Phosphorylation of c-Cbl and p85 PI3K driven by all-trans retinoic acid and CD38 depends on Lyn kinase activity

The leukocyte antigen CD38 is expressed after all-trans retinoic acid (ATRA) treatment in HL-60 myelogenous leukemia cells and promotes induced myeloid differentiation when overexpressed. We found that Vav1 and SLP-76 associate with CD38 in two cell lines, and that these proteins complex with Lyn, a Src family kinase (SFK) upregulated by ATRA. SFK inhibitors PP2 and dasatinib, which enhance ATRA-induced differentiation, were used to evaluate the involvement of Lyn kinase activity in CD38-driven signaling. Cells treated with ATRA for 48 h followed by one hour of PP2 incubation show SFK/Lyn kinase inhibition. We observed that Lyn inhibition blocked c-Cbl and p85/p55 PI3K phosphorylation driven by the anti-CD38 agonistic mAb IB4 in ATRA-treated HL-60 cells and untreated CD38 + transfectants. In contrast, cells cultured for 48 h following concurrent ATRA and PP2 treatment did not show Lyn inhibition, suggesting ATRA regulates the effects on Lyn. 48 h of co-treatment preserved CD38-stimulated c-Cbl and p85/p55 PI3K phosphorylation indicating Lyn kinase activity is necessary for these events. In contrast another SFK inhibitor (dasatinib) which blocks Lyn activity with ATRA co-treatment prevented ATRA-induced c-Cbl phosphorylation and crippled p85 PI3K phosphorylation, indicating Lyn kinase activity is important for ATRA-propelled events potentially regulated by CD38. We found that loss of Lyn activity coincided with a decrease in Vav1/Lyn/CD38 and SLP-76/Lyn/CD38 interaction, suggesting these molecules form a complex that regulates CD38 signaling. Lyn inhibition also reduced Lyn and CD38 binding to p85 PI3K, indicating CD38 facilitates a complex responsible for PI3K phosphorylation. Therefore, Lyn kinase activity is important for CD38-associated signaling that may drive ATRA-induced differentiation.     

Fibronectin promotes cervical cancer tumorigenesis through activating FAK signaling pathway

Cervical cancer is a cancer arising from the cervix, and it is the fourth most common cause of death in women. Overexpression of fibronectin 1 (FN1) was observed in many tumors and associated with the survival and metastasis of cancer cells. However, the mechanism by which FN1 promotes cervical cancer cell viability, migration, adhesion, and invasion, and inhibits cell apoptosis through focal adhesion kinase (FAK) signaling pathway remains to be investigated. Our results demonstrated that FN1 was upregulated in patients with cervical cancer and higher FN1 expression correlated with a poor prognosis for patients with cervical cancer. FN1 knockdown by small interfering RNA (siRNA) inhibited SiHa cell viability, migration, invasion, and adhesion, and promoted cell apoptosis. FN1 overexpression in CaSki cell promoted cell viability, migration, invasion, and adhesion, and inhibited cell apoptosis. Further, phosphorylation of FAK, a main downstream signaling molecule of FN1, and the protein expression of Bcl-2/Bax, matrix metalloproteinase 2 (MMP-2), matrix metalloproteinase 9 (MMP-9), and N-cadherin was upregulated in CaSki cells with FN1 overexpression, but caspase-3 protein expression was downregulated. The FAK phosphorylation inhibitor PF573228 inhibited FN1 overexpression-induced expression of those proteins in CaSki cells with FN1 overexpression. In vivo experiment demonstrated that FN1 knockdown significantly inhibited FN1 expression, phosphorylation of FAK, and tumor growth in xenograft from the nude mice. These results suggest that FN1 regulates the viability, apoptosis, migration, invasion, and adhesion of cervical cancer cells through the FAK signaling pathway and is a potential therapeutic target in the treatment of cervical cancer.     

The Src family kinase, Lyn, is activated in pancreatic acinar cells by gastrointestinal hormones/neurotransmitters and growth factors which stimulate its association with numerous other signaling molecules

Src family kinases (SFK) play a central signaling role for growth factors, cytokines, G-protein-coupled receptors and other stimuli. SFKs play important roles in pancreatic acinar cell secretion, endocytosis, growth, cytoskeletal integrity and apoptosis, although little is known of the specific SFKs involved. In this study we demonstrate the SFK, Lyn, is present in rat pancreatic acini and investigate its activation/signaling. Ca(2+)-mobilizing agents, cAMP-mobilizing agents and pancreatic growth factors activated Lyn. CCK, a physiological regulator of pancreatic function, rapidly activated Lyn. The specific SFK inhibitor, PP2, decreased Lyn activation; however, the inactive analogue, PP3, had no effect. Inhibition of CCK-stimulated changes in [Ca(2+)](i) decreased Lyn activation by 55%; GFX, a PKC inhibitor by 36%; and the combination by 95%. CCK activation of Lyn required stimulation of high and low affinity CCK(A) receptor states. CCK stimulated an association of Lyn with PKC-delta, Shc, p125(FAK) and PYK2 as well as with their autophosphorylated forms, but not with Cbl, p85, p130(CAS) or ERK 1/2. These results show Lyn is activated by diverse pancreatic stimulants. CCK's activation of Lyn is likely an important mediator of its ability to cause tyrosine phosphorylation of numerous important cellular mediators such as p125(FAK), PYK2, PKC-delta and Shc, which play central roles in CCK's effects on acinar cell function.     

Regulatory role for SRC and phosphatidylinositol 3-kinase in initiation of fibronectin matrix assembly

Fibronectin (FN) matrix assembly is a tightly regulated stepwise process that is initiated by interactions between FN and cell surface integrin receptors. These interactions activate many intracellular signaling pathways that regulate processes such as cell adhesion, migration, and survival. Here we demonstrate that cells lacking Src family kinases showed reduced ability to assemble FN fibrils as detected by immunofluorescence and by analysis of detergent extracts. The amount of FN matrix was further reduced by treatment with the phosphatidylinositol 3 (PI 3-kinase) inhibitor, wortmannin. CHOalpha5 cells, which are dependent on exogenous FN to initiate fibril formation, also showed significant reductions in matrix when treated with inhibitors of Src and PI 3-kinase. Combination of both inhibitors showed an additive inhibitory effect on assembly, which was concomitant with a loss of focal adhesion kinase phosphorylation. Decreased binding of the 70-kDa amino-terminal FN fragment at matrix assembly sites further supports a role for these kinases early during the process. We propose that these two signaling molecules, which lie downstream of integrins and focal adhesion kinase, are essential for efficient initiation of FN matrix assembly.     

Rapid trafficking of c-Src, a non-palmitoylated Src-family kinase, between the plasma membrane and late endosomes/lysosomes

Src-family kinases (SFKs) are co-expressed with multiple combinations of each member in a single cell and involved in various signalings. Recently, we showed by sucrose-density gradient fractionation that the subcellular distribution of c-Src is distinct from that of Lyn. However, little is known about the trafficking of c-Src in living cells. Here, we show by time-lapse monitoring combined with photobleaching techniques that c-Src, a non-palmitoylated SFK, is rapidly exchanged between the plasma membrane and intracellular organelles representing late endosomes/lysosomes possibly through its cytosolic release. Although Lyn, a palmitoylated SFK, is exocytosed to the plasma membrane via the Golgi apparatus along the secretory pathway, lack of palmitoylation directs Lyn away from the exocytotic transport to the c-Src-type trafficking between the plasma membrane and late endosomes/lysosomes. Intriguingly, c-Src and a non-palmitoylated Lyn mutant are efficiently delivered and immobilized to focal adhesions when their SH2 domains are able to mediate protein-protein interactions in place of intramolecular bindings. However, palmitoylation of Lyn inhibits its recruitment to focal adhesions. These results suggest that palmitoylation of SFKs is critical for SFK localization and trafficking and implicate that two distinct trafficking pathways for SFKs may be involved in SFKs' specific functions.     

Cellular settings mediating Src Substrate switching between focal adhesion kinase tyrosine 861 and CUB-domain-containing protein 1 (CDCP1) tyrosine 734

Reciprocal interactions between Src family kinases (SFKs) and focal adhesion kinase (FAK) are critical during changes in cell attachment. Recently it has been recognized that another SFK substrate, CUB-domain-containing protein 1 (CDCP1), is differentially phosphorylated during these events. However, the molecular processes underlying SFK-mediated phosphorylation of CDCP1 are poorly understood. Here we identify a novel mechanism in which FAK tyrosine 861 and CDCP1-Tyr-734 compete as SFK substrates and demonstrate cellular settings in which SFKs switch between these sites. Our results show that stable CDCP1 expression induces robust SFK-mediated phosphorylation of CDCP1-Tyr-734 with concomitant loss of p-FAK-Tyr-861 in adherent HeLa cells. SFK substrate switching in these cells is dependent on the level of expression of CDCP1 and is also dependent on CDCP1-Tyr-734 but is independent of CDCP1-Tyr-743 and -Tyr-762. In HeLa CDCP1 cells, engagement of SFKs with CDCP1 is accompanied by an increase in phosphorylation of Src-Tyr-416 and a change in cell morphology to a fibroblastic appearance dependent on CDCP1-Tyr-734. SFK switching between FAK-Tyr-861 and CDCP1-Tyr-734 also occurs during changes in adhesion of colorectal cancer cell lines endogenously expressing these two proteins. Consistently, increased p-FAK-Tyr-861 levels and a more epithelial morphology are seen in colon cancer SW480 cells silenced for CDCP1. Unlike protein kinase Cδ, FAK does not appear to form a trimeric complex with Src and CDCP1. These data demonstrate novel aspects of the dynamics of SFK-mediated cell signaling that may be relevant during cancer progression.     

Friction-controlled traction force in cell adhesion

The force balance between the extracellular microenvironment and the intracellular cytoskeleton controls the cell fate. We report a new (to our knowledge) mechanism of receptor force control in cell adhesion originating from friction between cell adhesion ligands and the supporting substrate. Adherent human endothelial cells have been studied experimentally on polymer substrates noncovalently coated with fluorescent-labeled fibronectin (FN). The cellular traction force correlated with the mobility of FN during cell-driven FN fibrillogenesis. The experimental findings have been explained within a mechanistic two-dimensional model of the load transfer at focal adhesion sites. Myosin motor activity in conjunction with sliding of FN ligands noncovalently coupled to the surface of the polymer substrates is shown to result in a controlled traction force of adherent cells. We conclude that the friction of adhesion ligands on the supporting substrate is important for mechanotransduction and cell development of adherent cells in vitro and in vivo.     

Roscovitine enhances All-trans retinoic acid (ATRA)-induced leukemia cell differentiation: Novel effects on signaling molecules for a putative Cdk2 inhibitor

All-trans retinoic acid (ATRA)-based differentiation therapy has been unsuccessful in treating t(15;17) negative acute myeloid leukemia (AML) patients, motivating interest in combination therapies using ATRA plus other agents. Using the t (15, 17) negative HL-60 human myeloblastic leukemia model, we find that the cyclin-dependent kinase (CDK) inhibitor, roscovitine, augments signaling by an ATRA-induced macromolecular signalsome that propels differentiation and enhances ATRA-induced differentiation. Roscovitine co-treatment enhanced ATRA-induced expression of pS259- pS289/296/301- pS621-c-Raf, pS217/221-Mek, Src Family Kinases (SFKs) Lyn and Fgr and SFK Y416 phosphorylation, adaptor proteins c-Cbl and SLP-76, Vav, and acetylated 14–3-3 in the signalsome. Roscovitine enhanced ATRA-induced c-Raf interaction with Lyn, Vav, and c-Cbl. Consistent with signalsome hyper-activation, roscovitine co-treatment enhanced ATRA-induced G1/0 arrest and expression of differentiation markers, CD11b, ROS and p47 Phox. Because roscovitine regulated Lyn expression, activation and partnering, a stably transfected Lyn knockdown was generated from wt-parental cells to investigate its function in ATRA-induced differentiation. Lyn-knockdown enhanced ATRA-induced up-regulation of key signalsome molecules, c-Raf, pS259-c-Raf, pS289/296/301-c-Raf, Vav1, SLP-76, and Fgr, but with essentially total loss of pY416-SFK. Compared to ATRA-treated wt-parental cells, differentiation markers p47 phox, CD11b, G1/G0 arrest and ROS production were enhanced in ATRA-treated Lyn-knockdown stable transfectants, and addition of roscovitine further enhanced these ATRA-inducible markers. The Lyn-knockdown cells expressed slightly higher c-Raf, pS259-c-Raf, pS289/296/301-c-Raf, and SLP-76 than wt-parental cells, and this was associated with enhanced ATRA-induced upregulation of Fgr and cell differentiation, consistent with heightened signaling, suggesting that enhanced Fgr may have compensated for loss of Lyn to enhance differentiation in the Lyn-knockdown cells.     

Real-time monitoring of hematopoietic cell interaction with fibronectin fragment

Real-time cell analysis (RTCA) system based on measurement of electrical microimpedance has been introduced to monitor adherent cell cultures. We describe its use for real-time analysis of hematopoietic cell adhesion to bone marrow stroma proteins. Cells growing in suspension do not generate any significant change in the microimpedance signal until the surface with embedded microelectrodes is coated with a cell-binding protein. We show that in this case, the microimpedance signal specifically reflects cell binding to the coated surface. The optimized method was used to monitor the effect of two histone deacetylase inhibitors, suberoylanilide hydroxamic acid (SAHA) and tubastatin A, on JURL-MK1 cell adhesion to cell-binding fragment of fibronectin (FNF). Both compounds were used in non-toxic concentrations and induced an increase in the cell adhesivity. The kinetics of this increase was markedly slower for SAHA although tubulin hyperacetylation occurred rapidly for any of the two drugs. The strengthening of cell binding to FNF was paralleled with a decrease of Lyn kinase activity monitored using an anti-phospho-Src family antibody. The inhibition of Src kinase activity with PP2 accordingly enhanced JURL-MK1 cell interaction with FNF. Actin filaments were present at the proximity of the plasma membrane and in numerous membrane protrusions. In some cells, F-actin formed clusters at membrane regions interacting with the coated surface and these clusters colocalized with active Lyn kinase. Our results indicate that the role of Src kinases in the regulation of hematopoetic cell adhesion signaling is similar to that of c-Src in adherent cells.     

Cleavage of focal adhesion kinase by different proteases during SRC-regulated transformation and apoptosis. Distinct roles for calpain and caspases

Integrin-associated focal adhesion complexes provide the main adhesive links between the cellular actin cytoskeleton and the surrounding extracellular matrix. In vitro, cells utilize a complex temporal and spatially regulated mechanism of focal adhesion assembly and disassembly required for cell migration. Recent studies indicate that members of both calpain and caspase protease families can promote limited proteolytic cleavage of several components of focal adhesions leading to disassembly of these complexes. Such mechanisms that influence cell adhesion may be deregulated under pathological conditions characterized by increased cell motility, such as tumor invasion. v-Src-induced oncogenic transformation is associated with loss of focal adhesion structures and transition to a less adherent, more motile phenotype, while inactivating temperature-sensitive v-Src in serum-deprived transformed cells leads to detachment and apoptosis. In this report, we demonstrate that v-Src-induced disassembly of focal adhesions is accompanied by calpain-dependent proteolysis of focal adhesion kinase. Furthermore, inhibitors of calpain repress v-Src-induced focal adhesion disruption, loss of substrate adhesion, and cell migration. In contrast, focal adhesion loss during detachment and apoptosis induced after switching off temperature-sensitive v-Src in serum-deprived transformed cells is accompanied by caspase-mediated proteolysis of focal adhesion kinase. Thus, calpain and caspase differentially regulate focal adhesion turnover during Src-regulated cell transformation, motility, and apoptosis.     

A new type of substratum adhesion structure in NRK cells revealed by correlated interference reflection and electron microscopy

The substratum adherent membrane of NRK cells in vitro has been studied using correlated interference reflection and surface replica electron microscopy. Structures visualized by interference reflection microscopy were identified in the subsequently prepared platinum-carbon replicas of the adherent membranes. Substratum adherent membranes were prepared using a hypotonic shock/cell shearing technique (lysis-squirting). Typically 20% of the original ventral membrane surface area and 50% of the original focal adhesion number were retained. Microfilament bundle termini, clathrin-coated sheets and pits, cytotic vesicles having a ridged surface and groups of membrane associated particles were well preserved in the replicas. Two types of isolated adhesion structures were found after lysis-squirting. In addition to typical elongate focal adhesions containing actin and vinculin, we report the existence of adherent membrane patches lacking microfilament bundles and negative for vinculin labelling, but coated with clathrin and identifiable in interference reflection microscopy as less dark than focal adhesions and having dot, U- or sinusoidal shapes.     

Cross talk of pp125(FAK) and pp59(Lyn) non-receptor tyrosine kinases to insulin-mimetic signaling in adipocytes

Signaling molecules downstream from the insulin receptor, such as the insulin receptor substrate protein 1 (IRS-1), are also activated by other receptor tyrosine kinases. Here we demonstrate that the non-receptor tyrosine kinases, focal adhesion kinase pp125(FAK) and Src-class kinase pp59(Lyn), after insulin-independent activation by phosphoinositolglycans (PIG), can cross talk to metabolic insulin signaling in rat and 3T3-L1 adipocytes. Introduction by electroporation of neutralizing antibodies against pp59(Lyn) and pp125(FAK) into isolated rat adipocytes blocked IRS-1 tyrosine phosphorylation in response to PIG but not insulin. Introduction of peptides encompassing either the major autophosphorylation site of pp125(FAK), tyrosine 397, or its regulatory loop with the twin tyrosines 576 and 577 inhibited PIG-induced IRS-1 tyrosine phosphorylation and glucose transport. PIG-induced pp59(Lyn) kinase activation and pp125(FAK) tyrosine phosphorylation were impaired by the former and latter peptide, respectively. Up-regulation of pp125(FAK) by integrin clustering diminished PIG-induced IRS-1 tyrosine phosphorylation and glucose transport in nonadherent but not adherent adipocytes. In conclusion, PIG induced IRS-1 tyrosine phosphorylation by causing (integrin antagonized) recruitment of IRS-1 and pp59(Lyn) to the common signaling platform molecule pp125(FAK), where cross talk of PIG-like structures and extracellular matrix proteins to metabolic insulin signaling may converge, possibly for the integration of the demands of glucose metabolism and cell architecture.     

Rho-ROCK signaling differentially regulates chondrocyte spreading on fibronectin and bone sialoprotein

The mammalian growth plate is a dynamic structure rich in extracellular matrix (ECM). Interactions of growth plate chondrocytes with ECM proteins regulate cell behavior. In this study, we compared chondrocyte adhesion and spreading dynamics on fibronectin (FN) and bone sialoprotein (BSP). Chondrocyte adhesion and spreading were also compared with fibroblasts to analyze potential cell-type-specific effects. Chondrocyte adhesion to BSP is independent of posttranslational modifications but is dependent on the RGD sequence in BSP. Whereas chondrocytes and fibroblasts adhered at similar levels on FN and BSP, cells displayed more actin-dependent spread on FN despite a 16x molar excess of BSP adsorbed to plastic. To identify intracellular mediators responsible for this difference in spreading, we investigated focal adhesion kinase (FAK)-Src and Rho-Rho kinase (ROCK) signaling. Although activated FAK localized to the vertices of adhered chondrocytes, levels of FAK activation did not correlate with the extent of spreading. Furthermore, Src inhibition reduced chondrocyte spreading on both FN and BSP, suggesting that FAK-Src signaling is not responsible for less cell spreading on BSP. In contrast, inhibition of Rho and ROCK in chondrocytes increased cell spreading on BSP and membrane protrusiveness on FN but did not affect cell adhesion. In fibroblasts, Rho inhibition increased fibroblast spreading on BSP while ROCK inhibition changed membrane protrusiveness of FN and BSP. In summary, we identify a novel role for Rho-ROCK signaling in regulating chondrocyte spreading and demonstrate both cell- and matrix molecule-specific mechanisms controlling cell spreading.     

PTP alpha regulates integrin-stimulated FAK autophosphorylation and cytoskeletal rearrangement in cell spreading and migration

We investigated the molecular and cellular actions of receptor protein tyrosine phosphatase (PTP) alpha in integrin signaling using immortalized fibroblasts derived from wild-type and PTP alpha-deficient mouse embryos. Defects in PTP alpha-/- migration in a wound healing assay were associated with altered cell shape and focal adhesion kinase (FAK) phosphorylation. The reduced haptotaxis to fibronectin (FN) of PTP alpha-/- cells was increased by expression of active (but not inactive) PTP alpha. Integrin-mediated formation of src-FAK and fyn-FAK complexes was reduced or abolished in PTP alpha-/- cells on FN, concomitant with markedly reduced phosphorylation of FAK at Tyr397. Reintroduction of active (but not inactive) PTP alpha restored FAK Tyr-397 phosphorylation. FN-induced cytoskeletal rearrangement was retarded in PTP alpha-/- cells, with delayed filamentous actin stress fiber assembly and focal adhesion formation. This mimicked the effects of treating wild-type fibroblasts with the src family protein tyrosine kinase (Src-PTK) inhibitor PP2. These results, together with the reduced src/fyn tyrosine kinase activity in PTP alpha-/- fibroblasts (Ponniah et al., 1999; Su et al., 1999), suggest that PTP alpha functions in integrin signaling and cell migration as an Src-PTK activator. Our paper establishes that PTP alpha is required for early integrin-proximal events, acting upstream of FAK to affect the timely and efficient phosphorylation of FAK Tyr-397.     

Conformational insights into the inhibitory mechanism of phyto-compounds against Src kinase family members implicated in psoriasis

Abstract

Psoriasis is a chronic immune mediated disorder of the skin. There is growing evidence that the Src family tyrosine kinases (SFK) are highly upregulated in psoriasis. The SFK are the key components of the signaling pathways triggering cell growth and differentiation in addition to the immune cascades. In the current work, the interactions between SFK and selective phyto-compounds were studied using molecular docking approach. Based on the results of docking and binding energy calculations quercetin was identified as potential lead compound. To get a deeper insight into the binding of quercetin with the SFK, a combined molecular dynamics and binding free energy calculations were performed. The binding of quercetin disrupted the intra-molecular contacts making the SFK compact except Src kinase. The MM/PBSA free energy decomposition analysis highlighted the significance of hydrophobic and polar residues which are involved in the binding of quercetin. An experimental validation was carried out against the activated forms of Fyn, Lyn and Src kinases, the top three proteins which showed high preference for quercetin. The flow cytometry analysis showed that the expression levels of Fyn, Lyn and Src kinases were dramatically increased in HaCaT cells. However, the treatment of quercetin at the concentration of 51.65 µM for 24?h markedly decreased their expression in HaCaT cells. Besides, similar results were also observed when the HaCaT cells were treated with the kinase inhibitor Ponitinib (1.43 µM) for 24?h.

Communicated by Ramaswamy H. Sarma     

Role of Src-family kinases in formation of the cortical actin cap at the dorsal cell surface

Protein-tyrosine phosphorylation is regulated by protein-tyrosine kinases and protein-tyrosine phosphatases (PTPs). Src-family tyrosine kinases (SFKs) participate in the regulation of the actin cytoskeleton. Actin filaments can be accumulated in a cap at the dorsal cell surface, which is called the cortical actin cap. Here, we show that SFKs play an important role in formation of the cortical actin cap. HeLa cells normally exhibit the cortical actin cap, one of the major sites of tyrosine phosphorylation. The cortical actin cap is disrupted by SFK inhibitors or overexpression of the Lyn SH3 domain. Csk-knockout cells form the cortical actin cap when the level of tyrosine phosphorylation is increased by Na₃VO₄, a PTP inhibitor, and the formation of the cortical actin cap is inhibited by SFK inactivation with re-introduction of Csk. SYF cells lacking SFKs minimally exhibit the cortical actin cap even in the presence of Na₃VO₄, and transfection with Lyn restores the cortical actin cap in the presence of Na₃VO₄. Disruption of the cortical actin cap by dominant-negative Cdc42 causes loss of tyrosine phosphorylation at the cell top. These results suggest that SFK(s) is involved in formation of the cortical actin cap, which may serve as a platform of tyrosine phosphorylation signaling.     

Transglutaminase stabilizes melanoma adhesion under laminar flow

To resist substantial wall shear stress (WSS) exerted by flowing blood, metastatic melanoma cells can form adhesive contacts with subendothelial extracellular matrix proteins, such as fibronectin (FN). Such contacts may be stabilized by transglutaminase catalyzed-crosslinkage of cell focal adhesion proteins. We analyzed human melanoma cell adhesion under flow by decreasing the flow (WSS) of melanoma cell suspensions and allowing them to adhere to immobilized wheat germ agglutinin or FN. At the wall shear adhesion threshold (WSAT), cell adherence was rapid with no rolling. Following cell adherence, we increased the flow and determined the wall shear detachment threshold (WSDeT). Cells spread and remained adherent on immobilized FN at high WSDeTs (≥ 32.5 dynes/cm²). The high resistance of adherent cells to shear forces suggested that transglutaminase-mediated crosslinking might be involved. Transglutaminase inhibitors monodansylcadaverine and INO-3178 decreased WSAT, and at low concentrations completely inhibited tumor cell spreading and promoted detachment at low WSDeTs (0.67 dynes/cm²). In static adhesion assays, transglutaminase inhibitors decreased cell adhesion to immobilized-FN in a dose-dependent manner and prevented the formation of crosslinked¹²⁵I-FN complex that failed to enter a SDS-polyacrylamide gradient gel. The data suggest that transglutaminase-catalyzed crosslinking, particularly in the presence of WSS, may be important in stabilizing cellular adhesive contacts during adhesion to immobilized-FN.     

Thy-1, via its GPI anchor, modulates Src family kinase and focal adhesion kinase phosphorylation and subcellular localization, and fibroblast migration, in response to thrombospondin-1/hep I

Normal fibroblast subpopulations have differential surface expression of the GPI-linked raft protein Thy-1, which correlates with differences in cellular adhesion and migration in vitro. Thrombospondin-1 (TSP-1) induces an intermediate state of adhesion in fibroblasts and other cells which facilitates migration. TSP-1 and the hep I peptide derived from the amino-terminal/heparin-binding domain of TSP-1 induce disassembly of cellular focal adhesions. Our lab previously reported that the induction of focal adhesion disassembly in fibroblasts by TSP-1 or by hep I requires surface expression of Thy-1, as well as lipid raft integrity and Src family kinase (SFK) signaling. We now report that TSP-1/hep I-induced fibroblast migration requires Thy-1 expression and FAK phosphorylation, and that following TSP-1/hep I stimulation, Thy-1 associates with FAK and SFK in a lipid raft-dependent manner. Furthermore, the GPI anchor of Thy-1, which localizes the protein to specific lipid raft microdomains, is necessary for hep I-induced FAK and SFK phosphorylation, focal adhesion disassembly, and migration. This is the first report of an association between Thy-1 and FAK. Thy-1 modulates SFK and FAK phosphorylation and subcellular localization, promoting focal adhesion disassembly and migration in fibroblasts, following exposure to TSP-1/hep I.     

Transglutaminase stabilizes melanoma adhesion under laminar flow.

To resist substantial wall shear stress (WSS) exerted by flowing blood, metastatic melanoma cells can form adhesive contacts with subendothelial extracellular matrix proteins, such as fibronectin (FN). Such contacts may be stabilized by transglutaminase catalyzed-cross-linkage of cell focal adhesion proteins. We analyzed human melanoma cell adhesion under flow by decreasing the flow (WSS) of melanoma cell suspensions and allowing them to adhere to immobilized wheat germ agglutinin or FN. At the wall shear adhesion threshold (WSAT), cell adherence was rapid with no rolling. Following cell adherence, we increased the flow and determined the wall shear detachment threshold (WSDeT). Cells spread and remained adherent on immobilized FN at high WSDeTs (greater than or equal to 32.5 dynes/cm2). The high resistance of adherent cells to shear forces suggested that transglutaminase-mediated crosslinking might be involved. Transglutaminase inhibitors monodansylcadaverine and INO-3178 decreased WSAT, and at low concentrations completely inhibited tumor cell spreading and promoted detachment at low WSDeTs (0.67 dynes/cm2). In static adhesion assays, transglutaminase inhibitors decreased cell adhesion to immobilized-FN in a dose-dependent manner and prevented the formation of crosslinked 125I-FN complex that failed to enter a SDS-polyacrylamide gradient gel. The data suggest that transglutaminase-catalyzed crosslinking, particularly in the presence of WSS, may be important in stabilizing cellular adhesive contacts during adhesion to immobilized-FN.