Stimulation of T cells through the CD3/T-cell receptor complex: role of cytoplasmic calcium, protein kinase C translocation, and phosphorylation of pp60c-src in the activation pathway.

Stimulation of T cells or the Jurkat T-cell line with soluble antibodies to the CD3/T-cell receptor complex causes mobilization of cytoplasmic Ca2+, which is blocked by pertussis toxin but not by ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, and translocation of protein kinase C activity from the cytoplasm to the membrane. Such stimulation also causes phosphorylation of pp60c-src at an amino-terminal serine residue. These activities are consistent with induction of phosphatidylinositol metabolism after antibody binding. Anti-CD3 stimulation with antibody in solution, however, does not cause Jurkat cells to release interleukin 2 and blocks rather than induces proliferation of T cells. Induction of interleukin 2 production by Jurkat cells and proliferation by normal T cells requires anti-CD3 stimulation with antibody on a solid support, such as Sepharose beads or a plastic dish. Thus, we examined phosphorylation of pp60c-src after stimulation of Jurkat cells with anti-CD3 in solution or on solid phase. Both of these caused serine phosphorylation of pp60c-src that was indistinguishable even after 4 h of stimulation. These results indicate that the mode of anti-CD3 stimulation (in solution or on solid phase) controls a cellular function that modifies the consequences of signal transduction through phosphatidylinositol turnover.     

Rho-ROCK-dependent ezrin-radixin-moesin phosphorylation regulates Fas-mediated apoptosis in Jurkat cells

Upon engagement by its ligand, the Fas receptor (CD95/APO-1) is oligomerized in a manner dependent on F-actin. It has been shown that ezrin, a member of the ERM (ezrin-radixin-moesin) protein family can link Fas to the actin cytoskeleton. We show herein that in Jurkat cells, not only ezrin but also moesin can associate with Fas. The same observation was made in activated human peripheral blood T cells. Fas/ezrin or moesin (E/M) association increases in Jurkat cells following Fas triggering and occurs concomitantly with the formation of SDS- and 2-ME-stable high molecular mass Fas aggregates. Ezrin and moesin have to be present together for the formation of Fas aggregates since down-regulation of either ezrin or moesin expression with small interfering RNAs completely inhibits Fas aggregate formation. Although FADD (Fas-associated death domain protein) and caspase-8 associate with Fas in the absence of E/M, subsequent events such as caspase-8 activation and sensitivity to apoptosis are decreased. During the course of Fas stimulation, ezrin and moesin become phosphorylated, respectively, on T567 and on T558. This phosphorylation is mediated by the kinase ROCK (Rho-associated coiled coil-containing protein kinase) I subsequently to Rho activation. Indeed, inhibition of either Rho or ROCK prevents ezrin and moesin phosphorylation, abrogates the formation of Fas aggregates, and interferes with caspase-8 activation. Thus, phosphorylation of E/M by ROCK is involved in the early steps of apoptotic signaling following Fas triggering and regulates apoptosis induction.     

The mitogenic activity of human T-cell leukemia virus type I is T-cell associated and requires the CD2/LFA-3 activation pathway.

The presence of a high number of activated T cells in the bloodstream and spontaneous proliferation of peripheral blood mononuclear cells in vitro are striking characteristics of human T-cell leukemia virus type I (HTLV-I) infection. The HTLV-I regulatory protein Tax and the envelope protein gp46 have been implicated in mediating the activation process. In this study, HTLV-I-producing cell lines and purified virus from the cell lines were examined for the ability to activate peripheral blood lymphocytes (PBLs) and Jurkat cells. Antisera and monoclonal antibodies against several cellular adhesion proteins involved in T-cell activation and against viral proteins were used to identify which molecules may be participating in the activation process. First, neither virus from a T-cell line, MT2, nor virus produced from the human osteosarcoma cell line HOS/PL was able to induce PBLs to proliferate. In contrast, both fixed and irradiated HTLV-I-producing T-cell lines induced proliferation of PBLs; HOS/PL cells did not activate PBLs. Second, HTLV-I-positive T-cell lines were capable of activating interleukin-2 mRNA expression in Jurkat cells. Induction of interleukin-2 expression was inhibited by anti-CD2 and anti-lymphocyte function-associated antigen 3 (LFA-3) monoclonal antibodies but not anti-human leukocyte antigen-DR, anti-CD4, anti-LFA-1, or anti-intercellular adhesion molecule 1. Similar results were obtained with PBLs as the responder cells. Furthermore, monoclonal antibodies and antisera against various regions of the HTLV-I envelope proteins gp46 and gp21 as well as p40tax did not block activation. These data indicate that HTLV-I viral particles are not intrinsically mitogenic and that infection of target T cells is not necessary for activation. Instead, the mitogenic activity is restricted to virus-producing T cells, requires cell-to-cell contact, and may be mediated through the LFA-3/CD2 activation pathway.     

Rab35 - A vesicular traffic-regulating small GTPase with actin modulating roles

The Rab family of GTPases are regulators of eukaryotic vesicular membrane traffic, while modulation of actin dynamics is a function conventionally associated with the Rho family of GTPases. Rab35 is a Rab protein with both plasma membrane and endosomal localization, and has been implicated in diverse processes that include T-cell receptor recycling, oocyte yolk protein recycling and cytokinesis. Rab35 regulates neurite outgrowth in neuronal-like cells, and can induce protrusions even in typically non-adherent Jurkat T-cells. Recent evidence indicates that Rab35’s activity, particularly the ability to mediate protrusive outgrowths, is due to its direct influence on actin dynamics. This can occur via activation of the Rho family of GTPases, or through the engagement of its effector fascin, an actin bundling protein.     

Simian immunodeficiency virus Nef protein delays the progression of CD4+ T cells through G1/S phase of the cell cycle

Human and simian immunodeficiency virus (HIV and SIV, respectively) infections are characterized by gradual depletion of CD4+ T cells. The underlying mechanisms of CD4+ T-cell depletion and HIV and SIV persistence are not fully determined. The Nef protein is expressed early in infection and is necessary for pathogenesis. Nef can cause T-cell activation and downmodulates cell surface signaling molecules. However, the effect of Nef on the cell cycle has not been well characterized. To determine the role of Nef in the cell cycle, we investigated whether the SIV Nef protein can modulate cell proliferation and apoptosis in CD4+ Jurkat T cells. We developed a CD4+ Jurkat T-cell line that stably expresses SIV Nef under the control of an inducible promoter. Alterations in cell proliferation were determined by flow cytometry using stable intracytoplasmic fluorescent dye 5- and 6-carboxyfluorescein diacetate succinimidyl ester and bromodeoxyuridine incorporation. Apoptotic cell death was measured by annexin V and propidium iodide staining. Our results demonstrated that SIV Nef inhibited Fas-induced apoptosis in these cells and that the mechanism involved upregulation of the Bcl-2 protein. SIV Nef suppressed CD4+ T-cell proliferation by inhibiting the progression of cells into S phase of the cell cycle. Suppression involved an upregulation of cyclin-dependent kinase inhibitors p21 and p27 and the downregulation of cyclin D1 and cyclin A. In summary, inhibition of apoptosis by Nef can lead to persistence of infected cells and can support viral replication. In addition, a Nef-mediated delay in cell cycle progression may contribute to CD4+ T-cell anergy/depletion seen in HIV and SIV disease.     

Activation of endogenous c-fos proto-oncogene expression by human T-cell leukemia virus type I-encoded p40tax protein in the human T-cell line, Jurkat.

We examined the ability of the trans-acting factor p40tax of human T-cell leukemia virus type I (HTLV-I), which is thought to be a crucial molecule in T-cell transformation by HTLV-I, to activate expression of a set of endogenous cellular genes related to T-cell proliferation. For this purpose we established a subclone (JPX-9) of Jurkat cells that was stably transfected with an expression plasmid containing the p40tax gene, whose expression is definitely dependent on heavy-metal ions. Expression of the interleukin-2 receptor alpha chain in JPX-9 cells was induced in response to the induction of p40tax expression, as has been demonstrated by others in transient transfection experiments with Jurkat cells. In addition, we found that significant enhancement of expression of the nuclear proto-oncogene c-fos was closely associated with expression of p40tax. Continous enhancement in the level of c-fos mRNA was observed in the presence of p40tax. In contrast, mRNA levels of other nuclear proto-oncogenes (c-myc, c-myb, and c-jun) were not appreciably effected by the expression of p40tax. These results suggest that (i) in addition to the interleukin-2-interleukin-2 receptor system, cellular genes such as c-fos, which regulate normal T-cell growth, are also activated directly or indirectly by p40tax and (ii) p40tax-induced modulation of gene expression plays a crucial role in T-cell transformation by HTLV-I.     

Regulation of proteasome-mediated protein degradation during oxidative stress and aging

Protein degradation is a physiological process required to maintain cellular functions. There are distinct proteolytic systems for different physiological tasks under changing environmental and pathophysiological conditions. The proteasome is responsible for the removal of oxidatively damaged proteins in the cytosol and nucleus. It has been demonstrated that proteasomal degradation increases due to mild oxidation, whereas at higher oxidant levels proteasomal degradation decreases. Moreover, the proteasome itself is affected by oxidative stress to varying degrees. The ATP-stimulated 26S proteasome is sensitive to oxidative stress, whereas the 20S form seems to be resistant. Non-degradable protein aggregates and cross-linked proteins are able to bind to the proteasome, which makes the degradation of other misfolded and damaged proteins less efficient. Consequently, inhibition of the proteasome has dramatic effects on cellular aging processes and cell viability. It seems likely that during oxidative stress cells are able to keep the nuclear protein pool free of damage, while cytosolic proteins may accumulate. This is because of the high proteasome content in the nucleus, which protects the nucleus from the formation and accumulation of non-degradable proteins. In this review we highlight the regulation of the proteasome during oxidative stress and aging.     

Somatostatin analog SMS 201995 inhibits proliferation in human leukemia T-cell line: relevance of the adenylyl cyclase stimulation

Octreotide SMS 201995 is a stable somatostatin (SRIF) analog with potent antiproliferative actions in numerous cell types including normal T lymphocytes. It is currently used in the clinical treatment of different malignancies. However, the possible beneficial actions of octreotide in T-cell leukemia have not been addressed before, although these cells express SRIF receptors. For instance, human leukemia Jurkat T cells have been shown to express a single SRIF receptor isotype: sst3 that can be pharmacologically targeted by octreotide. In this study, we therefore studied SMS 201995 effects on in vitro [(3)H-CH3]thymidine incorporation in Jurkat T cells. Our data show that octreotide inhibits the proliferation of Jurkat cells both in the absence and in the presence of mitogens. By contrast, SRIF28, an endogenous SRIF analog sharing with SMS 201995 an almost identical affinity for somatostatin sst3 receptors, increases [(3)H-CH3]thymidine uptake in both mitogen-activated and nonactivated cells. To assess the mechanisms of the opposite actions of these two analogs on leukemia T-cell proliferation, we next studied their effects on adenylyl cyclase activity in whole Jurkat cells. At least in the presence of mitogens, SMS 201995 significantly enhances the adenylyl cyclase activity whereas SRIF28 inhibits it. Taken together these data are in accordance with the current hypothesis according to which increase and decrease in cAMP production are required to allow the inhibition and stimulation of T-cell proliferation, respectively. They also point to a potential therapeutic benefit of SMS 201995 in the management of human T-cell leukemia.     

A single amino acid substitution in a proteasome subunit triggers aggregation of ubiquitinated proteins in stressed neuronal cells

Accumulation of ubiquitinated proteins in inclusions is common to various neurodegenerative disorders such as Parkinson's disease, Alzheimer's disease and amyotrophic lateral sclerosis, although it occurs in selective neurons in each disease. The mechanisms generating such abnormal aggregates and their role in neurodegeneration remain unclear. Inclusions appear in familial and non-familial cases of neurodegenerative disorders, suggesting that factors other than particular mutations contribute to protein accumulation and aggregation. Proteasome impairment triggered by aging or conditions such as oxidative stress may contribute to protein accumulation and aggregation in neurodegeneration. To test this hypothesis in mouse neuronal cells, we overexpressed a 20S proteasome beta5 subunit with an active site mutation. The N-terminal threonine to alanine substitution resulted in impairment of the chymotrypsin-like activity, which is a rate-limiting step in protein degradation by the proteasome. The Thr1Ala mutation was not lethal under homeostatic conditions. However, this single amino acid substitution significantly hypersensitized the cells to oxidative stress, triggering not only the accumulation and aggregation of ubiquitinated proteins, including synuclein, but also cell death. Our results demonstrate that this genetic manipulation of proteasome activity involving a single amino acid substitution causes the formation of protein aggregates in stressed neuronal cells independently of the occurrence of mutations in other cellular proteins. These results support the notion that proteasome disruption may be central to the development of familial as well as sporadic cases of neurodegeneration.     

Extracellular signal-regulated kinase mediates phosphorylation of tropomyosin-1 to promote cytoskeleton remodeling in response to oxidative stress: impact on membrane blebbing

Oxidative stress induces in endothelial cells a quick and transient coactivation of both stress-activated protein kinase-2/p38 and extracellular signal-regulated kinase (ERK) mitogen-activated protein kinases. We found that inhibiting the ERK pathway resulted, within 5 min of oxidative stress, in a misassembly of focal adhesions characterized by mislocalization of key proteins such as paxillin. The focal adhesion misassembly that followed ERK inhibition with the mitogen-activated protein kinase kinase (MEK) inhibitor PD098059 (2'-amino-3'-methoxyflavone) or with a kinase negative mutant of ERK in the presence of H(2)O(2) resulted in a quick and intense membrane blebbing that was associated with important damage to the endothelium. We isolated by two-dimensional gel electrophoresis a PD098059-sensitive phosphoprotein of 38 kDa that we identified, by mass spectrometry, as tropomyosin-1. In fact, H(2)O(2) induced a time-dependent phosphorylation of tropomyosin that was sensitive to inhibition by PD098059 and UO126 (1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butanediane). Tropomyosin phosphorylation was also induced by expression of a constitutively activated form of MEK1 (MEK(CA)), which confirms that its phosphorylation resulted from the activation of ERK. In unstimulated cells, tropomyosin-1 was found diffuse in the cells, whereas it quickly colocalized with actin and stress fibers upon stimulation of ERK by H(2)O(2) or by expression of MEK(CA). We propose that phosphorylation of tropomyosin-1 downstream of ERK by contributing to formation of actin filaments increases cellular contractility and promotes the formation of focal adhesions. Incidentally, ML-7 (1-[5iodonaphthalene-1-sulfonyl]homopiperazine, HCl), an inhibitor of cell contractility, inhibited phosphorylation of tropomyosin and blocked the formation of stress fibers and focal adhesions, which also led to membrane blebbing in the presence of oxidative stress. Our finding that tropomyosin-1 is phosphorylated downstream of ERK, an event that modulates its interaction with actin, may lead to further understanding of the role of this protein in regulating cellular functions associated with cytoskeletal remodeling.     

The actin cytoskeleton response to oxidants: from small heat shock protein phosphorylation to changes in the redox state of actin itself

Actin is the major constituent of the cytoskeleton of almost all the eukaryotic cells. In vitro experiments have indicated that oxidant-stressed nonmuscle mammalian cells undergo remarkable changes in their morphology and in the structure of the actin cytoskeleton, often resulting in plasma membrane blebbing. Although the microfilament network is one of the earliest targets of oxidative stress, the mechanism by which oxidants change both the structure and the spatial organization of actin filaments is still a matter of debate and far from being fully elucidated. Starting from the 2-fold role of oxidants as injurious by-products of cellular metabolism and essential participants in cell signaling and regulation, this review attempts to gather the most relevant information related to (i) the activation of mitogen-activated protein (MAP) kinase stress-activated protein kinase-2/p38 (SAPK2/p38) which, via MAP kinase-activated protein (MAPKAP) kinase 2/3, leads to the phosphorylation of the actin polymerization (F-actin) modulator 25/27 kDa heat shock protein (HSP25/27), whose phosphorylation is causally related to the regulation of microfilament dynamics following oxidative stress; (ii) the alteration of the redox state of actin or some actin regulatory proteins. The actin cytoskeleton response to oxidants is discussed on the basis of the growing body of evidence indicating the actin system as the most sensitive constituent of the cytoskeleton to the oxidant attack.     

Actin bodies in yeast quiescent cells: an immediately available actin reserve?

Most eukaryotic cells spend most of their life in a quiescent state, poised to respond to specific signals to proliferate. In Saccharomyces cerevisiae, entry into and exit from quiescence are dependent only on the availability of nutrients in the environment. The transition from quiescence to proliferation requires not only drastic metabolic changes but also a complete remodeling of various cellular structures. Here, we describe an actin cytoskeleton organization specific of the yeast quiescent state. When cells cease to divide, actin is reorganized into structures that we named “actin bodies.” We show that actin bodies contain F-actin and several actin-binding proteins such as fimbrin and capping protein. Furthermore, by contrast to actin patches or cables, actin bodies are mostly immobile, and we could not detect any actin filament turnover. Finally, we show that upon cells refeeding, actin bodies rapidly disappear and actin cables and patches can be assembled in the absence of de novo protein synthesis. This led us to propose that actin bodies are a reserve of actin that can be immediately mobilized for actin cables and patches formation upon reentry into a proliferation cycle.     

Menadione-induced bleb formation in hepatocytes is associated with the oxidation of thiol groups in actin

Incubation of isolated rat hepatocytes with menadione (2-methyl-1,4-naphthoquinone) or the thiol oxidant, diamide (azodicarboxylic acid bis(dimethylamide)), resulted in the appearance of numerous plasma membrane protrusions (blebs) preceding cell death. Analysis of the Triton X-100-insoluble fraction (cytoskeleton) extracted from treated cells revealed a dose- and time-dependent increase in the amount of cytoskeletal protein and a concomitant loss of protein thiols. These changes were associated with the disappearance of actin and formation of large-molecular-weight aggregates, when the cytoskeletal proteins were analyzed by polyacrylamide gel electrophoresis under nonreducing conditions. However, if the cytoskeletal proteins were treated with the thiol reductants, dithiothreitol or beta-mercaptoethanol, no changes in the relative abundance of actin or formation of large-molecular-weight aggregates were detected in the cytoskeletal preparations from treated cells. Moreover, addition of dithiothreitol to menadione- or diamide-treated hepatocytes protected the cells from both the appearance of surface blebs and the occurrence of alterations in cytoskeletal protein composition. Our findings show that oxidative stress induced by the metabolism of menadione in isolated hepatocytes causes cytoskeletal abnormalities, of which protein thiol oxidation seems to be intimately related to the appearance of surface blebs.     

The role of oxidative stress in the toxicity of pyridoxal isonicotinoyl hydrazone (PIH) analogues

Pyridoxal isonicotinoyl hydrazone (PIH) analogues are effective iron chelators in vivo and in vitro, and may be of value for the treatment of secondary iron overload. The sensitivity of Jurkat cells to Fe-chelator complexes was enhanced several-fold by the depletion of the antioxidant glutathione, indicating the role of oxidative stress in their toxicity. K562 cells loaded with eicosapentaenoic acid, a fatty acid particularly susceptible to oxidation, were also more sensitive to the toxic effects of the Fe complexes, and toxicity was proportional to lipid peroxidation. Thus Fe-chelator complexes cause oxidative stress, which may be a major component of their toxicity. As was the case for their Fe complexes, the toxicity of PIH analogues was enhanced by glutathione depletion of Jurkat cells and eicosapentaenoic acid-loading of K562 cells. Thus the toxicity of the chelators themselves is also enhanced by compromised cellular redox status. In addition, the toxicity of the chelators was diminished by culturing Jurkat cells under hypoxic conditions, which may limit the production of the reactive oxygen species that initiate oxidative stress. A significant part of the toxicity of the chelators may be due to intracellular formation of Fe-chelator complexes, which oxidatively destroy the cell.     

Regulation of T-cell activation by the cytoskeleton

To become activated, T cells must efficiently recognize antigen-presenting cells or target cells through several complex cytoskeleton-dependent processes, including integrin-mediated adhesion, immunological-synapse formation, cellular polarization, receptor sequestration and signalling. The actin and microtubule systems provide the dynamic cellular framework that is required to orchestrate these processes and ultimately contol T-cell activation. Here, we discuss recent advances that have furthered our understanding of the crucial importance of the T-cell cytoskeleton in controlling these aspects of T-cell immune recognition.     

Cytoskeletal organization affects cellular responses to cytochalasins: comparison of a normal line and its transformant

The relationships between cytoskeletal network organization and cellular response to cytochalasin D (CD) in a normal rat fibroblast cell line (Hmf-n) and its spontaneous transformant (tHmf-e), with markedly different cytoskeletal phenotypes, were compared (using immunofluorescence, electron microscopy, and DNAse I assay for actin content). Hmf-n have prominent, polar stress fiber (SF) arrays terminating in vinculin adhesion plaques whereas tHmf-e, which are apolar, epithelioid cells with dense plasma membrane-associated actin networks, lack SF and adhesion plaques. Hmf-n exposed to CD become markedly retracted and dendritic, SF-derived actin aggregates form large endoplasmic masses, and discrete tabular aggregates at the distal ends of retraction processes. Prolonged exposure leads to recession of process, cellular rounding, and development of large cystic vacuoles. tHmf-e cells exposed to similar doses of CD display a diagnostically different response; retraction is less drastic, cells retain broad processes containing scattered actin aggregates in discrete foci often associated with plasma membrane, large tabular aggregates are never found and processes persist throughout long exposure, vacuolation is uncommon. The CD-induced microfilamentous aggregates in Hmf-n are composed of short, kinky filament fragments forming a felt-like skein, often aggregates contain a more ordered array of roughly parallel fragments, while those of tHmf-e are very short, kinky, randomly orientated filaments imparting a distinctly granular nature to the mass. Total actin content and the amount of actin associated with detergent-resistant cytoskeletons increase following CD exposure in both cell types. Throughout exposure to CD, the actin-associated contractile proteins tropomyosin, myosin, and alpha-actinin co-localize within the actin aggregates in both cell types. Fodrin, the protein linking cortical actin to membrane, co-localizes with actin aggregates in tHmf-e cells and most, but not all, such aggregates in Hmf-n cells, consistent with their stress fiber derivation. Vinculin is lost from the tabular aggregates at the distal ends of retraction processes in Hmf-n cells concomitant with the fragmentation and contraction of SF. The aborized processes in both cells types contain strikingly similar axial cores of bundled vimentin filaments associated with passively compressed microtubules. The characteristic CD-induced distribution of actin filament aggregates and redistribution of vimentin in these cell types also occur when cells are allowed to respread from the rounded state in the presence of CD.     

Cytoskeletal forces during signaling activation in Jurkat T-cells

T-cells are critical for the adaptive immune response in the body. The binding of the T-cell receptor (TCR) with antigen on the surface of antigen-presenting cells leads to cell spreading and signaling activation. The underlying mechanism of signaling activation is not completely understood. Although cytoskeletal forces have been implicated in this process, the contribution of different cytoskeletal components and their spatial organization are unknown. Here we use traction force microscopy to measure the forces exerted by Jurkat T-cells during TCR activation. Perturbation experiments reveal that these forces are largely due to actin assembly and dynamics, with myosin contractility contributing to the development of force but not its maintenance. We find that Jurkat T-cells are mechanosensitive, with cytoskeletal forces and signaling dynamics both sensitive to the stiffness of the substrate. Our results delineate the cytoskeletal contributions to interfacial forces exerted by T-cells during activation.     

Stromal Cell-Derived Factor 1α Activates LIM Kinase 1 and Induces Cofilin Phosphorylation for T-Cell Chemotaxis

Stromal cell-derived factor 1 alpha (SDF-1alpha), the ligand for G-protein-coupled receptor CXCR4, is a chemotactic factor for T lymphocytes. LIM kinase 1 (LIMK1) phosphorylates cofilin, an actin-depolymerizing and -severing protein, at Ser-3 and regulates actin reorganization. We investigated the role of cofilin phosphorylation by LIMK1 in SDF-1alpha-induced chemotaxis of T lymphocytes. SDF-1alpha significantly induced the activation of LIMK1 in Jurkat human leukemic T cells and peripheral blood lymphocytes. SDF-1alpha also induced cofilin phosphorylation, actin reorganization, and activation of small GTPases, Rho, Rac, and Cdc42, in Jurkat cells. Pretreatment with pertussis toxin inhibited SDF-1alpha-induced LIMK1 activation, thus indicating that Gi protein is involved in LIMK1 activation. Expression of dominant negative Rac (DN-Rac), but not DN-Rho or DN-Cdc42, blocked SDF-1alpha-induced activation of LIMK1, which means that SDF-1alpha-induced LIMK1 activation is mediated by Rac but not by Rho or Cdc42. We used a cell-permeable peptide (S3 peptide) that contains the phosphorylation site (Ser-3) of cofilin to inhibit the cellular function of LIMK1. S3 peptide inhibited the kinase activity of LIMK1 in vitro. Treatment of Jurkat cells with S3 peptide inhibited the SDF-1alpha-induced cofilin phosphorylation, actin reorganization, and chemotactic response of Jurkat cells. These results suggest that the phosphorylation of cofilin by LIMK1 plays a critical role in the SDF-1alpha-induced chemotactic response of T lymphocytes.