Integrin signaling in the regulation of lymphocyte morphology

In normal lymphocytes an “inside-out” signal up-regulating integrin adhesion is followed by a ligand mediated “outside-in” signal for cell spreading. Although PKC mediates both events, distinct roles were found for different PLCs. The inhibition of phosphatidylinositol specific PLC decreased both cell adhesion and spreading on fibronectin in T cell receptor/CD28 activated peripheral blood T cells. However, inhibition of phosphatidylcholine specific PLC only blocked cell spreading and did not affect adhesion, indicating that “inside-out” signaling for the integrin α4β1 proceeds through phosphatidylinositol specific PLC and PKC, while the “outside-in” signal utilizes phosphatidylcholine specific PLC and PKC. Furthermore, β1 integrin chain mediated morphological changes in the T lymphocytic cell line HPB-ALL directly paralleled PKA activation, treatment of these cells with an inhibitory anti-β1 antibody blocked PKA activation and cell spreading, and this inhibition could be overcome by activating adenylate cyclase. Furthermore, inhibition of PKA was found to decrease the overall strength of cell adhesion or cellular avidity without affecting individual receptor affinity for soluble ligand. ^ When HPB-ALL cells interact with immobilized FN, two separate morphological phenotypes can be induced. Some cells flattened their cell body into a triangular shape and begin to migrate, while others extended a pseudopod from their stationary cell body. This second morphology recapitulates the shape changes observed during transendothelial migration. During these morphological changes, α4β1 integrins are internalized into endocytic vesicles that ultimately accumulate at the juncture between the cell body and an extending pseudopod. From this juncture, they are rapidly transported down the length of the pseudopod to its most distal end. ^ In addition to an accumulation of integrin containing vesicles, the pseudopod base was found to have increased amounts of the small GTPase RhoA and active PKA. The inhibition of PKA or RhoA resulted in lymphocytes with similar aberrant stellate morphologies. Furthermore, inhibition of PKA blocked the α4β1 mediated phosphorylation of RhoA. The co-localization of active PKA, RhoA and integrin containing endocytic vesicles indicates that integrin triggering can cause the rapid redistribution and activation of key signaling intermediates and raises the possibility that regulation of lymphocyte morphology by PKA and RhoA is through adhesion receptor recycling. ^

Loss of β1-integrin-deficient cells during the development of endoderm-derived epithelia

Beta1-integrins (beta1) represent cell surface receptors which mediate cell-matrix and cell-cell interactions. Fässler and Meyer described chimeric mice containing transgenic cells that express the LacZ gene instead of the beta1 gene. They observed beta1-negative cells in all germ layers at embryonic day E 8.5. Later in development, using a glucose phosphate isomerase assay of homogenized tissue samples, high levels of transgenic cells were found in skeletal muscle and gut, low levels in lung, heart, and kidney and none in the liver and spleen (Fässler and Meyer 1995). In order to study which cell types require beta1 during development of the primitive gut including its derivatives, chimeric fetuses containing 15 to 25% transgenic cells were obtained at days E 14.5 and E 15.5. They were LacZ (beta-galactosidase) stained "en bloc" and cross-sectioned head to tail. In esophagus, trachea, lung, stomach, hindgut, and the future urinary bladder, we observed various mesoderm-derived beta1-negative cells (e.g. fibroblasts, chondrocytes, endothelial cells, and smooth muscle cells) but no beta1-negative epithelial cells. Since the epithelia of lung, esophagus, trachea, stomach, hindgut, and urinary bladder are derived from the endodermal gut tube, we hypothesize that beta1 is essential for the development and/or survival of the epithelia of the fore- and hindgut and its derivatives.

Glomerular and renal vascular structural changes in alpha8 integrin-deficient mice

Integrins are matrix receptors that regulate cell-matrix interactions during development and in adult tissue. In the adult kidney, the alpha8 chain is specifically expressed in glomerular mesangial cells and vascular smooth muscle cells. alpha8-deficient (alpha8-/-) mice demonstrate reductions in renal mass, which can range from complete renal agenesis to the development of kidneys that are only slightly smaller than wild-type kidneys. No histologic abnormalities of these kidneys have been described. However, considering the prominent expression of alpha8 in glomeruli and renal vessels, it seemed unlikely that the kidneys of alpha8-/- mice would be completely normal. Therefore, the renal phenotype of adult alpha8-/- mice was investigated, for assessment of more subtle morphologic alterations in kidney tissue. alpha8-/- mice displayed a significant reduction in nephron number and an increase in glomerular volume, compared with wild-type control animals. Albuminuria was not different in wild-type and alpha8-/- mice. Quantitative morphologic analyses revealed that the glomeruli of alpha8-/- mice were hypercellular, with an increased number of mesangial cells, compared with wild-type mice. Mesangial matrix deposition (as demonstrated for collagen IV and the alpha8 ligand fibronectin) was expanded in alpha8-/- mice, compared with wild-type mice. Collagens I and III, which are not normally present in glomeruli, were detected in the glomeruli of alpha8-/- mice. Staining for other glomerular integrins demonstrated an increased abundance of the collagen receptor alpha2 integrin in alpha8-/- mice. The glomerular capillary length density was significantly greater in alpha8-/- mice than in wild-type mice. Cortical arterial vessel walls were not altered in alpha8-/- mice, but the capillaries of the peritubular network were widened. Despite the strong mesangial and vascular expression of alpha8, glomerular and renal vascular alterations in alpha8-/- mice were relatively mild. Only aged alpha8-/- mice demonstrated increased glomerular capillary widening, compared with control animals. The results suggest that the lack of alpha8 can be largely compensated for, at least in younger alpha8-/- mice. It is not yet clear whether the occurrence of collagens that are not normally present in glomeruli and the increased abundance of the collagen receptor alpha2 contribute to maintaining the glomerular structure in alpha8-/- mice. The compensatory mechanisms involved will be the subject of future research.

β2 integrin-mediated crawling on endothelial ICAM-1 and ICAM-2 is a prerequisite for transcellular neutrophil diapedesis across the inflamed blood-brain barrier

In acute neuroinflammatory states such as meningitis, neutrophils cross the blood-brain barrier (BBB) and contribute to pathological alterations of cerebral function. The mechanisms that govern neutrophil migration across the BBB are ill defined. Using live-cell imaging, we show that LPS-stimulated BBB endothelium supports neutrophil arrest, crawling, and diapedesis under physiological flow in vitro. Investigating the interactions of neutrophils from wild-type, CD11a(-/-), CD11b(-/-), and CD18(null) mice with wild-type, junctional adhesion molecule-A(-/-), ICAM-1(null), ICAM-2(-/-), or ICAM-1(null)/ICAM-2(-/-) primary mouse brain microvascular endothelial cells, we demonstrate that neutrophil arrest, polarization, and crawling required G-protein-coupled receptor-dependent activation of β2 integrins and binding to endothelial ICAM-1. LFA-1 was the prevailing ligand for endothelial ICAM-1 in mediating neutrophil shear resistant arrest, whereas Mac-1 was dominant over LFA-1 in mediating neutrophil polarization on the BBB in vitro. Neutrophil crawling was mediated by endothelial ICAM-1 and ICAM-2 and neutrophil LFA-1 and Mac-1. In the absence of crawling, few neutrophils maintained adhesive interactions with the BBB endothelium by remaining either stationary on endothelial junctions or displaying transient adhesive interactions characterized by a fast displacement on the endothelium along the direction of flow. Diapedesis of stationary neutrophils was unchanged by the lack of endothelial ICAM-1 and ICAM-2 and occurred exclusively via the paracellular pathway. Crawling neutrophils, although preferentially crossing the BBB through the endothelial junctions, could additionally breach the BBB via the transcellular route. Thus, β2 integrin-mediated neutrophil crawling on endothelial ICAM-1 and ICAM-2 is a prerequisite for transcellular neutrophil diapedesis across the inflamed BBB.

Nck-interacting Ste20 kinase couples Eph receptors to c-Jun N-terminal kinase and integrin activation.

The mammalian Ste20 kinase Nck-interacting kinase (NIK) specifically activates the c-Jun amino-terminal kinase (JNK) mitogen-activated protein kinase module. NIK also binds the SH3 domains of the SH2/SH3 adapter protein Nck. To determine whether Nck functions as an adapter to couple NIK to a receptor tyrosine kinase signaling pathway, we determined whether NIK is activated by Eph receptors (EphR). EphRs constitute the largest family of receptor tyrosine kinases (RTK), and members of this family play important roles in patterning of the nervous and vascular systems. In this report, we show that NIK kinase activity is specifically increased in cells stimulated by two EphRs, EphB1 and EphB2. EphB1 kinase activity and phosphorylation of a juxtamembrane tyrosine (Y594), conserved in all Eph receptors, are both critical for NIK activation by EphB1. Although pY594 in the EphB1R has previously been shown to bind the SH2 domain of Nck, we found that stimulation of EphB1 and EphB2 led predominantly to a complex between NIK/Nck, p62(dok), RasGAP, and an unidentified 145-kDa tyrosine-phosphorylated protein. Tyrosine-phosphorylated p62(dok) most probably binds directly to the SH2 domain of Nck and RasGAP and indirectly to NIK bound to the SH3 domain of Nck. We found that NIK activation is also critical for coupling EphB1R to biological responses that include the activation of integrins and JNK by EphB1. Taken together, these findings support a model in which the recruitment of the Ste20 kinase NIK to phosphotyrosine-containing proteins by Nck is an important proximal step in the signaling cascade downstream of EphRs.

Spatial proteomic and phospho-proteomic organization in three prototypical cell migration modes

BACKGROUND Tight spatio-temporal signaling of cytoskeletal and adhesion dynamics is required for localized membrane protrusion that drives directed cell migration. Different ensembles of proteins are therefore likely to get recruited and phosphorylated in membrane protrusions in response to specific cues. RESULTS HERE, WE USE AN ASSAY THAT ALLOWS TO BIOCHEMICALLY PURIFY EXTENDING PROTRUSIONS OF CELLS MIGRATING IN RESPONSE TO THREE PROTOTYPICAL RECEPTORS: integrins, recepor tyrosine kinases and G-coupled protein receptors. Using quantitative proteomics and phospho-proteomics approaches, we provide evidence for the existence of cue-specific, spatially distinct protein networks in the different cell migration modes. CONCLUSIONS The integrated analysis of the large-scale experimental data with protein information from databases allows us to understand some emergent properties of spatial regulation of signaling during cell migration. This provides the cell migration community with a large-scale view of the distribution of proteins and phospho-proteins regulating directed cell migration.

Purification and cloning of the novel phosphoprotein copine III and characterization of its associated kinase activity

The copines, named and first described by Creutz et al. (1998), comprise a two C2 domain-containing protein family that can aggregate phosphatidylserine membranes in a calcium-dependent manner. Although no enzymatic function has been attributed to copines, their carboxyl terminus shows homology to the A domain found in integrins that allows binding of magnesium ions. The secondary structure of A domains resembles a Rossmann fold, which can bind dinucleotides and is present in a number of intracellular enzymes. Due to a crossreacting activity of Mik b 1, an antibody to the IL-2R b chain, we were able to serendipitously clone human copine III (CIII). CIII is 65% identical to copine I (CI) and the 5 kb CIII transcript is expressed ubiquitously as determined by a multitissue Northern blot. A polyclonal antibody generated against the carboxyl terminus of CIII recognized CIII in immunoblots and immunoprecipitations. Phosphorylation of CIII was observed on serine and threonine residues, as determined by phosphoamino acid analysis. ^ Experiments were designed to determine whether or not any enzymatic activity, specifically kinase activity, was intrinsic to or associated with CIII. In vitro and in gel kinase assays were performed using transfected HA-tagged CI and CIII, immunoprecipitated endogenous CIII and purified endogenous CIII. The exogenous substrate MBP was phosphorylated in all in vitro kinase assays containing CIII protein purification and column chromatography expertise with me. ^

Collagen I modulates growth and gene expression in prostate cancer cells

Prostate cancer is the second leading cause of male cancer-related deaths in the United States. Interestingly, prostate cancer preferentially metastasizes to skeletal tissue. Once in the bone microenvironment, advanced prostate cancer becomes highly resistant to therapeutic modalities. Several factors, such as extracellular matrix (ECM) components, have been implicated in the spread and propagation of prostatic carcinoma. In these studies, we have utilized the PC3 cell line, derived from a human bone metastasis, to investigate the influence of the predominant bone ECM protein, type I collagen, on prostate cancer cell proliferation and gene expression. We have also initiated the design and production of ribozymes to specific gene targets that may influence prostate cancer bone metastasis. ^ Our results demonstrate that PC3 cells rapidly adhere and spread on collagen I to a greater degree than on fibronectin (FN) or poly-L-lysine (PLL). Flow cytometry analysis reveals the presence of the α1, α2 and α3 collagen binding integrin subunits. The use of antibody function blocking studies reveals that PC3 cells can utilize α2β 1 and α3β1 integrins to adhere to collagen I. Once plated on collagen I, the cells exhibit increased rates of proliferation compared with cells plated on FN or tissue culture plastic. Additionally, cells plated on collagen I show increased expression of proteins associated with progression through G1 phase of the cell cycle. Inhibitor studies point to a role for phosphatidylinositol 3-kinase (PI3K), MAP kinase (MAPK), and p70 S6 kinase in collagen I-mediated PC3 cell proliferation and cyclin D1 expression. To further characterize the effect of type I collagen on prostate cancer bone metastasis, we utilized a cDNA microarray strategy to monitor type I collagen-mediated changes in gene expression. Results of this analysis revealed a gene expression profile reflecting the increased proliferation occurring on type I collagen. Microarray analysis also revealed differences in the expression of specific gene targets that may impact on prostate cancer metastasis to bone. ^ As a result of our studies on the interaction of prostate cancer cells and the skeletal ECM, we sought to develop novel molecular tools for future gene therapy of functional knockdown experiments. To this end, we developed a series of ribozymes directed against the α2 integrin and at osteopontin, a protein implicated in the metastasis of various cancers, including prostate. These ribozymes should facilitate the future study of the mechanism of prostate cancer cell proliferation, and disease progression occurring at sites of skeletal metastasis where a type I collagen-based environment predominates. ^ Together these studies demonstrate the involvement of bone ECM proteins on prostate cancer cell proliferation and suggest that they may play a significant role on the growth of prostate metastases once in the bone microenvironment. ^

Integrin mediated growth and apoptosis in human gastric adenocarcinoma

Integrins are important as the primary cell adhesion molecule providing information about the extracellular microenvironment to the interior of the cell to influence cellular behavior such as differentiation, proliferation and apoptosis. Apoptotic death due to loss of adhesion is termed anoikis. In this study we have obtained a parental human gastric adenocarcinoma cell line that yielded two variant lines that had differing responses to lack of adhesion. The STAD.APO cell line undergoes apoptosis when denied adherence and the STAD.ARR cell line enters into cell cycle arrest under the identical suspended conditions. We have shown that cyclin A and cyclin D mRNA and protein are down regulated when cells are denied adherence for 24 hours in tissue culture wells previously coated with poly-HEMA. To test whether cyclin A was able to rescue cells from cell cycle arrest and/or anoikis by overriding the cell cycle machinery we transfected the full length cDNA in to each cell type. Surprisingly we found that anoikis and cell cycle arrest due to suspended conditions was not affected by overexpression of cyclin A protein, but that growth under adhered conditions was reduced compared to vector alone control transfectants. Further, we transfected other cell lines; ST7, gastric cancer, MDA-MB-4.35, breast cancer, and HPB T-cell leukemic and in no case were suspended culturing conditions overcome by cyclin A. This result indicates an additional level of regulation for the cell cycle machinery. Additionally, soluble collagen was shown to be able to save from anoikis and also from cell cycle arrest while the β1 specific mAb 33B6 was only able to save from anoikis. Immunofluorescent studies show that soluble collagen creates clusters of β1 with FAK and also β1 with actin in the STAD.ARR cells but does not in the STAD.APO cells. This result indicates that the phenotypes under suspended conditions between these cell lines may diverge at their requirements for integrin ligation. Additionally we characterized the nature of anoikis by showing cytochrome c release, caspase 3, p21 and p53 activation in STAD.APO cells. Thus, our results have implications in the understanding of integrin biology and neoplastic progression. ^

Requirements for Staphylococcus aureus in vitro cellular internalization

Staphylcoccus aureus is a prokaryotic organism capable of causing numerous superficial and severe human infections. Adhesion of S. aureus to host tissues or cells is believed to be a crucial event in S. aureus infections. Subsequently, S. aureus can seed into the bloodstream resulting in metastasis of the infection. Several reports show that S. aureus can be internalized by non-professional phagocytes, a process which has been proposed to be important in S. aureus dissemination. An intracellular residence has also been proposed to provide safe harbor to reservoirs of dormant bacteria contributing to the persistence of infection. This dissertation describes an investigation into the molecular mechanisms of S. aureus internalization into both fibroblast and epithelial cells. Bacterial requirements for internalization were found to be limited to expression of proteins that bind the extracellular matrix protein fibronectin. A previously unknown fibronectin-binding region in the S. aureus fibronectin-binding protein A was discovered after showing competitive inhibition of S. aureus internalization. This novel fibronectin-binding activity is characterized. Internalization also required cell-based factors. The presence of fibronectin and cell surface receptors of the β1 integrin class, which are known to bind and internalize fibronectin, were found to be necessary for optimal internalization of S. aureus. These results led to the conclusion that fibronectin acts as a bridge between the bacterium and integrins on the host cells. The internalization process exhibits features characteristic of integrin-mediated cell migration on fibronectin-coated surfaces. Both processes involved an active form of the β1 integrin subunit and the protein tyrosine kinase Src. Finally, a Src inhibitor previously shown to be effective in reducing osteoporosis in an in vivo rat model is capable of greatly reducing S. aureus internalization. ^

T cell adhesion regulation from clustering GM1 lipid rafts

Lipid rafts are small laterally mobile cell membrane structures that are highly enriched in lymphocyte signaling molecules. Lipid rafts can form from the assembly of specialized lipids and proteins through hydrophobic associations from saturated acyl chains. GM1 gangliosides are a common lipid raft component and have been shown to be essential in many T cell functions. Current lipid raft theory hypothesizes that certain aspects of T cell signaling can be initiated from the coalescence of these signaling-enriched lipid rafts to sites of receptor engagement. We have described how the specific aggregation of GM1 lipid rafts can cause a reorganization of cell surface molecular associations which include dynamic associations of β1 integrins with GM1 lipid rafts. These associations had pronounced effects on T cell adhesive and migratory states. We show that GM1 lipid raft aggregation can dramatically inhibit T cell migration and chemotaxis on the extracellular matrix constituent fibronectin. This inhibition of migration function was shown to be dependent on the src kinase Lck and PKC-regulated F-actin polymerization to extending pseudopods. Furthermore, GM1 lipid raft clustering could activate T cell adhesion-strengthening mechanisms. These include an increase in cellular rigidity, the creation of polymerized cortical F-actin structures, the induction of high affinity integrin states, an increase in surface area and symmetry of the contact plane, and resistance to shear flow detachment while adherent to fibronectin. This indicates that GM1 lipid raft aggregation defines a novel stimulus to regulate lymphocyte motility and cellular adhesion which could have important implications in T cell homing mechanisms. ^

Diversity in T cell costimulation by alpha4beta1 integrin

T cell activation requires antigen-specific T cell receptor signals that spatially and temporally coincide with a second costimulatory signal. CD28 and α4β1 integrin both function as T cell costimulators, but their individual mechanisms remain elusive. By directly comparing CD3-dependent functions and signaling pathways employed by these two costimulatory receptors, aspects of their individual signaling mechanisms are explored. We determined that CD28 and α4β1 integrins both use Src-family kinase Lck and MAPK Erk, but to different extents and functional ends. After identifying functional differences between CD28 and integrin costimulatory pathways, the focus of the study turned to integrin signaling in naïve and memory T cell subsets. CD45RO T cells are fully co-activated by natural β1 integrin ligands fibronectin (FN) and VCAM-1, β1 monoclonal antibody 33B6, as well as α4β1 monoclonal antibody 19H8 which binds a combinatorial epitope of the α4β1 heterodimer. While CD28 fully costimulates CD45RA T cells, the degree of activation from integrin ligands varies. FN costimulates CD3-dependent proliferation, IL-2 secretion, and early activation markers CD25 and CD69. However, β1 antibody 33B6, which binds to the same T cell integrins (α4β1 and α5β1) as natural ligand FN, failed to costimulate proliferation or IL-2 in the CD45RA subset, but retained the ability to regulate CD25 and CD69. Unique aspects of 19H8 signaling involve early Erk activation and IL-2 independent proliferation. Signaling defects through 33B6 ligation correlates with poor adhesion under fluid flow conditions, suggesting a cytoskeletal basis for signaling. All together, these data provide evidence for a mechanism of α4β1 integrin signaling and describe functional differences between naïve and memory T cells. ^

Tyrosine phosphorylation of paxillin and focal adhesion kinase by activation of muscarinic m3 receptors is dependent on integrin engagement by the extracellular matrix

The G protein-coupled m1 and m3 muscarinic acetylcholine receptors increase tyrosine phosphorylation of several proteins, including the focal adhesion-associated proteins paxillin and focal adhesion kinase (FAK), but the mechanism is not understood. Activation of integrins during adhesion of cells to extracellular matrix, or stimulation of quiescent cell monolayers with G protein-coupled receptor ligands including bradykinin, bombesin, endothelin, vasopressin, and lysophosphatidic acid, also induces tyrosine phosphorylation of paxillin and FAK and formation of focal adhesions. These effects are generally independent of protein kinase C but are inhibited by agents that prevent cytoskeletal assembly or block activation of the small molecular weight G protein Rho. This report demonstrates that tyrosine phosphorylation of paxillin and FAK elicited by stimulation of muscarinic m3 receptors with the acetylcholine analog carbachol is inhibited by soluble peptides containing the arginine–glycine–aspartate motif (the recognition site for integrins found in adhesion proteins such as fibronectin) but is unaffected by peptides containing the inactive sequence arginine–glycine–glutamate. Tyrosine phosphorylation elicited by carbachol, but not by cell adhesion to fibronectin, is reduced by the protein kinase C inhibitor GF 109203X. The response to carbachol is dependent on the presence of fibronectin. Moreover, immunofluorescence studies show that carbachol treatment induces formation of stress fibers and focal adhesions. These results suggest that muscarinic receptor stimulation activates integrins via a protein kinase C-dependent mechanism. The activated integrins transmit a signal into the cell’s interior leading to tyrosine phosphorylation of paxillin and FAK. This represents a novel mechanism for regulation of tyrosine phosphorylation by muscarinic receptors.

Resistance to endotoxic shock and reduced neutrophil migration in mice deficient for the Src-family kinases Hck and Fgr

Signal transduction through the leukocyte integrins is required for the processes of firm adhesion, activation, and chemotaxis of neutrophils during inflammatory reactions. Neutrophils isolated from knockout mice that are deficient in the expression of p59/61hck (Hck) and p58c-fgr (Fgr), members of the Src-family of protein tyrosine kinases, have been shown to be defective in adhesion mediated activation. Cells from these animals have impaired induction of respiratory burst and granule secretion following plating on surfaces that crosslink β2 and β3 integrins. To determine if the defective function of hck−/−fgr−/− neutrophils observed in vitro also results in impaired inflammatory responses in vivo, we examined responses induced by lipopolysaccharide (LPS) injection in these animals. The hck−/−fgr−/− mice showed marked resistance to the lethal effects of high-dose LPS injection despite the fact that high levels of serum tumor necrosis factor α and interleukin 1α were detected. Serum chemistry analysis revealed a marked reduction in liver and renal damage in mutant mice treated with LPS, whereas blood counts showed a marked neutrophilia that was not seen in wild-type animals. Direct examination of liver sections from mutant mice revealed reduced neutrophil migration into the tissue. These data demonstrate that defective integrin signaling in neutrophils, caused by loss of Hck and Fgr tyrosine kinase activity, results in impaired inflammation-dependent tissue injury in vivo.

Interleukin-2 induces β2-integrin-dependent signal transduction involving the focal adhesion kinase-related protein B (fakB)

β2 integrin molecules are involved in a multitude of cellular events, including adhesion, migration, and cellular activation. Here, we studied the influence of β2 integrins on interleukin-2 (IL-2)-mediated signal transduction in human CD4+ T cell lines obtained from healthy donors and a leukocyte adhesion deficiency (LAD) patient. We show that IL-2 induces tyrosine phosphorylation of a 125-kDa protein and homotypic adhesion in β2 integrin (CD18)-positive but not in β2-integrin-negative T cells. EDTA, an inhibitor of integrin adhesion, blocks IL-2-induced tyrosine phosphorylation of the 125-kDa protein but not other proteins in β2-integrin-positive T cells. Likewise, a β2 integrin (CD18) antibody selectively inhibits induction of the 125-kDa phosphotyrosine protein, whereas cytokine-mediated tyrosine phosphorylation of other proteins is largely unaffected. Immunoprecipitation experiments indicate that the IL-2-induced 125-kDa phosphotyrosine protein is the focal adhesion kinase-related protein B (fakB). Thus, IL-2 induces strong tyrosine phosphorylation of fakB in β2-integrin-positive but not in β2-integrin-negative T cells, and CD18 mAb selectively blocks IL-2-induced fakB-tyrosine phosphorylation in β2-integrin-positive T cells. In parallel experiments, IL-2 does not induce or augment tyrosine phosphorylation of p125FAK. In conclusion, our data indicate that IL-2 induces β2-integrin-dependent signal transduction events involving the tyrosine kinase substrate fakB.