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Results for “Focal Adhesion”

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PubMed2004

Transforming growth factor beta regulates cell-cell adhesion through extracellular matrix remodeling and activation of focal adhesion kinase in human colon carcinoma Moser cells.

Transforming growth factor (TGF) beta is a potent regulator of cell-matrix and cell-cell adhesions (collectively termed cellular adhesions). Cellular adhesions play crucial roles in controlling the differentiation of epithelial cells and in maintaining the integrity of the epithelium. Loss of TGF beta-responsiveness is thought to be an important early initiating event in the malignant progression of epithelial cancer. In the TGFbeta-responsive human colon adenocarcinoma Moser cells, TGFbeta promotes cellular adhesions and suppresses their malignant phenotype. TGFbeta promotes cell-matrix adhesion by inducing the synthesis of extracellular matrix (ECM) adhesion molecules and the expression of integrin receptors for these molecules (termed ECM remodeling). TGFbeta promotes cell-cell adhesion through the induction of E-cadherin expression, an epithelial associated homotypic cell-cell adhesion molecule, which also functions as a tumor suppressor in colon cancer. How TGFbeta regulates E-cadherin expression is not known. In this study, we showed that the induction of E-cadherin by TGFbeta was mediated through the activation of focal adhesion kinase (FAK), a major signaling molecule in focal adhesion contacts and that the activation of FAK was due to ECM remodeling and increased cell-matrix interactions. Thus, TGFbeta regulates cell-cell adhesion through its ability to remodel the ECM and to activate FAK through ECM remodeling.

Adenocarcinoma↗

PubMed1997

Thrombin-mediated focal adhesion plaque reorganization in endothelium: role of protein phosphorylation.

Endothelial cell (EC) gap formation and barrier function are subject to dual regulation by (1) axial contractile forces, regulated by myosin light chain kinase activity, and (2) tethering forces, represented by cell-cell and cell-substratum adhesions. We examined whether focal adhesion plaque proteins (vinculin and talin) and focal adhesion kinase, p125FAK (FAK), represent target regulatory sites involved in thrombin-mediated EC barrier dysfunction. Histologically, thrombin produced dramatic rearrangement of EC actin, vinculin, and FAK in parallel with the evolution of gap formation and barrier dysfunction. Vinculin and talin were in vitro substrates for phosphorylation by EC PKC, a key effector enzyme involved in thrombin-induced EC barrier dysfunction. Although vinculin and talin were phosphorylated in situ under basal conditions in 32P-labeled EC, thrombin failed to alter the basal level of phosphorylation of these proteins. Phosphotyrosine immunoblotting showed that neither vinculin nor talin was significantly phosphorylated in situ on tyrosine residues in unstimulated ECs, and this was not further increased after thrombin. In contrast, both thrombin and the thrombin receptor-activating peptide (TRAP) produced an increase in FAK phosphotyrosine levels (corrected for immunoreactive FAK content) present in EC immunoprecipitates. Ionomycin, which produces EC barrier dysfunction in a myosin light chain kinase-independent manner, was used to increase intracellular Ca2+ and evaluate the Ca2+ sensitivity of this observation. In contrast to thrombin, ionomycin effected a dramatic decrease in the phosphotyrosine-to-immunoreactive FAK ratios, suggesting distinct effects of the two agents on FAK phosphorylation and function. These data indicate that modulation of cell tethering via phosphorylation of focal adhesion proteins is complex, agonist-specific, and may be a relevant mechanism of EC barrier dysfunction in permeability models that do not depend on an increase in myosin 20-kD regulatory light chain phosphorylation.

Animals↗

PubMed2006

Mechanical forces alter zyxin unbinding kinetics within focal adhesions of living cells.

The formation of focal adhesions that mediate alterations of cell shape and movement is controlled by a mechanochemical mechanism in which cytoskeletal tensional forces drive changes in molecular assembly; however, little is known about the molecular biophysical basis of this response. Here, we describe a method to measure the unbinding rate constant k(OFF) of individual GFP-labeled focal adhesion molecules in living cells by modifying the fluorescence recovery after photobleaching (FRAP) technique and combining it with mathematical modeling. Using this method, we show that decreasing cellular traction forces on focal adhesions by three different techniques--chemical inhibition of cytoskeletal tension generation, laser incision of an associated actin stress fiber, or use of compliant extracellular matrices--increases the k(OFF) of the focal adhesion protein zyxin. In contrast, the k(OFF) of another adhesion protein, vinculin, remains unchanged after tension dissipation. Mathematical models also demonstrate that these force-dependent increases in zyxin's k(OFF) that occur over seconds are sufficient to quantitatively predict large-scale focal adhesion disassembly that occurs physiologically over many minutes. These findings demonstrate that the molecular binding kinetics of some, but not all, focal adhesion proteins are sensitive to mechanical force, and suggest that force-dependent changes in this biophysical parameter may govern the supramolecular events that underlie focal adhesion remodeling in living cells.

Amides↗

PubMed2006

Clinical significance of focal adhesion kinase in resectable pancreatic cancer.

Focal adhesion kinase (FAK) is a non-receptor, cytoplasmic protein tyrosine kinase that is involved in the regulation of cellular signaling, migration, apoptosis, and cell cycle progression. Previous reports have shown that FAK is expressed in various kinds of cancer tissues and cancer cell lines; however, no information is available about human pancreatic carcinoma specimens. Tissue such specimens were obtained from 50 patients who underwent pancreatic resection for pancreatic invasive ductal carcinoma at our institute from 1996 to 2002. Immunohistochemical analysis of FAK was performed in the resected specimens. Focal adhesion kinase expression in seven human pancreatic cancer cell lines was analyzed by reverse transcription polymerase chain reaction (PCR) analysis and Western blot analysis. Focal adhesion kinase expression was detected in 24 of 50 cases (48%). There was a statistically significant correlation between FAK expression and tumor size (P=0.004), although FAK expression did not significantly correlate with other factors such as tumor histological grade, lymph node metastasis, distant metastasis, histological stage, and overall survival. Reverse transcription PCR analysis and Western blot analysis showed that FAK was expressed in all seven pancreatic cancer cell lines. Focal adhesion kinase expression was not directly related to clinicopathological factors except tumor size in pancreatic carcinoma. Focal adhesion kinase expression may not be a prognostic marker for pancreatic cancer patients.

Aged↗

PubMed2001

Targeting Pyk2 to beta 1-integrin-containing focal contacts rescues fibronectin-stimulated signaling and haptotactic motility defects of focal adhesion kinase-null 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 beta 1-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 NH(2)-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 NH(2)-terminal and kinase domains of Pyk2 can functionally substitute for FAK in promoting FN-stimulated signaling and motility events when localized to beta-integrin-containing focal contact sites via interactions mediated by the FAK-CT domain.

Animals↗

PubMed2003

PIAS1-mediated sumoylation of focal adhesion kinase activates its autophosphorylation.

Focal adhesion kinase (FAK) is a protein tyrosine kinase enriched in focal adhesions, which plays a critical role in integrin-dependent cell motility and survival. The crucial step in its activation is autophosphorylation on Tyr-397, which promotes the recruitment of several enzymes including Src family kinases and the activation of multiple signaling pathways. We found in a yeast two-hybrid screen that the N-terminal domain of FAK interacted with protein inhibitor of activated STAT1 (PIAS1). This interaction was confirmed and shown to be direct using in vitro assays. PIAS1 was co-immunoprecipitated with FAK from transfected cells and brain extracts. PIAS1 has recently been recognized as a small ubiquitin-like modifier (SUMO) ligase. In the presence of PIAS1 and SUMO-1, FAK was sumoylated in intact cells, whereas PYK2, a closely related enzyme, was not. Sumoylation occurred on Lys-152, a residue conserved in FAK during evolution. Sumoylated FAK, like PIAS1, was recovered predominantly from the nuclear fraction. Sumoylation did not require the catalytic activity or autophosphorylation of FAK. In contrast, sumoylation increased dramatically the ability of FAK to autophosphorylate in intact cells and in immune precipitate kinase assays. Endogenous FAK was sumoylated in the presence of PIAS1 and SUMO-1 independently of cell adhesion, and autophosphorylation of sumoylated FAK was persistently increased in suspended cells. These observations show that sumoylation controls the activity of a protein kinase and suggest that FAK may play a novel role in signaling between the plasma membrane and the nucleus.

Amino Acid Sequence↗

PubMed2004

Force-induced focal adhesion translocation: effects of force amplitude and frequency.

Vascular endothelial cells rapidly transduce local mechanical forces into biological signals through numerous processes including the activation of focal adhesion sites. To examine the mechanosensing capabilities of these adhesion sites, focal adhesion translocation was monitored over the course of 5 min with GFP-paxillin while applying nN-level magnetic trap shear forces to the cell apex via integrin-linked magnetic beads. A nongraded steady-load threshold for mechanotransduction was established between 0.90 and 1.45 nN. Activation was greatest near the point of forcing (<7.5 microm), indicating that shear forces imposed on the apical cell membrane transmit nonuniformly to the basal cell surface and that focal adhesion sites may function as individual mechanosensors responding to local levels of force. Results from a continuum, viscoelastic finite element model of magnetocytometry that represented experimental focal adhesion attachments provided support for a nonuniform force transmission to basal surface focal adhesion sites. To further understand the role of force transmission on focal adhesion activation and dynamics, sinusoidally varying forces were applied at 0.1, 1.0, 10, and 50 Hz with a 1.45 nN offset and a 2.25 nN maximum. At 10 and 50 Hz, focal adhesion activation did not vary with spatial location, as observed for steady loading, whereas the response was minimized at 1.0 Hz. Furthermore, applying the tyrosine kinase inhibitors genistein and PP2, a specific Src family kinase inhibitor, showed tyrosine kinase signaling has a role in force-induced translocation. These results highlight the mutual importance of force transmission and biochemical signaling in focal adhesion mechanotransduction.

Animals↗

PubMed2001

Proteolysis of the docking protein HEF1 and implications for focal adhesion dynamics.

The dynamic regulation of focal adhesions is implicated in cellular processes of proliferation, differentiation, migration, and apoptosis. The focal adhesion-associated docking protein HEF1 is cleaved by caspases during both mitosis and apoptosis. Common to both of these cellular processes is the loss of focal adhesions, transiently during mitosis and permanently during apoptosis. The proteolytic processing of HEF1 during both mitosis and apoptosis therefore posits a general role for HEF1 as a sensor of altered adhesion states. In this study, we find that HEF1 undergoes proteolytic processing specifically in response to cellular detachment, while HEF1 proteolysis is prevented by specific integrin receptor ligation and focal adhesion formation. We show that overexpression of a C-terminal caspase-derived 28-kDa HEF1 peptide causes cellular rounding that is demonstrably separable from apoptosis. Mutation of the divergent helix-loop-helix motif found in 28-kDa HEF1 significantly reduces the induction of apoptosis by this peptide, while deletion of the amino-terminal 28 amino acids of 28-kDa HEF1 completely abrogates the induction of apoptosis. Conversely, these mutations have no effect on the rounding induced by 28-kDa HEF1. Finally, we detect a novel focal adhesion targeting domain located in the C terminus of HEF1 and show that this activity is necessary for HEF1-induced cell spreading. Together, these data suggest that proteolytic and other posttranslational modifications of HEF1 in response to loss of adhesion serve to modulate the disassembly of focal adhesions.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Gastrin-releasing peptide mediates its morphogenic properties in human colon cancer by upregulating intracellular adhesion protein-1 (ICAM-1) via focal adhesion kinase.

Gastrin-releasing peptide (GRP) and its receptor (GRPR) act as morphogens when expressed in colorectal cancer (CRC), promoting the assumption of a better differentiated phenotype by regulating cell motility in the context of remodeling and retarding tumor cell metastasis by enhancing cell-matrix attachment. Although we have shown that these processes are mediated by focal adhesion kinase (FAK), the downstream target(s) of GRP-induced FAK activation are not known. Since osteoblast differentiation is mediated by FAK-initiated upregulation of ICAM-1 (Nakayamada S, Okada Y, Saito K, Tamura M, Tanaka Y. J Biol Chem 278: 45368-45374, 2003), we determined whether GRP-induced activation of FAK alters ICAM-1 expression in CRC and, if so, determined the contribution of ICAM-1 to mediating GRP's morphogenic properties. Caco-2 and HT-29 cells variably express GRP/GRPR. These cells only express ICAM-1 when GRPR are present. In human CRC, GRPR and ICAM-1 are only expressed by better differentiated tumor cells, with ICAM-1 located at the basolateral membrane. ICAM-1 expression was only observed subsequent to GRPR signaling via FAK. To study the effect of ICAM-1 expression on tumor cell motility, CRC cells expressing GRP, GRPR, and ICAM-1 were cultured in the presence and absence of GRPR antagonist or monoclonal antibody to ICAM-1. CRC cells engaged in directed motility in the context of remodeling and were highly adherent to the extracellular matrix, only in the absence of antagonist or ICAM-1 antibody. These data indicate that GRP upregulation of ICAM-1 via FAK promotes tumor cell motility and attachment to the extracellular matrix.

Amino Acid Sequence↗

PubMed1999

Dibutyryl cyclic AMP-induced process formation in astrocytes is associated with a decrease in tyrosine phosphorylation of focal adhesion kinase and paxillin.

Focal adhesion kinase (FAK or pp125FAK) is a cytosolic protein tyrosine kinase which plays an important role in integrin-mediated signal transduction. Adhesion of cells to the substratum correlates with an increase in tyrosine phosphorylation of FAK as well as an associated protein, paxillin. In this report we show that the tyrosine phosphorylation of FAK and paxillin are decreased during dibutyryl cyclic AMP-induced (dB-cAMP) process formation in astrocytes. When astrocytes in suspension are treated with dB-cAMP, no alteration in morphology or tyrosine phosphorylation is observed, suggesting that both phenomena are linked and adhesion dependent. Furthermore, genistein, a tyrosine kinase inhibitor, can induce process formation in such cells, underscoring the significance of protein tyrosine kinases in maintaining the morphology of adherent cells. Finally, endothelin-1, a vasopeptide which is known to inhibit process formation in astrocytes, inhibited the tyrosine dephosphorylation of proteins associated with dB-cAMP treatment. These results suggest that the formation of asymmetric processes in astrocytes results from a coordinated set of alterations in the actin cytoskeleton as well as the adhesion of the cell to the substratum. Modification of the properties of such molecules is required for process formation and the dynamic modulation of astrocytic morphology in vitro and in vivo.

Actins↗

PubMedDate not supplied

Integrin-mediated signal transduction linked to Ras pathway by GRB2 binding to focal adhesion kinase.

The cytoplasmic focal adhesion protein-tyrosine kinase (FAK) localizes with surface integrin receptors at sites where cells attach to the extracellular matrix. Increased FAK tyrosine phosphorylation occurs upon integrin engagement with fibronectin. Here we show that adhesion of murine NIH3T3 fibroblasts to fibronectin promotes SH2-domain-mediated association of the GRB2 adaptor protein and the c-Src protein-tyrosine kinase (PTK) with FAK in vivo, and also results in activation of mitogen-activated protein kinase (MAPK). In v-Src-transformed NIH3T3, the association of v-Src, GRB2 and Sos with FAK is independent of cell adhesion to fibronectin. The GRB2 SH2 domain binds directly to tyrosine-phosphorylated FAK. Mutation of tyrosine residue 925 of FAK (YENV motif) to phenylalanine blocks GRB2 SH2-domain binding to FAK in vitro. Our results show that fibronectin binding to integrins on NIH3T3 fibroblasts promotes c-Src and FAK association and formation of an integrin-activated signalling complex. Phosphorylation of FAK at Tyr 925 upon fibronectin stimulation creates an SH2-binding site for GRB2 which may link integrin engagement to the activation of the Ras/MAPK signal transduction pathway.

3T3 Cells↗

PubMed1998

Adhesion to fibronectin's EDb domain induces tyrosine phosphorylation of focal adhesion proteins in Balb/c 3T3 cells.

Balb/c 3T3 cell adhesion on substrata coated with fibronectin's (FN) alternatively-spliced EDb, implicated in some tumor cell systems, and its neighboring type III repeats (III7 and III8) induced intracellular signaling coincident with morphological responses. These events were analysed using minigene-expressed proteins containing various permutations of type III repeats of FN. Cells adherent to the tri-repeat protein 7-EDb-8 were compared to those attached to the tri-repeat 8-9-10 which can interact with integrins through RGD and its synergistic sequences. Cell adhesion to 7-EDb-8 generated rapid tyrosine phosphorylation of several intracellular proteins (particularly the complex at 120-130 kD), with the overall phosphorylation level and its sensitivity to tyrosine kinase inhibitors similar to responses on the 8-9-10 tri-repeat. This similarity contrasted with the differential morphological responses of cells mediated by these proteins. Further analysis of the kinetics of phosphorylation through immunoblotting of two focal adhesion proteins, p125FAK and pl30cas, revealed a profile for Balb/c 3T3 adhesion to 7-EDb-8 distinct from that on 8-9-10. EDb mono-repeat was also more potent for inducing both limited cell spreading and FAK tyrosine phosphorylation than its neighboring repeats III7 or III8. Examination of cellular localization of FAK and vinculin showed that cells spread on the 7-EDb-8 substratum displayed vinculin-positive focal complex-like structures at the cell periphery, in contrast to the classical focal adhesions seen in 8-9-10-adherent cells. These results suggest that EDb induces cell signaling events, leading to tyrosine phosphorylation of focal adhesion proteins, through a mechanism different from that mediated by integrins recognizing sequences in III8-9-10. EDb-dependent signaling may have special significance in some tumor systems.

3T3 Cells↗

PubMedDate not supplied

[Focal adhesion kinase (FAK), a multifunctional protein].

Focal adhesion kinase (FAK) is a cytoplasmic protein tyrosine kinase localized to regions called focal adhesions. Many stimuli can induce tyrosine phosphorylation and activation of FAK, including integrins and growth factors. The major site of autophosphorylation, tyrosine 397, is a docking site for the SH2 domains of Src family proteins. The other sites of phosphorylation are phosphorylated by Src kinases. Phosphorylated FAK binds proteins of focal adhesion and can activate them directly or indirectly by phosphorylation. These activated proteins forming the FAK complex facilitate the generation of downstream signals necessary to regulate cell functions, like motility, survival and proliferation. Dysregulation of FAK could participate in the development of cancer. This review will focus upon the mechanisms by which FAK transmits biochemical signals and elicits biological effects.

Animals↗

PubMed2002

The structural basis of localization and signaling by the focal adhesion targeting domain.

The localization of focal adhesion kinase (FAK) to sites of integrin clustering initiates downstream signaling. The C-terminal focal adhesion targeting (FAT) domain causes this localization by interacting with talin and paxillin. FAT also mediates signaling through Grb2 via phosphorylated Y925. We report two crystal structures of the FAT domain. Large rearrangements of the structure are indicated to allow phosphorylation of Y925 and subsequent interaction with Grb2. Sequence homology and structural compatibility suggest a FAT-like fold for the C-terminal domains of CAS, Efs/Sin, and HEF1. A structure-based alignment including these proteins and the vinculin tail domain reveals a conserved region that could play a role in focal adhesion targeting. Previously postulated "paxillin binding subdomains" may contribute to structural integrity rather than directly to paxillin binding.

Adaptor Proteins, Signal Transducing↗

PubMed1994

Focal adhesion kinase and associated proteins.

Focal adhesion kinase (pp125FAK), a protein tyrosine kinase, has recently been suggested to regulate aspects of signalling induced by integrins and by certain growth factor and hormone receptors. New studies indicate that the domains of pp125FAK regulate its interactions with integrin subunits, other protein tyrosine kinases, and the focal adhesion associated protein paxillin. Through these interactions, pp125FAK may regulate signalling via different extracellular ligands.

Animals↗

PubMed2003

[Study of adhesion-related molecule beta1-integrin and focal adhesion kinase in chronic myeloid leukemia].

OBJECTIVE: To study the changes in the expressions of beta1-integrin receptor (CD29) and focal adhesion kinase (FAK) during the deterioration of chronic myeloid leukemia (CML), thereby explore the potential mechanism of interferon (IFN)-alpha in the treatment of CML through testing the quantitative changes of FAK. METHODS: Bone marrow mononuclear cells were tested for CD34, CD29 and FAK monoclonal antibody by flow cytometry in CML patients of the chronic phase and blast crisis and in normal subjects. In the presence or absence of IFN-alpha, the bone marrow mononuclear cells from CML patients of chronic phase and normal subjects were cultured for 48 h to determine the content of FAK in the cells with Western blotting. RESULTS: CD 29 expression on the surface of CD34+ cells scarcely differ between normal subjects and CML patients of chronic-phase, but the intracellular content of FAK was lower in the latter. The expression of beta1-integrin receptor on the surface of hematopoietic cells in CML patients with blast crisis was significantly higher than that in chronic-phase CML patients, but the intracellular FAK in the former patients was lower. No difference of FAK content was observed in normal mononuclear cells before and after IFN-alpha treatment, while the treatment increased FAK content in the mononuclear cells in chronic phase CML patients. CONCLUSION: The changes in the expressions of beta1-integrin receptor and FAK may play important roles during CML deterioration, and IFN-alpha may function to repair the defect in the beta1-integrin receptor pathway by restoring the cellular content of FAK.

Adult↗

PubMed1999

DFak56 is a novel Drosophila melanogaster focal adhesion kinase.

The mammalian focal adhesion kinase (FAK) family of nonreceptor protein-tyrosine kinases have been implicated in controlling a multitude of cellular responses to the engagement of cell surface integrins and G protein-coupled receptors. We describe here a Drosophila melanogaster FAK homologue, DFak56, which maps to band 56D on the right arm of the second chromosome. Full-length DFak56 cDNA encodes a phosphoprotein of 140 kDa, which shares strong sequence similarity not only with mammalian p125(FAK) but also with the more recently described mammalian Pyk2 (also known as CAKbeta, RAFTK, FAK2, and CADTK) FAK family member. DFak56 has intrinsic tyrosine kinase activity and is phosphorylated on tyrosine in vivo. As is the case for FAK, tyrosine phosphorylation of DFak56 is increased upon plating Drosophila embryo cells on extracellular matrix proteins. In situ hybridization and immunofluorescence staining analysis showed that DFak56 is ubiquitously expressed with particularly high levels within the developing central nervous system. We utilized the UAS-GAL4 expression system to express DFak56 and analyze its function in vivo. Overexpression of DFak56 in the wing imaginal disc results in wing blistering in adults, a phenotype also observed with both position-specific integrin loss of function and position-specific integrin overexpression. Our results imply a role for DFak56 in adhesion-dependent signaling pathways in vivo during D. melanogaster development.

Amino Acid Sequence↗

PubMed1997

Contact between human bone marrow stromal cells and B lymphocytes enhances very late antigen-4/vascular cell adhesion molecule-1-independent tyrosine phosphorylation of focal adhesion kinase, paxillin, and ERK2 in stromal cells.

Contact with bone marrow stromal cells is crucial for the normal growth and development of B-cell precursors. We have previously shown that human bone marrow stromal cell tyrosine kinase activity can be activated by direct contact with B-lymphoid cells (J Immunol 155:2359, 1995). In the present study, we show that increased tyrosine phosphorylation of focal adhesion kinase, paxillin, and extracellular-related kinase 2 (or p42 MAP kinase) accounted for the major changes occurring in stromal cell tyrosine phosphorylation after 5 to 10 minutes of contact with the RAMOS B-lymphoma cell line. Although adhesion of B-cell precursors to stromal cells is primarily mediated by very late antigen-4 (VLA-4) and vascular cell adhesion molecule-1 (VCAM-1), VLA-4-deficient and adhesion-deficient RAMOS cells were equally capable of stimulating stromal cell tyrosine phosphorylation. Similar changes in the tyrosine phosphorylation pattern of stromal cells were induced by contact with normal human B-cell precursors and several other B-lineage cell lines. After 5 to 30 minutes of contact with stromal cells, no change in protein tyrosine phosphorylation was detected in RAMOS or normal human B-cell precursors removed from stromal cells. Pretreatment of stromal cells with cytochalasin D abrogated contact-mediated enhancement of stromal cell tyrosine phosphorylation, suggesting that an intact cytoskeleton was essential. These results suggest that B-cell contact activates stromal cell signaling cascades that regulate cytoskeletal organization and transcription, independent of the interaction mediated by VLA-4 and VCAM-1.

B-Lymphocytes↗