1988; Altmann et al., 1989; Davignon et al, 1981; Akella and Hall, 1992), allogenic and autologous mixed lymphocyte reactions (Bagnasco et al., 1990) and recirculation and homing of lymphocytes through tissue endothelium (Hamann et al., 1988; Pals et al, 1988).     

Role of ICAM-3 in Intercellular Adhesion and Activation of T Lymphocytes

The immune system requires a fine regulation of intercellular communication for its normal function. There are several regulated molecular pathways involved in leukocyte cell interactions (Springer, 1990; Hynes, 1992). Among them, the interaction of the leukocyte integrin LFA-1 (CDlla/CD18) with its ligands provides multiple accessory adhesion signals of capital importance during different functions of the immune response, such as antigen presentation (Harding and Unanue, 1991), T-B lymphocyte interaction (Moy and Brian, 1992), cellular cytotoxicity (Makgoba et al., 1988; Altmann et al., 1989; Davignon et al, 1981; Akella and Hall, 1992), allogenic and autologous mixed lymphocyte reactions (Bagnasco et al., 1990) and recirculation and homing of lymphocytes through tissue endothelium (Hamann et al., 1988; Pals et al, 1988).     

Soluble Collagen VI Induces Tyrosine Phosphorylation of Paxillin and Focal Adhesion Kinase and Activates the MAP Kinase Erk2 in Fibroblasts

Signals from the extracellular matrix can modulate cellular differentiation and gene expression. We have shown previously that in contrast to other extracellular matrix molecules pepsin-solubilized collagen VI (CVI) can stimulate DNA synthesis of various mesenchymal cell types, apparently independent of integrin-mediated signal transduction. In order to further elucidate collagen VI-induced signaling events, we exposed mouse 3T3 fibroblasts and human HT1080 fibrosarcoma cells to soluble CVI. CVI induced tyrosine phosphorylation of proteins that associate with focal adhesions, such as paxillin, focal adhesion kinase (FAK), and p130CAS. Furthermore, it activated the mitogen-activated protein kinase, erk2. Kinetic analysis showed that these phosphorylations were transient, reaching a maximum after 5 min for transformed HT1080 cells and 30 min for 3T3 fibroblasts. These effects were partly inhibited by a beta1-integrin function blocking antibody and by single chains of CVI. Our results indicate that soluble fragments of native collagen VI, a ubiquitous component of the interstitial extracellular matrix, can mediate stimulation of DNA synthesis via tyrosine phosphorylation of paxillin, FAK, p130CAS, and erk2 in the absence of classical growth factors. Thus, CVI may serve as a matrix-derived sensor that allows for rapid reconstitution of a tissue defect by activating nearby mesenchymal cells.     

The Spontaneously Adhesive Leukocyte Function-associated Antigen-1 (LFA-1) Integrin in Effector T Cells Mediates Rapid Actin- and Calmodulin-dependent Adhesion Strengthening to Ligand under Shear Flow

Integrins in effector T cells are highly expressed and important for trafficking of these cells and for their effector functions. However, how integrins are regulated in effector T cells remains poorly characterized. Here, we have investigated effector T cell leukocyte function-associated antigen-1 (LFA-1) regulation in primary murine effector T cells. These cells have high LFA-1 integrin expression and display high spontaneous binding to intercellular adhesion molecule-1 (ICAM-1) ligand under static conditions. In addition, these cells are able to migrate spontaneously on ICAM-1. Atomic force microscopy measurements showed that the force required for unbinding of integrin-ligand interactions increases over time (0.5–20-s contact time). The maximum unbinding force for this interaction was ∼140 piconewtons at 0.5-s contact time, increasing to 580 piconewtons at 20-s contact time. Also, the total work required to disrupt the interaction increased over the 20-s contact time, indicating LFA-1-mediated adhesion strengthening in primary effector T cells over a very quick time frame. Effector T cells adhered spontaneously to ICAM-1 under conditions of shear flow, in the absence of chemokine stimulation, and this binding was independent of protein kinase B/Akt and protein kinase C kinase activity, but dependent on calcium/calmodulin signaling and an intact actin cytoskeleton. These results indicate that effector T cell integrins are highly expressed and spontaneously adhesive in the absence of inside-out integrin signaling but that LFA-1-mediated firm adhesion under conditions of shear flow requires downstream integrin signaling, which is dependent on calcium/calmodulin and the actin cytoskeleton.     

The Kindlin 3 Pleckstrin Homology Domain Has an Essential Role in Lymphocyte Function-associated Antigen 1 (LFA-1) Integrin-mediated B Cell Adhesion and Migration

The protein kindlin 3 is mutated in the leukocyte adhesion deficiency III (LAD-III) disorder, leading to widespread infection due to the failure of leukocytes to migrate into infected tissue sites. To gain understanding of how kindlin 3 controls leukocyte function, we have focused on its pleckstrin homology (PH) domain and find that deletion of this domain eliminates the ability of kindlin 3 to participate in adhesion and migration of B cells mediated by the leukocyte integrin lymphocyte function-associated antigen 1 (LFA-1). PH domains are often involved in membrane localization of proteins through binding to phosphoinositides. We show that the kindlin 3 PH domain has binding affinity for phosphoinositide PI(3,4,5)P₃ over PI(4,5)P₂. It has a major role in membrane association of kindlin 3 that is enhanced by the binding of LFA-1 to intercellular adhesion molecule 1 (ICAM-1). A splice variant, kindlin 3-IPRR, has a four-residue insert in the PH domain at a critical site that influences phosphoinositide binding by enhancing binding to PI(4,5)P₂ as well as by binding to PI(3,4,5)P₃. However kindlin 3-IPRR is unable to restore the ability of LAD-III B cells to adhere to and migrate on LFA-1 ligand ICAM-1, potentially by altering the dynamics or PI specificity of binding to the membrane. Thus, the correct functioning of the kindlin 3 PH domain is central to the role that kindlin 3 performs in guiding lymphocyte adhesion and motility behavior, which in turn is required for a successful immune response.     

K-252a Induces Tyrosine Phosphorylation of the Focal Adhesion Kinase and Neurite Outgrowth in Human Neuroblastoma SH-SY5Y Cells

Abstract: The protein kinase inhibitor K-252a has been shown to promote cholinergic activity in cultures of rat spinal cord and neuronal survival in chick dorsal root ganglion cultures. To determine the mechanism by which K-252a acts as a neurotrophic factor, we examined the effects of this molecule on a human neuroblastoma cell line, SH-SY5Y. K-252a induced neurite outgrowth in a dose-dependent manner. Coincident with neurite outgrowth was the early tyrosine phosphorylation of 125- and 140-kDa proteins. The phosphorylation events were independent of protein kinase C inhibition because down-regulation of protein kinase C by long-term treatment with phorbol ester did not prevent K-252a-induced tyrosine phosphorylation. Similarly, the protein kinase C inhibitors H7, GF-109203X, and calphostin C did not induce the phosphorylation. We have identified one of the phosphosubstrates as the pp125 focal adhesion protein tyrosine kinase (Fak). Induction of phosphorylation coincided with increased Fak activity and appeared to be independent of ligand/integrin interaction. The induction of Fak phosphorylation by K-252a was also observed in LA-N-5 cells and primary cultures of rat embryonic striatal cells but not in PC12 cells. The protein kinase C-independent induction of tyrosine phosphorylation and the identification of Fak as a substrate of K-252a-induced tyrosine kinase activity suggest that this compound mediates neurotrophic effects through a novel signaling pathway.     

Integrin Cross Talk: Activation of Lymphocyte Function-associated Antigen-1 on Human T Cells Alters α4β1- and α5β1-mediated Function

A regulated order of adhesion events directs leukocytes from the vascular compartment into injured tissues in response to inflammatory stimuli. We show that on human T cells, the interaction of the β2 integrin leucocyte function–associated antigen-1 (LFA-1) with its ligand intercellular adhesion molecule-1 (ICAM-1) will decrease adhesion mediated by α4β1 and, to a lesser extent, α5β1. Similar inhibition is also seen when T cells are exposed to mAb 24, which stabilizes LFA-1 in an active state after triggering integrin function through divalent cation Mg²⁺, PdBu, or T cell receptor/ CD3 complex (TCR/CD3) cross-linking. Such cross talk decreases α4β1 integrin–mediated binding of T cells to fibronectin and vascular cell adhesion molecule-1 (VCAM-1). In contrast, ligand occupancy or prolonged activation of β1 integrin has no effect on LFA-1 adhesion to ICAM-1. We also show that T cell migration across fibronectin, unlike adhesion, is mediated solely by α5β1, and is increased when the α4β1-mediated component of fibronectin adhesion is decreased either by cross talk or the use of α4-blocking mAb. The ability of mAb 24 Fab′ fragments to induce cross talk without cross-linking LFA-1 suggests signal transduction through the active integrin. These data provide the first direct evidence for cross talk between LFA-1 and β1 integrins on T cells. Together, these findings imply that activation of LFA-1 on the extravasating T cell will decrease the binding to VCAM-1 while enhancing the subsequent migration on fibronectin. This sequence of events provides a further level of complexity to the coordination of T cell integrins, whose sequential but overlapping roles are essential for transmigration.     

Proteomic Profiling of Endothelial Invasion Revealed Receptor for Activated C Kinase 1 (RACK1) Complexed with Vimentin to Regulate Focal Adhesion Kinase (FAK)

Angiogenesis is critical for many physiological and pathological processes. To identify molecules relevant to angiogenesis, we performed a proteomic screen comparing invading versus non-invading endothelial cells in three-dimensional collagen matrices. We found up-regulated levels of receptor for activated C kinase 1 (RACK1) and the intermediate filament protein vimentin that correlated with increased endothelial cell invasion. Because both RACK1 and vimentin have been linked to focal adhesion kinase (FAK), we investigated whether this pathway regulated invasion. RACK1 depletion reduced invasion responses, and this was associated with attenuated activation of FAK. Knockdown of vimentin significantly decreased levels of phosphorylated and total FAK. Treatment with a pharmacological inhibitor of FAK dose-dependently reduced invasion, indicating a crucial role for FAK activity during invasion. Because RACK1 and vimentin were both up-regulated with sphingosine 1-phosphate treatment, required for invasion, and regulated FAK, we tested whether they complexed together. RACK1 complexed with vimentin, and growth factors enhanced this interaction. In addition, RACK1, vimentin, and FAK formed an intermolecular complex in invading endothelial cultures in three dimensions in response to stimulation by sphingosine 1-phosphate and growth factors. Moreover, depletion of RACK1 decreased the association of vimentin and FAK, suggesting that RACK1 was required for stabilizing vimentin-FAK interactions during sprouting. Silencing of vimentin and RACK1 decreased cell adhesion and focal contact formation. Taken together, these results demonstrate that proangiogenic signals converge to enhance expression and association of RACK1 and vimentin, which regulated FAK, resulting in successful endothelial sprout formation in three-dimensional collagen matrices.     

The Arg-Gly-Asp Motif in the Cell Adhesion Molecule L1 Promotes Neurite Outgrowth via Interaction with the αvβ3 Integrin

The cell adhesion molecule L1 is a potent inducer of neurite outgrowth and it has been implicated in X-linked hydrocephalus and related neurological disorders. To investigate the mechanisms of neurite outgrowth stimulated by L1, attempts were made to identify the neuritogenic sites in L1. Fusion proteins containing different segments of the extracellular region of L1 were prepared and different neuronal cells were assayed on substrate-coated fusion proteins. Interestingly, both immunoglobulin (Ig)-like domains 2 and 6 (Ig2, Ig6) promoted neurite outgrowth from dorsal root ganglion cells, whereas neural retinal cells responded only to Ig2. L1 Ig2 contains a previously identified homophilic binding site, whereas L1 Ig6 contains an Arg-Gly-Asp (RGD) sequence. The neuritogenic activity of Ig6 was abrogated by mutations in the RGD site. The addition of RGD-containing peptides also inhibited the promotion of neurite outgrowth from dorsal root ganglion cells by glutathione S-transferase-Ig6, implicating the involvement of an integrin. The monoclonal antibody LM609 against α_vβ₃ integrin, but not an anti-β₁ antibody, inhibited the neuritogenic effects of Ig6. These data thus provide the first evidence that the RGD motif in L1 Ig6 is capable of promoting neurite outgrowth via interaction with the α_vβ₃ integrin on neuronal cells.     

Integrin-mediated Activation of Focal Adhesion Kinase Is Required for Signaling to Jun NH2-terminal Kinase and Progression through the G1 Phase of the Cell Cycle

The extracellular matrix exerts a stringent control on the proliferation of normal cells, suggesting the existence of a mitogenic signaling pathway activated by integrins, but not significantly by growth factor receptors. Herein, we provide evidence that integrins cause a significant and protracted activation of Jun NH2-terminal kinase (JNK), while several growth factors cause more modest or no activation of this enzyme. Integrin-mediated stimulation of JNK required the association of focal adhesion kinase (FAK) with a Src kinase and p130(CAS), the phosphorylation of p130(CAS), and subsequently, the recruitment of Crk. Ras and PI-3K were not required. FAK-JNK signaling was necessary for proper progression through the G1 phase of the cell cycle. These findings establish a role for FAK in both the activation of JNK and the control of the cell cycle, and identify a physiological stimulus for JNK signaling that is consistent with the role of Jun in both proliferation and transformation.     

The Microvesicle Component of HIV-1 Inocula Modulates Dendritic Cell Infection and Maturation and Enhances Adhesion to and Activation of T Lymphocytes

HIV-1 is taken up by immature monocyte derived dendritic cells (iMDDCs) into tetraspanin rich caves from which the virus can either be transferred to T lymphocytes or enter into endosomes resulting in degradation. HIV-1 binding and fusion with the DC membrane results in low level de novo infection that can also be transferred to T lymphocytes at a later stage. We have previously reported that HIV-1 can induce partial maturation of iMDDCs at both stages of trafficking. Here we show that CD45⁺ microvesicles (MV) which contaminate purified HIV-1 inocula due to similar size and density, affect DC maturation, de novo HIV-1 infection and transfer to T lymphocytes. Comparing iMDDCs infected with CD45-depleted HIV-1_BaL or matched non-depleted preparations, the presence of CD45⁺ MVs was shown to enhance DC maturation and ICAM-1 (CD54) expression, which is involved in DC∶T lymphocyte interactions, while restricting HIV-1 infection of MDDCs. Furthermore, in the DC culture HIV-1 infected (p24⁺) MDDCs were more mature than bystander cells. Depletion of MVs from the HIV-1 inoculum markedly inhibited DC∶T lymphocyte clustering and the induction of alloproliferation as well as limiting HIV-1 transfer from DCs to T lymphocytes. The effects of MV depletion on these functions were reversed by the re-addition of purified MVs from activated but not non-activated SUPT1.CCR5-CL.30 or primary T cells. Analysis of the protein complement of these MVs and of these HIV-1 inocula before and after MV depletion showed that Heat Shock Proteins (HSPs) and nef were the likely DC maturation candidates. Recombinant HSP90α and β and nef all induced DC maturation and ICAM-1 expression, greater when combined. These results suggest that MVs contaminating HIV-1 released from infected T lymphocytes may be biologically important, especially in enhancing T cell activation, during uptake by DCs in vitro and in vivo, particularly as MVs have been detected in the circulation of HIV-1 infected subjects.     

Cross-talk between Serine/Threonine Protein Phosphatase 2A and Protein Tyrosine Phosphatase 1B Regulates Src Activation and Adhesion of Integrin αIIbβ3 to Fibrinogen

Integrin α_IIbβ₃ signaling mediated by kinases and phosphatases participate in hemostasis and thrombosis, in part, by supporting stable platelet adhesion. Our previous studies indicate that the genetic manipulation of PP2Acα (α isoform of the catalytic subunit of protein phosphatase 2A) negatively regulate the adhesion of human embryonal kidney 293 cells expressing α_IIbβ₃ to fibrinogen. Here, we demonstrated that small interference RNA (siRNA) mediated knockdown of PP2Acα in 293 α_IIbβ₃ cells led to the dephosphorylation of Src Tyr-529, phosphorylation of Src Tyr-418 and an increased Src kinase activity. Conversely, overexpression of PP2Acα decreased the basal Src activity. Pharmacological inhibition of PP2Ac in human platelets or PP2Acα knockdown in primary murine megakaryocytes resulted in Src activation. PP2Acα-depleted 293 α_IIbβ₃ cells did not alter the serine (Ser) phosphorylation of Src but enhanced the Ser-50 phosphorylation of protein tyrosine phosphatase 1B (PTP-1B) with a concomitant increase in the PTP-1B activity. Src activation in the PP2Acα-depleted 293 α_IIbβ₃ cells was abolished by siRNA mediated knockdown of PTP-1B. Pharmacological inhibition of Src or knockdown of Src, PTP-1B blocked the enhanced activation of extracellular signal-regulated kinase (ERK1/2) and the increased adhesiveness of PP2Acα-depleted 293 α_IIbβ₃ cells to fibrinogen, respectively. Thus, inactivation of PP2Acα promotes hyperphosphorylation of PTP-1B Ser-50, elevates PTP-1B activity, which dephosphorylates Src Tyr-529 to activate Src and its downstream ERK1/2 signaling pathways that regulate α_IIbβ₃ adhesion. Moreover, these studies extend the notion that a cross-talk between Ser/Thr and Tyr phosphatases can fine-tune α_IIbβ₃ outside-in signaling.     

Induced Focal Adhesion Kinase (FAK) Expression in FAK-Null Cells Enhances Cell Spreading and Migration Requiring Both Auto- and Activation Loop Phosphorylation Sites and Inhibits Adhesion-Dependent Tyrosine Phosphorylation of Pyk2

Focal adhesion kinase (FAK) is a nonreceptor protein tyrosine kinase involved in integrin-mediated control of cell behavior. Following cell adhesion to components of the extracellular matrix, FAK becomes phosphorylated at multiple sites, including tyrosines 397, 576, and 577. Tyr-397 is an autophosphorylation site that promotes interaction with c-Src or Fyn. Tyr-576 and Tyr-577 lie in the putative activation loop of the kinase domain, and FAK catalytic activity may be elevated through phosphorylation of these residues by associated Src family kinase. Recent studies have implicated FAK as a positive regulator of cell spreading and migration. To further study the mechanism of adhesion-induced FAK activation and the possible role and signaling requirements for FAK in cell spreading and migration, we utilized the tetracycline repression system to achieve inducible expression of either wild-type FAK or phosphorylation site mutants in fibroblasts derived from FAK-null mouse embryos. Using these Tet-FAK cells, we demonstrated that both the FAK autophosphorylation and activation loop sites are critical for maximum adhesion-induced FAK activation and FAK-enhanced cell spreading and migration responses. Negative effects on cell spreading and migration, as well as decreased phosphorylation of the substrate p130(Cas), were observed upon induced expression of the FAK autophosphorylation site mutant. These negative effects appear to result from an inhibition of integrin-mediated signaling by the FAK-related kinase Pyk2/CAKbeta/RAFTK/CadTK.     

Dynamic Phosphorylation of Tyrosine 665 in Pseudopodium-enriched Atypical Kinase 1 (PEAK1) Is Essential for the Regulation of Cell Migration and Focal Adhesion Turnover

Pseudopodium-enriched atypical kinase 1 (PEAK1) is a recently described tyrosine kinase that associates with the actin cytoskeleton and focal adhesion (FA) in migrating cells. PEAK1 is known to promote cell migration, but the responsible mechanisms remain unclear. Here, we show that PEAK1 controls FA assembly and disassembly in a dynamic pathway controlled by PEAK1 phosphorylation at Tyr-665. Knockdown of endogenous PEAK1 inhibits random cell migration. In PEAK1-deficient cells, FA lifetimes are decreased, FA assembly times are shortened, and FA disassembly times are extended. Phosphorylation of Tyr-665 in PEAK1 is essential for normal PEAK1 localization and its function in the regulation of FAs; however, constitutive phosphorylation of PEAK1 Tyr-665 is also disruptive of its function, indicating a requirement for precise spatiotemporal regulation of PEAK1. Src family kinases are required for normal PEAK1 localization and function. Finally, we provide evidence that PEAK1 promotes cancer cell invasion through Matrigel by a mechanism that requires dynamic regulation of Tyr-665 phosphorylation.     

药物治疗老年高血压对hs-CRP和ICAM-1 水平的影响

目的：通过比较老年原发性高血压患者使用瑞舒伐他汀治疗前后的情况, 探讨瑞舒伐他汀对老年原发性高血压患者血清超 敏C反应蛋白（hs-CRP）和细胞间黏附分子-1（ICAM-1）水平的影响。方法：选取86 例老年高血压患者随机分为对照组和治疗组, 其中对照组进行采用氨氯地平治疗,而治疗组在对照组的基础上采用瑞舒伐他汀治疗,分别检测和比较两组患者的hs-CRP和ICAM- 1 水平,并进行统计学分析。结果：与治疗前相比,治疗后对照组与治疗组hs-CRP 和ICAM-1 均明显降低,而两组的hs-CRP 和ICAM-1 差异显著（t=3.1655,P<0.01;t=9.6983,P<0.01）,具有统计学意义。结论：瑞舒伐他汀能显著降低老年高血压患者的 hs-CRP 和ICAM-1 水平,减轻患者的炎性反应,具有重要的临床应用价值。     

Biological and Structural Characterization of Glycosylation on Ephrin-A1, a Preferred Ligand for EphA2 Receptor Tyrosine Kinase

The EphA2 receptor tyrosine kinase is overexpressed in a number of malignancies and is activated by ephrin ligands, most commonly by ephrin-A1. The crystal structure of the ligand-receptor complex revealed a glycosylation on the Asn-26 of ephrin-A1. Here we report for the first time the significance of the glycosylation in the biology of EphA2 and ephrin-A1. Ephrin-A1 was enzymatically deglycosylated, and its activity was evaluated in several assays using glioblastoma (GBM) cells and recombinant EphA2. We found that deglycosylated ephrin-A1 does not efficiently induce EphA2 receptor internalization and degradation, and does not activate the downstream signaling pathways involved in cell migration and proliferation. Data obtained by surface plasmon resonance confirms that deglycosylated ephrin-A1 does not bind EphA2 with high affinity. Mutations in the glycosylation site on ephrin-A1 result in protein aggregation and mislocalization. Analysis of Eph/ephrin crystal structures reveals an interaction between the ligand''s carbohydrates and two residues of EphA2: Asp-78 and Lys-136. These findings suggest that the glycosylation on ephrin-A1 plays a critical role in the binding and activation of the EphA2 receptor.     

Multiple Grb2-Mediated Integrin-Stimulated Signaling Pathways to ERK2/Mitogen-Activated Protein Kinase: Summation of Both c-Src- and Focal Adhesion Kinase-Initiated Tyrosine Phosphorylation Events

Fibronectin receptor integrin-mediated cell adhesion triggers intracellular signaling events such as the activation of the Ras/mitogen-activated protein (MAP) kinase cascade. In this study, we show that the nonreceptor protein-tyrosine kinases (PTKs) c-Src and focal adhesion kinase (FAK) can be independently activated after fibronectin (FN) stimulation and that their combined activity promotes signaling to extracellular signal-regulated kinase 2 (ERK2)/MAP kinase through multiple pathways upstream of Ras. FN stimulation of NIH 3T3 fibroblasts promotes c-Src and FAK association in the Triton-insoluble cell fraction, and the time course of FN-stimulated ERK2 activation paralleled that of Grb2 binding to FAK at Tyr-925 and Grb2 binding to Shc. Cytochalasin D treatment of fibroblasts inhibited FN-induced FAK in vitro kinase activity and signaling to ERK2, but it only partially inhibited c-Src activation. Treatment of fibroblasts with protein kinase C inhibitors or with the PTK inhibitor herbimycin A or PP1 resulted in reduced Src PTK activity, no Grb2 binding to FAK, and lowered levels of ERK2 activation. FN-stimulated FAK PTK activity was not significantly affected by herbimycin A treatment and, under these conditions, FAK autophosphorylation promoted Shc binding to FAK. In vitro, FAK directly phosphorylated Shc Tyr-317 to promote Grb2 binding, and in vivo Grb2 binding to Shc was observed in herbimycin A-treated fibroblasts after FN stimulation. Interestingly, c-Src in vitro phosphorylation of Shc promoted Grb2 binding to both wild-type and Phe-317 Shc. In vivo, Phe-317 Shc was tyrosine phosphorylated after FN stimulation of human 293T cells and its expression did not inhibit signaling to ERK2. Surprisingly, expression of Phe-925 FAK with Phe-317 Shc also did not block signaling to ERK2, whereas FN-stimulated signaling to ERK2 was inhibited by coexpression of an SH3 domain-inactivated mutant of Grb2. Our studies show that FN receptor integrin signaling upstream of Ras and ERK2 does not follow a linear pathway but that, instead, multiple Grb2-mediated interactions with Shc, FAK, and perhaps other yet-to-be-determined phosphorylated targets represent parallel signaling pathways that cooperate to promote maximal ERK2 activation.     

Flt3配体增强HCV核心-包膜E2融合基因DNA疫苗诱导的细胞免疫应答

建立一种可高效诱导细胞免疫应答 ,对丙型肝炎病毒 (HCV)感染可能起预防和治疗作用的DNA疫苗。将小鼠Flt3配体 (FL)信号肽和胞外段cDNA插入结构优化的HCV核心 包膜E2融合抗原DNA疫苗pST CE2t,构建成pST CE2t FL。将pST CE2t FL转染COS7细胞 ,Westernblot和ELISA检测表明该重组质粒能表达HCV核心 包膜E2融合抗原和可溶性小鼠FL。分别将pST CE2t、pST CE2t FL和空载体pCI neo肌肉注射接种BALB c小鼠 ,检测小鼠的体液和细胞免疫应答。结果表明两种DNA结构均能在小鼠体内诱生细胞和体液免疫应答 ,但pST CE2t诱导的体液免疫应答强于pST CE2t FL ,而后者诱导的细胞免疫应答明显强于前者。FL能明显增强HCV核心 包膜E2融合抗原DNA疫苗诱导的细胞免疫应答 ,对于发展HCV预防和治疗性疫苗有潜在的应用价值。     

Moesin Interacts with the Cytoplasmic Region of Intercellular Adhesion Molecule-3 and Is Redistributed to the Uropod of T Lymphocytes during Cell Polarization

During activation, T lymphocytes become motile cells, switching from a spherical to a polarized shape. Chemokines and other chemotactic cytokines induce lymphocyte polarization with the formation of a uropod in the rear pole, where the adhesion receptors intercellular adhesion molecule-1 (ICAM-1), ICAM-3, and CD44 redistribute. We have investigated membrane–cytoskeleton interactions that play a key role in the redistribution of adhesion receptors to the uropod. Immunofluorescence analysis showed that the ERM proteins radixin and moesin localized to the uropod of human T lymphoblasts treated with the chemokine RANTES (regulated on activation, normal T cell expressed, and secreted), a polarization-inducing agent; radixin colocalized with arrays of myosin II at the neck of the uropods, whereas moesin decorated the most distal part of the uropod and colocalized with ICAM-1, ICAM-3, and CD44 molecules. Two other cytoskeletal proteins, β-actin and α-tubulin, clustered at the cell leading edge and uropod, respectively, of polarized lymphocytes. Biochemical analysis showed that moesin coimmunoprecipitates with ICAM-3 in T lymphoblasts stimulated with either RANTES or the polarization- inducing anti–ICAM-3 HP2/19 mAb, as well as in the constitutively polarized T cell line HSB-2. In addition, moesin is associated with CD44, but not with ICAM-1, in polarized T lymphocytes. A correlation between the degree of moesin–ICAM-3 interaction and cell polarization was found as determined by immunofluorescence and immunoprecipitation analysis done in parallel. The moesin–ICAM-3 interaction was specifically mediated by the cytoplasmic domain of ICAM-3 as revealed by precipitation of moesin with a GST fusion protein containing the ICAM-3 cytoplasmic tail from metabolically labeled Jurkat T cell lysates. The interaction of moesin with ICAM-3 was greatly diminished when RANTES-stimulated T lymphoblasts were pretreated with the myosin-disrupting drug butanedione monoxime, which prevents lymphocyte polarization. Altogether, these data indicate that moesin interacts with ICAM-3 and CD44 adhesion molecules in uropods of polarized T cells; these data also suggest that these interactions participate in the formation of links between membrane receptors and the cytoskeleton, thereby regulating morphological changes during cell locomotion.