Cell adhesion to extracellular matrix (ECM) proteins can generate transmembrane signals important for cell survival and can promote directed cell migration events. In a variety of cell types, integrin stimulation by ECM proteins such as fibronectin (FN) leads to changes in intracellular protein tyrosine phosphorylation events. In fibroblasts, the focal adhesion kinase (FAK), a nonreceptor protein-tyrosine kinase (PTK), colocalizes with integrin receptors at sites of cell attachment to ECM proteins. FAK may associate directly with ␤ integrin cytoplasmic domains (44) or may cocluster with integrin receptors through interactions with other integrin-associated proteins (4,8,22). FAK tyrosine phosphorylation is stimulated by cell binding to ECM proteins (for a review, see reference 50), by overexpression of the ␤ integrin cytoplasmic domains (52) and also by other growth factor or serum mitogens (for a review, see reference 24). Since integrin receptors lack catalytic activity, FAK association and activation may be important for integrin-mediated signal transduction events (for a review, see reference 20). This hypothesis is supported by gene knockout results in which both the FN-and FAK-null mice die as a result of similar developmental gastrulation defects (15,25).In addition to integrin stimulation of FAK, ECM protein binding to cells can lead to changes in the tyrosine phosphorylation of a number of different signaling proteins, including p130Cas , Shc, and Cbl, as well as structural proteins such as paxillin and tensin. Integrin stimulation can also promote increases in intracellular calcium levels (51), protein kinase C activity (32, 56), and phosphatidylinositol (PI) 3-kinase activity (7, 28). One downstream target for integrin-initiated signaling events is the activation of the extracellular signal-regulated kinase 2/mitogen-activated protein (ERK2/MAP) kinase pathway (9,38,39,47,59). Although integrin-initiated signaling to ERK2 is dependent on the integrity of the actin cytoskeleton and involves the activation of both the Rho and the Ras families of small GTPase proteins (12,40), the integrin signaling pathways upstream of Ras have not been clearly defined.Attempts to delineate the molecular mechanisms of integrin-stimulated signaling to ERK2 have yielded potentially conflicting results. In NIH 3T3 fibroblasts, Grb2 transiently binds to a motif surrounding FAK Tyr-925 after FN stimula-* Corresponding author. Mailing address:

A successful immune response depends on the capacity of immune cells to travel from one location in the body to another–these cells are rapid migrators, travelling at speeds of μm/minute. Their ability to penetrate into tissues and to make contacts with other cells depends chiefly on the β2 integrin known as LFA-1. For this reason, we describe the control of its activity in some detail. For the non-immunologist, the fine details of an immune response often seem difficult to fathom. However, the behaviour of immune cells, known as leukocytes (Box 1), is subject to the same biological rules as many other cell types, and this holds true particularly for the functioning of the integrins on these cells. In this Commentary, we highlight, from a cell-biology point of view, the integrin-mediated immune-cell migration and cell-cell interactions that occur during the course of an immune response.

A successful immune response depends on the migration of lymphocytes into lymph nodes or inflamed tissues where they make contact with antigen-presenting cells. We are interested in how one member of the integrin family, leukocyte function-associated antigen-1 (LFA-1), controls the function and, in particular, the migration of immune cells. We find that this integrin operates not only as an adhesion receptor for T lymphoblasts (T cells) but also induces their migration in vitro at approximately 15 microm/min. Migration requires active myosin light chain kinase at the leading edge and Rho kinase at the trailing edge of the cell. Two active conformations of LFA-1 are differently distributed on the T-cell membrane and regulate independent aspects of migration. High-affinity LFA-1 is located in a midcell 'focal zone' and influences the speed of migration, whereas intermediate affinity LFA-1 controls leading edge adhesions. Manipulating LFA-1 conformation in vivo can be performed, for example, by creating the active conformation in a transgenic mouse, and this model gives further insight into the role of LFA-1 in migration. In humans, the beneficial effect of functioning CD18 integrins in combating infections in vivo is illustrated by rare patients displaying two forms of leukocyte adhesion deficiency. In summary, we speculate that T cells have evolved a mode of rapid migration that is of paramount importance in achieving the high-speed immune surveillance upon which depends the body's protection against diverse invaders from pathogens to cancer cells.

Focal adhesion kinase–null (FAK−/−) fibroblasts exhibit morphological and motility defects that are reversed by focal adhesion kinase (FAK) reexpression. The FAK-related kinase, proline-rich tyrosine kinase 2 (Pyk2), is expressed in FAK−/− cells, yet it exhibits a perinuclear distribution and does not functionally substitute for FAK. Chimeric Pyk2/FAK proteins were created and expressed in FAK−/− cells to determine the impact of Pyk2 localization to focal contacts. Whereas an FAK/Pyk2 COOH-terminal (CT) domain chimera was perinuclear distributed, stable expression of a Pyk2 chimera with the FAK-CT domain (Pyk2/FAK-CT) localized to focal contact sites and enhanced fibronectin (FN)-stimulated haptotactic cell migration equal to FAK-reconstituted cells. Disruption of paxillin binding to the FAK-CT domain (S-1034) inhibited Pyk2/FAK-CT localization to focal contacts and its capacity to promote cell motility. Paxillin binding to the FAK-CT was necessary but not sufficient to mediate the indirect association of FAK or Pyk2/FAK-CT with a β1-integrin–containing complex. Both FAK and Pyk2/FAK-CT but not Pyk2/FAK-CT S-1034 reconstituted FAK−/− cells, exhibit elevated FN-stimulated extracellular signal–regulated kinase 2 (ERK2) and c-Jun NH2-terminal kinase (JNK) kinase activation. FN-stimulated FAK or Pyk2/FAK-CT activation enhanced both the extent and duration of FN-stimulated ERK2 activity which was necessary for cell motility. Transient overexpression of the FAK-CT but not FAK-CT S-1034 domain inhibited both FN-stimulated ERK2 and JNK activation as well as FN-stimulated motility of Pyk2/FAK-CT reconstituted cells. These gain-of-function studies show that the NH2-terminal and kinase domains of Pyk2 can functionally substitute for FAK in promoting FN-stimulated signaling and motility events when localized to β-integrin–containing focal contact sites via interactions mediated by the FAK-CT domain.

Neutrophils and T cells play an important role in host protection against pulmonary infection caused by Streptococcus pneumoniae. However, the role of the integrins in recruitment of these cells to infected lungs is not well understood. In this study we used the twin approaches of mAb blockade and gene-deficient mice to investigate the relative impact of specific integrins on cellular recruitment and bacterial loads following pneumococcal infection. We find that both Mac-1 (CD11b/CD18) and α4β1 (CD49d/CD29) integrins, but surprisingly not LFA-1 (CD11a/CD18), contribute to two aspects of the response. In terms of recruitment from the circulation into lungs, neutrophils depend on Mac-1 and α4β1, whereas the T cells are entirely dependent on α4β1. Second, immunohistochemistry results indicate that adhesion also plays a role within infected lung tissue itself. There is widespread expression of ICAM-1 within lung tissue. Use of ICAM-1−/− mice revealed that neutrophils make use of this Mac-1 ligand, not for lung entry or for migration within lung tissue, but for combating the pneumococcal infection. In contrast to ICAM-1, there is restricted and constitutive expression of the α4β1 ligand, VCAM-1, on the bronchioles, allowing direct access of the leukocytes to the airways via this integrin at an early stage of pneumococcal infection. Therefore, integrins Mac-1 and α4β1 have a pivotal role in prevention of pneumococcal outgrowth during disease both in regulating neutrophil and T cell recruitment into infected lungs and by influencing their behavior within the lung tissue itself.

The identification of phosphoinositide-dependent kinase-1 (PDK-1) as an activating kinase for members of the AGC family of kinases has led to its implication as the activating kinase for cAMP-dependent protein kinase. It has been established in vitro that PDK-1 can phosphorylate the catalytic (C) subunit (19), but the Escherichia coli-expressed C-subunit undergoes autophosphorylation. To assess which of these mechanisms occurs in mammalian cells, a set of mutations was engineered flanking the site of PDK-1 phosphorylation, Thr-197, on the activation segment of the C-subunit. Two distinct requirements appeared for autophosphorylation and phosphorylation by PDK-1. Autophosphorylation was disrupted by mutations that compromised activity (Thr-201 and Gly-200) or altered substrate recognition (Arg-194). Conversely, only residues peripheral to Thr-197 altered PDK-1 phosphorylation, including a potential hydrophobic PDK-1 binding site at the C terminus. To address the in vivo requirements for phosphorylation, select mutant proteins were transfected into COS-7 cells, and their phosphorylation state was assessed with phospho-specific antibodies. The phosphorylation pattern of these mutant proteins indicates that autophosphorylation is not the maturation mechanism in the eukaryotic cell; instead, a heterologous kinase with properties resembling the in vitro characteristics of PDK-1 is responsible for in vivo phosphorylation of PKA.

Lymphocytes use the integrin leukocyte functionassociated antigen-1 (LFA-1) to cross the vasculature into lymph nodes (LNs), but it has been uncertain whether their migration within LN is also LFA-1 dependent. We show that LFA-1 mediates prolonged LN residence ashosts lacking the major LFA-1 ligand ICAM-1. Intra-vital two-photon microscopy revealed that LFA-1 þ / þ and LFA-1 À / À T cells reacted differently when probing the ICAM-1-expressing lymphatic network. While LFA-1T cells returned to the LN parenchyma with greater frequency, LFA-1 À / À T cells egressed promptly. This difference in exit behaviour was a feature of egress through all assessed lymphatic exit sites. We show that use of LFA-1 as an adhesion receptor amplifies the number of T cells returning to the LN parenchyma that can lead to increased effectiveness of T-cell response to antigen. Thus, we identify a novel function for LFA-1 in guiding T cells at the critical point of LN egress when they either exit or return into the LN for further interactions.